ProstaCaid Induces G2/M Cell Cycle Arrest and Apoptosis in Human and Mouse AndrogenDependent and Independent Prostate

e Cancer Cells
Jun Yan, PhD1, and Aaron E. Katz, MD1

Integrative Cancer Therapies 9(2) 186196 The Author(s) 2010 Reprints and permission: http://www. sagepub.com/journalsPermissions.nav DOI: 10.1177/1534735410371478 http://ict.sagepub.com

Abstract The anticancer effects of ProstaCaid, a novel integrative blend of vitamins, minerals, multiherb extracts, and derivatives, were tested in human and mouse androgendependent (AD) and independent (AI) prostate cancer cell lines. ProstaCaid shows growth inhibitory effects on both human and mouse AD prostate cancer cells (LNCaP and CASP 2.1) and AI prostate cancer cells (PC3 and CASP 1.1) in a dose-/time-dependent manner. Consistently, long-term treatment with ProstaCaid also reduced colony formation capacities of prostate cancer cells. Flow cytometry assays revealed that ProstaCaid induces G2/M arrest and apoptosis in LNCaP and PC3 cells after 72 hours of treatment. Immunoblotting assay demonstrated that 25 mg/mL of ProstaCaid treatment resulted in (1) the reduction of cyclin D1, cyclin B1, and Cdc2 expression in a time-dependent way; (2) increase in p21WAF1/Cip1 as early as 12 hours after the treatments in PC3 cells and reduction to base line at the 72-hour time point; and (3) repression of Bcl-2, BclxL, and induction of Bim as well as the cleavages of caspase-3 and poly(ADP-ribose) polymerase (PARP) at 72 hours of treatment, suggesting caspase-3-dependent apoptosis. Moreover, ProstaCaid suppressed activation of AKT and MAPK signaling pathways in PC3 and LNCaP cells by reducing phosphorylation levels of AKT, its downstream target S6 ribosomal protein and GSK3b, and ERK1/2, respectively. In summary, these findings strongly suggest that ProstaCaid may be a potential chemopreventive and therapeutic agent for both AD and, more importantly, AI prostate cancer. Keywords prostate cancer, herbal medicine, cell cycle arrest, apoptosis, AKT signal pathway, MAPK signal pathway

Introduction
Prostate cancer is one of the most common malignancies and the second leading cause of cancer-related death in men in the United States and in Western countries. Despite significant advances in prostate cancer treatment, chemotherapy using available cytotoxic anticancer drugs for advanced stage prostate cancer offers little survival benefit.1 It is now being increasingly recognized that natural herbal and phytochemical agents can be crucial in decreasing the morbidity and mortality associated with prostate cancer for both chemoprevention and therapy. Recent surveys demonstrated that approximately 40% of prostate cancer patients use various complementary and alternative medicine modalities as a component of therapy.2 ProstaCaid is a 33-ingredient comprehensive polyherbal preparation with supplements of vitamin C, vitamin D3, zinc, selenium, quercitin, 3,3-diinodolymethane (DIM), and lycopene. Herbal extracts include the extracts from turmeric root,

saw palmetto berry, grape skin, pomegranate, pumpkin seed, pygeum bark, sarsaparilla root, green tea, and Japanese knotweed. Hence, it is rich in natural polyphenols, including quercetin, resveratrol, epigallocatechin gallate (EGCG), and ellagic acid, which have previously demonstrated anticancer potential.3-7 The unique formula contains 3 medicinal mushrooms grown on an herbal-enhanced medium. The mushrooms included are Phellinus linteus, Ganoderma lucidum, and Coriolus versicolor, each with known anticancer properties.8-13 Therefore, ProstaCaid was designed based on constituents that exhibit antiprolifetaive, antioxidant, and apoptotic activities; however, its efficacy and the mechanisms
1

Department of Urology, Columbia University Medical Center, New York, NY, USA Corresponding Author: Jun Yan, Department of Urology, Columbia University Medical Center, 1130 St. Nicholas Ave, New York, NY 10032, USA Email: jy2326@columbia.edu

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Yan and Katz of action are yet to be examined. In this study, we look at the effectiveness of the preparation in suppressing several types of prostate cancer cell lines in culture and attempt to delineate the mechanism of action for justification in pursuing animal studies to determine efficacy in vivo. Activations of AKT and MAPK signal pathways are frequently detected in human prostate cancer specimens. Ablation of pten alleles in mouse prostate epithelia cells can induce prostate cancer through activation of PI3K/AKT signaling.14,15 Activation of AKT signaling can also synergize with MAPK kinase signaling to facilitate prostate cancer progression,16 while simultaneous inhibition of mTOR, a downstream target of AKT signaling and MAPK signaling, can significantly reduce tumor burden, especially for AI prostate cancers.17 To delineate the role of ProstaCaid in prostate cancer cell proliferation, we determined it to induce G2/M cell cycle arrest and apoptosis in both androgen-dependent (AD) and independent (AI) prostate cancer cell lines. Changes in cell cycle and apoptotic proteins were detected. Moreover, ProstaCaid shows the inhibition of AKT and MAPK signal pathways, suggesting its suppression of prostate cancer progression in both AD and, more importantly AI for which there is currently no curative therapy.

187 The preparation was dissolved in dimethyl sulfoxide and stored at -80C in aliquots after removal of insoluble pellet by centrifugation. Penicillin, streptomycin, RPMI 1640 medium, Dulbeccos modified Eagles medium, and fetal bovine serum were obtained from Invitrogen (Carlsbad, CA). Anti-p21Waf1/Cip1 antibody was purchased from BD Biosciences (San Diego, CA). Anti-Bcl2, BclxL, Bim, cyclin D1, cyclin B1, Cdc2, pAKT (Ser473), pERK1/2 (Thr202/Tyr204), pS6 ribosomal protein (Ser235/236), pGSK3b (Ser9), b-actin, total AKT1, GSK3b, poly(ADP-ribose) polymerase (PARP), and caspase-3 antibodies were obtained from Cell Signaling (Danvers, MA).

Cell Culture
LNCaP and PC3 cells were obtained from the American Type Culture Collection (Manassas, VA) and routinely maintained in 100-mm tissue culture dishes in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic antimycotic (Invitrogen, Carlsbad, CA). Two mouse prostate cancer cell lines, CASP2.1 and CASP1.1 were derived from the tumors of Nkx3.1;Pten;p27 compound mutant mice and kindly provided by Dr Cory Abate-Shen.18 These cells were maintained in Dulbeccos modified Eagles medium supplemented with 15% fetal bovine serum, 5% Nu-Serum IV (Invitrogen), epidermal growth factor (20 ng/ml; Sigma, St. Louis, MO), bovine pituitary extract (1X, Invitrogen), insulin/transferrin/selenium (1X, Invitrogen), nonessential amino acids (1X, Invitrogen), and antibiotic/antimycotic reagent (Invitrogen). All the cells were cultured at 37C in a humidified atmosphere of 95% air and 5% CO2.

Materials and Methods Materials

ProstaCaid (EcoNugenics, Inc, Santa Rosa, CA) contains the following active weight components: Curcuma longa root extract complex with enhanced bioavailability (BCM95) 20%; quercetin 15%; Coriolus versicolor, Ganoderma lucidum, Phellinus linteus mushroom mycelium blend 10%; Astragalus membranaceus root extract (5:1); Coix lacrymajobi 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 glabra extract (5:1); Taraxacum officinale herb (5:1) herbal blend 9%; Urtica dioica herb extract (5:1) 6%; beta 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%; a lipoic acid 3%; 3,3-DIM 3%; Cucurbita pepo seed 2%; Prunus africana bark extract (4:1) 2%; Camellia sinensis herb extract (40% EGCG, 95% phenols, 70% catechens) 1.5%; lycopene 0.6%; zinc 0.4%; vitamin D3 0.2%; resveratrol 0.2%; berberine 0.1%; boron 0.06%; selenium 0.004%. ProstaCaid is manufactured consistent with the FDA Good Manufacturing Practices regulation for dietary supplements as defined in 21 CFR111, for batch-to-batch consistency and quality control.

Cell Viability Assay

Cells were seeded at 4 103 cells/well in 96-well plates overnight before treatment with agent and then allowed to grow for an additional 3 days. MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] solution (10 mL; 5 mg/mL in PBS, USB Corporation, Cleveland, OH) was added to each well of the plates and incubated for 3 hours at 37C. MTT lysis buffer was then added to dissolve the formazan. The optical density was measured at 570 nm using a mQuant Microplate Spectrophotometer (Biotek, Winooski, VT). The optical density at 690 nm served as the reference. The percentage of viable cells was calculated as the relative optical density compared with the control.

Colony-Forming Assay
After 10-day incubation, colonies were fixed with 7:1 methanol:acetic acid and stained with 0.5% crystal violet (Sigma, St. Louis, MO). Only colonies with >50 cells were

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Figure 1. Photographs of human prostate cancer cell line, LNCaP and PC3 cells taken after 48 hours treatment with vehicle (A, C) and 25 mg/mL ProstaCaid (B, D) in complete media; scale bar, 200 mm

counted. Each condition per colony-forming assay was plated in triplicate.

Flow Cytometry Analysis

Cells were incubated with indicated concentrations of ProstaCaid for 72 hours before samples were fixed by 70% ethanol. Cells were incubated in RNase A containing PBS for 1 hour at 37C, followed by addition of propium iodine (Sigma, St. Louis, MO). Then, cells were analyzed using a FACScan flow cytometer.

overnight at 4C. The blots were washed and incubated with horseradish peroxidaseconjugated secondary antibodies for 1 hour and finally detected by ECL reagent (GE Healthcare, Buckinghamshire, UK). The blots were repeated 2-3 times to confirm consistency of results. Densitometry analysis was performed using NIH ImageJ software.

Results Growth Inhibitory Effects of ProstaCaid on Prostate Cancer Cells

As is shown in Figure 1, after 48 hours of treatment with ProstaCaid, changes in cellular morphologies (such as cell shrinkage, elongation, and roundup) were observed in both AD prostate cancer cells (LNCaP) and AI prostate cancer cells (PC3). The MTT assay indicates that the viability of LNCaP cells decreased in both a dose- and time-dependent manner by ProstaCaid with 0 to 25 mg/mL at 24, 48, and 72 hours, respectively. The IC50 value of ProstaCaid for LNCaP cells was 3.19 0.34 mg/mL at 72 hours (Figure 2A). ProstaCaid also showed growth inhibitory effects on AI PC3 cells. The IC50 value for PC3 cells is 8.72 1.49 mg/mL

Western Blotting Assay

Cells were lysed in Radioimmunoprecipitation Assay Buffer (RIPA) buffer containing protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN) and phosphotase inhibitor cocktail I (Sigma, St. Louis, MO). Cell lysates (20 mg) were resolved on sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred onto Polyvinylidene Fluoride (PVDF) membrane. After blotting in 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with primary antibodies at 1:500 to 1000 dilutions in Tween 20

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Figure 2. Effects of ProstaCaid on cell viability on 2 androgen-dependent prostate cancer cell lines, LNCaP (A) and CASP 2.1 (C), and 2 androgen-independent prostate cancer cell lines, PC3 (B) and CASP 1.1 (D). The cells were exposed to ProstaCaid at the indicated dosages for 24 hours, 48 hours, and 72 hours. The cell viability was determined by MTT assay.Vehicle-treated cells were arbitrarily set as 100% control viability. Results are expressed as mean standard errors; n = 4. *P < .05; **P < .01, compared with the vehicle-treated group at the same time point

at 72 hours, which is higher than that for LNCaP cells (Figure 2B). Furthermore, we tested ProstaCaid on 2 mouse prostate cancer cells (CASP2.1 and CASP1.1), which are derived from the tumors of Nkx3.1;Pten;p27 compound mutant mice.18 Prostate cancer cell line CASP2.1 is AD and CASP1.1 is AI. As shown in Figures 2C and 2D, the IC50 value of AD CASP2.1 cells for ProstaCaid is 11.90 2.16 mg/mL, a little lower than that of AI CASP1.1 cells (IC50 = 15.52 4.53 mg/mL). Overall, the MTT assay revealed that ProstaCaid (<25 mg/mL) shows growth inhibitory effects on both AD and AI prostate cancer cells within 72 hours.

and 25.0 mg/mL of ProstaCaid reduced colony formation efficiency from 100% to 31.3%, 1.44%, and 0%, respectively. PC3 cells also lost colony formation capacities under the treatment of ProstaCaid in a dose-dependent manner. We found that 6.3, 25.0, and 50.0 mg/mL of ProstaCaid reduced colonies from 100% in the control group to 49.2%, 0.31%, and 0%, respectively. Moreover, ProstaCaid also reduced colony numbers in 2 CASP cells (Figure 3).

ProstaCaid-Induced Cell Cycle Arrest and Apoptosis in Prostate Cancer Cells

Because both growth arrest and apoptosis account for the growth inhibitory effect of ProstaCaid, we carried out cell cycle analysis. The data demonstrated that treatment with 25.0 mg/mL ProstaCaid for 72 hours resulted in an arrest of LNCaP and PC3 cell lines at the G2/M phase (Figure 4, Table 1). The exposure to ProstaCaid resulted in 22.75% of LNCaP cells in the G2/M phase, compared with 19.29% of vehicle-treated cells. PC3 cells showed more striking G2/M

Long-Term Treatment With ProstaCaid Suppressed Colony Formation Capacities

To test the long-term growth inhibitory effects of ProstaCaid on prostate cancer cells, we performed a colony formation assay. As shown in Figure 3, LNCaP cells displayed loss of colony formation capacities after treatment with ProstaCaid. Compared with the vehicle-treated group, 3.1, 12.5,

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Figure 3. Effects of ProstaCaid on long-term cell viability by colony formation assay. A. Photographs of LNCaP, PC3, CASP2.1, and CASP1.1 prostate cancer cells. Cells were treated with ProstaCaid at indicated concentration or with vehicle only. The number of colonies was recorded after 10 to 12 days of treatment. B. The percentages of colony formation capacity were shown. Each point represents the average of triplicate experiments standard errors. *P < .05; **P < .01

arrest by ProstaCaid, from 27.97% in vehicle-treated cells to 54.27% in the experimental group. Moreover, ProstaCaid also induced the appearance of subG1 peak in LNCaP cells, from 1.50% in vehicle-treated cells to 20.33% in ProstaCaid-treated cells. Similarly, ProstaCaid also induced apoptosis in PC3 cells. The apoptotic rates increased from 0.61% in the control group to 9.67% in the experimental group. Overall, ProstaCaid can induce cell growth arrest and apoptosis in both LNCaP and PC3 cells. To further define the molecular mechanism underlying ProstaCaid-induced cell growth arrest and apoptosis, we performed a Western blotting assay using antibodies against cell cycle proteins (cyclin D1, cyclin B1, Cdc2, p21Waf1/Cip1) and apoptosis-related proteins (antiapoptosis proteins, Bcl2

and BclxL; proapoptotic protein Bim; and caspase-3 as well as its substrate PARP; Figure 5). Biochemical results demonstrated that 25 mg/mL of ProstaCaid decreased cyclin D1 expression and increased p21Waf1/Cip1 as early as 12 hours after the treatments in PC3 cells and reduced to base line at the 72-hour time point. ProstaCaid also decreased the expression of Cdc2 and cyclin B1 levels, 2 G2/M cell cycle progressionrelated proteins (Figure 5A). The reductions of Cdc2 and cyclin B1 levels were more obvious after 48 hours of treatment, which was consistent with cell cycle analysis data, indicating that 72 hours of treatment of ProstaCaid induced G2/M arrest. Two antiapoptotic proteins, Bcl2 and BclxL, were downregulated and 1 proapoptotic protein Bim was significantly induced at 72 hours of treatment.

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Figure 4. Effects of ProstaCaid on cell cycle in asynchronously growing LNCaP (A) and PC3 (B); growing cells in the log phase were incubated in a complete medium with 25 mg/mL ProstaCaid for 72 hours, stained with PI (30 mg/mL), and analyzed by flow cytometry Table 1. Cell Cycle Changes of Prostate Cancer Cells as Treated by ProstaCaida Cell Lines LNCaP PC3
a

Concentration (mg/mL) 0 25 0 25

G0/G1 (%) 72.33 1.22 65.00 0.93 58.82 1.63 29.27 0.56

S (%) 8.81 0.90 12.22 1.49 13.35 0.76 16.46 0.87

G2/M (%) 19.29 1.10 22.75 0.17b 27.97 1.27 54.27 0.90c

Sub G1 (%) 1.50 0.24 20.33 0.84c 0.61 0.18 9.67 0.79c

Data are expressed as the average of 3 independent experiments standard deviation. P < .05. c P < .01.
b

Consistently, cleavage of caspase-3 appeared after 72 hours of treatment with ProstaCaid as well as the cleavage of its downstream target, PARP. In the vehicle-treated group,

there were no striking changes in cyclin D1, cylcin B1, and Cdc2 expression and p21Waf1/Cip1 did not show upregulation during 12 to 24 hours. As for the apoptotic-related protein,

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Figure 5. ProstaCaid induced cell growth inhibition and apoptosis in PC3 cells. Western blotting analysis of (A) cell-cycle-related proteins (cyclin D1, cyclin B1, Cdc2, and p21Waf1/Cip1) and (B) apoptosis-related proteins (Bcl2, BclxL, Bim, caspase-3, and PARP) in PC3 cells; cells were treated with 25 mg/mL ProstaCaid and collected at the indicated time points, and 20 mg proteins were loaded for the assay. b-Actin was used as a loading control. Densitometry analysis was performed for each blot, and numbers are shown under each blot
Abbreviations: CF, cleaved form.

the vehicle-treated group did not show striking changes at the protein level, and no cleavage of caspase-3 and PARP appeared at 72 hours, indicating that their vehicle did not induce apoptosis. These data confirmed that ProstaCaid can induce cell growth arrest and apoptosis, suggesting that apoptosis may be caspase-3 dependent.

Discussion
Because the side effects of and resistance to many anticancer agents are serious problems in the treatment of cancer, it is necessary to develop better therapeutic drugs. ProstaCaid is a 33-ingredient comprehensive polyherbal preparation with supplements of vitamin C, vitamin D3, zinc, selenium, quercetin, DIM, and lycopene. In this study, we document that ProstaCaid induced G2/M cell cycle arrest and apoptosis in both AD (LNCaP and CASP2.1) and AI (PC3 and CASP1.1) prostate cancer cell lines. Both short-term treatment by MTT assay and long-term treatment by colony formation assay revealed that ProstaCaid efficiently inhibited prostate cancer cell growth, regardless of their androgen dependence. Of note, the IC50 values of AD prostate cancer cells (LNCaP and CASP2.1) were lower than that of 2 other AI prostate cancer cells (PC3 and CASP1.1). Consistently, flow cytometry assay revealed that 25 mg/mL ProstaCaid induced an apoptotic rate in LNCaP cells that was more than in PC3 cells, although ProstaCaid can induce significant G2/M arrest in PC3 cells compared with that in LNCaP cells. The anticancer activity of ProstaCaid may be ascribed to its polyphenolic flavonoids and curcuminoids derived from various herbs as well as other supplements, such as DIM. The preparation contains supplements such as quercetin (15%), Curcuma longa root extract complex with enhanced bioavailability (BCM-95; 20%), DIM (3%), and resveratrol (0.2%). Some of these components have shown a strong doseand time-dependent growth inhibition and apoptotic death in prostate cancer cells; 25 mM of quercetin inhibited about

ProstaCaid Suppressed the Activations of AKT and MAPK Signal Pathways

Deregulation of AKT and MAPK signal pathways were frequently detected during prostate carcinogenesis. Because pten alleles were deleted in PC3 and LNCaP cells, resulting in loss of PTEN expression, PI3K/AKT signaling was constitutively active in these cell lines.19-21 First, we used PC3 cells to further define molecular alterations after ProstaCaid treatment. Western blotting assay showed that 25 mg/mL of ProstaCaid can gradually suppress phosphorylation of both AKT and ERK1/2 during 72 hours of treatment (Figure 6A). Moreover, phosphorylated ribosomal protein S6 and GSK3b, 2 of the AKT downstream targets, also showed the reduction after 72 hours of treatment with ProstaCaid, whereas there was not much change in the vehicle treatment group. We also found inhibition of AKT and MAPK signaling in LNCaP cells with 25 mg/mL of ProstaCaid but not in the vehicle treatment group (Figure 6B). The data shown here indicate that the mechanisms we found here are not cell type dependent. In summary, ProstaCaid at 25 mg/mL suppresses the activation of PI3K/AKT and MAPK signal pathways in 2 human prostate cancer cell lines for 72 hours.

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Figure 6. ProstaCaid inhibited AKT and MAPK signal pathways in human prostate cancer cells, PC3 (A) and LNCaP (B). AKT and MAPK signal pathways (phosphorylated AKT at serine 473, total AKT, phosphorylated ERK1/2 at theronine 202 and tyrosine 204, and phosphorylated S6 ribosomal protein at serine 235 and 236, GSK3b at serine 9). Cells were treated with 25 mg/mL ProstaCaid and collected at the indicated time points, and 20 mg proteins were loaded for the assay. b-Actin was used as a loading control. Densitometry analysis was performed for each blot, and numbers are shown under each blot

50% PC3 cell growth for 72 hours. At 24 hours, 50 mM and 100 mM quercetin induced G2/M arrest and apoptosis, manifested by the decrease in G2/M-related proteins (Cdc2 and cyclin B1), induction of p21, reduction of antiapoptotic proteins (Bcl2 and BclxL), and induction of cleavage of caspase-3.22 In LNCaP cells, quercetin suppressed androgen receptor function and induced apoptosis through the dissociation of Bax from BclxL and the activation of caspase families in LNCaP.3,23 DIM has been shown to inhibit cell proliferation, induce apoptosis, and suppress angiogenesis as well as invasion in both AD and AI prostate cell lines, partly because of a downregulation of AR, AKT, and the nuclear factor kB (NF-kB).24-27 The treatment of LNCaP and C4-2B prostate cancer cells with B-DIM showed maximal inhibition seen at 50 mM, with IC50 around 10 mM.25 DIM also enhances taxotere-induced apoptosis in AI prostate cancer cells through downregulation of survivin, an apoptosis inhibitor, that is associated with both prostate cancer progression and drug resistance.28 Resveratrol, an active component in grape skin, can delay LNCaP tumor growth and inhibit steroid hormonedependent pathway and PI3K/ AKT pathway in vitro and in vivo.4 Also, 24-hour treatment with 5 mM resveratrol showed more than 25% cell growth inhibition, whereas it did not show cytotoxicity on normal human prostate epithelial cells.29 Our study demonstrated

that ProstaCaid can inhibit cell proliferation by suppressing AKT activation and inducing apoptotic death of PC3 cells through caspase-3 activation. These findings suggested that in ProstaCaid, polyphenols and supplements could be bioactive compounds. Of note, the combination of several polyphenols has been investigated in several studies. The synergistic effects of phytochemicals EGCG, genistein, and quercetin have been shown on AR, tumor suppressor p53, and NQO1 in CWR22Rv1 prostate cancer cells.30 A pilot clinical study showed that the combination of curcumin and quercetin appeared to reduce the number and size of ileal and rectal adenomas in patients with familial adenomatous polyposis, without appreciable toxicity.31 These studies suggested that a combinatorial approach for prostate cancer prevention and treatment is feasible and more beneficial for patients than a single agent. It has been reported that PI3K/AKT plays an important role in prostate carcinogenesis. PTEN is a phosphoinositide3,4,5 trisphosphate phosphatase, which inhibits PI3K activity.32 Loss of PTEN expression was detected in about 30% to 60% of human prostate cancer.33,34 Moreover, ablation of pten alleles in prostate epithelial cells can spontaneously induce prostate cancer in mice.14,15 Furthermore, both activation of AKT and MAPK signal pathways strikingly induced prostate

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194 cancer, and pharmacological inhibition of both pathways efficiently suppressed androgen refractory prostate cancer, which indicates that AKT and MAPK signal pathways are important drug targets for prostate cancer.16,17 The results in Figure 5 demonstrate that ProstaCaid can suppress activation of both AKT and MAPK pathways in PC3 cells. Moreover, the inhibition of the AKT pathway was further confirmed by the reduction of phosphorylation levels of S6 and GSK3b, 2 of the AKT downstream targets. Dephosphorylation of GSK3b at ser9 increased its kinase activity and eventually reduced cyclin D1 stability.35 The activation of the PI3K/ Akt pathway leads to increased expression of Bcl2.36,37 Bim is a proapoptotic BH3-only Bcl-2 family member, binding to prosurvival Bcl-2 proteins, thereby releasing Bax or Bak proteins to promote apoptosis. The reduction of Bim has been attributed to the activation of both prosurvival MAPK signaling and AKT signaling.17,38 We found that ProstaCaid suppressed 2 antiapoptotic proteins, Bcl2 and BclxL, and induction of proapoptotic protein Bim after 72 hours of treatment. These changes in Bcl2 family proteins facilitate the induction of apoptosis, which was further confirmed by cleavage of caspase-3 and PARP. In this study, we have also shown that ProstaCaid-treated LNCaP and PC3 cells were arrested in the G2/M phase of the cell cycle (Figure 4 and Table 1). Cell cycle arrest in PC3 cells by ProstaCaid was accompanied by a reduction in the level of cyclin B1 and Cdc2 (Figure 5A). It is reasonable to postulate that ProstaCaid may affect activity of Cdc2/cyclin B1 kinase by reducing this complex formation. Cdc2 could be dephosphorylated by Cdc25C and become inactive or be phosphorylated by protein kinase, such as Wee1, and then converted into an inactive form.39,40 However, more studies are needed in the future to test it and to define its upstream events in PC3 cells. The bioavailability of certain botanical extracts such as curcumin from turmeric root has been poor, and the physiological activities have yet to be translated into clinical benefits. The bioavailability of ProstaCaid is not evaluated in this study and will be determined in future in vivo studies, but it is noted the preparation does include a bioenhanced curcumin preparation, BCM-95, thought to be more absorbable.41 In summary, data from our studies indicate that ProstaCaid has anti-cancer activities in both AD and AI prostate cancer cells at very low concentrations (25 mg/mL). It also suggests that ProstaCaid inhibits cell growth and survival, at least through the inhibition of AKT and MAPK signaling. The effect on AI cell lines is especially of importance as there is presently no curative therapy for hormone refractory prostate cancer. These findings warrant further investigation in animal models and humans to determine the potential role of ProstaCaid in vivo, either as a chemopreventative agent or in the management of the disease.

Integrative Cancer Therapies 9(2) More studies are needed to clarify the mechanism of ProstaCaid as well as the synergistic effects of its bioactive compounds in the near future. Acknowledgments
The authors would like to thank Dr Isaac Eliaz for his contribution to the study and for developing the formula, Dr Daniel Sliva, Dr Geovanni Espinosa, and Barry Wilk for their contribution in helping with this study. We would also like to thank Dr Cory Abate-Shen for the kind gifts of CASP cell lines.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the authorship and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research and/or authorship of this article: this work was supported by a sponsored research grant from EcoNugenics, Inc, Santa Rosa, CA.

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