Cancro e terapie naturali
Le cellule cancerose appaiono come cellule immature che si dividono costantemente, non adempiono le loro funzioni naturali e invadono il tessuto circostante. Queste cellule privano le cellule normali vicine dei loro elementi nutritivi essenziali, causando un grave esaurimento nel paziente affetto da cancro. Le cellule cancerose sono in grado di spostarsi e impiantarsi in qualsiasi parte dell’organismo causando crescite abnormi o tumori. Il cancro viene diviso a seconda del tipo di tessuto da cui nasce.
L’importanza di una diagnosi precoce nel trattamento del cancro è fondamentale. E’ l’unica possibilità di curare questa malattia con risultati positivi. Attualmente si ritiene che il cancro all’intestino inizi il suo decorso anche 20 anni prima di diventare una malattia conclamata ed essere notato. E’ importantissimo fare i test che permettono di scoprirlo. Si dovrebbe essere sempre attenti ai sette sintomi premonitori segnalati dalla American Cancer Society: sanguinamento o secrezioni insolite, comparsa di protuberanze o gonfiori, tosse rauca, difficoltà nel deglutire e nel digerire, cambiamenti nelle abitudini intestinali o della vescica, cicatrizzazione lenta, modificazione di una verruca o di un neo. Esistono dei kit che permettono di effettuare da soli un’analisi per il tumore all’intestino. I sintomi e la loro gravità variano in relazione al tipo e alla localizzazione del cancro.
E’ stato scoperto che l’obesità femminile è un fattore che aumenta il rischio di tumore uterino, cervicale, mammario e alla cistifellea. I grassi influenzano gli ormoni femminili stimolando la divisione cellulare che a sua volta dà inizio al processo cancerogeno. Negli uomini l’obesità aumenta il rischio di tumore colorettale. E’ stato riportato che gli uomini che si sono sottoposti a vasectomia hanno tre volte più possibilità di ammalarsi di tumore alla prostata.
Nel trattamento delle escrescenze cancerose e dei tumori sono stati usati la chirurgia, le radiazioni e alcuni farmaci. Le operazioni chirurgiche rimuovono la neoplasia originale e quelle secondarie. I farmaci, benché non siano in grado di curare completamente il cancro, vengono usati per ridurre la neoplasia o per ritardare l’apparizione di escrescenze secondarie. Le radiazioni vengono spesso usate per distruggere le cellule cancerose e per impedirne la diffusione.
Le sostanze nutritive possono essere d’aiuto. Gli effetti collaterali provocati dalla terapia ai raggi e dalla chemioterapia, come il vomito e la diarrea, possono essere diminuiti o evitati con le vitamine C, E e il complesso B. La vitamina E dovrebbe essere assunta prima dei pasti. E’ importante iniziare l’assunzione di queste vitamine alcuni giorni prima dell’inizio del trattamento. Lo stress psicologico del cancro aumenta notevolmente il fabbisogno di vitamina C e di vitamine del complesso B. Per assicurare un immediato assorbimento nel sangue, le vitamine, se possibile, dovrebbero essere somministrate sotto forma di iniezioni.
I raggi X e i trattamenti con altri raggi indeboliscono il sistema immunitario e distruggono le vitamine A, C, E, K, B e gli acidi grassi insaturi: grandi quantità di vitamina E proteggono la vitamina A e gli acidi grassi insaturi. Nel corso della distruzione del tessuto maligno vi è la creazione di sottoprodotti dannosi. Il fegato è in grado di neutralizzare queste sostanze se sono presenti quantità sufficienti di vitamina C, E, proteine e l’aminoacido metionina. La vitamina E può prevenire le bruciature dalle radiazioni, dare sollievo al dolore e ridurre le cicatrici.
Negli animali si sviluppano tumori spontanei, soprattutto della tiroide, dovuti ad alimentazione carente di iodio. La carenza di iodio è legata anche al cancro del seno nella donna. Si ritiene che la carenza di ferro esponga i malati della sindrome di Plummer-Vinson (un disturbo che colpisce le donne di mezza età caratterizzato da spaccature intorno alla bocca e ulcere alla lingua e all’esofago) ad un maggior rischio di tumore all’esofago e allo stomaco. Una carenza di zinco può portare al tumore alla prostata, all’esofago e al carcinoma broncogeno. I danni al fegato di qualsiasi tipo, aumentano la predisposizione al cancro.
Le ricerche eseguite sugli animali hanno mostrato che le vitamine A, C, E, B3 e B6 inibiscono la crescita delle cellule tumorali stimolando il sistema immunitario del corpo ed eliminando i radicali liberi. I lipotropi proteggono le cellule impedendo loro di trasformarsi in cellule cancerose. Anche il SOD (superossido dismutasi) distrugge i radicali liberi. I radicali liberi sono sostanze chimiche prodotte dal corpo quando viene esposto a radiazioni, contaminanti alimentari, grassi rancidi e inquinamento atmosferico. Questi danneggiano parti della cellula umana, sopratutto DNA e RNA, che dirigono in parte le azioni di ogni cellula. Quando questo processo viene disturbato, può svilupparsi il cancro. Anche le seguenti sostanze possono avere un effetto protettivo. L’aminoacido L-arginina inibisce lo sviluppo dei tumori. E’ stato scoperto che la vitamina K protegge contro gli effetti di alcune sostanze cancerogene. L’acido folico in grandi quantità, è stato usato nel trattamento di cellule cervicali precancerose e (insieme alla vitamina B12) di cellule bronchiali precancerose nei fumatori. L’acido folico per via orale previene la rottura dei cromosomi legati al manifestarsi di tumori diminuendo il rischio di tumore. Mantenere la flora intestinale in buono stato, con quantità generose di yogurt e acidophilus, può aiutare a prevenire il cancro.
E’ stato scoperto che il calcio insieme alla vitamina D ha un effetto preventivo in individui normali o ad alto rischio (quelli che in famiglia hanno casi di tumore colorettale). Il germanio svolge un’attività anticancro insieme al sistema immunitario. E’ importante consumare alimenti ricchi di oligoelementi per prevenire l’invasione delle sostanze cancerogene. I terreni arricchiti di molibdeno proteggono dal cancro all’esofago. Anche la vitamina B2 (riboflavina) protegge da questo tipo di tumore. Il selenio insieme allo iodio svolge un ruolo protettivo nei confronti di diversi tipi di tumore. Dosi supplementari di rame somministrate ad animali di laboratorio hanno ritardato in modo significativo lo sviluppo del cancro.
La timosina, un gruppo di ormoni, può rimpicciolire i tumori. Anche il DMSO (dimetilsolfossido) può essere d’aiuto nel trattamento di alcuni tumori. Si spera che una delle più promettenti scoperte in ambito tumorale, il DHEA, possa un giorno non solo prevenire il cancro ma anche curarlo. Questa sostanza è prodotta dalle ghiandole surrenali ed è da essa che derivano tutti gli altri ormoni che partecipano al funzionamento dell’organismo. Il fattore di trasferimento, ossia l’estrazione di cellule sane da un donatore sano impiantate nell’organismo di una persona malata, si è mostrato efficace in combinazione con altre terapie. Ci sono prove che l’aspirina possa essere efficace nel trattamento di alcune forme tumorali. Bisognerebbe evitare di assumere integratori di ferro perché tendono ad inibire le capacità anti cancro dei macrofagi e l’attività delle cellule T ed E.
Si raccomanda una dieta a basso contenuto di grassi, soprattutto di grassi saturi (20% del fabbisogno energetico quotidiano) e un aumento dei carboidrati (65% delle calorie). Bisognerebbe evitare o limitare la caffeina e limitare l’assunzione di alcolici a 3 o 5 volte alla settimana.
Una buona dieta anti-cancro è interessante non solo per i malati ma anche per chi vuole prevenire la malattia. E’ essenziale privilegiare tutti gli alimenti integrali e non raffinati evitando attentamente gli alimenti industriali. Un consumo eccessivo di grassi incoraggia lo sviluppo di cellule cancerose. Le carni rosse e i derivati del latte dovrebbero essere consumati con estrema moderazione, mentre si consiglia di aumentare il consumo di verdure crocifere come i cavolini di Bruxelles e i broccoli. Sono necessarie anche le verdure a foglia verde e la frutta e la verdura di colore arancio scuro.
L’uso dell’olio di pesce ha rallentato la progressione del tumore al seno. Si consiglia di consumare due o tre volte alla settimana pesce grasso cotto alla griglia. Anche lo yogurt e i prodotti a base di soia, come il tofu, possono rallentare lo sviluppo di tumori. Sono consigliati anche i succhi di frutta di colore scuro e le combinazioni con succhi di verdura come carote, bietole, cavoli e asparagi. Gli alimenti ricchi di potassio come i cereali integrali, la frutta secca, i legumi e i semi di girasole contengono altre sostanze che aiutano a combattere il tumore (Le persone che soffrono di insufficienza renale non dovrebbero prendere il potassio o consumare quantità eccessive di alimenti che lo contengono).
Le mandorle sono ricche di laetrile, una sostanza ritenuta (anche se non ci sono conferme ufficiali) anticancerogena. Le diete vegetariane e quelle macrobiotiche sono benefiche. E’ consigliabile sostituire il sale con il kelp, usare le melasse o il succo d’acero per dolcificare gli alimenti e la farina integrale invece di quella bianca. Si raccomanda di bere solo acqua filtrata.
Gli oli rancidi o scaldati più volte sono cancerogeni. Bisogna cercare di consumare oli che hanno un buon equilibrio di acidi grassi omega 3 e omega 6. Oltre all’olio di pesce, l’olio di lino contiene tre volte più omega 3 e può essere mischiato con altri oli ricchi di omega 6, come l’olio di mais e di cartamo, per raggiungere un buon equilibrio. L’olio di colza ha un buon equilibrio dei due acidi grassi. E’ consigliabile evitare alimenti affumicati, sottaceto o conservati con nitrati.
Si ritiene che gli alimenti cotti sul barbecue che vengono bruciacchiati possano scatenare reazioni cancerogene nell’organismo. E’ ormai provato che le persone che fumano o bevono hanno una maggiore incidenza del cancro. L’acetaldeide, una sostanza chimica, presente nel fumo delle sigarette e prodotta nel fegato a partire dall’alcool, è cancerogena e produttrice di radicali liberi. E’ anche responsabile della distruzione della cisteina, una sostanza antiossidante.
Le erbe e le altre sostanze naturali possono essere d’aiuto. L’astragalo, combatte gli effetti collaterali delle terapie antitumorali, incluse le radiazioni. Il polline inibisce il cancro. La fucoidina contenuta nelle alghe marine è attiva come sostanza anticancro. Un’alga chiamata spirulina contiene beta-carotene e altre sostanze naturali anticancro. La cipolla e l’aglio (entrambi contengono bioflavonoidi) sono conosciuti come alimenti anticancerogeni. La bardana e il fo-ti sono utilizzati in Cina per curare varie forme di tumori.
La “larrea divaricata” ha effetti antiossidanti. Anche il centonchio, ricco di vitamina C può essere d’aiuto. L’echinacea distrugge le cellule cancerose perché contiene macrofagi che stimolano il sistema immunitario. I Wobe-Mugos (enzimi dell’ananas e del pancreas) hanno proprietà anti-cancro. Il ginseng viene usato nella cura del cancro. L’idraste contiene solfato di berberina, una sostanza anti cancro. Vengono usate anche la consolida (sotto forma di impacchi), l’equiseto, l’infusione di Jason Winter, l’ortica, la carnivora, la suma, il rafano nero, il pau d’arco (anche sotto forma di impacchi), il tarassaco e la liquirizia.
Per il trattamento dei tumori sono state usate anche senecione e camedrio (impacchi), i germogli di grano e d’orzo, e la chinina. Il vischio bianco stimola il sistema immunitario e inibisce lo sviluppo del tumore. Gli estratti dei funghi shiitake e rei-shi hanno proprietà anti tumorali. La spirulina ricca di beta-carotene e altre sostanze naturali è una sostanza anti-cancro.
Le sostanze nutritive e gli altri elementi naturali possono rappresentare un aiuto. Sono consigliati i clisteri per tenere il colon libero da sostanze che potrebbero altrimenti diventare tossiche. Si consiglia anche di evitare le radiazioni sia quelle provenienti dal forno a microonde che i raggi X, e di sedersi ad una distanza di almeno 2 metri dal televisore. Le pentole usate per cucinare dovrebbero essere di vetro o ricoperte di ceramica. L’esposizione a sostanze chimiche come la pittura fresca, gli spray, i detersivi e i pesticidi favorisce la formazione di radicali liberi; il corpo spreca energie combattendo le sostanze tossiche invece di combattere il cancro.
L’attività fisica è importante. L’inattività è legata al cancro. Gli studi mostrano che le persone fisicamente attive hanno una minore incidenza di tumori rispetto a quelle che non praticano un’attività fisica regolarmente (vedi la Parte II).
Per il malato di cancro in fase terminale, il fabbisogno specifico di cibo dipende dal punto in cui si trova il tumore. Di solito comunque si consiglia una dieta ad alto tenore proteico e calorico per sostenere e riparare le cellule normali. Il ferro (quello di tipo eme contenuto negli alimenti) è essenziale nella dieta per prevenire l’anemia, che è frequentemente una complicazione del cancro.
ELEMENTI NUTRITIVI CHE POSSONO ESSERE EFFICACI NELLA CURA DEL CANCRO
Organi Sostanza Quantità*
Tiroide Iodio
Generale Tutte le sostanze antiossidanti Vedi alimenti ricchi di sostanze nutritive nella Parte VIII
Vitamina A 50.000-100.000 UI al dì per 10 giorni o sino alla fine della cura
Beta-carotene 10.000 UI al dì
Complesso B 100 mg al dì
Vitamina B1 500 mg al dì
Vitamina B2 100 mg al dì
Vitamina B6 300 mg al dì
Vitamina B12 in losanghe o iniezioni
Niacina 100-300 mg
Acido folico
Acido pantotenico 50-400 mg
Colina 500-1000 mg
PABA meno di 400 mg al dì
RNA/DNA
Vitamina C con Bioflavonoidi 500-1000 mg, sino a 10.000 mg nel corso della giornata
Vitamina D
Vitamina E Sino a 1200 UI al dì
Vitamina K
Coenzima Q10 100 mg al dì
SOD Secondo le dosi prescritte
DHEA
DMSO
Cromo
Germanio 200 mg al dì
Ferro
Calcio 2000 mg al dì
Fosforo
Iodio
Magnesio 1000 mg al dì
Molibdeno
Potassio
Selenio 200 mcg al dì
Zinco 100 mg al dì
Zolfo
Proteine
L-arginina
L-carnitina Secondo le dosi prescritte
L-cisteina Secondo le dosi prescritte
L-taurina Secondo le dosi prescritte
L-metionina Secondo le dosi prescritte
Acidi grassi insaturi
Kelp 5 compresse al dì
COMPUTER E NEURONI
Mescolare computer e neuroni sembra ancora un’idea lontana e quasi fantascientifica. Ma un recente esperimento dell’Università del Wisconsin dimostra che è possibile influenzare il comportamento dei neuroni, facendoli crescere e muovere attorno a nanomembrane costituite da minuscoli tubi semiconduttori di silicio e germanio. Si tratta di un tipo di interazione tra la tecnologia e i neuroni, che potrebbe avere molte ripercussioni anche per chi ha lesioni spinali.
Nuovi esperimenti su neuroni e semiconduttori
Il mix tra computer e neuroni è l’oggetto di studio di alcuni studenti laureati presso l’Università del Wisconsin che, guidati da Minrui Yu e Yu Huang, hanno pubblicato uno studio ACS Nano, dal titolo complicato di “Semiconductor Nanomembrane Tubes: Three-Dimensional Confinement for Controlled Neurite Outgrowth”. In questa ricerca gli scienziati mostrano che sono stati in grado di far crescere dei gruppi di neuroni attraverso piccoli tubi fatti di semi-conduttori e materiali di silicio e germanio. I neuroni sono le cellule che costituiscono il tessuto nervoso. Grazie alle loro proprietà fisiologiche e chimiche i neuroni sono in grado di ricevere, integrare e trasmettere impulsi nervosi.
Anche se questa ricerca innovativa su computer e neuroni non può far presagire nel breve periodo dei cyborg o che il cervello umano venga strettamente intrecciato con parti di computer, tuttavia questo studio apre la porta alla possibilità di rigenerare le cellule nervose danneggiate a causa di una malattia o di un infortunio.
Il team, guidato da Justin Williams, un ingegnere biomedico, ha creato tubi di varie dimensioni e forme, abbastanza piccoli perché una cellula nervosa possa infilarcisi, ma non così grandi che la stessa cellula possa entrarci completamente dentro. Le provette sono state poi rivestite con le cellule nervose di topo e quindi gli scienziati hanno studiato l’evoluzione della situazione per vedere come avrebbero reagito. Invece di rimanere ferme, le cellule nervose hanno iniziato a inviare filamenti attraverso i piccolissimi tubi, come se fossero in cerca di un cammino verso qualcosa o come se stessero cercando di andare da qualche altra parte. In alcuni casi le cellule hanno effettivamente seguito il contorni dei tubi, il che significa, in teoria, che i nervi possono essere coltivati in strutture.
Le prospettive future della ricerca sui neuroni
Gli scienziati sapevano già da qualche tempo che i neuroni hanno una funzione di ricerca, ma non sono ancora sicuri su cosa stiano cercando o se sia solo un movimento casuale da parte loro. Con la creazione di neuroni che seguono percorsi pre-pianificati attraverso piccoli tubi, il team di ricerca spera di trovare la risposta a questa domanda. L’idea è anche quella di installare dei dispositivi di ascolto per registrare le emissioni elettriche dei nervi, cosa che potrebbe in teoria portare a registrare le “conversazioni” tra le cellule nervose.
La speranza, naturalmente, in questo tipo di ricerca su computer e neuroni, è che si possa trovare un modo per connettere un computer di qualche tipo a un gruppo di cellule nervose per ristabilire la comunicazione che è stata interrotta. Il computer in questo caso potrebbe servire come un relè di sorta, permettendo a chi non può più camminare, per esempio, a causa di lesioni spinali o di una malattia, di riacquistare le proprie capacità motorie precedenti. A questo proposito, questa ricerca è ancora più rivelatrice di quanto possa sembrare a prima vista, perché i minuscoli tubi che sono stati creati, sono molto simili alla mielina, la guaina isolante che ricopre alcune parti delle cellule nervose normali. Sono simili alla mielina sia dal punto di vista fisico che elettrico.
Inoltre, in questo esperimento sui neuroni è possibile controllare il diametro del tubo perché sia vicino a quello di un assone, fornendo un contatto limitato con la membrana dell’assone e isolandola potenzialmente dalla soluzione extracellulare.
•
• GERMANIO
•
•
• È stato scoperto solo nel 1945. A seguito di sperimentazioni, si è dimostrato altamente utile nel prevenire patologie tumorali.
Il germanio organico si è dimostrato infatti efficace nel combattere le malattie degenerative, il diabete, l’ ipertensione, la cirrosi epatica, l’epilessia e persino il cancro.
L’importanza principale di questo minerale nel campo della nutrizione deriva dalla sua proprietà di aumentare l’ossigenazione delle cellule.
Il germanio presente nelle Bacche di Goji è riconosciuto per le sue caratteristiche molecolari; i suoi atomi presentano una struttura molto rara, unica si può dire:
Dei suoi 32 elettroni, ognuno dei 4 che ruotano nella parte più esterna dell’ atomo tendono a saltare fuori dall’orbita quando viene avvicinato da atomi di altre sostanze.
Nel corpo umano, le cellule cancerogene, hanno un aspetto diverse rispetto alle cellule sane. Il potenziale elettrico della membrana esterna delle cellule tumorali è insolitamente elevato se comparato con quello delle cellule sane. Questo è uno dei motivi per il quale le cellule cancerogene si moltiplicano molto velocemente.
Quando si assume il germanio, come nel caso di mangiare Bacche di Goji o bere il succo di Goji, i suoi atomi con la loro azione aggressiva, privano le cellule tumorali dei loro elettroni riducendo il potenziale elettrico della malattia devitalizzandola. Questa è nota clinicamente come “reazione di deidrogenizzazione” e sembra che abbia un grande potenziale per rallentare o fermare le attività delle cellule del cancro.
Il Germanio contenuto nelle bacche di Goji è stato anche riconosciuto per evitare gli effetti causati dal trattamento con radiazioni. Alcune fonti mediche arrivano a dire che le malattie causate dalle radiazioni possono essere addirittura evitate quando il germanio viene somministrato regolarmente nel tempo.
I raggi gamma (utilizzati in radioterapia) emettono elettroni che distruggono le cellule tumorali, ma purtroppo, distruggono anche i globuli rossi e bianchi, portando gravi malattie per alcuni pazienti addirittura la morte. Gli atomi di germanio si fissano saldamente ai globuli rossi prottegendoli da attacchi di elettroni provenienti dall’esterno e respingendoli con la loro struttura.
Il germanio quindi si rivela fondamentale per migliorare l’ossigenizzazione cellularee proprio per questo motivo è considerato un potente antiossidante.
La sua somministrazione è raccomandata in tutti i casi di patologie croniche degenerative, sia cardiovascolari che del metabolismo in genere ( diabete , dislipidemie, e cancro).
Oltre a ciò esso possiede anche altri benefici:
•
Favorisce la rimozione di metalli tossici del corpo, come il piombo, il cadmio ed il mercurio e contrasta gli effetti delle radiazioni ionizzanti.
• Diminuisce i valori del glucosio ematico, trigliceridi ed un aumento del rapporto HDL-LDL del colesterolo, con un aumento dell’emoglobina.
• E’ efficace nei casi di artrite remautoide.
• Utile in terapia medica in caso di danni ischemici e infarti.
Le bacche di Goji sono una grande risorsa di questo importante minerale che è molto raro trovare in natura, infatti integratori alimentari di germanio organico possono essere assai costosi.
Il germanio contenuto nelle Bacche di Goji non deve essere considerato una cura definitiva al cancro, esso può nettamente aiutare ad aumentare l’immuno resistenza del nostro organismo, ma per casi gravi è sempre meglio consultare un medico specialista.
Germanio organico
germanio Prodotti – Germanio comprare – germanio acquistare on-line – germanio ordine – Negozio informazioni germanio
Germanio organico (Ge-132TM), non deve essere confuso con il suo omologo materiale non-organico del settore dei semiconduttori, è un oligoelemento che si verifica nella crosta terrestre in concentrazione relativamente bassa di 7 a 1 milione.
Il Germanio organico è stato specificatamente studiato in Giappone dopo Kuzihiko Dr. Asai ha scoperto che alcune piante officinali come il ginseng, shiitake, aglio e naturalmente clorella hanno concentrazioni elevate di germanio organico, il che spiegherebbe molte delle loro modalità di azione terapeutica.
Per sviluppare la corsa dal Dr. Asai nel suo istituto, da lui fondato, gli studi sull’uso di Ge-132TM, l’efficacia di dosi giornaliere di 100-300 mg si sono dimostrati associati a diverse patologie: artrite, allergie alimentari, ipercolesterolemia, Candida albicans, infezioni virali croniche e cancro.
Il Germanio organico è anche spesso utilizzato in Giappone per il controllo del dolore.
Il germanio sembra agire attraverso meccanismi diversi: si lega all ‘ossigeno, per migliorare la respirazione cellulare. Essa ha la sua attività antivirale e anti micotica. E ‘in grado di attivare i macrofagi e le cellule tumorali naturale (la famosa Natural Killer Cells).
Permette anche, e forse soprattutto, la produzione naturale di interferone, e di conseguenza ad aumentare la proliferazione delle cellule generative di anticorpi.
La teoria prevalente tra gli investigatori è che il germanio organico agisce principalmente a stimolare le difese naturali dell’organismo.
Vi è anche un prodotto sicuro che è completamente eliminata dall’organismo in meno di 48 ore dopo il suo assorbimento.
Le parti interessate alla ricerca per 40 anni
Dato che la produzione di sintesi riuscita di germanio organico un’intensa attività di ricerca su questa sostanza nella biochimica, neuro-chimica, patologia, farmacologia, oncologia ed immunologia. numerosi risultati delle ricerche hanno confermato un effetto sorprendente di germanio organico. In aggiunta alle carbossietil Germanio sesquiossido-Ge-132 ci sono due altri composti organici germanio, il Sanumgerman (nome chimico: lattato citrato Germanio) e SpiriGermanium. E veniamo alla domanda:
Che cosa significa germanio organico?
Il nostro sistema immunitario ci protegge da batteri patogeni, virus, funghi, protozoi e sostanze chimiche tossiche e metalli pesanti. Il nostro fisico immunità è dovuta al gioco delicato di varie cellule specializzate e gli organi, che è controllata dal sistema endocrino ormonale e possono essere influenzati mentalmente. germanio Org apparentemente stimola il sistema immunitario e portare le sue varie componenti in equilibrio.
Gli scienziati Suzuki, Brtkiewicz e Pollard entrato nelle loro ricerche sul presupposto che ciò potrebbe contribuire stimolato dal sistema immunitario Ge132 contro alcuni tipi di cancro. Il Germanio organico appare indiretto effetto anti-tumorale a stimolando cellule T circolanti per la produzione di linfochine (interferone gamma, probabilmente). The linfochine, a sua volta attiva i macrofagi a riposo, che alla fine sopprimere la crescita di tumori. Tutti questi risultati indicano che rafforza il nostro sistema immunitario da cima a fondo, mettendo in equilibrio e di sostegno di un modo naturale per la salute.
come è germanio organico – sembra germanio organico – dove posso ottenere germanio organico?
Il Germanio organico è stato associato con il cancro, AIDS, indebolimento del sistema immunitario, la mancanza di ossigeno nel sangue e la cella – combinato. Eppure, quasi nessuno sa che in Germania Germanio organico, alcune persone possono ancora sapere che c’è un elemento, o è il germanio organico come un semiconduttore utilizzati nella microelettronica. Ma quasi nessuno lo conosce, che germanio organico è stato usato da oltre 30 anni per il trattamento di diverse malattie possano essere – con risultati di ricerca che a sedere. curare il cancro e le cliniche di AIDS negli Stati Uniti, i pazienti con germanio organico sono realtà. In Giappone, ci fu un intensa attività di ricerca sul germanio organico, ma anche in Germania stava facendo ricerche sulla voce che deve il suo nome al tedesco Clemens esploratore Winkle.
Conoscenza degli effetti benefici del biologico germanio organico attraverso sab. Forse perché le grandi compagnie farmaceutiche non sono interessati ad un mezzo a buon mercato che hanno rafforza significativamente il sistema immunitario? “Che cosa è germanio organico, germanio organico sembra, germanio realizzati da Sanum / SanumGermanium viene dalla Germania e viene utilizzato solo per l’esportazione. GE 132 (Ge 32), un germanio organico negli Stati Uniti è molto più concentrata, ma è disponibile solo in gli Stati Uniti. Dove posso trovare germanio organico, quali condizioni devo soddisfare per ricevere Germanio organico? Tutte queste domande sono nel libro: Organic Germanio – Massaggi Spa del ponte chiaro al I
Germanio e cancro:
Germanio a volte può fondersi Cancro
Germanio rende il dolore
Germanio può prevenire metastasi (Dr. Haruo Sato – Università Tohuku), se non impedire
Statistiche disponibili per il tumore del polmone (21 casi) – 500mg
Dr. Asai anche avuto un cancro alla gola è stato rimosso chirurgicamente convenzionale (solo il tumore), ma si difese contro le radiazioni e pienamente a conoscenza di “essere” Germanio e sconfitto, come i suoi clienti, il cancro di successo.
Leucemia – 500mg
AIDS – HIV: Germanio (organico) può aiutare (2 articoli, di cui uno doppio)
Cancro: germanio (Ge-132) può arrestare la crescita dei tumori (due studi)
CFS: Germanio può aiutare (2 studi)
depressione immunitaria, indebolimento del sistema immunitario: Germanio può aiutare (2 studi)
Osteoporosi: Germanio può aiutare (uno studio sperimentale)
Pain: Germanio può contribuire attraverso la promozione di Morphinanalgesie (uno studio di Animal Experiment)
Germanio organico scoperto dal giapponese Kazuhiko Asai ingegnere minerario è stato sia nel carbone e nelle piante medicinali locali. Ciò ha comportato un germanio outsider nella medicina ortomolecolare, con un auto-esperimento.
Dr. Asai era malato negli anni Sessanta da artrite reumatoide grave di artrite. I medicinali e l’agopuntura non ha migliorato la sua vita. Intuitivamente, il paziente stesso ha sviluppato trattati con composti organici germanio 132, un cosiddetto Carboxyethylsesquoxid. 10 giorni è rimasta invariata male la sua condizione, poi migliorare rapidamente, il dolore passò, e le articolazioni sono più mobili.
Germanio e sistema immunitario
Germanio ha un effetto stimolante sul sistema immunitario, tra cui aumenta la produzione di interferone gamma. Aumenta l’utilizzo dell’ossigeno da parte delle cellule in modo che lo stato di tessuti e organi malati migliorata. Aiuta il corpo a normalizzare le funzioni fisiologiche di base, come ad esempio riduce patologicamente elevati di pressione arteriosa a livelli sani – ma non inferiori.
Rende il sangue meno viscoso e denso, migliorando così la circolazione di “gambe del fumatore”.
Essa colpisce le endorfine come sostanze endogene al dolore, antidolorifici effetto di per sé e migliora anche l’efficacia di altri farmaci analgesici.
Si lega metalli come il cadmio e il mercurio, così vicino a lui che lei con il togliere dal corpo e lo liberarono dal inquinanti.
Questa diversità rende gli effetti della (biologico!) Germanio in un importante mezzo di terapia, soprattutto in quanto è in composti organici della medicina ortomolecolare del tutto innocuo.
Benché conforme 400-600 milligrammi al giorno, prendendo in Giappone, molti pazienti l’esperienza quotidiana cinque grammi e più di uno, senza effetti collaterali indesiderati.
1967 è riuscito a sintetizzare il dottor Asai organici germanio inorganico. In seguito a questa scoperta, l’innocuità perfetta internamente amministrati germanio organico è stato trovato in studi su animali, e ben presto fondò un ospedale, venga trattata in un grande successo con germanio organico a questo giorno. Lì, tutti i pazienti sono trattati con la medicina convenzionale “caduto dal tavolo operatorio” sono: oncologi guardando perplesso di tumori cancerosi che non esistono più; Herz-/Hirninfarkte scappare incredibilmente senza danni; reumatismi sono spazzati via; nascite scadenza problema e di morire è facilitato. Non da ultimo, la malattia di Lyme patologie essere contenuto con successo. Citazione dal libro di Akai è “Bio-Germanio: una speranza per il trattamento molti pazienti ‘un caso: una donna di 50 anni iniziò 15 anni fa con” acuto disabilità sensoriale e non poteva camminare a causa di “disturbi motori gravi non di più. La visione era gradualmente e nell’occhio di sinistra era praticamente ciechi. “Aveva dato due volte al giorno 2 grammi di germanio organico e gocce germanio-eye. Dopo due mesi hanno cominciato a camminare con le stampelle, dopo tre mesi aveva bisogno di un solo piano. Il caso è stato un “sub-acuta (mielo-Optico) con diagnosi di neuropatia, ma non è il minimo dubbio che si trattava di un corso della malattia.
germanio organico è costituito da un anello di sei atomi di germanio, che sono circondato da 12 atomi di ossigeno vicino. Il germanio è come un collante che tiene insieme i 12 atomi di ossigeno in uno spazio confinato, ma non reagiscono con l’organismo. Questo, di ossigeno legato germanio non viene rilasciato nell’organismo, ma serve soprattutto come spazzino per H-ioni, derivanti dal processo di combustione organismica: il cibo è bruciato dal corpo, mentre l’anidride carbonica (CO2) e l’idrogeno si formano (H2 ). L’anidride carbonica trova la sua strada dal corpo per essere esalata attraverso i polmoni. Al contrario, l’idrogeno si combina con l’ossigeno per via inalatoria (!) Per l’acqua e viene eliminata attraverso i reni e la pelle. Molte malattie – tra cui la malattia di Lyme a causa di ipossia cronica e in quanto incide sul tragico quando gran parte di ossigeno per via inalatoria non può servire il metabolismo cellulare, perché non vi è stato convertito da ioni idrogeno avidi di acqua e escreto o edema è memorizzato.
La lattina molti, contribuire al germanio atomi di ossigeno legato benefico in questa situazione, perché legano l’idrogeno libero, in quanto fanno Liberare che l’ossigeno inalato libero accesso alle cellule e quindi sviluppare il suo effetto tonificante. consente Così il germanio organico intensificato la respirazione dei tessuti, che sarebbe in particolare lo stato di malattia altrimenti difficili da raggiungere – e presto il paziente ottiene una pelle rosea e arti caldo. Il fatto che l’acqua non necessari da ioni di idrogeno e ossigeno si crea, il corpo è disidratato in modo sano o prodotti chimici. Mal di non più bagnata, i processi di riparazione sono senza ostacoli
invece. Lo ione idrogeno associazione ha anche l’effetto che sono patogeni per l’uomo senza eccessive ambiente acquoso patologico non è la sopravvivenza.
L’ossigeno germanio-bound cattura non solo gli ioni di idrogeno, ma anche i radicali liberi e altri veleni cellulare. Di particolare rilievo: il cadmio e il mercurio sono messi in evidenza di germanio organico.
Un prodotto relativamente nuovo medico è germanio organico. In Giappone fin dagli anni settanta, di sintesi e di ampia diffusione, non è disponibile qui. Mentre era travestito da tempo disponibile nei negozi di alimentazione tedesco come supplemento dietetico, ma poi improvvisamente scomparso dalla scena. Così, anche un antisettico, che non possono essere distribuiti, perché l’industria farmaceutica non si desidera un buon antisettico riduce le vendite antibiotico? Anche in questo brutto con il pretesto di proteggere la popolazione, deve diminuire in Germania lobbying interessi della vittima.
Akai l’aveva notato, che il carbone nero, che consiste principalmente di tronchi d’albero, un contenuto di germanio molto più elevato è il carbone, che consiste principalmente di foglie e semi. Specialmente in zone urbane come steli vegetali, quindi depositato sul germanio in forma organica e probabilmente ha la funzione, batteri / Fungus e prevenire così la morte dell’albero.
Marciume della pianta, torna il germanio nel terreno e viene memorizzato da piante e nuovi usi. Se l’albero è raccolto, ma, come nel caso di ginseng, il suolo impoverito di germanio, e un tempo per crescere nello stesso luogo nessun albero Ginseng – il circuito di germanio è interrotto. (Per gli effetti di guarigione di germanio ginseng è probabilmente coinvolto anche.) Piante di riso crescere più rapidamente e più spesso quando sono forniti con Germanio organico unico e non ci sono gelate meglio senza aggiunta di germanio.
In un piatto vaccinati con diversi ceppi di batteri Petri è stato quando germanio organico è stato aggiunto solo un singolo risultato di muffa dopo un po ‘sofisticato, dal germanio integrato nel suo metabolismo e quindi mantenuto i batteri.
Queste note spettacolo nel suo complesso, che il germanio è usato dalle piante per mantenere la loro circolazione micro e provare se stessi come più elevata rispetto a forme di vita inferiore. Il principio di integrazione di semiconduttori metallici, la recitazione antisettico garantire la sopravvivenza dell’organismo, il sintetico, germanio organico fino all’estremo. A differenza dei negozi di impianto l’organismo umano, il germanio organico non uno, ma separa dopo venti ore insieme con i radicali e gli inquinanti intrappolati privi di effetti collaterali sui reni.
Un tumore cancro viene caricato sulla superficie eccessivamente positivo. Se germanio nuoto in dosi elevate, proprio questo con la sua alta carica negativa, gli ioni con carica positiva dal muro e destabilizza il tumore bio cancro elettricamente fino a quando non si disintegra ed è dovuto il problema della rimozione di grandi quantità di detriti cellulari. L’effetto sorprendente metastasi di soppressione di germanio è presumibilmente sulla base delle sue proprietà bioelettrica: Il sangue è come altamente viscoso e la migrazione di cellule cancerose può stabilire ovunque. Si dà loro la più bella capillari e linfatici, che sarà ugualmente fatto negli ambienti intrisi di germanio. In particolare, gli organi, il sangue sarà naturalmente trarre grandi vantaggi da una terapia germanio.
Così, vi sono notevoli successi nel polmone e cancro del fegato. Germanio riduce in genere il dolore e gli effetti collaterali di terapie antitumorali convenzionali. Il risultato chirurgico è nettamente migliorata e ha lanciato un recupero rapido. Risultato che germanio organico stimola l’arricchimento di ossigeno basale processi metabolici, per nulla tossicità a lungo termine è disponibile un trattamento successi spettacolari in molte diverse malattie tra cui il cancro, è stato descritto a livello atomico e di ricerca, è probabilmente anche come trattamento permanente per La malattia di Lyme adatto.
L’effetto di arricchimento di ossigeno del germanio
Il Germanio organico favorisce l’ossigenazione del corpo ed è anche un antiossidante efficace. Queste sono qualità che molti effetti positivi di questo oligoelemento per molti processi metabolici richiedono reciprocità nel corpo. Anche se l’effetto di germanio organico relativo alla fornitura di ossigeno non è stato sufficientemente determinato, che vanno dalla ricerca clinica come base per la ricerca sulle cellule in arrivo.
Il Germanio organico promuove l’apporto di ossigeno
In una cultura di germanio organico riduce la domanda di ossigeno degli organi negli animali con la mancanza di ossigeno ha effettivamente prolungare la vita.
Esperimenti in laboratorio di biochimica Tohoku University per studiare gli effetti di germanio organico sono state effettuate sul consumo di ossigeno nel fegato e il diaframma di topi ha mostrato una riduzione del consumo di ossigeno.
Dr. Asais teoria afferma che germanio organico svolge lo stesso ruolo nel corpo come l’ossigeno e contemporaneamente aumenta l’apporto di ossigeno al corpo. C’è un rapporto tra il rifornimento di ossigeno, la viscosità del sangue e il flusso sanguigno. L’ossigeno più disponibile, più il sangue diminuisce la viscosità e il flusso di sangue di tutti gli organi sarà migliorata.
Il Germanio organico protegge da monossido di carbonio-asfissia, ictus, e la malattia di Raynaud. Con la cattura di germanio organico in dosi terapeutiche spesso una sensazione di calore e una sensazione di formicolio si fa sentire. Dr. Asai attribuito questo fenomeno al consumo di ossigeno. Nei pazienti affetti da malattie circolatorie, come ad esempio la malattia di Raynaud, che può portare alla necrosi dei tessuti e amputazioni in comune, è stata rilevata dopo la somministrazione di germanio organico, un netto miglioramento.
Il Germanio organico atti di malattie degli occhi e ferite, ustioni soprattutto. Il Germanio organico è stato usato con successo per curare le malattie degli occhi diversi, come il glaucoma, cataratta, distacco di retina, infiammazione della retina e ustioni utilizzati. Non si sa quanto queste proprietà terapeutiche del germanio organico based. Sembra però essere basata sul presupposto che il germanio organico causati da ossigeno migliorata in queste condizioni, ha contribuito alla guarigione.
Il Germanio organico può riunire un trattamento hyperen ossigeno nella sclerosi multipla e altre malattie degenerative, un miglioramento significativo. The naturopata Jan de Vries descrive nel suo libro? Solo su appuntamento? s trattamento di successo con il rifornimento di ossigeno aumentata nella sclerosi multipla. De Vries è arrivato nel 1975 insieme con il Dr. Asai, ed egli usa germanio organico nella sua pratica. Poi si raccoglie materiale per un libro di storie di casi sul trattamento germanio-in cancro e leucemia. Egli scrive:? Un trattamento di ossigeno iperbarico è possibile? effetto in combinazione con Germanio – un incredibile miglioramento nelle condizioni del paziente?
spesso migliora un paziente che soffre di sclerosi multipla ed è trattata con ossigeno iperbarico, anche la vista, per una buona visione dipende da un Sauerstoftversorgung sufficiente il corpo “.
La struttura del germanio organico, di un pertinente molti ioni negativi di ossigeno cristallina alla rete dovrebbe essere in grado di sostituire l’ossigeno e l’attrazione e l’eliminazione acidificanti ioni di idrogeno? e quindi la disintossicazione del sangue? . Consentire
Durante il metabolismo ossidativo Se trasportati nel trasporto di elettroni, gli elettroni in forma di elettroni e, infine, la combinazione di idrogeno e ossigeno in acqua. Per mancanza di ossigeno può causare la perdita di elettroni ad un accumulo di ioni idrogeno positivi, che provoca quindi un acidificazione del sangue. Ge-132 ha caricato negativamente gli ioni di ossigeno per eliminare questi ioni idrogeno e quindi disintossicare il sangue.
Come il germanio organico trasportato elettroni, si può servire durante il metabolismo ossidativo come una fuga di elettroni, e sostenere la produzione di energia del corpo senza ossigeno supplementare. Il sistema di trasporto degli elettroni può essere paragonata a una catena di eliminare. Con una carenza di accettori di elettroni, l’intero processo si ferma, proprio come una lotta a catena, in cui il secchio dell’acqua non può essere passato perché una persona è mancante. Il Germanio organico ha dimostrato di essere vettore di elettroni eccellente, ma dà un contributo importante per l’efficacia del processo globale di ossidazione, è l’energia prodotta alla fine per il corpo.
osservazione personale (G. Schaffar):
Germanio influsso estremamente positivo sulla performance visiva, che confermano molti clienti. deve svolgere un ruolo centrale nella difesa immunitaria, in quanto stimola la sintesi di interferone
è usato per curare il cancro. Viene in molti prima di acque termali (Lourdes, ad esempio).
Requisiti media: 1-3mg;
Germanio Sandra Ph.D.Thorsons Goodman, 1988
Germanio ha antimutagenic, anche le radiazioni viso, disintossica il corpo da metalli pesanti. contribuiscono anche agli effetti negativi di 300mg/Tag irradiazione cobalto, dopo 100mg introduttiva, molto bene. Essa allevia il dolore nei pazienti con carcinoma, reumatismi e angina.
Germanio ha antimutagenic, anche le radiazioni viso, disintossica il corpo da metalli pesanti. contribuiscono anche agli effetti negativi di 300mg/Tag irradiazione cobalto, dopo 100mg introduttiva, molto bene. Essa allevia il dolore nei pazienti con carcinoma, reumatismi e angina.
Dosi di Sanumgerman e Ge-132: 20mg – 1500 mg, 500 mg di solito è dato per malattie gravi a 1000mg/Tag, ad esempio, altre fonti di pazienti con carcinoma 1000 a 5000mg.
In Europa, generalmente somministrato 200-1200mg/Tag; per raggiungere il limite più basso al quale vengono considerati effetti è di 20 mg. Nessuna tossicità 3.4g/kg (topi) e 10g/kg (ratti).
Dr. Asai conduce l’effetto travolgente di germanio organico grazie alla sua capacità di utilizzare l’ossigeno nelle cellule molto più efficiente. L’autore è silenziosa, ma non l’importanza di una corretta regolazione dell’equilibrio acido-base (deacidificazione) e l’adattamento emozionale dei pazienti.
Cancro e terapie naturali
Le cellule cancerose appaiono come cellule immature che si dividono costantemente, non adempiono le loro funzioni naturali e invadono il tessuto circostante. Queste cellule privano le cellule normali vicine dei loro elementi nutritivi essenziali, causando un grave esaurimento nel paziente affetto da cancro. Le cellule cancerose sono in grado di spostarsi e impiantarsi in qualsiasi parte dell’organismo causando crescite abnormi o tumori. Il cancro viene diviso a seconda del tipo di tessuto da cui nasce.
L’importanza di una diagnosi precoce nel trattamento del cancro è fondamentale. E’ l’unica possibilità di curare questa malattia con risultati positivi. Attualmente si ritiene che il cancro all’intestino inizi il suo decorso anche 20 anni prima di diventare una malattia conclamata ed essere notato. E’ importantissimo fare i test che permettono di scoprirlo. Si dovrebbe essere sempre attenti ai sette sintomi premonitori segnalati dalla American Cancer Society: sanguinamento o secrezioni insolite, comparsa di protuberanze o gonfiori, tosse rauca, difficoltà nel deglutire e nel digerire, cambiamenti nelle abitudini intestinali o della vescica, cicatrizzazione lenta, modificazione di una verruca o di un neo. Esistono dei kit che permettono di effettuare da soli un’analisi per il tumore all’intestino. I sintomi e la loro gravità variano in relazione al tipo e alla localizzazione del cancro.
E’ stato scoperto che l’obesità femminile è un fattore che aumenta il rischio di tumore uterino, cervicale, mammario e alla cistifellea. I grassi influenzano gli ormoni femminili stimolando la divisione cellulare che a sua volta dà inizio al processo cancerogeno. Negli uomini l’obesità aumenta il rischio di tumore colorettale. E’ stato riportato che gli uomini che si sono sottoposti a vasectomia hanno tre volte più possibilità di ammalarsi di tumore alla prostata.
Nel trattamento delle escrescenze cancerose e dei tumori sono stati usati la chirurgia, le radiazioni e alcuni farmaci. Le operazioni chirurgiche rimuovono la neoplasia originale e quelle secondarie. I farmaci, benché non siano in grado di curare completamente il cancro, vengono usati per ridurre la neoplasia o per ritardare l’apparizione di escrescenze secondarie. Le radiazioni vengono spesso usate per distruggere le cellule cancerose e per impedirne la diffusione.
Le sostanze nutritive possono essere d’aiuto. Gli effetti collaterali provocati dalla terapia ai raggi e dalla chemioterapia, come il vomito e la diarrea, possono essere diminuiti o evitati con le vitamine C, E e il complesso B. La vitamina E dovrebbe essere assunta prima dei pasti. E’ importante iniziare l’assunzione di queste vitamine alcuni giorni prima dell’inizio del trattamento. Lo stress psicologico del cancro aumenta notevolmente il fabbisogno di vitamina C e di vitamine del complesso B. Per assicurare un immediato assorbimento nel sangue, le vitamine, se possibile, dovrebbero essere somministrate sotto forma di iniezioni.
I raggi X e i trattamenti con altri raggi indeboliscono il sistema immunitario e distruggono le vitamine A, C, E, K, B e gli acidi grassi insaturi: grandi quantità di vitamina E proteggono la vitamina A e gli acidi grassi insaturi. Nel corso della distruzione del tessuto maligno vi è la creazione di sottoprodotti dannosi. Il fegato è in grado di neutralizzare queste sostanze se sono presenti quantità sufficienti di vitamina C, E, proteine e l’aminoacido metionina. La vitamina E può prevenire le bruciature dalle radiazioni, dare sollievo al dolore e ridurre le cicatrici.
Negli animali si sviluppano tumori spontanei, soprattutto della tiroide, dovuti ad alimentazione carente di iodio. La carenza di iodio è legata anche al cancro del seno nella donna. Si ritiene che la carenza di ferro esponga i malati della sindrome di Plummer-Vinson (un disturbo che colpisce le donne di mezza età caratterizzato da spaccature intorno alla bocca e ulcere alla lingua e all’esofago) ad un maggior rischio di tumore all’esofago e allo stomaco. Una carenza di zinco può portare al tumore alla prostata, all’esofago e al carcinoma broncogeno. I danni al fegato di qualsiasi tipo, aumentano la predisposizione al cancro.
Le ricerche eseguite sugli animali hanno mostrato che le vitamine A, C, E, B3 e B6 inibiscono la crescita delle cellule tumorali stimolando il sistema immunitario del corpo ed eliminando i radicali liberi. I lipotropi proteggono le cellule impedendo loro di trasformarsi in cellule cancerose. Anche il SOD (superossido dismutasi) distrugge i radicali liberi. I radicali liberi sono sostanze chimiche prodotte dal corpo quando viene esposto a radiazioni, contaminanti alimentari, grassi rancidi e inquinamento atmosferico. Questi danneggiano parti della cellula umana, sopratutto DNA e RNA, che dirigono in parte le azioni di ogni cellula. Quando questo processo viene disturbato, può svilupparsi il cancro. Anche le seguenti sostanze possono avere un effetto protettivo. L’aminoacido L-arginina inibisce lo sviluppo dei tumori. E’ stato scoperto che la vitamina K protegge contro gli effetti di alcune sostanze cancerogene. L’acido folico in grandi quantità, è stato usato nel trattamento di cellule cervicali precancerose e (insieme alla vitamina B12) di cellule bronchiali precancerose nei fumatori. L’acido folico per via orale previene la rottura dei cromosomi legati al manifestarsi di tumori diminuendo il rischio di tumore. Mantenere la flora intestinale in buono stato, con quantità generose di yogurt e acidophilus, può aiutare a prevenire il cancro.
E’ stato scoperto che il calcio insieme alla vitamina D ha un effetto preventivo in individui normali o ad alto rischio (quelli che in famiglia hanno casi di tumore colorettale). Il germanio svolge un’attività anticancro insieme al sistema immunitario. E’ importante consumare alimenti ricchi di oligoelementi per prevenire l’invasione delle sostanze cancerogene. I terreni arricchiti di molibdeno proteggono dal cancro all’esofago. Anche la vitamina B2 (riboflavina) protegge da questo tipo di tumore. Il selenio insieme allo iodio svolge un ruolo protettivo nei confronti di diversi tipi di tumore. Dosi supplementari di rame somministrate ad animali di laboratorio hanno ritardato in modo significativo lo sviluppo del cancro.
La timosina, un gruppo di ormoni, può rimpicciolire i tumori. Anche il DMSO (dimetilsolfossido) può essere d’aiuto nel trattamento di alcuni tumori. Si spera che una delle più promettenti scoperte in ambito tumorale, il DHEA, possa un giorno non solo prevenire il cancro ma anche curarlo. Questa sostanza è prodotta dalle ghiandole surrenali ed è da essa che derivano tutti gli altri ormoni che partecipano al funzionamento dell’organismo. Il fattore di trasferimento, ossia l’estrazione di cellule sane da un donatore sano impiantate nell’organismo di una persona malata, si è mostrato efficace in combinazione con altre terapie. Ci sono prove che l’aspirina possa essere efficace nel trattamento di alcune forme tumorali. Bisognerebbe evitare di assumere integratori di ferro perché tendono ad inibire le capacità anti cancro dei macrofagi e l’attività delle cellule T ed E.
Si raccomanda una dieta a basso contenuto di grassi, soprattutto di grassi saturi (20% del fabbisogno energetico quotidiano) e un aumento dei carboidrati (65% delle calorie). Bisognerebbe evitare o limitare la caffeina e limitare l’assunzione di alcolici a 3 o 5 volte alla settimana.
Una buona dieta anti-cancro è interessante non solo per i malati ma anche per chi vuole prevenire la malattia. E’ essenziale privilegiare tutti gli alimenti integrali e non raffinati evitando attentamente gli alimenti industriali. Un consumo eccessivo di grassi incoraggia lo sviluppo di cellule cancerose. Le carni rosse e i derivati del latte dovrebbero essere consumati con estrema moderazione, mentre si consiglia di aumentare il consumo di verdure crocifere come i cavolini di Bruxelles e i broccoli. Sono necessarie anche le verdure a foglia verde e la frutta e la verdura di colore arancio scuro.
L’uso dell’olio di pesce ha rallentato la progressione del tumore al seno. Si consiglia di consumare due o tre volte alla settimana pesce grasso cotto alla griglia. Anche lo yogurt e i prodotti a base di soia, come il tofu, possono rallentare lo sviluppo di tumori. Sono consigliati anche i succhi di frutta di colore scuro e le combinazioni con succhi di verdura come carote, bietole, cavoli e asparagi. Gli alimenti ricchi di potassio come i cereali integrali, la frutta secca, i legumi e i semi di girasole contengono altre sostanze che aiutano a combattere il tumore (Le persone che soffrono di insufficienza renale non dovrebbero prendere il potassio o consumare quantità eccessive di alimenti che lo contengono).
Le mandorle sono ricche di laetrile, una sostanza ritenuta (anche se non ci sono conferme ufficiali) anticancerogena. Le diete vegetariane e quelle macrobiotiche sono benefiche. E’ consigliabile sostituire il sale con il kelp, usare le melasse o il succo d’acero per dolcificare gli alimenti e la farina integrale invece di quella bianca. Si raccomanda di bere solo acqua filtrata.
Gli oli rancidi o scaldati più volte sono cancerogeni. Bisogna cercare di consumare oli che hanno un buon equilibrio di acidi grassi omega 3 e omega 6. Oltre all’olio di pesce, l’olio di lino contiene tre volte più omega 3 e può essere mischiato con altri oli ricchi di omega 6, come l’olio di mais e di cartamo, per raggiungere un buon equilibrio. L’olio di colza ha un buon equilibrio dei due acidi grassi. E’ consigliabile evitare alimenti affumicati, sottaceto o conservati con nitrati.
Si ritiene che gli alimenti cotti sul barbecue che vengono bruciacchiati possano scatenare reazioni cancerogene nell’organismo. E’ ormai provato che le persone che fumano o bevono hanno una maggiore incidenza del cancro. L’acetaldeide, una sostanza chimica, presente nel fumo delle sigarette e prodotta nel fegato a partire dall’alcool, è cancerogena e produttrice di radicali liberi. E’ anche responsabile della distruzione della cisteina, una sostanza antiossidante.
Le erbe e le altre sostanze naturali possono essere d’aiuto. L’astragalo, combatte gli effetti collaterali delle terapie antitumorali, incluse le radiazioni. Il polline inibisce il cancro. La fucoidina contenuta nelle alghe marine è attiva come sostanza anticancro. Un’alga chiamata spirulina contiene beta-carotene e altre sostanze naturali anticancro. La cipolla e l’aglio (entrambi contengono bioflavonoidi) sono conosciuti come alimenti anticancerogeni. La bardana e il fo-ti sono utilizzati in Cina per curare varie forme di tumori.
La “larrea divaricata” ha effetti antiossidanti. Anche il centonchio, ricco di vitamina C può essere d’aiuto. L’echinacea distrugge le cellule cancerose perché contiene macrofagi che stimolano il sistema immunitario. I Wobe-Mugos (enzimi dell’ananas e del pancreas) hanno proprietà anti-cancro. Il ginseng viene usato nella cura del cancro. L’idraste contiene solfato di berberina, una sostanza anti cancro. Vengono usate anche la consolida (sotto forma di impacchi), l’equiseto, l’infusione di Jason Winter, l’ortica, la carnivora, la suma, il rafano nero, il pau d’arco (anche sotto forma di impacchi), il tarassaco e la liquirizia.
Per il trattamento dei tumori sono state usate anche senecione e camedrio (impacchi), i germogli di grano e d’orzo, e la chinina. Il vischio bianco stimola il sistema immunitario e inibisce lo sviluppo del tumore. Gli estratti dei funghi shiitake e rei-shi hanno proprietà anti tumorali. La spirulina ricca di beta-carotene e altre sostanze naturali è una sostanza anti-cancro.
Le sostanze nutritive e gli altri elementi naturali possono rappresentare un aiuto. Sono consigliati i clisteri per tenere il colon libero da sostanze che potrebbero altrimenti diventare tossiche. Si consiglia anche di evitare le radiazioni sia quelle provenienti dal forno a microonde che i raggi X, e di sedersi ad una distanza di almeno 2 metri dal televisore. Le pentole usate per cucinare dovrebbero essere di vetro o ricoperte di ceramica. L’esposizione a sostanze chimiche come la pittura fresca, gli spray, i detersivi e i pesticidi favorisce la formazione di radicali liberi; il corpo spreca energie combattendo le sostanze tossiche invece di combattere il cancro.
L’attività fisica è importante. L’inattività è legata al cancro. Gli studi mostrano che le persone fisicamente attive hanno una minore incidenza di tumori rispetto a quelle che non praticano un’attività fisica regolarmente (vedi la Parte II).
Per il malato di cancro in fase terminale, il fabbisogno specifico di cibo dipende dal punto in cui si trova il tumore. Di solito comunque si consiglia una dieta ad alto tenore proteico e calorico per sostenere e riparare le cellule normali. Il ferro (quello di tipo eme contenuto negli alimenti) è essenziale nella dieta per prevenire l’anemia, che è frequentemente una complicazione del cancro.
ELEMENTI NUTRITIVI CHE POSSONO ESSERE EFFICACI NELLA CURA DEL CANCRO
Organi Sostanza Quantità*
Tiroide Iodio
Generale Tutte le sostanze antiossidanti Vedi alimenti ricchi di sostanze nutritive nella Parte VIII
Vitamina A 50.000-100.000 UI al dì per 10 giorni o sino alla fine della cura
Beta-carotene 10.000 UI al dì
Complesso B 100 mg al dì
Vitamina B1 500 mg al dì
Vitamina B2 100 mg al dì
Vitamina B6 300 mg al dì
Vitamina B12 in losanghe o iniezioni
Niacina 100-300 mg
Acido folico
Acido pantotenico 50-400 mg
Colina 500-1000 mg
PABA meno di 400 mg al dì
RNA/DNA
Vitamina C con Bioflavonoidi 500-1000 mg, sino a 10.000 mg nel corso della giornata
Vitamina D
Vitamina E Sino a 1200 UI al dì
Vitamina K
Coenzima Q10 100 mg al dì
SOD Secondo le dosi prescritte
DHEA
DMSO
Cromo
Germanio 200 mg al dì
Ferro
Calcio 2000 mg al dì
Fosforo
Iodio
Magnesio 1000 mg al dì
Molibdeno
Potassio
Selenio 200 mcg al dì
Zinco 100 mg al dì
Zolfo
Proteine
L-arginina
L-carnitina Secondo le dosi prescritte
L-cisteina Secondo le dosi prescritte
L-taurina Secondo le dosi prescritte
L-metionina Secondo le dosi prescritte
Acidi grassi insaturi
Kelp 5 compresse al dì
Enzimi proteolitici Al momento dei pasti
Aglio
3 capsule 3 volte al dì
Induction of immunopotentiation activity by a protein-bound polysaccharide, PSK (review).
Sakagami H, Aoki T, Simpson A, Tanuma S.
Source
First Department of Biochemistry, School of Medicine, Showa University, Tokyo, Japan.
Abstract
A protein-bound polysaccharide, PSK, extracted from the mycelium of Coriolus versicolor (Fr.) Quel, has been recognized for its host-mediated induction of antitumor and antimicrobial activities in mice. Intravenous administration of PSK, in association with OK-432 (Picibanil), transiently induced endogenous production of a cytotoxic factor (CF) (possibly tumor necrosis factor, TNF) in normal mice. The ability to produce CF depended greatly on both dose and interval between administration of the PSK and OK-432. Although PSK has been reported to contain several active ingredients, unfractionated PSK has been used in almost all experiments performed so far. We recently reported that, of the four subfractions separated by successive filtration through membrane filters, only the highest molecular weight fraction F4 (MW greater than 200 kD) induced significant antimicrobial activity in mice. PSK stimulated the NBT-reducing activity of mouse peritoneal macrophages and the iodination (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood polymorphonuclear cells (PMN). Among the subfractions of PSK, the highest molecular weight fraction F4, and the fraction precipitated at pH 4.0-4.5 (Fr. 4), stimulated macrophage NBT-reducing activity and PMN iodination most. In contrast, natural and chemically modified glucans had little or no stimulating activity. PSK, F4 or Fr. 4 additively or synergistically stimulated TNF-induced cytotoxicity against L-929 cells, differentiation of human myelogenous leukemia cell lines toward monocytes/macrophages, and iodination of human peripheral blood PMN. The active PSK subfractions significantly reduced the down regulation of specific 125I-TNF or 125I-IFN-gamma binding to cellular receptors. These data suggest that (i) immunopotentiation activity of PSK might be ascribed, at least in part, to stimulation of cytokine action and production, and (ii) PSK might have some unique structural features.
PMID:
2064356
[PubMed – indexed for MEDLINE]
• Stimulation of human peripheral blood polymorphonuclear cell iodination by PSK subfractions.[Anticancer Res. 1990]
Stimulation of human peripheral blood polymorphonuclear cell iodination by PSK subfractions.
Sakagami H, Kim F, Konno K. Anticancer Res. 1990 May-Jun; 10(3):697-702.
• Functional maturation of monocytes/macrophages induced by PSK subfractions.[Anticancer Res. 1991]
Functional maturation of monocytes/macrophages induced by PSK subfractions.
Kurakata Y, Sakagami H, Sato A, Kikuchi K, Takeda M, Asano K, Sato T. Anticancer Res. 1991 Sep-Oct; 11(5):1767-72.
• Stimulation of interferon-gamma-induced human myelogenous leukemic cell differentiation by high molecular weight PSK subfraction.[Anticancer Res. 1990]
Stimulation of interferon-gamma-induced human myelogenous leukemic cell differentiation by high molecular weight PSK subfraction.
Kim F, Sakagami H, Tanuma S, Konno K. Anticancer Res. 1990 Jan-Feb; 10(1):55-8.
• Review Anticancer effects and mechanisms of polysaccharide-K (PSK): implications of cancer immunotherapy.[Anticancer Res. 2002]
Review Anticancer effects and mechanisms of polysaccharide-K (PSK): implications of cancer immunotherapy.
Fisher M, Yang LX. Anticancer Res. 2002 May-Jun; 22(3):1737-54.
• Review Antitumor, antiviral and immunopotentiating activities of pine cone extracts: potential medicinal efficacy of natural and synthetic lignin-related materials (review).[Anticancer Res. 1991]
Review Antitumor, antiviral and immunopotentiating activities of pine cone extracts: potential medicinal efficacy of natural and synthetic lignin-related materials (review).
Sakagami H, Kawazoe Y, Komatsu N, Simpson A, Nonoyama M, Konno K, Yoshida T, Kuroiwa Y, Tanuma S. Anticancer Res. 1991 Mar-Apr; 11(2):881-8.
See reviews…See all…
Cited by 1 PubMed Central article
• Chemopreventive effect of PSP through targeting of prostate cancer stem cell-like population.[PLoS One. 2011]
Chemopreventive effect of PSP through targeting of prostate cancer stem cell-like population.
Luk SU, Lee TK, Liu J, Lee DT, Chiu YT, Ma S, Ng IO, Wong YC, Chan FL, Ling MT. PLoS One. 2011; 6(5):e19804. Epub 2011 May 16.
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• Induction of immunopotentiation activity by a protein-bound polysaccharide, PSK …
Induction of immunopotentiation activity by a protein-bound polysaccharide, PSK (review).
Anticancer Res. 1991 Mar-Apr ;11(2):993-9.
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• Mechanisms of action of novel agents for prostate cancer chemoprevention.
Mechanisms of action of novel agents for prostate cancer chemoprevention.
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The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell lines via arrest of cell cycle and induction of apoptosis
Eva Jiménez-Medina1, Enrique Berruguilla1, Irene Romero1, Ignacio Algarra2, Antonia Collado3, Federico Garrido1 and Angel Garcia-Lora1*
• * Corresponding author: Angel Garcia-Lora angel.miguel.exts@juntadeandalucia.es
Author Affiliations
1 Servicio de Análisis Clínicos e Inmunologia, Hospital Universitario Virgen de las Nieves, Universidad de Granada, Av. de las Fuerzas Armadas 2, 18014 Granada, Spain
2 Departamento de Ciencias de la Salud, Universidad Jaén, Jaén, Spain
3 Unidad de Investigación, Hospital Universitario Virgen de las Nieves, Granada, Spain
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BMC Cancer 2008, 8:78 doi:10.1186/1471-2407-8-78
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2407/8/78
Received: 5 November 2007
Accepted: 24 March 2008
Published: 24 March 2008
© 2008 Jiménez-Medina et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
BACKGROUND
Protein-bound polysaccharide (PSK) is derived from the CM-101 strain of the fungus Coriolus versicolor and has shown anticancer activity in vitro and in in vivo experimental models and human cancers. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the adjuvant treatment of gastric, esophageal, colorectal, breast and lung cancers. These studies have suggested the efficacy of PSK as an immunomodulator of biological responses. The precise molecular mechanisms responsible for its biological activity have yet to be fully elucidated.
METHODS
The in vitro cytotoxic anti-tumour activity of PSK has been evaluated in various tumour cell lines derived from leukaemias, melanomas, fibrosarcomas and cervix, lung, pancreas and gastric cancers. Tumour cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of PSK on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in PSK-treated cells.
RESULTS
PSK showed in vitro inhibition of tumour cell proliferation as measured by BrdU incorporation and viable cell count. The inhibition ranged from 22 to 84%. Inhibition mechanisms were identified as cell cycle arrest, with cell accumulation in G0/G1 phase and increase in apoptosis and caspase-3 expression. These results indicate that PSK has a direct cytotoxic activity in vitro, inhibiting tumour cell proliferation. In contrast, PSK shows a synergistic effect with IL-2 that increases PBL proliferation.
CONCLUSION
These results indicate that PSK has cytotoxic activity in vitro on tumour cell lines. This new cytotoxic activity of PSK on tumour cells is independent of its previously described immunomodulatory activity on NK cells.
Background
A number of bioactive molecules, including antitumour substances, have been identified in various mushroom species. Polysaccharides are the best known and most potent of these and have antitumour and immunomodulating properties [1-5]. PSK, a protein-bound polysaccharide obtained from Basidiomycetes, also known as Krestin, has been used as an agent in the treatment of cancer in Asia for over 30 yrs [6-8]. PSK is derived from the fungus Coriolus versicolor and has documented anticancer activity in vitro in experimental models [9] and in human clinical trials. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the treatment of gastric, esophageal, colorectal, breast and lung cancers [10,11]. These studies have suggested the efficacy of PSK as an immunomodulator of biological response.
Previous reports indicated that PSK might act in different ways: as antioxidant [5,12,13]; as inhibitor of metalloproteinases and other enzymes involved in metastatic processes [14] and as inhibitor of the action of various carcinogens in vulnerable cell lines. However its most important and widely reported property is its immunomodulatory capacity. PSK may act to increase leukocyte activation and response via upregulation of key cytokines. Thus, natural killer (NK) and lymphocyte-activated killer (LAK) cell activation has been demonstrated in vivo and in vitro [15,16]. Our group demonstrated that PSK is capable of inhibiting metastatic colonization in vivo in some experimental fibrosarcomas, and that this effect is mediated by activation of NK cells [17,18]. Moreover, the NK cell line NKL, derived from a large granular lymphocyte leukaemia [19], is activated in vitro by PSK [16]. This activation may replace IL-2 in inducing the proliferation and cytotoxicity of NKL cells. The signal transduction pathways involved in the responses to IL-2 or PSK are different: IL-2 increases PKCα and ERK3 expression and decreases ERK2 expression, whereas PSK decreases PKCα expression and increases ERK3 expression [20]. PSK also enhances CRE binding activity, while IL-2 increases SP-1 and modifies GAS/ISRE, IRF-1 and STAT5 [21]. In addition, PSK and IL-2 have been shown to bind to different receptor on NKL cells [22].
The direct in vitro effect of PSK on the proliferation of tumour cell lines was compared with its effect on PBLs. PSK had cytotoxic activity on tumour cell lines, inhibiting proliferation, producing cell cycle arrest and cell accumulation in G0/G1phase and inducing apoptosis.
Methods
PROTEIN-BOUND POLYSACCHARIDE K
Protein-bound polysaccharide K (PSK) was kindly provided by Kureha Chemical Ind. Co. (Tokyo, Japan). It is prepared by extracting cultured mycelia of Coriolus versicolor with hot water. The precipitate is separated from the clear supernatant with saturated ammonium sulfate, then desalted and dried [23]. Protein-bound polysaccharide K was dissolved in RPMI medium or water and heated at 50°C for 20–30 min until a clear solution appeared. The PSK preparation was filter-sterilized and diluted in culture medium or water to the desired concentration. Protein-bound polysaccharide K was previously titrated in NKL cells [16] and the working dilution was 100 μg/mL. PSK extract digested with neuraminidase was also tested, digesting 100 μg of PSK with 4 μl (Sigma) and incubating for 3 h at 37°C. Our group previously showed that PSK is composed of two bands of very high molecular weight [22]. After digestion with neuraminidase, these bands are reduced to a single band of about 12 kd. These results indicate that PSK is probably composed of a single 12-kd protein, and that this protein is highly glycosylated [22]. Two different extracts of PSK were also used: one rich in sugars and other rich in proteins.
CELL LINES AND CELL CULTURE
The following tumour cell lines were studied: B16 murine melanoma, B9 murine MCA-induced fibrosarcoma, Ando-2 human melanoma, AGS human gastric cancer, A-549 human lung cancer, Hela human cervical adenocarcinoma and Jurkat T lymphoma leukemia. The NKL studied was established from PBLs of a patient with LGL leukemia [19]. All cell lines were obtained from the American Type Culture Collection (Manassas, USA) except for the B9 cell line, which was generated at our laboratory, and the Ando-2 and NKL cell lines, kindly provided by P. Coulie (Unite de Genetique Cellulaire, Louvain University, Brussels, Belgium), F. X. Real (Instituto Municipal de Investigaciones Medicas, Barcelona, Spain) and Dr. M. Lopet-Botet (Universidad Pompeu-Fabra, Barcelona, Spain), respectively.
Cell lines derived from solid tumours were grown at 37°C in a humidified atmosphere of 5% CO2 in DMEM culture medium (Gibco, Paisley UK) supplemented with 10% heat-inactivated foetal bovine serum (Life Technologics, Milan Italy), antibiotics and glutamine. Jurkat T cell leukemia was cultured in RPMI 1640 with 10% heat-inactivated fetal bovine serum. The NKL cell line was cultured in RPMI 1640 with 10% heat-inactivated human AB serum (Sigma Chemical, St Louis, MO; USA) and human recombinant IL-2 (100 U/ml; purity > 97%, specific activity, 2 × 106 U/mg) (Roche, Nutley, NJ; USA).
IN VITRO CYTOTOXICITY ASSAYS
The effect of PSK on tumour cell proliferation was assessed by measuring BrdU incorporation with the BrdU colorimetric ELISA Cell Proliferation Kit (Roche Diagnostic). Cells were plated in 96-well microculture plates (5 × 103 cells/well). Every 48 h, the culture medium was replaced and PSK was added. After 48–96 h, BrdU labelling reagent was added and cultured for a further 1–3 h. Assays were also performed by counting viable cells using Trypan Blue. Briefly, cancer cell lines were seeded into culture tissue-flask (1.5–2 × 105/culture tissue-flask) and incubated for 24 h at 37°C in a humidified atmosphere of 5% CO2. Cells were then treated with 100 μg/ml of PSK in the culture medium, which was replaced every 48 h. After 4–6 days, cells were collected by centrifugation and a small sample of cell suspension was diluted in 0.4% Trypan Blue, counting cells in a haemocytometer chamber. Each cell sample was counted in this way at least three times and each assay was repeated at least three times.
LYMPHOCYTE AND NKL PROLIFERATION ASSAY
Human lymphocytes were isolated from venous blood by the Ficoll-Hystopaque separation method. Proliferation of PBLs was analyzed in vitro using 5-bromo-2′-deoxyuridine (BrdU) labelling of DNA-synthesizing cells with the above-mentioned kit. PBLs were seeded in 96-well microculture plates at a cell density of 5 × 104 per well. Two different concentrations of PSK were used, 100 μg/ml and 50 μg/ml. Concanavalin A (5 μg/ml, Sigma) and IL-2 were used as positive controls. PSK was also used in combination with IL-2 or Concanavalin A. After 48 h of culture in presence or absence of PSK, BrdU labelling reagent (final concentration 10 μM) was added and cells were cultured for 24 h. Cells were then fixed for 30 min and incubated with anti-BrdU for 1 h at 37°C. 100 μl of tetramethyl-benzidine (TMB) was used as substrate. Optical densities were determined at 370 nm by means of an ELISA microplate reader (Biotek, Power-Wave XS). Controls were the culture medium, cells cultured only in medium and cells incubated with anti-BrdU in absence of BrdU. All experiments were repeated at least three times.
CELL CYCLE DISTRIBUTION ANALYSIS
Briefly, cells were plated in six-well plates (5 × 105 per well) or in culture tissue-flask (15 × 105) and continuously exposed for 4 days to 100 μg/ml of PSK. The DNA synthesis rate was examined by BrdU incorporation method using FITC BrdU Flow Kit (BD Pharmingen) according to manufacturer’s instructions. BrdU was then detected by DNase cell treatment using FITC-conjugated anti-BrdU antibody. Cells were washed with 1 ml 1 × BD Perm/Wash Buffer, and 20 μl 7-amino-actinomycin D was added. Analysis was performed with 50000 cells using Cell Quest Software and FACScan flow cytometer (Becton-Dickinson).
ANNEXIN V BINDING ASSAY TO DETECT APOPTOTIC CELLS
After treatment of cancer cells with PSK for four days, cells were detached from the culture tissue-flask with PBS containing 3 mM EDTA. These cells were then collected together with floating cells, washed twice with cold PBS and resuspended in binding buffer at a concentration of 1 × 106 cells per ml; 100 μl of solution was incubated for 30 min at 4°C with 5 μl of Annexin V-PE antibody (BD Biosciences), and 5 μl of 7-amino-actynomycin D was then added. Cells were incubated for 15 min in darkness, and 400 μl of staining buffer was added before flow cytometry analysis. Apoptosis was analyzed by quadrant statistics as follows: Annexin V- and 7-AAD-negative cells are alive; Annexin V-positive and 7-AAD-negative cells are in early stages of apoptosis; Annexin V-negative and 7-AAD-positice cells are dead but not by apoptosis; and Annexin V-positive and 7-AAD-positive cells are in mid- or end-stage apoptosis.
ASSAY FOR ACTIVE CASPASE-3 EXPRESSION
FITC conjugated monoclonal anti-active-caspase-3 antibody (BD Biosciences) was used to determine whether the protease caspase-3 is involved in PSK-induced apoptosis. After 4-day treatment with PSK, cancer cells were washed twice with cold PBS and fixed and permeabilized in Cytofix/Cytoperm buffer. Then, cells were incubated with FITC-conjugated monoclonal rabbit anti-active human-caspase-3 antibody for 30 min. Cells were washed twice and 500 μl of 1 × Perm Wash Buffer was added before analysis by flow cytometry.
STATISTICAL ANALYSIS
Values are expressed as means ± SD. Student’s t-test was used for statistical comparisons, considering a significance value of P < 0.05.
Results
PSK INHIBITS IN VITRO TUMOUR CELL PROLIFERATION
Tumour cell lines were cultured in 96-well plate for 48–72 h (2.5–5 × 103 cells) in medium alone (control) or with PSK (100 μg/ml or 50 μg/ml) for 4–6 days. Cell proliferation was then measured by BrdU incorporation (absorbance), which was significantly lower in PSK-treated versus untreated tumour cells (Fig. 1). AGS and A549 cell lines showed a strong decrease in absorbance after treatment with 100 μg/ml PSK that was less marked after treatment with 50 μg/ml of PSK. Inhibition of proliferation was around 65% in melanoma cell lines B16 and Ando-2, lower in Hela and Jurkat cell lines and lowest (20%) in B9 murine fibrosarcoma (Fig. 1). PSK-treated tumour cells showed morphological changes (rounded and granulated morphology, increased vacuolisation, cell shrinkage) and a large number of the cells detached from culture flasks.
Figure 1. Effect of PSK on tumour cell line proliferation. B16, A549, Hela, AGS, Jurkat, B9 and Ando-2 tumour cell lines were treated or not with 50 μg/ml or 100 μg/ml of PSK for 72–96 h. Tumour cells (2.5 to 5 × 104) were plated in quadruplicate in 96-well plates. The proliferation of tumour cells was determined by BrdU incorporation and absorbance measurement. All cell lines analysed showed inhibition of proliferation. Each column represents the mean of five independent experiments ± SD. P < 0.001 versus control.
These assays were repeated in cell culture flasks (1.5–2.5 × 105cells), and viable cells were counted in Haemacytometer chamber using Trypan Blue. As shown in Table 1, there was a significant decrease in the final number of viable cells, with a proliferation inhibition of 22%–84% versus control cells. There was an excellent correlation between the results obtained with the two assays (absorbance and cell count). In Hela tumour cells, proliferation inhibition was higher after treatment with 50 μg/ml versus 100 μg/ml of PSK. In all other tumour cell lines, proliferation inhibition was similar or higher at 100 μg/ml PSK.
Table 1. Proliferation of tumour cell lines treated with PSK
PSK INCREASES IN VITRO PROLIFERATION OF IL-2-STIMULATED LYMPHOCYTES
A dose-response analysis was performed to determine the in vitro effect of PSK on human PBLs. PBLs (5 × 104) were plated in 96-well tissue plate for 48–72 h with eight serially diluted extractions ranging from 500 μg/ml (concentration n°8) to 3.9 μg/ml (concentration n°1). Concentration n°0 represents cells cultured in medium alone. BrdU incorporation during DNA synthesis was then measured by ELISA. Optical densities were very similar between treated and untreated PBLs (data not shown). However, simultaneous treatment of PBLs with IL-2 (100 U/ml) + PSK (100 μg/ml) produced a higher proliferation rate (4.5-fold) versus PBLs treated with IL-2 alone (3-fold) (Fig. 2). Untreated and Concanavalin A-treated PBLs served as controls (Fig. 2).
Figure 2. Effect of PSK on PBL proliferation. PBLs were cultured with PSK or IL-2 or with PSK+IL-2. PBLs (50 × 104 cells) were seeded in quadruplicate in 96-well plates. The proliferation was determined by BrdU incorporation and absorbance measurement. PSK showed a synergistic effect with IL-2, increasing PBL proliferation. PSK alone did not induce PBLs proliferation. Each column represents the mean of five independent experiments ± SD. *P < 0.001 versus control.
EFFECT OF DIFFERENT VARIANTS OF PSK
Tumour cell proliferation inhibition was compared among different PSK variants. Neuraminidase treatment digests glicosylated proteins. A549 tumour cell line was cultured in medium alone (control) or with PSK (100 μg/ml) or neuraminidase-treated PSK (100 μg/ml) for 4–6 days and then counted using trypan blue. No significant differences in proliferation inhibition were found between PSK and neuraminidase-treated PSK (Fig. 3a). The same results were found for sugar-rich and protein-rich PSK variants as for PSK (data not shown).
Figure 3. In vitro activity of neuraminidase treated-PSK. a) A549 tumour cell line was cultured with neuraminidase-treated PSK or PSK. A549 tumour cells (20 × 104) were seeded in culture flask and treated with PSK for 96 h, estimating cell viability by means of Trypan blue exclusion. Both agents produced a similar inhibition ofproliferation. b) NKL cell line was cultured with PSK or neuraminidase treated-PSK and proliferation was determined by BrdU incorporation and absorbance measurement. Both agents induced a similar increase of proliferation. Each column represents the mean of five independent experiments ± SD. *P < 0.001 versus control.
It was previously reported that PSK induces proliferation and activation of NKL cells [16]. Treatment of NKL cells with 100 μg/ml PSK or neuraminidase-treated PSK for 96 h induced a similar increase in their proliferation (Fig. 3b), which was slightly higher than that obtained after culture of NKL with IL-2 alone (Fig. 3b). Induction of NKL proliferation was slightly lower in sugar-rich and protein-rich PSK variants; this difference was not significant (data not shown)
CELL CYCLE PHASE DISTRIBUTION ANALYSIS OF PSK-TREATED CELLS
Mechanisms of PSK cytotoxic activity were analysed by flow cytometry in order to study the effect on cell cycle phase distribution. Culture of AGS tumour cell line with 100 μg/ml of PSK produced total cell cycle arrest with cell accumulation in G0/G1 phase and no cells in S phase (Fig. 4). Cell cycle phase distributions were: 32.2% G0/G1, 31.1% S and 16.2% G2/M in control AGS cells and 60.8% G0/G1, 0% S and 14.1% G2/M in PSK-treated AGS cells. Similar results were found in Ando-2, A549 and B16 tumour cell lines (Table 2). Results in B9 fibrosarcoma showed a slowing rather than an arrest of the cell cycle (Fig.4), with a partial accumulation in G0/G1 phase (49.15% untreated cells and 63.17% PSK-treated cells) at the expense of a decrease in S phase (20.54% vs. 15.14%) and G2/M phase (12.6% vs. 6.96%). Similar results were found in Hela and Jurkat tumour cells (Table 2). These results indicate that PSK produces arrest or slowing of the cell cycle according to the tumour cell histology.
Table 2. Effect of PSK on cell cycle distribution of tumor cell lines.
Figure 4. Cell cycle analysis of cells treated with PSK. Tumour cell lines were treated with PSK for 96 h. Cell cycle distribution was determined by flow cytometry using BrdU incorporation and 7-AAD. Data indicate the percentage of cells in each phase of cell cycle. Results are representative of three independent experiments.
ANALYSIS OF APOPTOSIS IN TUMOUR CELLS TREATED WITH PSK
Cancer cell lines were treated with 100 μg/ml PSK for 4 days to examine the capacity of PSK to induce apoptosis. Untreated or PSK-treated cancer cells were incubated with Annexin V-PE in a buffer containing 7-amino-actinomycin (7-AAD) and analyzed by flow cytometry. Figure 5 depicts representative results for AGS and B9 tumour cells. PSK increased apoptosis from 4.32% (untreated cells) to 37.52% in AGS cells but not in B9 tumour cells (untreated cells, 11.37% vs. PSK-treated cells, 12.11%). Table 3 depicts the results for other tumour cell lines, showing that PSK induces apoptosis in A549, B16 and Ando-2 tumour cells.
Figure 5. Apoptosis analysis of cells treated with PSK. AGS cell line was untreated or treated with 50 μg/ml of PSK for 96 h. Cells were double-stained with annexin V and 7AAD and analyzed by flow cytometry. PSK produced apoptosis in AGS tumour cell line. B9 tumour cell line was also cultured with PSK but apoptosis was not detected in this tumour cell line. All experiments were performed at least three times and gave similar results.
Table 3. Apoptosis induction in cancer cell lines after treatment with PSK for 4 days.
EXPRESSION OF ACTIVE HUMAN CASPASE-3
Caspases are the main enzymes involved in the apoptotic pathway and the participation of active caspase-3 in PSK-induced apoptosis was evaluated. Tumour cells were treated with PSK (100 μg/ml) for 4 days, then permeabilized, fixed and stained for active human caspase-3 and analyzed by flow cytometry. In the AGS cell line, untreated cells were negative for presence of active-caspase-3, whereas around 36% of PSK-treated cells showed detectable active caspase-3 (Fig. 6). However, in tumour cell lines in which PSK did not produce apoptosis, e.g., B9 tumour cells, no caspase-3 expression was detected after PSK treatment (Fig. 6). Table 4 depicts the results obtained with the other tumour cell lines analysed.
Figure 6. Caspase-3 expression in tumour cell lines treatedwith PSK. AGS and B9 tumour cell lines were treated with 50 μg/ml of PSK and expression was analysed by flow cytometry using FITC conjugated monoclonal anti-active-caspase-3 antibody. Data indicate the percentage of cells positive for presence of active-caspase-3. PSK produced increased caspase-3 expression in AGS but not in B9 tumour cell lines. Results are representative of three experiments.
Table 4. Expression of caspase-3 in cancer cell lines after treatment with PSK for 4 days.
Discussion
Several clinical assays have reported the anti-tumour properties of PSK and its synergestic effect in combined therapies [9,24,25]. Our group previously reported the immunomodulatory activity of PSK on NK cells, producing in vitro proliferation and activation of NKL cells [16,20,21]. In the present study, we have identified a new cytotoxic anti-tumour activity of PSK. This activity varied according to the histological origin of the tumour cell lines under study, with inhibition rates ranging from 84% to 22% (Table 1). The highest profileration inhibition rates were found in AGS (84%) and A549 (80%) cell lines (gastric and lung cancer, respectively). PSK was previously reported to be effective in adjuvant immunotherapy for patients after curative resection of gastric cancer [25], and this effect was attributed to its immunomodulatory activity on NK cells [26]. Our group previously reported that PSK mediates induction of the NKL cell proliferation and activation. The present results suggest that PSK may also exert a direct antitumour cytotoxic activity. Inhibition was around 65% in melanoma cell lines Ando-2 (human) and B16 (mice) and was lowest (22%) in the B9 murine fibrosarcoma cell line. Deglycosylation of PSK by neuraminidase treatment did not modify its cytotoxic effect on tumour cell lines. The sugar-rich and protein-rich PSK variants showed identical results to those of PSK in their inhibition of proliferation of tumour cell lines in vitro. These results indicate that the cytotoxic properties are in a compound that is present in all three variants studied and does not vary among them.
Interestingly, PSK had the opposite effect on lymphocytes. Thus, PSK, in synergy with IL-2, induced proliferation of PBLs. PSK also induced proliferation and activation of NKL cells, producing an effect similar to that of IL-2. Hence, PSK has a cytotoxic effect on tumour cells and a mitotic effect on lymphocytes and NK cells.
The cell cycle was arrested or slowed by PSK according to the histological origin of the tumour cells. PSK is known to increase docetaxel-induced apoptosis of NOR-P human pancreatic cancer cells [27] and of Namalwa Burkitt lymphoma cells [28]. PSK induced apoptosis in the AGS cell line but not in all tumour cell lines analysed and induced caspase-3 expression in some tumour cell lines but not all. These results indicate that PSK may induce cytotoxic activity by different molecular mechanisms according to the histology of tumour.
The molecular mechanisms implicated in PSK-induced proliferation and activation of NKL cells have been widely described, showing that PSK and IL-2 bind to different receptors on NKL cells and induce different signal transduction pathways [20-22]. The present results indicate that the anti-tumour properties of PSK observed in clinical trials might be due to a dual biological activity: 1) a direct cytotoxic activity on tumour cells and 2) an immunomodulatory activity largely produced by NK cell activation. A similar dual activity has also been described in a Calendula extract, LACE, which produces an in vitro cytotoxic activity and in vivo immunomodulatory effect on tumour cell lines, including human and mouse melanioma cells, increasing the number and activation of CD4+, CD19+ and NKT cells [29]. PSK suppressed in vivo metastases in spontaneous metastasis assays of mouse fibrosarcoma, melanoma, rat hepatoma AH60C and mouse colon cancer 26 [17,30,31]via NK cell activation. Based on the present findings, it can be hypothesised that this anti-metastatic capacity may also derive from the cytotoxic component of PSK.
Research into the biological mechanisms underlying the anti-tumour effect of PSK is ongoing. We can now add a direct cytotoxic effect on tumour cells to the previously described immunomodulatory effect of this polysaccharide. Greater knowledge of the molecular mechanisms implicated in PSK anti-tumour activity may improve cancer immunotherapy, leading to the application of new anti-tumour protocols.
Conclusion
PSK shows in vitro growth inhibition of various tumour cell lines, producing cell cycle arrest/slowing, apoptosis and induction of caspase-3 expression. In combination with IL-2, PSK induces proliferation of PBLs. The biological activity of PSK appears to include both an immunomodulatory effect on NK cells and a cytotoxic effect on tumour cells
Abbreviations
PBLs: Peripheral Blood Lymphocytes; LACE: laser-activated calendula extract; 7-AAD: 7-amino-actinomicin D; BrdU: 5-Bromo-2-deoxyuridine; Concan.: Concanavalin A; LAK: lymphocyte-activated killer; NK: Natural killer; TMB: tetramethyl-benzidine.
Competing interests
Materials for these studies were partially supported by a grant from Kureha Chemical Industry (Japan), which manufactures PSK. The authors declare that they have no other competing interest.
Authors’ contributions
EJM, EB and IR performed the assays. IA and AC helped in some experiments. FG and AGL designed the study and drafted the manuscript. All authors have read and approved the final manuscript.
Acknowledgements
The authors thank I. Linares for technical assistance. AGL was supported by FIS Postdoctoral Research Contract CP03/00111. Studies were partially supported by a grant from Kureha Chemical Industry (Japan).
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Mechanisms of action of novel agents for prostate cancer chemoprevention.
Singh RP, Agarwal R.
Source
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Cancer Center, Denver 80262, USA.
Abstract
Despite advances in the understanding of prostate cancer (PCa) growth and development, it is still the leading incidence of cases and the second leading cause of mortality due to cancer in men. The problem of early diagnosis compounded with the emergence of androgen independence during commonly used anti-androgen therapy of PCa, have been discouraging for optimal therapeutic response. Recently, many chemopreventive agents, including silibinin, inositol hexaphosphate, decursin, apigenin, acacetin, grape seed extract, curcumin, and epigallocatechin-3 gallate have been identified in laboratory studies, which could be useful in the management of PCa. In vivo pre-clinical studies have indicated chemopreventive effect of many such agents in PCa xenograft and transgenic mouse models. The molecular targets of these agents include cell signaling, cell-cycle regulators, and survival/apoptotic molecules, which are implicated in uncontrolled PCa growth and progression. Furthermore, angiogenic and metastatic targets, including vascular endothelial growth factor, hypoxia-inducing factor-1alpha, matrix metalloproteinase, and urokinase-type plasminogen activator are also modulated by many chemopreventive agents to suppress the growth and invasive potential of PCa. This review focuses on novel PCa chemopreventive observations in laboratory studies, which could provide the rationale for the prospective use of chemopreventive agents in translational studies.
PMID:
16954429
[PubMed – indexed for MEDLINE]
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Publication Types, MeSH Terms, Substances, Grant Support
PUBLICATION TYPES
• Research Support, N.I.H., Extramural
• Review
MESH TERMS
• Angiogenesis Inhibitors/therapeutic use
• Anticarcinogenic Agents/therapeutic use*
• Cell Cycle/drug effects
• Cell Survival/drug effects
• Chemoprevention
• Humans
• Male
• Neoplasm Invasiveness
• Prostatic Neoplasms/blood supply
• Prostatic Neoplasms/pathology
• Prostatic Neoplasms/prevention & control*
• Protein-Tyrosine Kinases/physiology
• Receptors, Androgen/physiology
• Signal Transduction
SUBSTANCES
• Angiogenesis Inhibitors
• Anticarcinogenic Agents
• Receptors, Androgen
• Protein-Tyrosine Kinases
GRANT SUPPORT
• R01 CA102514/CA/NCI NIH HHS/United States
• R01 CA104286/CA/NCI NIH HHS/United States
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Psp (Coriolus versicolor ) e K prostata
Chemopreventive Effect of PSP Through Targeting of Prostate Cancer Stem Cell-Like Population
Recent evidence suggested that prostate cancer stem/progenitor cells (CSC) are responsible for cancer initiation as well as disease progression. Unfortunately, conventional therapies are only effective in targeting the more differentiated cancer cells and spare the CSCs. Here, we report that PSP, an active component extracted from the mushroom Turkey tail (also known as Coriolus versicolor), is effective in targeting prostate CSCs. We found that treatment of the prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. Meanwhile, PSP treatment not only suppressed the ability of PC-3 cells to form prostaspheres under non-adherent culture conditions, but also inhibited their tumorigenicity in vivo, further proving that PSP can suppress prostate CSC properties. To investigate if the anti-CSC effect of PSP may lead to prostate cancer chemoprevention, transgenic mice (TgMAP) that spontaneously develop prostate tumors were orally fed with PSP for 20 weeks. Whereas 100% of the mice that fed with water only developed prostate tumors at the end of experiment, no tumors could be found in any of the mice fed with PSP, suggesting that PSP treatment can completely inhibit prostate tumor formation. Our results not only demonstrated the intriguing anti-CSC effect of PSP, but also revealed, for the first time, the surprising chemopreventive property of oral PSP consumption against prostate cancer.
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Formal Correction: This article has been formally corrected to address the following errors.
A panel in Figure 1 is missing. The correct Figure 1 can be viewed here: http://www.plosone.org/co… (read formal correction)
There are multiple errors in the legends of Figure 1 and Figure 2. The correct legends are:
Figure 1. PSP down-regulates prostate CSC markers in PC-3 cells.
(A) Western blotting of prostate CSC markers CD44 and CD133 in PC-3 cells after… (read formal correction)
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Abstract
Introduction
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Sze-Ue Luk2#, Terence Kin-Wah Lee3#, Ji Liu1, Davy Tak-Wing Lee2, Yung-Tuen Chiu2, Stephanie Ma3, Irene Oi-Lin Ng3, Yong-Chuan Wong2, Franky Leung Chan4, Ming-Tat Ling1*
1 Australian Prostate Cancer Research Centre-Queensland and Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia, 2 Department of Anatomy, The University of Hong Kong, Hong Kong, Hong Kong SAR, 3 Department of Pathology, The University of Hong Kong, Hong Kong, Hong Kong SAR, 4 School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong SAR
Abstract Top
Recent evidence suggested that prostate cancer stem/progenitor cells (CSC) are responsible for cancer initiation as well as disease progression. Unfortunately, conventional therapies are only effective in targeting the more differentiated cancer cells and spare the CSCs. Here, we report that PSP, an active component extracted from the mushroom Turkey tail (also known as Coriolus versicolor), is effective in targeting prostate CSCs. We found that treatment of the prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. Meanwhile, PSP treatment not only suppressed the ability of PC-3 cells to form prostaspheres under non-adherent culture conditions, but also inhibited their tumorigenicity in vivo, further proving that PSP can suppress prostate CSC properties. To investigate if the anti-CSC effect of PSP may lead to prostate cancer chemoprevention, transgenic mice (TgMAP) that spontaneously develop prostate tumors were orally fed with PSP for 20 weeks. Whereas 100% of the mice that fed with water only developed prostate tumors at the end of experiment, no tumors could be found in any of the mice fed with PSP, suggesting that PSP treatment can completely inhibit prostate tumor formation. Our results not only demonstrated the intriguing anti-CSC effect of PSP, but also revealed, for the first time, the surprising chemopreventive property of oral PSP consumption against prostate cancer.
Citation: Luk S-U, Lee TK-W, Liu J, Lee DT-W, Chiu Y-T, et al. (2011) Chemopreventive Effect of PSP Through Targeting of Prostate Cancer Stem Cell-Like Population. PLoS ONE 6(5): e19804. doi:10.1371/journal.pone.0019804
Editor: Kelvin Yuen Kwong Chan, Tsan Yuk Hospital, Hospital Authority, China
Received: December 23, 2010; Accepted: April 16, 2011; Published: May 16, 2011
Copyright: © 2011 Luk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Vice Chancellor Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: mingtat.ling@qut.edu.au
# These authors contributed equally to this work.
Introduction Top
Prostate cancer (PCa) is the most common male malignancy in western countries and represents a major disease burden in the world. When diagnosed at an advanced stage where surgery is no longer feasible, the only frontline treatment available is hormone ablation therapy. Unfortunately, the majority of PCa patients eventually relapse and develop hormone refractory PCa (HRPC), a fatal and terminal stage regarded as incurable [1].
Chemoprevention is an ideal strategy for battling prostate cancer, and a number of chemotherapeutic agents or natural food supplements are currently being tested for their potential of inhibiting prostate cancer development. For example, finasteride, a 5-alpha reductase specific inhibitor, has been shown to reduce prostate cancer incidence by 25% in a clinical trial [2]. Similarly, dutasteride, an analog of finasteride, was also reported to significantly inhibit prostate cancer development [3]. Despite of the promising result, the side-effects associated with the finasteride treatment remains the major concern for it to be used widely for prostate chemoprevention. Therefore, bioactive food compounds such as epigallocatechin-3-gallate or resveratrol [4], [5], [6] represents an attractive alternative for prostate cancer chemoprevention, mainly due to their relatively low toxicity. Unfortunately, most of the previous studies have produced inconclusive results regarding their chemopreventive potential.
Recent identification of prostate cancer stem cells (CSCs) [7] has provided a new insight into prostate carcinogenesis. The ability of these cancer stem cells to self-renew and differentiate into bulk cancer cells suggested that they may be the origin of prostate cancer [7]. Moreover, the highly resistant nature of these CSCs to different chemotherapies suggested that CSCs may also contribute to treatment failure and disease relapse [8]. Interestingly, a number of bioactive food compounds have recently been shown to have anti-CSC effect. For example, we recently reported that gamma-tocotrienol extracted from palm oil inhibits prostasphere formation ability and tumorigenicity of prostate cancer cells [9], suggesting that gamma-tocotrienol is effective in suppressing prostate CSC properties. In addition, a triterpene extracted from fruits was also found to inhibit the self-renewal ability of liver CSCs and sensitize the liver tumor to cisplatin treatment [10]. These findings highlight the potential of bioactive food compounds as CSC targeting agent either for the prevention or for the treatment of prostate cancer.
Here, we demonstrated that the polysaccharopeptide (PSP) extracted from Turkey tail (known as Coriolus versicolor or Yun-zhi) targets prostate CSCs in vitro and suppresses tumor formation in vivo. Treatment of prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. Meanwhile, formation of prostasphere, a major property of prostate CSCs, was completely suppressed in PC-3 cells in the presence of PSP. Furthermore, PSP pre-treatment significantly suppressed tumor initiation of PC-3 cells in immunocompromised mice, suggesting that PSP suppresses the tumorigenicity of the PC-3 cells. More importantly, oral feeding of transgenic mice (TgMAP) that spontaneously develop prostate tumor with PSP was found to completely inhibit prostate tumor formation. Our findings support that PSP may be a potent chemopreventive agent against prostate cancer, possibly through targeting of the prostate CSC population.
Results Top
Effects of PSP on CSC marker expression
PSP has previously shown to possess anti-cancer properties [11], [12], [13], although the underlying mechanisms are still unclear. To test if the anti-cancer effect of PSP is through targeting of CSC properties, we first investigated if PSP treatment affects the expression of prostate CSC markers in PC-3 cell line, which has been reported to contain CSCs. PC-3 cells were treated with 250 and 500 µg/ml of PSP for either 48 or 72 hr, and the expression of CSC markers such as CD133 or CD44 was examined by western blotting. As shown in Figure 1B, protein expression of CD133 was significantly down-regulated after PSP treatment in a time and dose-dependent manner. Downregulation of CD44 was also observed after PSP treatment, although the effect was less obvious. However, examination of the mRNA level of both CD133 and CD44 revealed that the downregulation of both proteins by PSP is not due to inhibition of gene transcription (data no shown). Nevertheless, these data suggest that PSP may be effective in targeting the putative prostate CSCs.
Figure 1. 1PSP down-regulates prostate CSC markers in PC-3 cells.
A) Western blotting of prostate CSC markers CD44 and CD133 in PC-3 cells after PSP treatment. Note that PSP significantly down-regulates both stem cell markers in a dose- and time-dependent manner. B) Viability of PC-3 cells after treatment with 5, 25, 125, 250 and 500 µg/ml of PSP for 48 or 72 hr was measured with MTT assay. Results are presented as mean ± s.d. C) Flow cytometry analysis of PC-3 cells after treatment with 250 µg/ml of PSP for 72 hr. Note that no significant difference in cell cycle distribution was observed. D) Western blotting results for apoptotic markers (left panel) and stem cell maintenance proteins (right panel) in PC-3 cells after PSP treatment. Note that no changes in Bax and Bcl-2 or cleavage of PARP were detected.
doi:10.1371/journal.pone.0019804.g001
To test if the down-regulation of CSC marker expression by PSP is due to a decrease in cell viability, PC-3 cells treated with PSP at different dosages (0, 5, 25, 125, 250 and 500 µg/ml) for 24, 48 or 72 hr were examined by MTT assay. Interestingly, 48 hr of PSP treatment did not have any observable effects on cell viability, even though the same treatments was found to significantly suppress the expression of CSC markers (Figure 1B & 1C). Meanwhile, PSP treatment also failed to induce cell cycle arrest or apoptosis, as evidenced by the lack of sub-G1 population in the result of flow cytometry analysis (Figure 1D). This was further confirmed by examination of apoptosis-associated proteins (i.e. Bax, Bcl-2 and cleaved PARP) (Figure 1E). However, the Akt/β-catenin pathway, which is responsible for the enrichment of CSCs in breast cancer, was drastically inhibited by PSP treatment. As shown in Figure 1D, activation of AKT by phosphorylation at ser 473 was inhibited by PSP at both doses, which was accompanied by complete disappearance of β-catenin expression (Figure 1E).
PSP inhibits prostasphere formation of prostate cancer cells under non-adherent culture conditions
The ability to form prostaspheres in non-adherent culture is one of the characteristics of prostate CSCs [14], [15], [16]. To confirm that PSP treatment can inhibit prostate CSC properties, prostasphere formation of PC-3 was studied in the presence or absence of PSP. As shown in Figure 2A, culturing of both PC-3 and DU145 cells for 14 days under non-adherent conditions results formation of prostaspheres, further confirming the presence of a stem-like population within both cell lines. Strikingly, addition of PSP into the medium drastically inhibited prostasphere formation in both cell lines. In particular, no prostaspheres was found in either cell line in the presence of 500 mg/ml of PSP, suggesting that PSP treatment significantly eliminated the prostate CSCs. To further proved that PSP is effective in inhibiting prostasphere formation, primary prostaspheres with enriched CSC population were dissociated and re-seeded into non-adherent culture condition to allow for the formation of secondary prostaspheres. Consistent with the result from the primary spheroid formation assay, PSP treatments significantly suppress the number of prostaspheres found in each cell line, although the higher dosage of PSP (500 mg/ml) was unable to completely eliminate all the secondary prostaspheres. Nonetheless, these results suggest that PSP is effective in suppressing the CSC properties of prostate cancer cells.
Figure 2. 1Effects of PSP on CSC properties.
A) Spheroid formation assay was performed with PC-3 and Du145 cells. Two hundred of cells were seeded onto polyHEMA pre-coated plates and treated with either 500 µg/mL of PSP or vehicle for 14 days. The number of prostaspheres formed was counted, and the result was presented as the mean ± s.d. Note that γ-T3 treatment efficiently suppresses the spheroid formation ability of PC-3 cells. Image of the prostaspheres was captured under microscope. Note that no prostaspheres can be found in cells treated with 500 µg/mL of PSP. (B) PSP inhibited the formation of secondary prostaspheres. Primary prostaspheres were dissociated and re-seeded into polyHEMA pre-coated plate. PSP was added 24 hr after the plating. Note that prostasphere formation was inhibited by more than 70% and 90% in the presence of 250 µg/mL and 500 µg/mL of PSP respectively. * P<0.001, ** P<0.0001, t test.
doi:10.1371/journal.pone.0019804.g002
PSP significantly reduces the tumorigenicity of prostate cancer cells in vivo
Since CSC is responsible for cancer initiation, it is possible that PSP treatment may inhibit the tumor formation ability of PC-3 cells in vivo. To test this hypothesis, we first treated PC-3 cells that stably expressing the luciferase protein (PC-3-Luc) with PSP for 72 hr before orthotopically injected them into the SCID mice. As examined by bioluminescence imaging, all of the mice that were injected with vehicle-pre-treated PC-3-luc cells formed tumors two weeks after the implantation (Figure 3A). Intriguingly, three out of the eight mice that were injected with PSP pre-treated PC-3-luc cells failed to develop tumors even at week four post implantation (Figure 3A&B). The lack of tumors in the PSP-pre-treated group was further confirmed by examination of the mouse prostate glands at the end of the experiment (Figure 3C). Taken together, our results suggested that PSP was effective in reducing the tumorigenic potential of prostate cancer cells, which is an essential characteristic of CSCs.
Figure 3. PSP inhibits tumorigenicity of PC-3 cells in vivo.
A) Bioluminescent images of SCID mice orthotopically injected with PC-3-luc cells for two weeks. SCID mice in the upper row were injected with vehicle-treated PC-3-luc cells, whereas mice in the bottom row were injected with PSP-treated PC-3-luc cells. B) Table summarizes the percentages of mice developing detectable tumors at week 2. Approximately 40% of mice in the PSP pretreated group did not form detectable tumors, whereas 100% tumor formation was found in the control group (p = 0.07). C) Selected ex vivo images of the prostate from both groups. Note that in PSP-treated mice with negative luciferase signal, no visible tumor were found in the prostate tissue.
doi:10.1371/journal.pone.0019804.g003
Oral administration of PSP fails to inhibit prostatic intraepithelial neoplasia (PIN) development in TgMAP mice
The effect of PSP on prostate CSCs supports the hypothesis that it may have a chemopreventive effect against prostate cancer. To test this hypothesis, we have employed a transgenic mouse model that spontaneously develops adenocarcinoma of the prostate (TgMAP) [17], [18]. The TgMAP mice develop PIN between 16–20 weeks of age and progress to prostate adenocarcinoma after week 24 (Figure 4A). Because PIN is considered as the pre-malignant lesion and the most important risk factor of prostate cancer [19], we first tested if PSP administration affected PIN development in TgMAP mice. Five TgMAP mice (14-weeks old, at least 2 weeks before PIN development) were fed with 200 mg/kg of PSP for 4 weeks. Four mice of the same age were fed with water only for the same period of time. All mice were sacrificed at 20 weeks old and prostatic tissues were collected and sectioned for histology. As shown in Figure 4B&C, no differences were observed between the control and PSP-treated TgMAP mice regarding to the gross anatomy and the histology of the prostate gland. At low power magnification, tissue sections from both groups retained glandular structures, and at high power, PIN was detectable in both the control and PSP-treated mice. These results suggest that 4 weeks of PSP consumption was unable to stop the development of PIN.
Figure 4. Effect of PSP on PIN development in the TgMAP transgenic mouse model.
A) Time frame of the PIN and PCa development in TgMAP mice and the schedule of the PSP treatment. Fourteen-week old TgMAP mice were treated with 200 mg/kg of PSP by oral gavage feeding for 4 weeks and sacrificed at the time when PIN has developed (20 weeks old). The table summarizes the results of the histology examination of the prostate from the vehicle- and PSP-treated TgMAP mice. C) Representative photos of the Hematoxylin & Eosin staining of the prostatic tissues from the TgMAP mice. Note that both control- and PSP-treated TgMAP mice developed prostatic intraepithelial neoplasia (PIN), as indicated by the arrows.
doi:10.1371/journal.pone.0019804.g004
Prolonging PSP consumption inhibits prostate cancer development in TgMAP mice
The failure to inhibit PIN formation by PSP treatment may due to insufficient dose or treatment length. We therefore tested if a higher dose and longer period of PSP consumption may affect prostate tumor formation using the same model. Five TgMAP mice (8-weeks old) were fed with 300 mg/kg of PSP for a total of 20 weeks (Figure 5A). Four mice at the same age were again fed with water only for the same period of time. All mice were sacrificed at 28 weeks old when prostate tumors were formed, with prostatic tissues collected and sectioned for histology. As shown in Figure 5B, tumors were found in different sections of the prostate gland from all of the mice that were fed with water only. Surprisingly, examination of all of the prostate section revealed that none of the mice that were fed with PSP bare any prostate tumors (Figure 5C), suggesting that PSP treatment completely inhibited prostate tumor formation in the TgMAP mice. Meanwhile, whereas three of the PSP-fed mice were found to have PIN, the other two mice were found to have completely normal prostates (Figure 5D). Furthermore, consistent with the low toxicity of PSP, long term consumption appears to have no side effect on the mice, as judged by the body weight changes and physical signs (data not shown) (Figure 5E). These findings strongly suggest that oral intake of PSP may be a safe and effective chemopreventive agent against prostate cancer.
Figure 5. Effect of PSP on prostate tumor development of the TgMAP transgenic mouse model.
A) Outline of the schedule for PSP treatment. Eight-week old TgMAP mice were treated with 300 mg/kg of PSP by oral gavage feeding for 20 weeks and sacrificed at age of 28 weeks. B & C) Representative photos of the Hematoxylin & Eosin staining of the prostate tissues from the vehicle and PSP-treated TgMAP mice. Note that tumors were found in all of the mice that were treated with vehicle only but were absent in all the PSP-treated mice. D) The table summarizes the results of the histology examination of the prostate tissues from the vehicle and PSP-treated TgMAP mice. *P<0.05 compared to control treatment by Fisher’s exact test. E) Average body weight of the mice during the PSP treatment.
doi:10.1371/journal.pone.0019804.g005
Discussion Top
PSP has previously been demonstrated to induce apoptosis and inhibit growth of a wide-range of cancer cells which includes breast [20], [21], [22], liver [23] and prostate cancer [24], although the mechanisms underlying its anti-cancer effects remain poorly understood. Here, we demonstrated for the first time that PSP has anti-CSC effects, as evidenced by the downregulation of CSC markers and the suppression of prostasphere and tumor formation.
Prostate CSCs were first identified by Collins et al. in 2005 using CD44+/alpha2beta1hi/CD133+ as the cell surface markers [25]. Using similar approaches, CSCs have also been identified in prostate cancer cell lines such as LNCaP [26], DU145 [26], [27] and PC-3 [28], [29]. These prostate CSCs not only express high level of CD133 and CD44, but are also highly tumorigenic when compared to the non-CSC population. The fact that PSP can significantly suppress the expression of both CD133 and CD44, as well as the tumorigenicity of PC-3 cells, clearly indicates the effectiveness of PSP in targeting prostate CSCs. As demonstrated by Hsieh et al [24], PSP is effective on induction of apoptosis and inhibition of cell proliferation in LNCaP cells. However, its effect were much less prominent in androgen independent prostate cancer cell lines such as PC-3. This is indeed consistent with our finding, which showed that PSP can suppress CSC properties without inducing any detectable cell cycle arrest or apoptosis. Nevertheless, the finding that both Akt phosphorylation and β-catenin expression were also down-regulated by PSP (Figure 1E) suggests that PSP may act by inactivating the Pten/Akt/β-catenin pathway to inhibit CSC renewal. This recently identified stem cell maintenance pathway was shown to play a key role in the regulation of prostate and mammary stem cell populations [16], [30]. Aberrant activation of the Akt/β-catenin pathway through the knockdown of Pten was found to enrich the mammary stem cell population, leading to the induction of hyperplastic lesions in the mouse [30]. Similarly, knockdown of Pten in prostate cancer cells was also found to enhance prostasphere formation ability and tumorigenicity of the cells [16]. Therefore, the the loss of “stemness” of prostate CSCs after PSP treatment may be due to down-regulation of the Pten/AKT/β-catenin pathway.
One of the key properties of stem cells is their ability to form spheres in non-adherent, serum-free conditions [31]. Indeed, spheroid formation assays have recently been used to identify and to enrich putative CSCs [16], [32], [33], [34]. Consistent with previous studies, both prostate cancer cell lines PC-3 and DU145 were able to form prostaspheres in non-adherent culture [16], suggesting the presence of CSCs within these cell lines. These primary prostaspheres, which are resistant to chemotherapeutic drugs [9], are highly sensitive to PSP treatment (Figure 2A). In addition, the secondary prostaspheres were significantly inhibited in a dose-dependent manner (Figure 2B), supporting that PSP is effective in eliminating prostate CSCs in vitro.
Prostate CSCs is believed to be origin of prostate tumor, which have the ability to self-renew and differentiate into the bulk tumor [35]. The fact that PSP pretreatment can significantly inhibit the tumorigenicity of PC-3 cells (Figure 3) not only highlights the anti-CSC effect of PSP, but also suggests that PSP may have chemopreventive effects against prostate cancer. We tested this hypothesis using a recently developed transgenic mouse model of prostate cancer (TgMAP) [17], [18]. The stepwise development of the prostate tumor (from low grade PIN to gross tumor) in the TgMAP mouse highly mimics the pathogenesis of human prostate cancer, although it may not totally reflect the complex nature of prostate carcinogenesis. Nonetheless, it allowed us to develop an optimal PSP treatment dosage and time frame. Whereas four weeks of PSP oral consumption at 200 mg/kg failed to produce any differences in PIN development, complete inhibition of prostate tumor formation was achieved after 20 weeks of oral PSP feeding at 300 mg/Kg. Meanwhile, the suppression of PIN formation by PSP further suggested that the chemopreventive effect of PSP may due to suppression of the tumor initiation at early stage. The extremely low toxicity and the highly potent anti-CSC effect of PSP warrants further evaluation of its chemopreventive effect in human clinical trials.
In summary, we have demonstrated, for the first time, that PSP treatment not only inhibits CSC properties, but also effectively suppresses prostate tumor formation. Our results suggest that PSP may be an effective agent for prostate cancer chemoprevention.
Materials and Methods Top
Polysaccharopeptide (PSP)
PSP extracted from Yun-zhi was kindly provided by Wonder Herb Health Products, Ltd. The PSP powder was dissolved in autoclaved Milli Q water at a concentration of 30 mg/ml by mixing in a rotator at 4°C overnight. The PSP solution was stored at 4°C. For cell culture study, PSP stock was sterilized with 0.2 µm filtration prior to use. In the animal study, PSP was fed directly to mice.
Cell lines and culture conditions
Prostate cancer cell lines PC-3 and DU145 (ATCC, Rockville, MD) were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 1% (w/v) penicillin-streptomycin (Invitrogen, Carlsbad, CA) and 5% fetal bovine serum (Invitrogen, Carlsbad, CA). All cell lines were kept at 37°C in a 5% CO2 environment. Luciferase-expressing PC-3 cell line was generated in our previous study.
TgMAP prostate tumor model
TgMAP C57/BL6 mice at week 8 and 14 were administered with PSP at 200 mg/kg for 4 weeks (n = 5) or 300 mg/Kg for 20 weeks (n = 5) respectively by oral gavage feeding (5 days per week). Control group were fed with water only for the same period of time. Mice were sacrificed (at the age of 20 weeks for 200 mg/Kg treatment group and 28 weeks for the 300 mg/Kg treatment group) and prostate tissues were collected, fixed in 10% formalin and embedded in paraffin. The whole prostate was cut into 4 µm sections and one in every five consecutive sections was stained with H&E. Histology examination was performed by Dr. K.W. Chan (pathologist, HKU). Statistical difference was determined by Fisher’s exact test and was considered as significant if p<0.05. Animal ethics was approved by the Committee on the Use of Live Animals for Teaching and Research (CULATR) with the approval no. of 1694-08. All animal handling procedures were carried out according to the guidelines of the Committee on the Use of Live Animals in Teaching and Research (CULATR), The University of Hong Kong.
Spheroid formation assay
The spheroid formation assay was modified from a previously reported protocol [36]. Briefly, PCa cells (200 cells per line) were seeded onto 12-well polyHEMA (Sigma)-coated plates. Cells were grown in DMEM/F12 medium (Invitrogen, Carlsbad, CA) for 14 days supplemented with 4 µg/mL insulin (Sigma), B27 (Invitrogen), 20 ng/mL EGF (Sigma), and 20 ng/mL basic FGF (Invitrogen) with PSP at either 250 µg/mL and 500 µg/mL. For serial passage of primary spheres, the primary spheres were treated with PSP for the above doses for 72 h and subsequently collected, dissociated with trypsin, and resuspended in DMEM/F12 medium with the above supplements. Each experiment was repeated in triplicate, and each data point represents the mean and standard derivation. Statistical difference was determined by Student’s t-test and was considered as significant if p<0.05.
Cell viability assay
Cell viability upon PSP treatment was measured by a 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described previously [37]. Briefly, cells were seeded on 96-well plates and treated with different concentrations of PSP for the indicated time. At the end of the treatment, MTT (Sigma, St. Louis, MO) was added to each well, and wells were incubated for 4 hr at RT. DMSO was then added to each well to dissolve the formazan crystals. The plate was incubated for a further 5 min at RT, and the optical density (OD) was measured at a wavelength of 570 nm on a Labsystem multiscan microplate reader (Merck Eurolab, Dietikon, Switzerland). All individual wells were analyzed in triplicate. The percentage of cell viability was presented as the OD ratio between the treated and untreated cells at the indicated concentrations.
Western Blotting
Detailed experimental procedures have been described previously [37]. Briefly, cells were lysed with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% deoxycholic acid, 1% NP-40, 0.1% SDS) with protease inhibitors (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mM PMSF) and the protein concentrations were determined using a DC Protein Assay Kit (Bio-Rad, Hercules, CA). Proteins were resolved in SDS- polyacrylamide gel by electrophoresis and then transferred onto Hybond-P PVDF membrane (Amersham Biosciences, Piscataway, NJ). The membranes were blocked by 10% non-fat dry milk in TBS-T or 3% non-fat dry milk in TBS and incubated with primary antibodies at room temperature against Akt (ser 473), Bcl-2, PARP (Cell signaling, Technology Inc, Beverly, MA), CD133 (Miltenyi Biotec, Auburn, CA), CD44, β-catenin and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr at room temperature. After washing with TBS-T, the membrane was incubated with either anti-mouse or anti-rabbit IgG secondary antibodies, and the signals were visualized using the ECL plus western blotting system (Amersham, Piscataway, NJ).
Cell cycle analysis
Cells were fixed with 1 ml ice cold 70% ethanol at 4°C. After fixation, cell pellets were collected by centrifugation, resuspended with 500 µl PBS, and then incubated at 4°C a day before performing flow cytometry. On the next day, cells were stained with propidium iodide (50 µg/ml) and RNase (1 µg/ml) for 30 min. Cell cycle analysis was performed on a flow cytometer EPICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter, Miami, FL).
Orthotopic implantation of PC-3-luc cells
The orthotopic model was established with procedures described previously [38]. Briefly, eight-week-old CB-17 SCID mice were anesthetized and placed under a dissecting microscope. An incision at the midline of the abdomen was made, exposing the dorsal prostate at the base of the bladder. Equal amounts of viable PC3-luc cells (2.5×104) with or without prior PSP treatment were injected into the dorsal prostates of the mice. The organs were replaced, and the abdomen was closed. Tumor development was monitored by measuring the bioluminescent signal every two weeks for six weeks after tumor implantation. Mice were sacrificed at the end of the experiment and prostate tissues were collected for physical examination. Statistical difference was determined by a two-tailed t-test and was considered significant if p<0.05. All surgical and animal handling procedures were carried out according to the guidelines of the Committee on the Use of Live Animals in Teaching and Research (CULATR), The University of Hong Kong.
Author Contributions Top
Conceived and designed the experiments: M-TL. Performed the experiments: S-UL TK-WL JL DT-WL Y-TC FLC SM. Analyzed the data: IO-LN Y-CW. Wrote the paper: S-UL M-TL.
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PSP (Coriolus versicolor e cancro alla prostata)
Un fungo commestibile usato da secoli in Cina e in altri paesi asiatici per le sue qualita’ medicinali, la ‘coda di tacchino’ o Coriolus versicolor, si e’ rivelato altamente efficace nel combattere il cancro alla prostata. Scienziati dell’Universita’ di tecnologia del Queensland (Qut), in Australia, hanno dimostrato che il composto detto polisaccaropeptide (Psp), estratto dal fungo, ha avuto un’efficacia del 100% nel sopprimere lo sviluppo del cancro alla prostata in topi di laboratorio, colpendo le cellule staminali del tumore stesso e sopprimendo la sua formazione.
In una relazione sulla rivista della Public Library of Science, PLoS One, Patrick Ling dell’Istituto per la salute biomedica e l’innovazione della Qut, scrive che i risultati rappresentano un passo importante nel combattere una malattia tra le piu’ diffuse e letali. ‘Cio’ che volevamo dimostrare era se quel composto puo’ arrestare dall’inizio lo sviluppo dei tumori alla prostata… In passato altri inibitori hanno mostrato in sperimentazioni di ricerca un’efficacia del 70%, mentre con il Psp abbiamo osservato un’efficacia del 100%, per di piu’ senza alcun effetto collaterale’.
Ling aggiunge che le terapie convenzionali sono efficaci solo contro certe cellule cancerose, ma non quelle staminali, che danno inizio al cancro e fanno progredire la malattia. Il composto inoltre potra’ migliorare l’efficacia dei trattamenti correnti. ‘Il problema maggiore di tali trattamenti e’ che vi sono sempre dei tumori soffici residui, che resistono alle terapie. Ora potremo eliminare quei tumori residui, colpendo le cellule staminali, e cosi’ rafforzare la sopravvivenza d’insieme dei pazienti’, scrive.
In Asia il Coriolus versicolor e’ ambito da molti secoli per le sue efficacissime sostanze biovitali, per il conseguente valore fisiologico-nutrizionale e per l’effetto stimolante del sistema immunitario, soprattutto grazie all’elevata percentuale di polisaccaridi con legami proteici fra cui il Psp.
Mushroom compound heals cancer stem cells and prevents tumors
NaturalNews) Incredible new research out of Australia has shown that a compound called polysaccharopeptide (PSP), which comes from a type of mushroom called “Turkey Tail,” is 100 percent effective at targeting prostate cancer stem cells and suppressing tumor formation. The research, which has been published in the online journal PLoS ONE, represents the first to show that a natural substance is totally and completely effective in every single trial.
For the study, Dr. Patrick Ling, senior researcher from the Australian Prostate Cancer Research Centre in Queensland and the Institute for Biomedical Health & Innovation (IHBI) at QUT, and his colleagues fed PSP for 20 weeks to mice with prostate cancer. Compared to another group of prostate cancer mice not given PSP, which subsequently developed prostate tumors, the PSP group remained completely free of tumors.
“The findings are quite significant,” said Dr. Ling. “What we wanted to demonstrate was whether [PSP] could stop the development of prostate tumors in the first place. In the past, other inhibitors tested in research trials have been shown to be up to 70 percent effective, but we’re seeing 100 percent of this tumor prevented from developing with PSP.”
Turkey Tail mushrooms are native to many northern forests around the world, and they have been highly studied for their medicinal benefits. Particularly in China and Japan, Turkey Tail mushrooms are already used as anti-cancer medicine, as well as an antimicrobial, anti-malarial, and immunomodulating natural treatment. And besides PSP, Turkey Tail mushrooms contain many other anti-cancer compounds like beta-glucan-proteins, polysaccharide K (PSK), and ergosterol derivatives, all of which provide substantial health benefits.
“Our findings support that PSP may be a potent preventative agent against prostate cancer, possibly through targeting of the prostate cancer stem cell population,” added Dr. Ling.
Turkey Tail mushroom extracts with high levels of PSP and many other anti-cancer compounds can be found at most natural grocers, health food shops, and online vitamin and supplement venders.
Editor’s Note: NaturalNews is strongly against the use of all forms of animal testing. We fully support implementation of humane medical experimentation that promotes the health and well-being of all living creatures.
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Learn more: http://www.naturalnews.com/032574_Turkey_Tail_cancer.html#ixzz1iLzlBzce
The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell lines via arrest of cell cycle and induction of apoptosis
Eva Jiménez-Medina1, Enrique Berruguilla1, Irene Romero1, Ignacio Algarra2, Antonia Collado3, Federico Garrido1 and Angel Garcia-Lora1*
• * Corresponding author: Angel Garcia-Lora angel.miguel.exts@juntadeandalucia.es
Author Affiliations
1 Servicio de Análisis Clínicos e Inmunologia, Hospital Universitario Virgen de las Nieves, Universidad de Granada, Av. de las Fuerzas Armadas 2, 18014 Granada, Spain
2 Departamento de Ciencias de la Salud, Universidad Jaén, Jaén, Spain
3 Unidad de Investigación, Hospital Universitario Virgen de las Nieves, Granada, Spain
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BMC Cancer 2008, 8:78 doi:10.1186/1471-2407-8-78
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2407/8/78
Received: 5 November 2007
Accepted: 24 March 2008
Published: 24 March 2008
© 2008 Jiménez-Medina et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
BACKGROUND
Protein-bound polysaccharide (PSK) is derived from the CM-101 strain of the fungus Coriolus versicolor and has shown anticancer activity in vitro and in in vivo experimental models and human cancers. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the adjuvant treatment of gastric, esophageal, colorectal, breast and lung cancers. These studies have suggested the efficacy of PSK as an immunomodulator of biological responses. The precise molecular mechanisms responsible for its biological activity have yet to be fully elucidated.
METHODS
The in vitro cytotoxic anti-tumour activity of PSK has been evaluated in various tumour cell lines derived from leukaemias, melanomas, fibrosarcomas and cervix, lung, pancreas and gastric cancers. Tumour cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of PSK on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in PSK-treated cells.
RESULTS
PSK showed in vitro inhibition of tumour cell proliferation as measured by BrdU incorporation and viable cell count. The inhibition ranged from 22 to 84%. Inhibition mechanisms were identified as cell cycle arrest, with cell accumulation in G0/G1 phase and increase in apoptosis and caspase-3 expression. These results indicate that PSK has a direct cytotoxic activity in vitro, inhibiting tumour cell proliferation. In contrast, PSK shows a synergistic effect with IL-2 that increases PBL proliferation.
CONCLUSION
These results indicate that PSK has cytotoxic activity in vitro on tumour cell lines. This new cytotoxic activity of PSK on tumour cells is independent of its previously described immunomodulatory activity on NK cells.
Background
A number of bioactive molecules, including antitumour substances, have been identified in various mushroom species. Polysaccharides are the best known and most potent of these and have antitumour and immunomodulating properties [1-5]. PSK, a protein-bound polysaccharide obtained from Basidiomycetes, also known as Krestin, has been used as an agent in the treatment of cancer in Asia for over 30 yrs [6-8]. PSK is derived from the fungus Coriolus versicolor and has documented anticancer activity in vitro in experimental models [9] and in human clinical trials. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the treatment of gastric, esophageal, colorectal, breast and lung cancers [10,11]. These studies have suggested the efficacy of PSK as an immunomodulator of biological response.
Previous reports indicated that PSK might act in different ways: as antioxidant [5,12,13]; as inhibitor of metalloproteinases and other enzymes involved in metastatic processes [14] and as inhibitor of the action of various carcinogens in vulnerable cell lines. However its most important and widely reported property is its immunomodulatory capacity. PSK may act to increase leukocyte activation and response via upregulation of key cytokines. Thus, natural killer (NK) and lymphocyte-activated killer (LAK) cell activation has been demonstrated in vivo and in vitro [15,16]. Our group demonstrated that PSK is capable of inhibiting metastatic colonization in vivo in some experimental fibrosarcomas, and that this effect is mediated by activation of NK cells [17,18]. Moreover, the NK cell line NKL, derived from a large granular lymphocyte leukaemia [19], is activated in vitro by PSK [16]. This activation may replace IL-2 in inducing the proliferation and cytotoxicity of NKL cells. The signal transduction pathways involved in the responses to IL-2 or PSK are different: IL-2 increases PKCα and ERK3 expression and decreases ERK2 expression, whereas PSK decreases PKCα expression and increases ERK3 expression [20]. PSK also enhances CRE binding activity, while IL-2 increases SP-1 and modifies GAS/ISRE, IRF-1 and STAT5 [21]. In addition, PSK and IL-2 have been shown to bind to different receptor on NKL cells [22].
The direct in vitro effect of PSK on the proliferation of tumour cell lines was compared with its effect on PBLs. PSK had cytotoxic activity on tumour cell lines, inhibiting proliferation, producing cell cycle arrest and cell accumulation in G0/G1phase and inducing apoptosis.
Methods
PROTEIN-BOUND POLYSACCHARIDE K
Protein-bound polysaccharide K (PSK) was kindly provided by Kureha Chemical Ind. Co. (Tokyo, Japan). It is prepared by extracting cultured mycelia of Coriolus versicolor with hot water. The precipitate is separated from the clear supernatant with saturated ammonium sulfate, then desalted and dried [23]. Protein-bound polysaccharide K was dissolved in RPMI medium or water and heated at 50°C for 20–30 min until a clear solution appeared. The PSK preparation was filter-sterilized and diluted in culture medium or water to the desired concentration. Protein-bound polysaccharide K was previously titrated in NKL cells [16] and the working dilution was 100 μg/mL. PSK extract digested with neuraminidase was also tested, digesting 100 μg of PSK with 4 μl (Sigma) and incubating for 3 h at 37°C. Our group previously showed that PSK is composed of two bands of very high molecular weight [22]. After digestion with neuraminidase, these bands are reduced to a single band of about 12 kd. These results indicate that PSK is probably composed of a single 12-kd protein, and that this protein is highly glycosylated [22]. Two different extracts of PSK were also used: one rich in sugars and other rich in proteins.
CELL LINES AND CELL CULTURE
The following tumour cell lines were studied: B16 murine melanoma, B9 murine MCA-induced fibrosarcoma, Ando-2 human melanoma, AGS human gastric cancer, A-549 human lung cancer, Hela human cervical adenocarcinoma and Jurkat T lymphoma leukemia. The NKL studied was established from PBLs of a patient with LGL leukemia [19]. All cell lines were obtained from the American Type Culture Collection (Manassas, USA) except for the B9 cell line, which was generated at our laboratory, and the Ando-2 and NKL cell lines, kindly provided by P. Coulie (Unite de Genetique Cellulaire, Louvain University, Brussels, Belgium), F. X. Real (Instituto Municipal de Investigaciones Medicas, Barcelona, Spain) and Dr. M. Lopet-Botet (Universidad Pompeu-Fabra, Barcelona, Spain), respectively.
Cell lines derived from solid tumours were grown at 37°C in a humidified atmosphere of 5% CO2 in DMEM culture medium (Gibco, Paisley UK) supplemented with 10% heat-inactivated foetal bovine serum (Life Technologics, Milan Italy), antibiotics and glutamine. Jurkat T cell leukemia was cultured in RPMI 1640 with 10% heat-inactivated fetal bovine serum. The NKL cell line was cultured in RPMI 1640 with 10% heat-inactivated human AB serum (Sigma Chemical, St Louis, MO; USA) and human recombinant IL-2 (100 U/ml; purity > 97%, specific activity, 2 × 106 U/mg) (Roche, Nutley, NJ; USA).
IN VITRO CYTOTOXICITY ASSAYS
The effect of PSK on tumour cell proliferation was assessed by measuring BrdU incorporation with the BrdU colorimetric ELISA Cell Proliferation Kit (Roche Diagnostic). Cells were plated in 96-well microculture plates (5 × 103 cells/well). Every 48 h, the culture medium was replaced and PSK was added. After 48–96 h, BrdU labelling reagent was added and cultured for a further 1–3 h. Assays were also performed by counting viable cells using Trypan Blue. Briefly, cancer cell lines were seeded into culture tissue-flask (1.5–2 × 105/culture tissue-flask) and incubated for 24 h at 37°C in a humidified atmosphere of 5% CO2. Cells were then treated with 100 μg/ml of PSK in the culture medium, which was replaced every 48 h. After 4–6 days, cells were collected by centrifugation and a small sample of cell suspension was diluted in 0.4% Trypan Blue, counting cells in a haemocytometer chamber. Each cell sample was counted in this way at least three times and each assay was repeated at least three times.
LYMPHOCYTE AND NKL PROLIFERATION ASSAY
Human lymphocytes were isolated from venous blood by the Ficoll-Hystopaque separation method. Proliferation of PBLs was analyzed in vitro using 5-bromo-2′-deoxyuridine (BrdU) labelling of DNA-synthesizing cells with the above-mentioned kit. PBLs were seeded in 96-well microculture plates at a cell density of 5 × 104 per well. Two different concentrations of PSK were used, 100 μg/ml and 50 μg/ml. Concanavalin A (5 μg/ml, Sigma) and IL-2 were used as positive controls. PSK was also used in combination with IL-2 or Concanavalin A. After 48 h of culture in presence or absence of PSK, BrdU labelling reagent (final concentration 10 μM) was added and cells were cultured for 24 h. Cells were then fixed for 30 min and incubated with anti-BrdU for 1 h at 37°C. 100 μl of tetramethyl-benzidine (TMB) was used as substrate. Optical densities were determined at 370 nm by means of an ELISA microplate reader (Biotek, Power-Wave XS). Controls were the culture medium, cells cultured only in medium and cells incubated with anti-BrdU in absence of BrdU. All experiments were repeated at least three times.
CELL CYCLE DISTRIBUTION ANALYSIS
Briefly, cells were plated in six-well plates (5 × 105 per well) or in culture tissue-flask (15 × 105) and continuously exposed for 4 days to 100 μg/ml of PSK. The DNA synthesis rate was examined by BrdU incorporation method using FITC BrdU Flow Kit (BD Pharmingen) according to manufacturer’s instructions. BrdU was then detected by DNase cell treatment using FITC-conjugated anti-BrdU antibody. Cells were washed with 1 ml 1 × BD Perm/Wash Buffer, and 20 μl 7-amino-actinomycin D was added. Analysis was performed with 50000 cells using Cell Quest Software and FACScan flow cytometer (Becton-Dickinson).
ANNEXIN V BINDING ASSAY TO DETECT APOPTOTIC CELLS
After treatment of cancer cells with PSK for four days, cells were detached from the culture tissue-flask with PBS containing 3 mM EDTA. These cells were then collected together with floating cells, washed twice with cold PBS and resuspended in binding buffer at a concentration of 1 × 106 cells per ml; 100 μl of solution was incubated for 30 min at 4°C with 5 μl of Annexin V-PE antibody (BD Biosciences), and 5 μl of 7-amino-actynomycin D was then added. Cells were incubated for 15 min in darkness, and 400 μl of staining buffer was added before flow cytometry analysis. Apoptosis was analyzed by quadrant statistics as follows: Annexin V- and 7-AAD-negative cells are alive; Annexin V-positive and 7-AAD-negative cells are in early stages of apoptosis; Annexin V-negative and 7-AAD-positice cells are dead but not by apoptosis; and Annexin V-positive and 7-AAD-positive cells are in mid- or end-stage apoptosis.
ASSAY FOR ACTIVE CASPASE-3 EXPRESSION
FITC conjugated monoclonal anti-active-caspase-3 antibody (BD Biosciences) was used to determine whether the protease caspase-3 is involved in PSK-induced apoptosis. After 4-day treatment with PSK, cancer cells were washed twice with cold PBS and fixed and permeabilized in Cytofix/Cytoperm buffer. Then, cells were incubated with FITC-conjugated monoclonal rabbit anti-active human-caspase-3 antibody for 30 min. Cells were washed twice and 500 μl of 1 × Perm Wash Buffer was added before analysis by flow cytometry.
STATISTICAL ANALYSIS
Values are expressed as means ± SD. Student’s t-test was used for statistical comparisons, considering a significance value of P < 0.05.
Results
PSK INHIBITS IN VITRO TUMOUR CELL PROLIFERATION
Tumour cell lines were cultured in 96-well plate for 48–72 h (2.5–5 × 103 cells) in medium alone (control) or with PSK (100 μg/ml or 50 μg/ml) for 4–6 days. Cell proliferation was then measured by BrdU incorporation (absorbance), which was significantly lower in PSK-treated versus untreated tumour cells (Fig. 1). AGS and A549 cell lines showed a strong decrease in absorbance after treatment with 100 μg/ml PSK that was less marked after treatment with 50 μg/ml of PSK. Inhibition of proliferation was around 65% in melanoma cell lines B16 and Ando-2, lower in Hela and Jurkat cell lines and lowest (20%) in B9 murine fibrosarcoma (Fig. 1). PSK-treated tumour cells showed morphological changes (rounded and granulated morphology, increased vacuolisation, cell shrinkage) and a large number of the cells detached from culture flasks.
Figure 1. Effect of PSK on tumour cell line proliferation. B16, A549, Hela, AGS, Jurkat, B9 and Ando-2 tumour cell lines were treated or not with 50 μg/ml or 100 μg/ml of PSK for 72–96 h. Tumour cells (2.5 to 5 × 104) were plated in quadruplicate in 96-well plates. The proliferation of tumour cells was determined by BrdU incorporation and absorbance measurement. All cell lines analysed showed inhibition of proliferation. Each column represents the mean of five independent experiments ± SD. P < 0.001 versus control.
These assays were repeated in cell culture flasks (1.5–2.5 × 105cells), and viable cells were counted in Haemacytometer chamber using Trypan Blue. As shown in Table 1, there was a significant decrease in the final number of viable cells, with a proliferation inhibition of 22%–84% versus control cells. There was an excellent correlation between the results obtained with the two assays (absorbance and cell count). In Hela tumour cells, proliferation inhibition was higher after treatment with 50 μg/ml versus 100 μg/ml of PSK. In all other tumour cell lines, proliferation inhibition was similar or higher at 100 μg/ml PSK.
Table 1. Proliferation of tumour cell lines treated with PSK
PSK INCREASES IN VITRO PROLIFERATION OF IL-2-STIMULATED LYMPHOCYTES
A dose-response analysis was performed to determine the in vitro effect of PSK on human PBLs. PBLs (5 × 104) were plated in 96-well tissue plate for 48–72 h with eight serially diluted extractions ranging from 500 μg/ml (concentration n°8) to 3.9 μg/ml (concentration n°1). Concentration n°0 represents cells cultured in medium alone. BrdU incorporation during DNA synthesis was then measured by ELISA. Optical densities were very similar between treated and untreated PBLs (data not shown). However, simultaneous treatment of PBLs with IL-2 (100 U/ml) + PSK (100 μg/ml) produced a higher proliferation rate (4.5-fold) versus PBLs treated with IL-2 alone (3-fold) (Fig. 2). Untreated and Concanavalin A-treated PBLs served as controls (Fig. 2).
Figure 2. Effect of PSK on PBL proliferation. PBLs were cultured with PSK or IL-2 or with PSK+IL-2. PBLs (50 × 104 cells) were seeded in quadruplicate in 96-well plates. The proliferation was determined by BrdU incorporation and absorbance measurement. PSK showed a synergistic effect with IL-2, increasing PBL proliferation. PSK alone did not induce PBLs proliferation. Each column represents the mean of five independent experiments ± SD. *P < 0.001 versus control.
EFFECT OF DIFFERENT VARIANTS OF PSK
Tumour cell proliferation inhibition was compared among different PSK variants. Neuraminidase treatment digests glicosylated proteins. A549 tumour cell line was cultured in medium alone (control) or with PSK (100 μg/ml) or neuraminidase-treated PSK (100 μg/ml) for 4–6 days and then counted using trypan blue. No significant differences in proliferation inhibition were found between PSK and neuraminidase-treated PSK (Fig. 3a). The same results were found for sugar-rich and protein-rich PSK variants as for PSK (data not shown).
Figure 3. In vitro activity of neuraminidase treated-PSK. a) A549 tumour cell line was cultured with neuraminidase-treated PSK or PSK. A549 tumour cells (20 × 104) were seeded in culture flask and treated with PSK for 96 h, estimating cell viability by means of Trypan blue exclusion. Both agents produced a similar inhibition ofproliferation. b) NKL cell line was cultured with PSK or neuraminidase treated-PSK and proliferation was determined by BrdU incorporation and absorbance measurement. Both agents induced a similar increase of proliferation. Each column represents the mean of five independent experiments ± SD. *P < 0.001 versus control.
It was previously reported that PSK induces proliferation and activation of NKL cells [16]. Treatment of NKL cells with 100 μg/ml PSK or neuraminidase-treated PSK for 96 h induced a similar increase in their proliferation (Fig. 3b), which was slightly higher than that obtained after culture of NKL with IL-2 alone (Fig. 3b). Induction of NKL proliferation was slightly lower in sugar-rich and protein-rich PSK variants; this difference was not significant (data not shown)
CELL CYCLE PHASE DISTRIBUTION ANALYSIS OF PSK-TREATED CELLS
Mechanisms of PSK cytotoxic activity were analysed by flow cytometry in order to study the effect on cell cycle phase distribution. Culture of AGS tumour cell line with 100 μg/ml of PSK produced total cell cycle arrest with cell accumulation in G0/G1 phase and no cells in S phase (Fig. 4). Cell cycle phase distributions were: 32.2% G0/G1, 31.1% S and 16.2% G2/M in control AGS cells and 60.8% G0/G1, 0% S and 14.1% G2/M in PSK-treated AGS cells. Similar results were found in Ando-2, A549 and B16 tumour cell lines (Table 2). Results in B9 fibrosarcoma showed a slowing rather than an arrest of the cell cycle (Fig.4), with a partial accumulation in G0/G1 phase (49.15% untreated cells and 63.17% PSK-treated cells) at the expense of a decrease in S phase (20.54% vs. 15.14%) and G2/M phase (12.6% vs. 6.96%). Similar results were found in Hela and Jurkat tumour cells (Table 2). These results indicate that PSK produces arrest or slowing of the cell cycle according to the tumour cell histology.
Table 2. Effect of PSK on cell cycle distribution of tumor cell lines.
Figure 4. Cell cycle analysis of cells treated with PSK. Tumour cell lines were treated with PSK for 96 h. Cell cycle distribution was determined by flow cytometry using BrdU incorporation and 7-AAD. Data indicate the percentage of cells in each phase of cell cycle. Results are representative of three independent experiments.
ANALYSIS OF APOPTOSIS IN TUMOUR CELLS TREATED WITH PSK
Cancer cell lines were treated with 100 μg/ml PSK for 4 days to examine the capacity of PSK to induce apoptosis. Untreated or PSK-treated cancer cells were incubated with Annexin V-PE in a buffer containing 7-amino-actinomycin (7-AAD) and analyzed by flow cytometry. Figure 5 depicts representative results for AGS and B9 tumour cells. PSK increased apoptosis from 4.32% (untreated cells) to 37.52% in AGS cells but not in B9 tumour cells (untreated cells, 11.37% vs. PSK-treated cells, 12.11%). Table 3 depicts the results for other tumour cell lines, showing that PSK induces apoptosis in A549, B16 and Ando-2 tumour cells.
Figure 5. Apoptosis analysis of cells treated with PSK. AGS cell line was untreated or treated with 50 μg/ml of PSK for 96 h. Cells were double-stained with annexin V and 7AAD and analyzed by flow cytometry. PSK produced apoptosis in AGS tumour cell line. B9 tumour cell line was also cultured with PSK but apoptosis was not detected in this tumour cell line. All experiments were performed at least three times and gave similar results.
Table 3. Apoptosis induction in cancer cell lines after treatment with PSK for 4 days.
EXPRESSION OF ACTIVE HUMAN CASPASE-3
Caspases are the main enzymes involved in the apoptotic pathway and the participation of active caspase-3 in PSK-induced apoptosis was evaluated. Tumour cells were treated with PSK (100 μg/ml) for 4 days, then permeabilized, fixed and stained for active human caspase-3 and analyzed by flow cytometry. In the AGS cell line, untreated cells were negative for presence of active-caspase-3, whereas around 36% of PSK-treated cells showed detectable active caspase-3 (Fig. 6). However, in tumour cell lines in which PSK did not produce apoptosis, e.g., B9 tumour cells, no caspase-3 expression was detected after PSK treatment (Fig. 6). Table 4 depicts the results obtained with the other tumour cell lines analysed.
Figure 6. Caspase-3 expression in tumour cell lines treatedwith PSK. AGS and B9 tumour cell lines were treated with 50 μg/ml of PSK and expression was analysed by flow cytometry using FITC conjugated monoclonal anti-active-caspase-3 antibody. Data indicate the percentage of cells positive for presence of active-caspase-3. PSK produced increased caspase-3 expression in AGS but not in B9 tumour cell lines. Results are representative of three experiments.
Table 4. Expression of caspase-3 in cancer cell lines after treatment with PSK for 4 days.
Discussion
Several clinical assays have reported the anti-tumour properties of PSK and its synergestic effect in combined therapies [9,24,25]. Our group previously reported the immunomodulatory activity of PSK on NK cells, producing in vitro proliferation and activation of NKL cells [16,20,21]. In the present study, we have identified a new cytotoxic anti-tumour activity of PSK. This activity varied according to the histological origin of the tumour cell lines under study, with inhibition rates ranging from 84% to 22% (Table 1). The highest profileration inhibition rates were found in AGS (84%) and A549 (80%) cell lines (gastric and lung cancer, respectively). PSK was previously reported to be effective in adjuvant immunotherapy for patients after curative resection of gastric cancer [25], and this effect was attributed to its immunomodulatory activity on NK cells [26]. Our group previously reported that PSK mediates induction of the NKL cell proliferation and activation. The present results suggest that PSK may also exert a direct antitumour cytotoxic activity. Inhibition was around 65% in melanoma cell lines Ando-2 (human) and B16 (mice) and was lowest (22%) in the B9 murine fibrosarcoma cell line. Deglycosylation of PSK by neuraminidase treatment did not modify its cytotoxic effect on tumour cell lines. The sugar-rich and protein-rich PSK variants showed identical results to those of PSK in their inhibition of proliferation of tumour cell lines in vitro. These results indicate that the cytotoxic properties are in a compound that is present in all three variants studied and does not vary among them.
Interestingly, PSK had the opposite effect on lymphocytes. Thus, PSK, in synergy with IL-2, induced proliferation of PBLs. PSK also induced proliferation and activation of NKL cells, producing an effect similar to that of IL-2. Hence, PSK has a cytotoxic effect on tumour cells and a mitotic effect on lymphocytes and NK cells.
The cell cycle was arrested or slowed by PSK according to the histological origin of the tumour cells. PSK is known to increase docetaxel-induced apoptosis of NOR-P human pancreatic cancer cells [27] and of Namalwa Burkitt lymphoma cells [28]. PSK induced apoptosis in the AGS cell line but not in all tumour cell lines analysed and induced caspase-3 expression in some tumour cell lines but not all. These results indicate that PSK may induce cytotoxic activity by different molecular mechanisms according to the histology of tumour.
The molecular mechanisms implicated in PSK-induced proliferation and activation of NKL cells have been widely described, showing that PSK and IL-2 bind to different receptors on NKL cells and induce different signal transduction pathways [20-22]. The present results indicate that the anti-tumour properties of PSK observed in clinical trials might be due to a dual biological activity: 1) a direct cytotoxic activity on tumour cells and 2) an immunomodulatory activity largely produced by NK cell activation. A similar dual activity has also been described in a Calendula extract, LACE, which produces an in vitro cytotoxic activity and in vivo immunomodulatory effect on tumour cell lines, including human and mouse melanioma cells, increasing the number and activation of CD4+, CD19+ and NKT cells [29]. PSK suppressed in vivo metastases in spontaneous metastasis assays of mouse fibrosarcoma, melanoma, rat hepatoma AH60C and mouse colon cancer 26 [17,30,31]via NK cell activation. Based on the present findings, it can be hypothesised that this anti-metastatic capacity may also derive from the cytotoxic component of PSK.
Research into the biological mechanisms underlying the anti-tumour effect of PSK is ongoing. We can now add a direct cytotoxic effect on tumour cells to the previously described immunomodulatory effect of this polysaccharide. Greater knowledge of the molecular mechanisms implicated in PSK anti-tumour activity may improve cancer immunotherapy, leading to the application of new anti-tumour protocols.
Conclusion
PSK shows in vitro growth inhibition of various tumour cell lines, producing cell cycle arrest/slowing, apoptosis and induction of caspase-3 expression. In combination with IL-2, PSK induces proliferation of PBLs. The biological activity of PSK appears to include both an immunomodulatory effect on NK cells and a cytotoxic effect on tumour cells
Abbreviations
PBLs: Peripheral Blood Lymphocytes; LACE: laser-activated calendula extract; 7-AAD: 7-amino-actinomicin D; BrdU: 5-Bromo-2-deoxyuridine; Concan.: Concanavalin A; LAK: lymphocyte-activated killer; NK: Natural killer; TMB: tetramethyl-benzidine.
Competing interests
Materials for these studies were partially supported by a grant from Kureha Chemical Industry (Japan), which manufactures PSK. The authors declare that they have no other competing interest.
Authors’ contributions
EJM, EB and IR performed the assays. IA and AC helped in some experiments. FG and AGL designed the study and drafted the manuscript. All authors have read and approved the final manuscript.
Acknowledgements
The authors thank I. Linares for technical assistance. AGL was supported by FIS Postdoctoral Research Contract CP03/00111. Studies were partially supported by a grant from Kureha Chemical Industry (Japan).
PSP (Coriolus versicolor e cancro alla prostata)
Un fungo commestibile usato da secoli in Cina e in altri paesi asiatici per le sue qualita’ medicinali, la ‘coda di tacchino’ o Coriolus versicolor, si e’ rivelato altamente efficace nel combattere il cancro alla prostata. Scienziati dell’Universita’ di tecnologia del Queensland (Qut), in Australia, hanno dimostrato che il composto detto polisaccaropeptide (Psp), estratto dal fungo, ha avuto un’efficacia del 100% nel sopprimere lo sviluppo del cancro alla prostata in topi di laboratorio, colpendo le cellule staminali del tumore stesso e sopprimendo la sua formazione.
In una relazione sulla rivista della Public Library of Science, PLoS One, Patrick Ling dell’Istituto per la salute biomedica e l’innovazione della Qut, scrive che i risultati rappresentano un passo importante nel combattere una malattia tra le piu’ diffuse e letali. ‘Cio’ che volevamo dimostrare era se quel composto puo’ arrestare dall’inizio lo sviluppo dei tumori alla prostata… In passato altri inibitori hanno mostrato in sperimentazioni di ricerca un’efficacia del 70%, mentre con il Psp abbiamo osservato un’efficacia del 100%, per di piu’ senza alcun effetto collaterale’.
Ling aggiunge che le terapie convenzionali sono efficaci solo contro certe cellule cancerose, ma non quelle staminali, che danno inizio al cancro e fanno progredire la malattia. Il composto inoltre potra’ migliorare l’efficacia dei trattamenti correnti. ‘Il problema maggiore di tali trattamenti e’ che vi sono sempre dei tumori soffici residui, che resistono alle terapie. Ora potremo eliminare quei tumori residui, colpendo le cellule staminali, e cosi’ rafforzare la sopravvivenza d’insieme dei pazienti’, scrive.
In Asia il Coriolus versicolor e’ ambito da molti secoli per le sue efficacissime sostanze biovitali, per il conseguente valore fisiologico-nutrizionale e per l’effetto stimolante del sistema immunitario, soprattutto grazie all’elevata percentuale di polisaccaridi con legami proteici fra cui il Psp.
Mushroom compound heals cancer stem cells and prevents tumors
NaturalNews) Incredible new research out of Australia has shown that a compound called polysaccharopeptide (PSP), which comes from a type of mushroom called “Turkey Tail,” is 100 percent effective at targeting prostate cancer stem cells and suppressing tumor formation. The research, which has been published in the online journal PLoS ONE, represents the first to show that a natural substance is totally and completely effective in every single trial.
For the study, Dr. Patrick Ling, senior researcher from the Australian Prostate Cancer Research Centre in Queensland and the Institute for Biomedical Health & Innovation (IHBI) at QUT, and his colleagues fed PSP for 20 weeks to mice with prostate cancer. Compared to another group of prostate cancer mice not given PSP, which subsequently developed prostate tumors, the PSP group remained completely free of tumors.
“The findings are quite significant,” said Dr. Ling. “What we wanted to demonstrate was whether [PSP] could stop the development of prostate tumors in the first place. In the past, other inhibitors tested in research trials have been shown to be up to 70 percent effective, but we’re seeing 100 percent of this tumor prevented from developing with PSP.”
Turkey Tail mushrooms are native to many northern forests around the world, and they have been highly studied for their medicinal benefits. Particularly in China and Japan, Turkey Tail mushrooms are already used as anti-cancer medicine, as well as an antimicrobial, anti-malarial, and immunomodulating natural treatment. And besides PSP, Turkey Tail mushrooms contain many other anti-cancer compounds like beta-glucan-proteins, polysaccharide K (PSK), and ergosterol derivatives, all of which provide substantial health benefits.
“Our findings support that PSP may be a potent preventative agent against prostate cancer, possibly through targeting of the prostate cancer stem cell population,” added Dr. Ling.
Turkey Tail mushroom extracts with high levels of PSP and many other anti-cancer compounds can be found at most natural grocers, health food shops, and online vitamin and supplement venders.
Editor’s Note: NaturalNews is strongly against the use of all forms of animal testing. We fully support implementation of humane medical experimentation that promotes the health and well-being of all living creatures.
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