prostacaidtm inhibits tumor growth in a xenograft model of human prostate cancer

7/30/2019 ProstaCaidTM inhibits tumor growth in a xenograft model of human prostate cancer

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INTERNATIONAL JOURNAL OF ONCOLOGY 40: 1339-1344, 2012

Abstract. We have recently demonstrated that the dietary

supplement ProstaCaid (PC) inhibits growth and invasive

behavior of PC-3 human prostate cancer cells in vitro. In the

present study, we evaluated toxicity and whether PC suppresses

growth of prostate cancer in a xenograft model of human pros-

tate cancer cells implanted in mice. Here, we show that an oral

administration of PC (100, 200 and 400 mg/kg) did not affect

body weight or activity of liver enzymes (ALT, AST) and did not

show any sign of toxicity in liver, spleen, kidney, lung and heart

tissues in mice. In addition, PC treatment resulted in the inhibi-

tion of tumor volumes (1024.6378.6 vs. 749.3234.3, P


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JIANG et al: ProstaCaid in vivo1340

containing penicillin (50 U/ml), streptomycin (50 U/ml) and

10% fetal bovine serum (FBS). Medium and supplements

came from Invitrogen (Grand Island, NY, USA). FBS was

obtained from Hyclone (Logan, UT, USA). ProstaCaid (PC)

a 33 ingredient comprehensive poly-herbal and nutrition

preparation containing the following active weight compo-

nents: Curcuma longa root extract complex with enhanced

bioavailability (BCM-95

) 20%, quercetin 15%, Coriolusversicolor, Ganoderma lucidum, Phellinus linteus mush-

room mycelium blend 10% [Astragalus membranaceus root

extract (5:1), Coix lacryma-jobi seed extract (5:1), Coptis

japonica rhizome extract (10:1),Eleutherococcus senticosus

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

Scutellaria barbata root extract (10:1), Smilax glabrae extract

(5:1), Taraxacum ofcinale herb (5:1)] herbal blend 9%, Urtica

dioica herb extract (5:1) 6%, sitostererol 6%, Serenoa repens

berry 5%,Brassica oleracea var. italic herb extract (22:1) 4%,

Punica granatum fruit (40% ellagic acid) 4%, Vitis vinifera

fruit skin extract (10:1) 4%, vitamin C 4%, lipoic acid 3%,

3,3'-diinodlylmethane (DIM) 3%, Cucurbita pepo seed 2%,

Prunus africana bark extract (4:1) 2%, Camellia sinensis

herb extract (40% EGCG; 95% phenols; 70% catechins) 1.5%,

lycopene 0.6%, Zinc 0.4%, vitamin D3 0.2%, resveratrol 0.2%,

berberine 0.1%, boron 0.06%, selenium 0.004%, was supplied

by the EcoNugenics, Inc. (Santa Rosa, CA, USA). PC stock

solution was prepared by dissolving PC in sterile water and

stored at 4C.

Toxicology studies. Toxicity of PC was evaluated in the 6 weeks

old male nude mice (Harlan, Indianapolis, IN, USA). The mice

were acclimatized for 1 week, and treated with PC (0, 100,

200 and 400 mg/kg of body weight, n=10 per group) by intra-

gastrical gavage 5 times/week for additional 4 weeks. The bodyweight was evaluated three times per week. At the end of the

experiment animals were euthanized by CO inhalation. Blood

was collected and a gross pathology examination performed.

Liver, spleen, kidney, lung and heart were harvested, xed in10% neutral-buffered formalin at 4C for 24 h followed tissueprocessing overnight and then embedded in parafn. Five-micrometer sections were stained with hematoxylin and eosin

(H&E). The levels of alanine aminotransferase (ALT), aspar-

tate aminotransferase (AST), alkaline phosphatase (ALP),

albumin and total protein were determined at the Department

of Pathology and Laboratory Medicine, Indiana University

(Indianapolis, IN, USA).

Human prostate tumor xenograf t experiments. PC-3 cells

(3x106) in 0.2 ml DMEM were implanted subcutaneously in

the right ank on the ventral side of the 6 weeks old male nudemice (Harlan). After 1-2 weeks of implantation with tumor cells,

when tumors reached ~20-30 mm3, the animals were random-

ized into control and treatment groups (15 animals per group).

The animals received intragastrical gavage 5 times/week with

water (control) or 400 mg PC/kg of body weight (treatment) for

additional 6 weeks The tumor size was measured using calipers

and the tumor volume was estimated by the formula: tumor

volume (mm3) = (LxW2)x1/2, where L is the length and W is

the width of the tumor. At the end of the experiment (Day 42),

the tumors were snap frozen and stored separately in liquid

nitrogen. The protocol for animal experiments was approved

by the Animal Research Committee at the Methodist Hospital

(protocol no. 2010-05) according to the NIH guidelines for the

Care and Use of Laboratory Animals.

Quantitative RT-PCR. The quantitative real-time polymerase

chain reaction (qRT-PCR) was performed using the ABI PRISM

7900HT Fast Real-Time PCR System (Applied Biosystems)

according to the manufacturer's instructions. Total RNAwas isolated from tumors with RNAeasy (Qiagen, Valencia,

CA). The RNA samples were reverse transcribed into cDNA

(RT-PCR) using random hexamer primers and TaqMan reverse

transcription kit (Applied Biosystems). The cDNA (100 ng per

sample) was subjected to qPCR analysis in quadruplicate using

forward and reverse primers, TaqMan Universal Master Mix

and probe (10 ml per reaction) in fast optical 96-well plates.

The data were analyzed using the ABI PRISM 7900 relative

quantication (DDCt) study software (Applied Biosystems).In this study we have used primers CAV1, CCND1, CDKN1A,

E2F1, ELAC2, IGF2, MAPK6, NR2F2, PCNA and PLAU

genes with -actin as internal control (Applied Biosystems).

The gene expression levels are normalized to -actin and are

presented as arbitrary fold changes compared between control

and treated groups.

Statistical analysis. Toxicology analyses of plasma were

summarized using median (min, max) and compared across

groups using Kruskal-Wallis tests and Mann-Whitney U tests

with signicance level adjusted using the Bonferroni correc-tion. The changes in tumor volume in the control and treatment

group were assessed by a random effects mixed model. Tumor

weights were evaluated by the Mann-Whitney U test. RT-PCR

data are presented as means SD and statistically evaluated

by Student's t-test. The value of P


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INTERNATIONAL JOURNAL OF ONCOLOGY 40: 1339-1344, 2012 1341

nude mice (24). In conclusion, dietary supplementation with

PC (0-400 mg/kg) was not toxic in tested animals.

PC suppressed tumor growth in a xenograft model of human

prosta te cancer. Based on our data demonstrating that PC

is not toxic in vivo, we have employed an animal model of

human prostate PC-3 cancer cells xenografted in nude mice.

PC-3 cells were subcutaneously injected into male nude mice,

and when the forming tumors reached the size approximately

30 mm3, the mice were divided into the control group (water)

and the treatment group (400 mg PC/kg of body weight). Therewere no changes in the tumor volumes for the rst 3 weeks ofthe treatment (Fig. 3A). However, after 3 weeks the tumors

in the treatment group were smaller, and we observed a

signicant difference in the change in tumor volume over timebetween control and PC treatment groups (P


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of the individual components. Indeed, another polybotanical

agent TBS-101 (containing extracts from Panax ginseng,

cranberry, green tea, grape skin, grape seed, G. lucidum

and chamomile) was not toxic and suppressed tumor growth

in mice (25). In addition, the use of different biologically

active components in PC can also focus on the specic targetmolecules and signaling pathways which are associated with

prostate cancer.

Figure 3. Effect of PC on the tumor size and tumor weight. PC-3 cells were injected subcutaneously into male nude mice and treated with water (control) or

400 mg PC/kg of body weight 5 times/week for daily for 42 days (n=15 mice per group) as described in Materials and methods. (A) Tumor volumes. Data

are the mean SD and the changes in tumor volumes were statistically evaluated by a random effects mixed model (P


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prostacaidtm inhibits tumor growth in a xenograft model of human prostate cancer

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