Androgen receptor signalling impairs docetaxel efficacy in castration-resistant prostate cancer.

Androgen receptor (AR) signalling drives neoplastic growth and therapy resistance in prostate cancer. Recent clinical data show that docetaxel combined with androgen deprivation therapy improves outcome in hormone-sensitive disease. We studied whether testosterone and AR signalling interferes with docetaxel treatment efficacy in castration-resistant prostate cancer (CRPC). We found that testosterone supplementation significantly impaired docetaxel tumour accumulation in a CRPC model, resulting in decreased tubulin stabilisation and antitumour activity. Furthermore, testosterone competed with docetaxel for uptake by the drug transporter OATP1B3. Irrespective of docetaxel-induced tubulin stabilisation, AR signalling by testosterone counteracted docetaxel efficacy. AR-pathway activation could also reverse long-term tumour regression by docetaxel treatment in vivo. These results indicate that to optimise docetaxel efficacy, androgen levels and AR signalling need to be suppressed. This study lends evidence for continued maximum suppression of AR signalling by combining targeted therapeutics with docetaxel in CRPC.

Potential of Moringa oleifera in the Treatment of Benign Prostate Hyperplasia: Role of Antioxidant Defence Systems.

OBJECTIVE: This study sought to evaluate the protective effect of ethanolic leaf extract of Moringa oleifera on testosterone-induced benign prostatic hyperplasia (BPH) in male Sprague-Dawley rats. MATERIALS AND METHODS: BPH was induced in rats by the administration of testosterone propionate (3 mg/kg, s.c., in olive oil) for 4 weeks. M. oleifera (50, 100, or 200 mg/kg), celecoxib (20 mg/kg), or M. oleifera (50 mg/kg) + celecoxib (20 mg/kg) were orally administered daily 15 min before testosterone. On day 29, blood was collected to measure the levels of serum testosterone and prostate-specific antigen before the animals were sacrificed. The prostates were weighed, assayed, and histologically examined. RESULTS: M. oleifera significantly reduced the testosterone-induced increase in prostate weight (20.16%), prostate index (65.85%), serum testosterone (72.86%), and prostate-specific antigen (48.49%). Testosterone caused a significant increase in malondialdehyde (73%) as well as a reduction in glutathione (62.5%), superoxide dismutase (50%), and catalase (64%) activities which were attenuated by M. oleifera with a peak effect obtained at 100 mg/kg. The disruption of prostate histoarchitecture by testosterone was also ameliorated by M. oleifera. CONCLUSION: M. oleifera prevented testosterone-induced BPH through enhancement of antioxidant defence mechanisms, and hence could be used as an adjunct in the treatment of BPH.

Expressed prostatic secretion biomarkers improve stratification of NCCN active surveillance candidates: performance of secretion capacity and TMPRSS2:ERG models.

PURPOSE: Active surveillance is a viable patient option for prostate cancer provided that a clinical determination of low risk and presumably organ confined disease can be made. To standardize risk stratification schemes the NCCN (National Comprehensive Cancer Network®) provides guidelines for the active surveillance option. We determined the effectiveness of expressed prostatic secretion biomarkers for detecting occult risk factors in NCCN active surveillance candidates. MATERIALS AND METHODS: Expressed prostatic secretion specimens were obtained before robot-assisted radical prostatectomy. Secretion capacity biomarkers, including total RNA and expressed prostatic secretion specimen volume, were measured by standard techniques. RNA expression biomarkers, including TXNRD1 mRNA, prostate specific antigen mRNA, TMPRSS2:ERG fusion mRNA and PCA3 mRNA, were measured by quantitative reverse-transcription polymerase chain reaction. RESULTS: Of the 528 patients from whom expressed prostatic secretions were collected 216 were eligible for active surveillance under NCCN guidelines. Variable selection on logistic regression identified 2 models, including one featuring types III and VI TMPRSS2:ERG variants, and one featuring 2 secretion capacity biomarkers. Of the 2 high performing models the secretion capacity model was most effective for detecting cases in this group that were up-staged or up-staged plus upgraded. It decreased the risk of up-staging in patients with a negative test almost eightfold and decreased the risk of up-staging plus upgrading about fivefold while doubling the prevalence of up-staging in the positive test group. CONCLUSIONS: Noninvasive expressed prostatic secretion testing may improve patient acceptance of active surveillance by dramatically reducing the presence of occult risk factors among those eligible for active surveillance under NCCN guidelines.

Cutaneous metastasis of prostatic adenocarcinoma: a cautionary tale.

With the exception of skin cancer, prostatic adenocarcinoma represents the most common cancer among men in the United States and the second most common cause of cancer mortality. Mortality is often associated with metastatic disease, which in the case of prostatic adenocarcinoma typically involves bones and only rarely affects the skin. Although clinical history and examination, laboratory tests and routine pathology can suggest the prostate as a source of metastatic disease, immunohistochemistry - specifically, for prostate-specific antigen (PSA) - is often used to help establish the diagnosis. We report a case of cutaneous metastatic prostatic adenocarcinoma presenting in the inguinal region of a 78-year-old man 5 years after his initial diagnosis. The case is unusual in that the clinical appearance mimicked a vascular proliferation and in that the metastatic prostatic adenocarcinoma failed to express PSA. Rather, expression of prostatic acid phosphatase was observed.

Selenium enrichment of broccoli sprout extract increases chemosensitivity and apoptosis of LNCaP prostate cancer cells.

BACKGROUND: Broccoli is a Brassica vegetable that is believed to possess chemopreventive properties. Selenium also shows promise as an anticancer agent. Thus, selenium enrichment of broccoli has the potential to enhance the anticancer properties of broccoli sprouts. METHOD: Selenium-enriched broccoli sprouts were prepared using a sodium selenite solution. Their anticancer properties were evaluated in human prostate cancer cell lines and compared with those of a control broccoli sprout extract. RESULTS: Selenium-enriched broccoli sprouts were superior to normal broccoli sprouts in inhibiting cell proliferation, decreasing prostate-specific antigen secretion, and inducing apoptosis of prostate cancer cells. Furthermore, selenium-enriched broccoli sprouts but, not normal broccoli sprouts, induced a downregulation of the survival Akt/mTOR pathway. CONCLUSION: Our results suggest that selenium-enriched broccoli sprouts could potentially be used as an alternative selenium source for prostate cancer prevention and therapy.

Tea polyphenols decrease serum levels of prostate-specific antigen, hepatocyte growth factor, and vascular endothelial growth factor in prostate cancer patients and inhibit production of hepatocyte growth factor and vascular endothelial growth factor in vitro.

The purpose of this study was to determine the effects of short-term supplementation with the active compounds in green tea on serum biomarkers in patients with prostate cancer. Twenty-six men with positive prostate biopsies and scheduled for radical prostatectomy were given daily doses of Polyphenon E, which contained 800 mg of (-)-epigallocatechin-3-gallate (EGCG) and lesser amounts of (-)-epicatechin, (-)-epigallocatechin, and (-)-epicatechin-3-gallate (a total of 1.3 g of tea polyphenols), until time of radical prostatectomy. Serum was collected before initiation of the drug study and on the day of prostatectomy. Serum biomarkers hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-I, IGF binding protein-3 (IGFBP-3), and prostate-specific antigen (PSA) were analyzed by ELISA. Toxicity was monitored primarily through liver function enzymes. Changes in serum components were analyzed statistically using the Wilcoxon signed rank test. Cancer-associated fibroblasts were treated with EGCG, and HGF and VEGF protein and mRNA levels were measured. HGF, VEGF, PSA, IGF-I, IGFBP-3, and the IGF-I/IGFBP-3 ratio decreased significantly during the study. All of the liver function tests also decreased, five of them significantly: total protein, albumin, aspartate aminotransferase, alkaline phosphatase, and amylase. The decrease in HGF and VEGF was confirmed in prostate cancer-associated fibroblasts in vitro. Our results show a significant reduction in serum levels of PSA, HGF, and VEGF in men with prostate cancer after brief treatment with EGCG (Polyphenon E), with no elevation of liver enzymes. These findings support a potential role for Polyphenon E in the treatment or prevention of prostate cancer.

Baseline urinary phytoestrogen levels and the natural history of untreated, localised prostate cancer in a British population.

AIM: To determine whether urinary concentrations of phytoestrogens are associated with the rate of disease progression in men with untreated, localised prostate cancer. PATIENTS AND METHODS: Patients with untreated, localised prostatic adenocarcinoma on a prospective clinical study of active surveillance had urine samples collected at baseline. Patients underwent monitoring with serial PSA levels and repeat octant prostate biopsies. Disease progression was defined as either adverse histology on repeat biopsy (primary Gleason grade >or= 4, or >50% positive cores) or radical treatment for PSA velocity >1 ng/mL/year. Time to disease progression was analysed with respect to baseline urinary levels of genistein, enterolactone, daidzein and equol, assayed using liquid chromatography/tandem mass spectrometry. RESULTS: 191 patients were evaluable, with a median follow-up of 2.5 years. 71 patients experienced disease progression. No significant association was seen between time to disease progression and baseline urinary levels of daidzein (p=0.85), genistein (p=0.81), enterolactone (p=0.085) or equol (p=0.33). No significant association was seen between adverse histology on repeat biopsy and urinary levels of either daidzein (p=0.85), genistein (p=0.58), enterolactone (p=0.88) or equol (p=0.71). There was no significant correlation between PSA velocity and urinary levels of daidzein (p=0.90), genistein (p=0.98), enterolactone (p=0.10) or equol (p=0.60). CONCLUSION: These data do not support the hypothesis that phytoestrogens prevent disease progression in men with localised prostate cancer.

Down-regulation of androgen-receptor and PSA by phytochemicals.

The androgen receptor (AR) signaling pathway continues to be active in hormone resistant prostate cancer (HRPC) and can inappropriately activate transcription. Consequently the AR is a therapeutic target for HRPC. We reported that PC-SPES is active against HRPC, partly due to its actions in down-regulating AR protein expression and modulating cell cycle. Further investigation has identified five active anticancer compounds. This study describes the effects of three of these compounds (oridonin, isoliquiritigenin and wogonin) on cell proliferation, cell apoptosis, cell cycle parameters, AR and PSA protein expression. In each case, these compounds have independent activities which may partly contribute to the biological activity of PC-SPES.

Saw Palmetto induces growth arrest and apoptosis of androgen-dependent prostate cancer LNCaP cells via inactivation of STAT 3 and androgen receptor signaling.

PC-SPES is an eight-herb mixture that has an activity against prostate cancer. Recently, we purified Saw Palmetto (Serenoa repens) from PC-SPES and found that Saw Palmetto induced growth arrest of prostate cancer LNCaP, DU145, and PC3 cells with ED50s of approximately 2.0, 2.6, and 3.3 microl/ml, respectively, as measured by mitochondrial-dependent conversion of the the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Saw Palmetto induced apoptosis of LNCaP cells in a time- and dose-dependent manner as measured by TUNEL assays. Also, Saw Palmetto increased the expression of p21waf1 and p53 protein in LNCaP cells. In addition, we found that Saw Palmetto down-regulated DHT- or IL-6-induced expression of prostate specific antigen in conjunction with down-regulation of the level of androgen receptor in the nucleus as measured by Western blot analysis. Moreover, Saw Palmetto down-regulated the IL-6-induced level of the phosphorylated form of STAT 3 in LNCaP cells. Furthermore, Saw Palmetto inhibited the growth of LNCaP cells present as tumor xenografts in BALB/c nude mice without adverse effect. These results indicate that Saw Palmetto might be useful for the treatment of individuals with prostate cancer.

Androgen receptor or estrogen receptor-beta blockade alters DHEA-, DHT-, and E(2)-induced proliferation and PSA production in human prostate cancer cells.

BACKGROUND: Dehydroepiandrosterone (DHEA) is an endogenous steroid that is metabolized to androgens and/or estrogens in the human prostate. DHEA levels decline with age, and use of DHEA supplements to retard the aging process is of unproved effectiveness and safety. LNCaP and LAPC-4 prostate cancer cells were used to determine whether DHEA-modulated proliferation and prostate specific antigen (PSA) production were mediated via the androgen receptor (AR) and/or ERbeta. METHODS: Cells were treated with DHEA, DHT, or E(2) and antagonists to AR (Casodex-bicalutamide) or ER (ICI 182,780) or siRNA to the respective receptors. Proliferation was assessed by MTT assay and PSA mRNA and protein secretion were measured by quantitative real-time PCR and ELISA. Associations of AR and ERbeta were analyzed by co-immunoprecipitation studies and fluorescent confocal microscopy. RESULTS: DHEA-, T-, and E(2)-induced proliferation of LNCaP cells was blunted by Casodex but not by ICI treatment. In LNCaP cells, Casodex and ICI suppressed hormone-induced PSA production. In LAPC-4 cells, DHT-stimulated PSA mRNA was inhibited by Casodex and ICI, and the minimal stimulation by DHEA was inhibited by ICI. Use of siRNAs confirmed involvement of AR and ERbeta in hormone-induced PSA production while AR-ERbeta co-association was suggested by immunoprecipitation and nuclear co-localization. CONCLUSIONS: These findings support involvement of both AR and ERbeta in mediating DHEA-, DHT-, and E(2)-induced PSA expression in prostate cancer cells.

Zanthoxyli Fructus induces growth arrest and apoptosis of LNCaP human prostate cancer cells in vitro and in vivo in association with blockade of the AKT and AR signal pathways.

Zanthoxyli Fructus belongs to the family of oranges and is used as a seasoning in Asian countries including Japan. This study found that a water extract of Zanthoxyli Fructus possessed anti-tumor activity against a wide variety of cancer cells including those from prostate (LNCaP, DU145, PC-3), breast (MCF-7, T47D, MDA-MB231), lung (NCI-H460, -H520), as well as leukemia (HL-60, NB4, Jurkat) in vitro, as measured by the trypan blue exclusion test. Importantly, Zanthoxyli Fructus slowed the proliferation of LNCaP, DU145, and MDA-MB231 cells present as xenografts in BALB/c nude mice without adverse effects. Further studies explored the molecular mechanism by which Zanthoxyli Fructus inhibited the proliferation of androgen-dependent human prostate cancer LNCaP cells because Zanthoxyli Fructus possessed the strongest anti-tumor activity against these cells. Zanthoxyli Fructus blocked androgen receptor (AR) signaling in conjunction with down-regulation of nuclear levels of AR and induced apoptosis of these cells, as measured by the reporter assay, Western blot analysis, and TUNEL assay, respectively. As expected, Zanthoxyli Fructus also decreased the level of the AR-target molecule, prostate-specific antigen in these cells. Furthermore, Zanthoxyli Fructus inhibited AKT kinase and down-regulated levels of cyclin D1 protein, as measured by the AKT kinase assay with GSK-3alpha/beta as a substrate and Western blot analysis, respectively. Taken together, Zanthoxyli Fructus might be useful as an adjunctive therapeutic agent for the treatment of individuals with a variety of cancer types.

Potent antiandrogen and androgen receptor activities of an Angelica gigas-containing herbal formulation: identification of decursin as a novel and active compound with implications for prevention and treatment of prostate cancer.

Androgen and androgen receptor (AR)-mediated signaling are crucial for the development of prostate cancer. Identification of novel and naturally occurring phytochemicals that target androgen and AR signaling from Oriental medicinal herbs holds exciting promises for the chemoprevention of this disease. In this article, we report the discovery of strong and long-lasting antiandrogen and AR activities of the ethanol extract of a herbal formula (termed KMKKT) containing Korean Angelica gigas Nakai (AGN) root and nine other Oriental herbs in the androgen-dependent LNCaP human prostate cancer cell model. The functional biomarkers evaluated included a suppression of the expression of prostate-specific antigen (PSA) mRNA and protein (IC50, approximately 7 microg/mL, 48-hour exposure) and an inhibition of androgen-induced cell proliferation through G1 arrest and of the ability of androgen to suppress neuroendocrine differentiation at exposure concentrations that did not cause apoptosis. Through activity-guided fractionation, we identified decursin from AGN as a novel antiandrogen and AR compound with an IC50 of approximately 0.4 microg/mL (1.3 micromol/L, 48-hour exposure) for suppressing PSA expression. Decursin also recapitulated the neuroendocrine differentiation induction and G1 arrest actions of the AGN and KMKKT extracts. Mechanistically, decursin in its neat form or as a component of AGN or KMKKT extracts inhibited androgen-stimulated AR translocation to the nucleus and down-regulated AR protein abundance without affecting the AR mRNA level. The novel antiandrogen and AR activities of decursin and decursin-containing herbal extracts have significant implications for the chemoprevention and treatment of prostate cancer and other androgen-dependent diseases.

Chemoprevention of prostate cancer by cholecalciferol (vitamin D3): 25-hydroxylase (CYP27A1) in human prostate epithelial cells.

The 20-30 year latency period for prostate cancer provides an important opportunity to prevent the development of invasive cancer. A logical approach for chemoprevention to reduce incidence is to identify agents, such as, vitamin D, which can inhibit cell proliferation and induce differentiation, are safe, and readily available to the public at low cost. Epidemiological evidence suggests that vitamin D deficiency is associated with increased risk for prostate cancer. We examined the ability and mechanisms of action of cholecalciferol (vitamin D(3)), a precursor of the most biologically active hormone calcitriol, to block or reverse premalignant changes. The immortalized, non-tumorigenic, RWPE-1 human prostate epithelial cell line, was used. Results show that cholecalciferol, at physiological levels: (i) inhibits anchorage-dependent growth (ii) induces differentiation by increasing PSA expression and (iii) exerts its effects by up-regulating vitamin D receptor (VDR), retinoid-X receptors (RXRs), and androgen receptor (AR). Furthermore, we discovered that human prostate epithelial cells constitutively express appreciable levels of 25-hydroxylase CYP27A1 protein, the enzyme which catalyzes the conversion of cholecalciferol to 25(OH)D(3), and that CYP27A1 is up-regulated by cholecalciferol. Recent studies show that human mitochondrial CYP27A1 can also catalyze 1alpha-hydroxylation of 25(OH)D(3) to calcitriol. The presence of 25-hydroxylase in human prostate epithelial cells has not previously been shown. Since human prostate epithelial cells have the necessary enzymes and the rare ability to locally convert cholecalciferol to the active hormone calcitriol, we propose that they are a prime target for chemoprevention of prostate cancer with cholecalciferol whose safety is well established as a supplement in vitamins and fortified foods.

[In vitro effect of zilongjin on prostate cancer cell line LNCaP].

OBJECTIVE: To investigate the effect of Zilongjin (ZLJ) on human androgen-dependent type of prostate cancer cell line LNCaP. METHODS: MTT assay, flow cytometry and fluorescence microscopy were used to observe the effect of ZLJ in anti-proliferation, cell cycle arresting and apoptosis induction. RT-PCR was used to examine the effect of ZLJ on expressions of prostate marker gene (PSA), androgen receptor (AR), apoptosis related genes (bcl-2 and bax), and Western blot assay was used to detect the effect on protein expression of bcl-2 and bax. RESULTS: ZLJ could cause apparent inhibition on proliferation, induce G0/G1 phase arresting and apoptosis in time- and dose-dependent manner on LNCaP cells. The concentration for inhibiting cell growth by 50% (IC50) in 72 hrs was 0.79 mg/ml. ZLJ could down-regulate the expression of PSA, AR, bcl-2 genes and lower bcl-2 protein expression, but showed ineffective on bax protein expression. CONCLUSION: ZLJ displays its anti-tumor effects by way of inhibiting the cell proliferation, arresting the G0/G1 phase, inducing apoptosis, down-regulating PSA, AR, bcl-2 gene expression and lowering bcl-2 protein expressions.

PC-SPES: Molecular mechanism to induce apoptosis and down-regulate expression of PSA in LNCaP human prostate cancer cells.

PC-SPES is an eight-herbal mixture which has activity against prostate cancer cells and can reduce the serum level of prostate specific antigen (PSA) in more than 80% of individuals with prostate cancer. We conducted this study to begin to clarify the molecular mechanism by which PC-SPES inhibited the growth of prostate cancer cells and down-regulated expression of PSA. Western blot analysis, luciferase reporter assay using a variety of promoters of the PSA gene and the isolated androgen receptor response elements (ARE), as well as electrophoretic mobility shift assay (EMSA) were employed to study the effect of PC-SPES on DHT-induced expression of PSA in LNCaP androgen-dependent human prostate cancer cells. Also, Western blot analysis and luciferase reporter assay using 12-0-tetradecanoylphorbol-13-acetate response elements were employed to study the ability of PC-SPES to activate the c-Jun NH2-terminal kinase (JNK)/c-Jun/AP-1 signal pathway in these cells. Reporter studies showed that PC-SPES inhibited DHT-induced PSA promoter/enhancer-luciferase activity via inhibition of ARE transcriptional activity. Western blot analysis showed that PC-SPES down-regulated DHT-induced expression of PSA without decreasing DHT-induced nuclear level of AR. EMSA demonstrated that PC-SPES inhibited the binding of DHT-activated AR to ARE. Moreover, we found that PC-SPES phosphorylated JNK, increased levels of phosphorylated and unphosphorylated forms of c-Jun, and enhanced AP-1 transcriptional activity in LNCaP cells. Interestingly, when LNCaP cells were stably tranfected with the dominant negative JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), these cells no longer underwent apoptosis and growth inhibition in the presence of PC-SPES. But, PC-SPES still decreased levels of PSA in the LNCaP-JIP-1 cells. Taken together, PC-SPES inhibited binding of DHT-activated AR to AREs of PSA gene resulting in down-regulation of ARE transcriptional activity and expression of PSA, and this occurred independently of the JNK/c-Jun/AP-1 signal pathway. Also, PC-SPES activated the JNK/c-Jun/AP-1 signal pathway resulting in growth arrest and apoptosis of prostate cancer cells.

Inhibition of proliferation and expression of AR/PSA by herbal supplement Equiguard in LNCaP cells cultured in androgen-proficient FBS and androgen-deficient charcoal-stripped FBS is correlated with increased serine-15 phosphorylation of the tumor suppressor gene p53.

Use of dietary supplements and botanical products is widely accepted by patients diagnosed with prostate cancer (CaP) as a primary or complementary form of treatment for their medical conditions in the U.S. Yet, the majority of these products have not been rigorously studied with regard to scientific mechanism(s). Because many of the available products are mixtures of multiple extracts derived from plants, some of which are not necessarily native to the U.S., we consider mechanistic studies under defined laboratory conditions to be valuable and essential, not only from the standpoint of standardization and possible contamination with the products, but also in providing insights and scientific evidence for the clinical efficacy some of these products purportedly demonstrate. In previous studies from this laboratory, Equiguard, a composite supplement consisting of standardized extracts from nine Chinese herbs, which was originally formulated to correct physiological decline in kidney functions associated with age, was fortuitously found to display anti-CaP properties. Using a panel of CaP cells, we showed that ethanol extracts of Equiguard significantly inhibited cancer cell growth, induced apoptosis, lowered expression of the androgen receptor (AR), decreased intracellular and secreted prostate-specific antigen (PSA) levels and completely abolished the colony forming activities of CaP cells. Since responsiveness to Equiguard was observed in cells mimicking the androgen-dependent (AD) and androgen-independent (AI) states of CaP, our results raise the interesting possibility that this herbal supplement may potentially prevent, delay or circumvent the onset of AI, and thereby induce chronic instead of terminal CaP. Since androgen ablation therapy (chemical or surgical castration) is the mainstay for localized CaP, we questioned whether Equiguard might still exert the aforementioned activities in experimental settings modeled after androgen ablation. Accordingly, we studied the effects of Equiguard in LNCaP cells, cultured in androgen-proficient (FBS) or -deficient (CS-FBS) media that simulate the hormonal status pre- and post-castration in vivo. Extracts of Equiguard were effective in reducing colony formation, proliferation and PCNA expression of cells cultured in CS-FBS. Moreover, within a concentration range of Equiguard, the prostate-specific genes, PSA and AR, were affected to a similar extent in cells cultured either in FBS or CS-FBS, and were correlated with increased phosphorylation at serine-15 of the tumor suppressor gene p53. These results are consistent with the interpretation that the anti-proliferative and gene modulatory properties of Equiguard are largely independent of the status of androgens in the culture media.

Gene profiling and promoter reporter assays: novel tools for comparing the biological effects of botanical extracts on human prostate cancer cells and understanding their mechanisms of action.

The use of botanical mixtures is commonplace in patients with prostate cancer, yet the majority of these products have not been rigorously tested in clinical trials. Here we use PC-SPES, a combination of eight herbs that has been shown to be effective in clinical trials in patients with prostate cancer, as a model system to demonstrate 'proof of principle' as to how gene expression profiling coupled with promoter assays can evaluate the effect of herbal cocktails on human prostate cancer. In addition, we demonstrate how such approaches may be used for standardization of herbal extract activity by comparing the gene profile of PC-SPES with that of PC-CARE, a product with a similar herbal composition. Since prior studies have shown that PC-SPES contains estrogenic organic compounds, and such compounds are known to affect prostate cancer, an important issue is whether these are the primary drivers of the gene profile. Our data suggest that gene expression profiles of LNCaP human prostate cancer cells in response to PC-SPES are different from those found when diethylstilbestrol (DES), a synthetic estrogen, is used, suggesting that the estrogenic moieties within PC-SPES do not drive this expression signature. In contrast, the expression profile of PC-CARE was almost identical to that of DES, highlighting that mixtures containing similar herbal compositions do not necessarily result in similar biological activities. Interestingly, these three agents cause similar in vitro morphological changes and growth effects on LNCaP. To validate the expression profiling data, we evaluated the protein expression and promoter activity of prostate-specific antigen (PSA), a gene induced by PC-SPES but repressed by DES. In order to gain a mechanistic understanding of how PC-SPES and DES affect PSA expression differently, LNCaP cells were transiently transfected with wild-type and mutagenized PSA promoter, ARE concatemers and appropriate controls. We provide evidence that androgen response elements (ARE) II and III within the promoter region are responsible for the suppressive effects of DES and stimulatory effects of PC-SPES. In addition, we show that the effects on PSA transcription are ARE specific in the case of DES while PC-SPES affects this promoter nonspecifically. In conclusion, expression profiling coupled with mechanistic target validation yield valuable clues as to the mode of action of complex botanical mixtures and provides a new way to compare objectively mixtures with similar components either for effect or quality assurance prior to their use in clinical trials.

Mechanism of action of herbal supplement PC-SPES: elucidation of effects of individual herbs of PC-SPES on proliferation and prostate specific gene expression in androgen-dependent LNCaP cells.

PC-SPES is a herbal mixture used by prostate cancer patients as an alternative form of treatment. Since PC-SPES is derived from eight individual herbs, each with distinct as well as overlapping properties, it is of interest to investigate whether a particular herb in the formulation principally accounts for the biological properties of PC-SPES. We tested the ability of extracts from individual herbs, using amounts estimated to be equivalent to that present in the herbal mixture, to suppress LNCaP cell growth and/or lower PSA expression, in comparison with cells treated with PC-SPES. Cells were incubated with 0, 1, and 5 microl/ml of single herbal extract for 72 h and proliferation/viability was measured by trypan blue exclusion. LNCaP cells treated with 5 microl/ml ethanol extracts of PC-SPES showed a 72-80% reduction in cell growth, and had a similar decrease in cell viability. These results contrasted with cells incubated with 5 microl/ml of individual herbal extract, which suppressed growth in the following order: Dendranthema morifolium Tzvel (85.2% reduction) > Panax pseudo-ginseng (80.9%) > Glycyrrhiza uralensis Fisch (73%) > Rabdosia rubescens Hara (70.8%) > Scutellaria baicalensis Georgi (66.5%) > Ganoderma lucidum Karst (63.5%) > Isatis indigotica Fort (50.0%) > Serenoa repens (14.5%). Analysis of efficacy of individual herbs to control intracellular/secreted PSA levels and the expression of AR and PSA revealed that only Glycyrrhiza uralensis Fisch, Scutellaria baicalensis Georgi and Serenoa repens lowered intracellular and secreted PSA, while the remaining herbs actually increased PSA expression. Also, no uniform response in AR/PSA was observed in individual herb treated cells, contrary to PC-SPES, which elicited a coordinated change in AR/PSA. Lack of concordance between changes in prostate cell growth and prostate specific gene expression makes it unlikely that the activity of a single herb can account for the overall effects of PC-SPES.

Growth of heterotopic LNCaP prostate cancer tumor in nude mice is not affected by dietary calcium.

BACKGROUND: We attempted to provide experimental evidence linking increased dietary calcium to progression of prostate cancer, as suggested by some epidemiological studies, using a heterotopic prostate cancer nude mice model. METHODS: Twenty heterotopic LNCaP prostate cancer tumor bearing nude mice were randomly assigned to one of the four groups: (I) high fat/low calcium diet, (II) high fat and high calcium diet, (III) high fat diet fortified with Vitamin D3, and (IV) high fat and high calcium diet fortified with Vitamin D3. In addition to weekly animal weights and tumor size measurements, the serum prostate specific antigen (PSA), 25-hydroxy Vitamin D3, calcium, phosphorus, total protein, albumin (to account for bound calcium) [1], and serum alkaline phosphatase (a measure of bone loss) [2] were determined at the termination of experiments. RESULTS: Although the serum calcium and 25-hydroxy Vitamin D3 were significantly higher in groups III and IV compared to groups I and II (P < 0.05), there was no significant difference between the tumor growth rates, final tumor weights (P = 0.9), and the serum PSA levels (P = 0.94) between the four groups. CONCLUSIONS: The results suggest that dietary calcium does not significantly affect the growth of heterotopic LNCaP prostate cancer in nude mice.

Cell growth and gene modulatory activities of Yunzhi (Windsor Wunxi) from mushroom Trametes versicolor in androgen-dependent and androgen-insensitive human prostate cancer cells.

The incidence of prostate cancer varies greatly throughout the world; it is highest in African-Americans and lowest in the Asian populations of China, India, and Japan. Geographical differences in both prevalence of latent prostate cancer and mortality have been postulated to be influenced by diverse tumor-promoting and protective factors, both environmental and dietary. Prostate cancer is a tumor with an extremely long latency; the pattern of prostate tumorigenesis, in terms of the display and sequence of appearance of particular molecular or biochemical features, or morphological changes, characterizing different stages of the carcinogenic process, is expected to be heterogeneous. Some insights into tumor heterogeneity and progression can be obtained from studies using cell lines, particularly those derived from different anatomical sites. The present study aims to investigate whether hormone-responsive LNCaP and androgen-refractory JCA-1, PC-3, and DU-145 prostate cancer cells are responsive to Yunzhi (YZ), a proprietary dietary supplement prepared from extracts of Trametes versicolor, also known as Coriolus versicolor (a mushroom consumed by Chinese for its purported health benefits), and to elucidate its mechanism of action. Ethanolic extracts (70%) of YZ significantly reduced LNCaP cell growth, down-regulated the levels of secreted PSA, but had less effects on the expression of intracellular PSA and did not affect levels of the androgen receptor. In androgen-unresponsive prostate cancer cells, YZ had a much less pronounced suppressive effect on proliferation of PC-3 and DU-145 cells, compared to LNCaP, and was inactive against JCA-1 cells. Western blot analyses show that the expression of Rb, a key regulatory protein in G1/S transition, and PCNA, integrally involved in mammalian cell DNA replication, were significantly reduced by treatment with YZ in PC-3 and DU-145 cells, respectively. In contradiction, none of these biochemical parameters were affected in JCA-1 cells under identical treatment conditions. Further analysis shows that YZ increased the levels of signal transducer and activator family of transcription factors STAT 1 and STAT 3 in JCA-1 and not LNCaP cells. The greater sensitivity of LNCaP cells to this polysaccharopeptide raises the possibility that YZ may be considered as an adjuvant therapy in the treatment of hormone responsive prostate cancer; additionally, it may have chemopreventive potential to restrict prostate tumorigenic progression from the hormone-dependent to the hormone-refractory state.