suppression of growth and invasive behavior of human prostate cancer cells by prostacaidtm:...

7/30/2019 Suppression of growth and invasive behavior of human prostate cancer cells by ProstaCaidTM: Mechanism of acti

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INTERNATIONAL JOURNAL OF ONCOLOGY 38: 1675-1682, 2011

Abstract. Since te use of dietary supplements as alternative

treatments or adjuvant terapies in cancer treatment is growing,

a scinic ricaion o hir ioogica aciiy and h daidmecanisms of teir action are necessary for te acceptance of

dietary supplements in conventional cancer treatments. In te

present study we ave evaluated te anti-cancer effects of dietary

supplement ProstaCaid (PC) wic contains mycelium from

medicinal musrooms (Ganoderma lucidum, Coriolus versi-

color, Phellinus linteus), saw pamo rry, pomgrana,pumpkin seed, green tea [40% epigallocatecin-3-gallate

(EGCG)], Japanese knotweed (50% resveratrol), extracts of

turmeric root (BCM-95), grap skin, pygum ark, sarsapariaroo, Scuaria araa, uhro roo, Jo's ars, asragausroo, skucap, dandion, copis roo, roccoi, and singingn, wih purid iamin C, iamin D3, snium, qurcin,

cirus ioaonoid compx, sitosterolzinc, lycopene, lipoicacid, oron, rrin and 3.3'-diinodoymhan (DIM). Wshow ha PC ramn rsud in h inhiiion o cproliferation of te igly invasive uman ormone refractory

(independent) PC-3 prostate cancer cells in a dose- and time-

dependent manner wit IC50 56.0, 45.6 and 39.0 g/ml for 24,

48 and 72 h, rspciy. DNA-microarray anaysis dmon-srad ha PC inhiis proiraion hrough h moduaionof expression ofCCND1, CDK4, CDKN1A, E2F1, MAPK6

and PCNA genes. In addition, PC also suppresses metastatic

haior o PC-3 y h inhiiion o c adhsion, c migraionand cell invasion, wic was associated wit te down-regulation

of expression ofCAV1, IGF2, NR2F1, and PLAUgenes andsuppressed secretion of te urokinase plasminogen activator

(uPA) from PC-3 cells. In conclusion, te dietary supplement

PC is a promising naura compx wih h poncy o inhiiinvasive uman prostate cancer.

Introduction

Prostate cancer is one of te leading causes of cancer-related

dah in Amrican mn du o is unprdica hormonaindependence and igly metastatic nature (1). Prostate cancers

usually progress from androgen-dependent to androgen-

independent penotype wit igly metastatic properties (2-4).

Tus, te metastasis of prostate cancer remains te primary

issue in improving prostate cancer patient survival. Moreover,

hormon aaion hrapy and chmohrapy or adancdsag prosa cancr sm no o or mor n in improingpatient survival rate (5,6). Terefore, tere is an urgent need

or h idnicaion o nw hrapis wih ani-cancr csin igly metastatic prostate cancers. Recent epidemiologic

and experimental studies sow tat natural agents ave potential

cemopreventive and cemoterapeutic action for prostate

cancr. Naura hra and phyochmica agns ar ingrecognized as an alternative terapy of prostate cancer patients

(7,8).

ProstaCaid (PC) is a dietary supplement consisting of a

33-ingrdin comprhnsi poyhra and nurin prparaionwhich inhiis arran c proiraion and inducs apoposisin androgen dependent and independent uman and mouse

prostate cancer cell lines (9). PC contains mycelium from medi-

cinal musrooms (Ganoderma lucidum, Coriolus versicolorand Phellinus linteus), wic separately demonstrated anti-

cancer properties (7,10-12). Ganoderma lucidum (G. lucidum)

(Ling Zi, Reisi) is a popular medicinal musroom used as a

traditional medicine in Cina, Korea and Japan for more tan

2,000 years to prevent or treat different diseases, including

cancer (13,14). Te anti-cancer properties ofG. lucidum ave

n ariud o h poysaccharids, which ar rsponsifor te modulation of te immune system, or triterpenes, wic

demonstrate cytotoxic activity against a variety of cancer cells

incuding ras, prosa, ung, coon, sarcoma, hpaoma andleukemia cells (13-15). G. lucidum has n shown o inhiiproiraion y c cyc arrs a h G2/M phas and inducdapoposis in human prosa cancr cs y down-rguaionof transcription factors NF-B (16), resulting in modulating

te expression of NF-B-regulated Bcl-2 and Bcl-xl. G. lucidum

Suppression of growth and invasive behavior of human prostate

cancer cells by ProstaCaidTM: Mechanism of activity

JIAhUA JIANG1, ISAAC ELIAZ

2and DANIel SlIvA1,3,4

1Cancr Rsarch laoraory, Mhodis Rsarch Insiu, Indiana Unirsiy Hah, 1800 N Capio A, e504,

Indianapolis, IN 46202;2Amiaha Mdica Cinic and Haing Cnr, 7064 Corin Cour, Sasopo, CA; 3Dparmn

of Medicine and4Indiana University Simon Cancer Center, Indiana University Scool of Medicine, Indianapolis IN, USA

Received January 17, 2011; Accepted Marc 2, 2011

DOI: 10.3892/ijo.2011.996

Correspondence to: Dr Dani Sia, Cancr Rsarch laoraory,Metodist Researc Institute, 1800 N Capitol Ave, E504, Indianapolis,

IN 46202, USA

E-mail: [email protected]

Key words: ProstaCaidTM

, plasminogen activator, prostate cancer


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JIANG et al: GROWtH AND INvASIve beHAvIOR Of HUMAN PROStAte CANCeR CellS1676

as also demonstrated anti-invasive and anti-angiogenic

propris which wr mdiad y h supprssion o scrionof plasminogen activator (uPA), vascular endotelial growt

acor (veGf), and ransorming growh acor-1 (TGF-1)from prostate cancer cells, respectively (17,18). Coriolus

versicolor (C. versicolor) (Yunzi) is a medicinal musroom

traditionally used in Asia to treat cancers as well as improve

immunomodulatory activities (19). C. versicolor containsioogicay aci srucuray dirn proin-ound poy-saccaride-K (PSK, Krestin) and polysaccaropeptide (PSP),

which ha n shown o inhii h proiraion o ariouscancer cells including prostate cancer cells (12,20,21). In addition

to te anti-cancer properties, extracts ofC. versicolor demon-

strated strong immunomodulatory effects suc as elevated

IL-2, natural killer cell activity and T-cell proliferation

(11,22). In vivo studies sowed tat oral administration ofC.

versicolor xrac or PSP o nud mic signicany supprssdte growt of inoculated prostate cancer cells (12,20,23,24).

Altoug te detailed mecanisms of action ofC. versicolor

on h growh o cancr cs rmains o addrssd, rcnstudies indicate tat PSK and PSP ofC. versicolor caused cell

cyc arrs a h G0/G1 phas, inducd apoposis, and inhiidmetastasis of prostate cancer cells (11,12,23,24). Phellinus

linteus (P. linteus) was mainly used in Asian countries for te

treatment of various uman malignancies including prostate

cancr (7,25). Ahough h major ioogicay aci compo-nents in P. linteus are polysaccarides (25), P. linteus also

contains a polysaccaride-protein complex (PPC) wic

stimulated te tumoricidal activities of macropages and

natural killer (NK) cells, and induced te proliferation of

B cells in vitro (26). In addition, P. linteus inhiis growh andinduces apoptosis of invasive prostate cancer cells in vitro

(7,10,27,28), and sensitizes advanced prostate cancer cells toapoptosis in a xenograft model of prostate cancer (10).

In addition, some of te natural compounds in PC demon-

strated a direct effect on prostate cancer cells. For example,

resveratrol is a natural polypenol present in various plants

wic ave demonstrated anti-inflammatory, anti-oxidant,

anti-invasive, and cardioprotective properties (29-32). Previous

sudis showd ha rsraro inhiid growh and incrasdapoptosis in prostate cancer cells (33,34) and a dimetyl ester

driai o rsraro (Prosin) aso inhiid MMP-9and metylacyl-CoA recemase of prostate cancer cells, two

metastatic markers for te invasion and metastasis of prostate

cancr cs (35). viamin D3 posssss ani-proirai,anti-invasive, anti-migration, anti-metastasis, and anti-

angiogenesis effect on prostate cancer cells (36,37), wic are

mediated troug te arrest of cell cycle and te down-regulation

o xprssion o caoin and inhiiion o MMP-9 aciiy(38,39). Epigallocatecin-3-gallate (EGCG), a major poly-

penolic component in te green tea, induced cell cycle arrest

and apoptosis in androgen-dependent and -independent uman

prosa cancr c ins (40-43). Moror, eGCG inhiidMMP-2 and MMP-9 via suppression of activation of mitogen-

aciad proin kinas (MAPK) and aso inhiidinammaion-riggrd MMP-2 aciaion and inasion in amurine TRAMP model of prostate cancer (44,45). Te molecular

mchanisms rsponsi or h ani-inasi aciiy oEGCG were associated wit down-regulation of activation of

c-Jun and NF-B signaling (43,45).

In te present study, we evaluated anti-proliferative and

anti-invasive properties of a dietary supplement PC on igly

invasive uman ormone refractory (independent) prostate

cancr cs PC-3. Hr, w show ha PC inhiis PC-3 proir-ation and modulates expression of prostate cancer-related

iomarkr gns. In addiion, PC aso supprsss inasihaior o PC-3 cs y h inhiiion o c adhsion,

migration and invasion. Our results demonstrate a novelmchanism o acion o PC in h inhiiion o growh andinasi haior o prosa cancr cs.

Materials and methods

Cell culture and reagents. Te uman prostate cancer cell

in PC-3 was oaind rom AtCC (Manassas, vA, USA).PC-3 cs wr mainaind in DMeM/f-12 mdium conainingpenicillin (50 U/ml), streptomycin (50 U/ml), and 10% fetal

oin srum (fbS). Mdium and suppmns cam romInirogn (Grand Isand, NY, USA). fbS was oaind romhyclone (Logan, UT, USA). ProstaCaid (PC) a 33-ingredient

comprhnsi poy-hra and nuriion prparaion conainingte following active weigt components: Curcuma longa root

xrac compx wih nhancd ioaaiaiiy (bCM-95)20%, qurcin 15%, Coriolus versicolor, Ganoderma lucidum,Phellinus linteus musroom myceliumnd 10% [Astragalusmembranaceus root extract (5:1), Coix lacryma-jobi seed extract

(5:1), Coptis japonica rizomeextract (10:1),Eleutherococcus

senticosus root extract (5:1), Scutellaria baicalensis root extract

(5:1), Scutellaria barbata root extract (10:1), Similax glabrae

extract (5:1), Taraxacum ofcinale hr (5:1)] hra nd9%, Urtica dioica hr xrac (5:1) 6%, sitostererol 6%,Serenoa repens rry 5%, Brassica oleracea ar. iaic hr

extract (22:1) 4%, Punica granatum fruit (40% Ellagic acid) 4%,Vitis vinifera rui skin xrac (10:1) 4%, viamin C 4%, ipoic acid 3%, 3.3'-diinodymhan (DIM) 3%, Cucurbita

pepo seed 2%, Prunus africana ark xrac (4:1) 2%,Camellia sinensis hr xrac (40% eGCG; 95% phnos; 70%cachns) 1.5%, ycopn 0.6%, Zinc 0.4%, viamin D3 0.2%,rsraro 0.2%, rrin 0.1%, oron 0.06%, snium0.004%, was suppid y h ecoNugnics, Inc. (Sana Rosa,CA, USA). PC sock souion was prpard y dissoing PCin dimhy-suphoxid (DMSO) a a concnraion o 25 mg/m and sord a 4C.

Cell proliferation. C proiraion was drmind y htetrazolium salt metod (MTT metod), according to te

manuacurr's insrucions (Promga, Madison, WI, USA).briy, PC-3 cs wr cuurd in a 96-w pa and radwit PC (0-80 g/ml) for 24, 48 and 72 . At te end of te

incuaion priod, h cs wr harsd and asorpionwas determined wit an ELISA plate reader at 570 nm, as

priousy dscrid (46). Daa poins rprsn man SD inte representative experiment of triplicate determinations.

Simiar rsus wr oaind in wo indpndn xprimns.

DNA microarrays. PC-3 cells were treated wit PC (0-80 g/ml)

for 24 and total RNA isolated wit RNAeasy (Qiagen,

vancia, CA). this RNA was usd or h auaion o prosacancer genes wit Oligo GEArray human Prostate Cancer

biomarkrs Microarray according o h manuacurr's prooco


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INTERNATIONAL JOURNAL OF ONCOLOGY 38: 1675-1682, 2011 1677

(SAbioscincs, frdrick, MD, USA), as priousy dscrid(47). th od chang o gn xprssion was drmind yGEArray expression analysis suite (SABiosciences).

Cell adhesion, migration and invasion assays. Cell adesion

was performed wit Cytomatrix Adesion Strips coated wit

human roncin (Chmicon Inrnaiona, tmcua, CA,

USA). briy, PC-3 cs wr rad wih PC (0-80 g/m)for 24 , arvested, and counted. Cell adesion was determined

ar 1.5 h o incuaion a 37C (46). C migraion o PC-3cells treated wit PC (0-80 g/ml) was assessed in Transwell

chamrs in h Ducco's modid eag mdium: nurinmixur f-12 (DMeM/f12) mdium conaining 10% aoin srum (fbS) (46). Inasion o PC-3 cs rad wih PC(0-80 g/m) was assssd in transw chamrs coad wih100 o Marig (bD bioscincs, bdord, MA, USA)diud 1:3 wih DMeM/f12, ar 24 h o incuaion (46).

uPA secretion. DMeM/f12 mdia rom PC-3 cs rad wihPC (0-80 g/ml) for 24 were collected and concentrated, and

h scrion o uPA was dcd y Wsrn o anaysiswih ani-uPA aniody (Oncogn Rsarch Producs,Camridg, MA, USA), as dscrid (46). Quanicaion ouPA scrion was prormd y masuring opica dnsiis oauoradiograms wih HP-Scanj 550c and anayzd yUN-SCAN-It sowar (Sik Scinic, Orm, Ut, USA).

Reverse transcription-polymerase chain reaction (RT-PCR).

PC-3 cells were treated wit different concentrations of PC

(0-80 g/ml) for 24 . Te total RNA from PC-3 cells was

isoad y RNasy mini ki (Qiagn, vancia, CA, USA)according to instruction of manufacture. RT-PCR was performed

as priousy dscrid (48). briy, PCR or CDK4,CNKN1A and e2f1 was run or 30 cycs a 95C or dnau-raion or 45 sc, 60C or annaing or 45 sc and 72C orxnsion or 1 min. PCR or CAv1 was run or 38 cycs a95C or dnauraion or 45 sc, 60C or annaing or 1 minand 72C or xnsion or 1 min. th primr squncs orCDK4 wr 5'-tGGtGAGGGtGGGGtGAGG-3' (sns)and 5'-tGGCCACtGtGGGGAtCACG-3' (anisns); hprimr squncs or CDKN1A wr 5'-CCtGCCCtCAtGGCCCCtCt-3' (sns) and 5'-tGGGACCCtCACCCCCACAG-3' (anisns); h primr squncs or e2f1 wr 5'-GGCCGtCCtCCCAGCCtGtt-3' (sns) and 5'-CCCACGCGC

ACACAtGGACt-3' (anisns); h primr squncs orCAv1 wr 5'-CGCCCtCtGCtGCCAGAACC-3' (sns) and5'-GGCCCGtGGCtGGAtGAAAA-3' (anisns); and hprimr squncs or -acin wr 5'-ACGAGtCCGGCCCCtCCAtC-3' (sns) and 5'-GGGGGCACGAAGGCtCAtCA-3' (anisns). th na Rt-PCR producs (10 ) wrrun on a 1.5% agaros g conaining hidium romid andquanid using imagr fuor Chm HD2 (C bioscincs,Santa Clara, CA, USA). Te results are presented as te ratio of

a spcic arg gn o -actin.

Statistical analysis. Daa ar prsnd as h mans SD.Saisica comparison wn h conro group (0 g/m oPC) and groups wit different PC doses were carried out using

on-way anaysis o arianc (ANOvA). P


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corrsponds o 128 ng/m, iamin D3 o 160 ng/m, and eGCGto 464 ng/ml at te igest used dose of 80 g/ml of PC in our

experiments.

Effect of PC on the invasive behavior of prostate cancer cells.

Tumor invasion and metastasis are multifaceted processes

including cell adesion, proteolytic degradation of tissue

arrirs, c migraion, inasion, and angiognsis (43,53).Inasi haior o prosa cancr cs is associad wihhir aiiy o migra and inad h surround issus and ismediated troug uPA/uPAR complex (43,53,54). To investi-

ga i PC has an inhiiory c on inasi haior o

figur 2. ec o PC on inasi haior o prosa cancr cs. (A) C adhsion. PC-3 cs wr rad wih PC (0-80 g/m) or 24 h and c adhsiono roncin drmind as dscrid in Marias and mhods. each ar rprsns h man SD o hr xprimns. *P


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igly invasive prostate cancer cells, PC-3 cells were pretreated

wih PC (0-80 g/m) or 24 h and hir adhsion o roncin

was drmind on srips coad wih human roncin asdscrid in Marias and mhods. As sn in fig. 2A,adhsion o PC-3 cs o roncin was markdy supprssdy h PC ramn y 28.3 and 59.9% a 40 and 80 g/m,respectively. Te effect of PC on migratory potential of prostate

cancer cells was evaluated in PC-3 cells pretreated wit PC

(0-80 g/ml) for 1 and cell migration was determined after

addiiona 24 h o incuaion. As xpcd, PC signicanydcrasd h migraion ra o PC-3 cs y 29.7 and 58.5%at 40 and 80 g/ml, respectively (Fig. 2B). Cell invasion is

anoter key factor involved in cancer progression and meta-

stasis (3,43,46). To examine te effect of PC on te invasive

aiiy o PC-3 cs, c inasion assays wr prormd intransw chamrs coad wih Marig as dscrid inMaterials and metods. As seen in Fig. 2C, PC markedly

inhiid inasion o PC-3 cs in a dos-rspons mannr y

28.3 and 48.7% at 40 and 80 g/ml, respectively. In order to

evaluate te molecular mecanism of action of PC on te

invasion of prostate cancer cells, conditioned media from PC-3cells treated wit PC (0-80 g/ml) were collected and secretion

o uPA was drmind y Wsrn o anaysis. As xpcd,PC markedly decreased secretion of uPA from PC-3 cells

(fig. 2D). this osraion is consisn wih our priousreport demonstrating te anti-invasive effect ofG. lucidum in

uman prostate cancer cells troug te mecanisms including

uPA/uPAR signaing (18,46). As in h inhiiion o proir-aion y PC dscrid ao, h concnraion oG. lucidumin PC was markedly lower (19.5 g/mg PC wic corresponds

o h na concnraion oG. lucidum at 1.56 g/ml at teigest used dose of 80 g/ml of PC), tan in te original experi-

ments wit individual G. lucidum extracts (0.5-2.5 mg/ml) (18).

Effect of PC on the gene expression proles of prostate cancer-

related biomarkers in prostate cancer cells. In order to evaluate

figur 3. ec o PC on h mRNA xprssion on prosa cancr iomarkrs in prosa cancr cs. th mRNA xprssion o (A) p21, (b) CDK4, (C)e2f1 and (D) CAv1 in PC-3 cs was drmind y Rt-PCR anaysis. toa RNA was isoad rom PC-3 cs rad wih hic or PC (80 g/m) andRt-PCR was prormd wih spcic primrs as dscrid in Marias and mhods. Rt-PCR or -actin was used as an internal loading control. Te resultsar xprssd as h rai xprssion raios o spcic mRNA o -acin. each ar rprsns h man SD o hr xprimns. *P


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weter anti-proliferative and anti-invasive effects of PC are

associad wih h xprssion o gns priousy idnid

in prosa cancr, w usd cDNA microarray anaysis wihhuman prosa cancr iomarkr gns. PC-3 cs wr rad

wih PC (0-80 g/m) or 24 h and cDNA microarray anaysisprormd as dscrid in Marias and mhods. As sn inta I, PC up-rguad h xprssion o CDKN1A, and

down-regulated expression ofCAV1, CCND1, CDK4, E2F1,ELAC2,IGF2,MAPK6,NR2F2 and PLAUgenes in te PC-3

cs. furhrmor, w ha conrmd h xprssion o somgns y Rt-PCR. PC-3 cs wr rad wih PC (80 g/m)for 24 . Total RNA was isolated and RT-PCR analysis was

prormd. Consisn wih h DNA microarray daa, PC

signicany inducd h xprssion o h CDKN1A mRNAand down-rguad h xprssion o CDK4 mRNA and

CAv1 mRNA (fig. 3). Inrsingy, xprssion o e2f1 mRNAwas no changd y h PC ramn. thror, PC rguas

h c cyc progrssion nwork hrough inding o c cycrguaors such as cycin D1, R, and h ranscripion acor

e2f1 (49,55-58). th c progrssion is rguad y cycins,cycin dpndn kinass (Cdks) and Cdk inhiiors such as

p15, p16, 21, and p27; cycin D1 (CCND1) and CDK4 orm acompx o accra c cyc progrssion, whi Cdk inhii-tors slow cell cycle progression (48,55,56,59,60). Terefore,

h up-rguaion o CDKN1A (p21) and down-rguaion oCCND1 and CDK4 genes will cause cell cycle arrest at G1/G0

pase. Up-regulation of p21 induced strong downstream

inhiiion o CDK4 and cycin D1 and hypophosphory-

aion o R, urhr ading o h inhiiion o ranscripion

acor e2f1. Nrhss, p21 can ind o proiraing cnuclear antigen (product ofPCNA gene) (61) and PCNA regulated

te expression of ERK3/MAPK6 (product ofMAPK6gene)which ac c iaiiy and rgua h c cyc (62).

thus, h inducion o p21 aso rsud in h inhiiion oPNCA expression, leading to te reduction of ERK3 protein.

In addition to te cell cycle regulatory genes, PC treatment

aso moduad xprssion o ohr gns priousy idnidin prostate cancer CAV-1,IGF2,ELAC2 and PLAU(ta I).

For example, Caveolin-1 (product ofCAV-1 gene) is a major

structural component of caveolae, specialized in plasma

mmran inaginaions inod in ndocyosis, c adhsion,and signal transduction (63). Furter, Caveolin-1 is over-

expressed in advanced prostate cancer were it promotes

migration, invasion, and angiogenesis in prostate cancer cells

(63,64). Te precise role of te insulin-like growt factor 2

(IGF2) on progression of tumor remains unclear. however,

polymorpism of teIGF2 gene is associated wit increased

prostate cancer risk (65,66) and an activation of autocrine IGF2

loop is linked to te neoplastic progression (67). uPA (product

ofPLAUgene) and its receptor (uPAR) are important in cancer

adesion, migration, and invasion. uPA interacts wit uPAR,

wic furter form te multi-complex wit integrin receptor

31 or v3 and rgua h inasi haior (adhsion,migration and invasion) of cancer cells (18,43,68,69). Altoug

suggested polymorpism of te elaC omolog-2/ereditary

prostate cancer (ELAC2/HPC2) gene and prostate cancer risk

dmonsrad conicing rsus in a ariy o sudis, a rcnmeta-analysis sowed tat ELAC2 is associated wit prostate

cancer risk (70,71).

In summary, our data clearly demonstrate tat PC modulates

xprssion o spcic gns rad o prosa cancr growhand inasinss, and spcia ingrdins in h PC may conriuo h inhiiion o prosa cancr cs hrough disinc signaingpatways.

In conclusion, ProstaCaid is a novel dietary supplement tat

contains multiple ingredients wic sow an anti-proliferation

effect on androgen-dependent and -independent prostate cancercs. Our rsus show ha PC inhiis proiraion andinasi haior o prosa cancr cs y h moduaion ote expression of genes associated wit prostate cancer. Our

data suggest tat PC as multiple targets for its terapeutic effect

and h ioogica aciiy o PC is mdiad y h addii orsynergistic effects of its individual ingredients. In summary, PC

may ave potential clinical application for an alternative prostate

cancer terapy.

Acknowledgements

this sudy was suppord y a rsarch gran rom ecoNugnics,Inc., Sana Rosa, CA. W woud ik o hank barry Wik orhis conriuion o his sudy. On o h auhors, I. eiaz,acknowledges is interest as te formulator and owner of

EcoNugenics, Inc.

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