Cultured Prostate Cancer Cells Research Articles

Serenoa repens extract targets mitochondria and activates the intrinsic apoptotic pathway in human prostate cancer cells

To investigate the effects of Serenoa repens extract (Sr) in human PC3 and LNCaP prostate cancer and MCF7 breast cancer cells, with specific emphasis on the role of the mitochondrial apoptotic pathway, as the molecular pathway through which Sr, a natural product of plant origin, induces death of prostate cancer cells in culture is still unknown. Cellular and mitochondrial structure and function, and the cell cycle, were analysed using light, electron and fluorescence microscopy, spectrophotometry and flow cytometry. Apoptosis was evaluated using biochemical and cytohistochemical methods. Cells treated with Sr underwent massive vacuolization and cytosolic condensation, followed by cell death only in the prostate lines. Within minutes of adding Sr to prostate cells, it caused opening of the permeability transition pore (PTP), which led to complete mitochondrial depolarization within 2 h, and to the appearance of small, pycnotic mitochondria. Release of cytochrome c and SMAC/Diablo to the cytosol was detectable after 4 h of treatment, while caspase 9 activation and poly(ADP-ribose) polymerase 1 cleavage occurred at 16 h, followed by appearance of a sub-G1 peak and apoptosis at 24 h. Sr selectively induces apoptotic cell death of prostate cancer cells through the intrinsic pathway, and activation of the mitochondrial PTP might play a central role in this process.

Commentary on Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model

The AKT/mammalian target of rapamycin (AKT/mTOR) and ERK MAPK signaling pathways have been shown to cooperate in prostate cancer progression and the transition to androgen-independent disease. We have now tested the effects of combinatorial inhibition of these pathways on prostate tumorigenicity by performing preclinical studies using a genetically engineered mouse model of prostate cancer. We report here that combination therapy using rapamycin, an inhibitor of mTOR, and PD0325901, an inhibitor of MAPK kinase 1 (MEK; the kinase directly upstream of ERK), inhibited cell growth in cultured prostate cancer cell lines and tumor growth particularly for androgen-independent prostate tumors in the mouse model. We further showed that such inhibition leads to inhibition of proliferation and up-regulated expression of the apoptotic regulator Bcl-2-interacting mediator of cell death (Bim). Furthermore, analyses of human prostate cancer tissue microarrays demonstrated that AKT/mTOR and ERK MAPK signaling pathways are often coordinately deregulated during prostate cancer progression in humans. We therefore propose that combination therapy targeting AKT/mTOR and ERK MAPK signaling pathways may be an effective treatment for patients with advanced prostate cancer, in particular those with hormone-refractory disease.

CAMP and Pyk2 interact to regulate prostate cells proliferation and function

In cultured prostate cancer cells cAMP blocks proliferation and induces neuroendocrine differentiation. Pyk2 expression inversely correlates with malignancy of prostate cancer. The aim of this study was to investigate the interaction between cAMP and Pyk2 in the prostate. EPN cells, a line derived from human normal prostate expressing Pyk2, and EPN-PKM3 cells, an EPN clone bearing a Pyk2 kinase-negative mutant, were adopted as model system. cAMP inhibited cell growth in both prostate cell lines, and activated Pyk2, but not ERK1/2, in EPN cells. cAMP treatment, abolished the activation of AKT1, an important component of the pro-survival pathway, in the EPN cells but not in EPN-PKM3 cells. Finally, upon cAMP treatment, EPN and EPN-PKM3 cells exhibited different expression patterns of HOX genes, an important network controlling cell identity. These data demonstrated for the first time that Pyk2 and cAMP interact in regulating prostate cell functions and in "keeping" prostate identity.

Inhibition of prostaglandin synthesis and actions contributes to the beneficial effects of calcitriol in prostate cancer

Our research is aimed at obtaining a better understanding of the molecular mechanisms of the anti-proliferative and cancer preventive effects of calcitriol with the goal of developing strategies to improve the treatment of prostate cancer (PCa). In PCa cells calcitriol inhibits the synthesis and biological actions of prostaglandins (PGs) by three actions: (i) the inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (ii) the upregulation of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs and (iii) decreasing the expression of EP and FP PG receptors that are essential for PG signaling. Since PGs have been shown to promote carcinogenesis and progression of multiple cancers, we hypothesize that the inhibition of the PG pathway contributes to the ability of calcitriol to prevent or inhibit PCa development and growth. We have shown that the combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of the growth of PCa cell cultures and this combination therapy offers a potential therapeutic strategy. These findings led us to embark on a clinical trial combining the non-selective NSAID naproxen with calcitriol in men with early recurrent PCa. The results indicate that the combination of high dose weekly calcitriol with naproxen slows the rate of rise (doubling time) of PSA in most patients indicating the slowing of disease progression. Further studies are warranted to determine the role of this combination therapy in the management of recurrent PCa.

Ligand-Independent Androgen Receptor Variants Derived from Splicing of Cryptic Exons Signify Hormone-Refractory Prostate Cancer

Suppression of androgen production and function provides palliation but not cure in men with prostate cancer (PCa). Therapeutic failure and progression to hormone-refractory PCa (HRPC) are often accompanied by molecular alterations involving the androgen receptor (AR). In this study, we report novel forms of AR alteration that are prevalent in HRPC. Through in silico sequence analysis and subsequent experimental validation studies, we uncovered seven AR variant transcripts lacking the reading frames for the ligand-binding domain due to splicing of "intronic" cryptic exons to the upstream exons encoding the AR DNA-binding domain. We focused on the two most abundantly expressed variants, AR-V1 and AR-V7, for more detailed analysis. AR-V1 and AR-V7 mRNA showed an average 20-fold higher expression in HRPC (n = 25) when compared with hormone-naive PCa (n = 82; P < 0.0001). Among the hormone-naive PCa, higher expression of AR-V7 predicted biochemical recurrence following surgical treatment (P = 0.012). Polyclonal antibodies specific to AR-V7 detected the AR-V7 protein frequently in HRPC specimens but rarely in hormone-naive PCa specimens. AR-V7 was localized in the nuclei of cultured PCa cells under androgen-depleted conditions, and constitutively active in driving the expression of canonical androgen-responsive genes, as revealed by both AR reporter assays and expression microarray analysis. These results suggest a novel mechanism for the development of HRPC that warrants further investigation. In addition, as expression markers for lethal PCa, these novel AR variants may be explored as potential biomarkers and therapeutic targets for advanced PCa.

Identification of extracellular δ‐catenin accumulation for prostate cancer detection

Prostate cancer is the second leading cause of cancer death in men, and early detection is essential to reduce mortality and increase survival. delta-Catenin is a unique beta-catenin superfamily protein primarily expressed in the brain but is upregulated in human prostatic adenocarcinomas. Despite its close correlation with the disease, it is unclear whether delta-catenin presents the potential in prostate cancer screening because it is an intracellular protein. In this study, we investigated the hypothesis of delta-catenin accumulation in the urine of prostate cancer patients and its potential pathways of excretion into extracellular milieu. Prostate cancer cell cultures, human tissue biopsies, and voided urines were characterized to determine extracellular delta-catenin accumulation and co-isolation with exosomes/prostasomes. We identified delta-catenin in culture media and in the stroma of human prostate cancer tissues. In PC-3 cells in culture, delta-catenin was partially co-localized and co-isolated with raft-associated membrane protein caveolin-1 and glycosylphosphatidylinositol-anchored protein CD59, suggesting its potential excretion into extracellular milieu through exosome/prostasome associated pathways. Interference with endocytic pathway using wortmannin did not block prostasome excretion, but delta-catenin overexpression promoted the extracellular accumulation of caveolin-1. delta-Catenin, caveolin-1, and CD59 were all detected in cell-free human voided urine prostasomes. delta-Catenin immunoreactivity was significantly increased in the urine of prostate cancer patients (P < 0.0005). This study demonstrated, for the first time, the extracellular accumulation of delta-catenin in urine supporting its potential utility for non-invasive prostate cancer detection.

Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach

Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes. Although many substrates for MMP-9, including components of the extracellular matrix, soluble mediators such as chemokines, and cell surface molecules have been identified, we undertook a more comprehensive proteomics-based approach to identify new substrates to further understand how MMP-9 might contribute to tumor metastasis. Previous proteomics approaches to identify protease substrates have depended upon differential labeling of each sample. Instead we used a label-free quantitative proteomics approach based on ultraperformance LC-ESI-high/low collision energy MS. Conditioned medium from a human metastatic prostate cancer cell line, PC-3ML, in which MMP-9 had been down-regulated by RNA interference was compared with that from the parental cells. From more than 200 proteins identified, 69 showed significant alteration in levels after depletion of the protease (>+/-2-fold), suggesting that they might be candidate substrates. Levels of six of these (amyloid-beta precursor protein, collagen VI, leukemia inhibitory factor, neuropilin-1, prostate cancer cell-derived growth factor (PCDGF), and protease nexin-1 (PN-1)) were tested in the conditioned media by immunoblotting. There was a strong correlation between results by ultraperformance LC-ESI-high/low collision energy MS and by immunoblotting giving credence to the label-free approach. Further information about MMP-9 cleavage was obtained by comparison of the peptide coverage of collagen VI in the presence and absence of MMP-9 showing increased sensitivity of the C- and N-terminal globular regions over the helical regions. Susceptibility of PN-1 and leukemia inhibitory factor to MMP-9 degradation was confirmed by in vitro incubation of the recombinant proteins with recombinant MMP-9. The MMP-9 cleavage sites in PN-1 were sequenced. This study provides a new label-free method for degradomics cell-based screening leading to the identification of a series of proteins whose levels are affected by MMP-9, some of which are clearly direct substrates for MMP-9 and become candidates for involvement in metastasis.

Fas-mediated killing of primary prostate cancer cells is increased by mitoxantrone and docetaxel

Therapies for prostate cancer based on Fas (CD95) modulation have been under active development at the preclinical stage using immortalized cell lines. To address clinical applicability, the potential of 11 cultures of primary prostate cancer cells to be killed by Fas-mediated apoptosis was investigated. In addition, the effect of the chemotherapeutic agents mitoxantrone and docetaxel on this killing was determined. Apoptosis was induced in patient-derived, primary prostate cancer cells using effector cells engineered by recombinant lentivirus infection to express Fas ligand (FasL) and measured by 51Cr release assays. All cultured prostate cells were found to undergo Fas-mediated killing; cytotoxicity ranged from 12% to 87% after 6 h. These cells were significantly more sensitive to FasL-mediated killing than PC-3 cells. The basal expression of Fas or the expression of five inhibitors of apoptosis (c-FLIP, survivin, cellular inhibitors of apoptosis protein 1 and 2, and bcl-2) was not found to correlate with susceptibility to Fas-mediated killing. Both mitoxantrone and docetaxel were able to induce Fas receptor expression on primary prostate cancer cells, which translated into a 1.5- to 3-fold enhancement of apoptosis mediated by FasL. Whereas mitoxantrone increased the Fas-induced apoptotic response of all cultured prostate cells tested, docetaxel pretreatment was found to preferentially enhance the killing of bcl-2-expressing cells. These findings show that cultured primary prostate cancer cells are sensitive to Fas-mediated apoptosis. Furthermore, the incidence of apoptosis was found to be improved by combining Fas-mediated therapy with standard chemotherapeutic agents. These findings may have significant implications for prostate cancer therapy.

Entwicklung eines dreidimensionalen Prostatakarzinomzellkulturmodells

Much prostate cancer research is based on cell culture results. Recent genomic studies found major differences between primary prostate cancer tissue and established prostate cancer cell lines, which calls into question the clinical relevance of study results based on cell cultures.Using primary cultures of prostate cancer cells from prostatectomy specimens seems to be a reasonable solution, but primary cell cultures are much more difficult to establish. In this study, a primary cell culture model was combined with an invasion assay. With this combination it was possible not only to select invasive cell clones from the primary culture but also to culture these cells in a three-dimensional model, forming spheroids. A further characterization of this cell population was done by comparative genomic hybridization, showing numerous genetic alterations. The presented cell culture model offers, for the first time, an opportunity to isolate invasive growing cells from primary prostate cancer tissue and cultivate these cells for further analyses.

KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis

Metastatic prostate cancer (PCa) is one of the leading causes of death from cancer in men. The molecular mechanisms underlying the transition from localized tumor to hormone-refractory metastatic PCa remain largely unknown, and their identification is key for predicting prognosis and targeted therapy. Here we demonstrated that increased expression of a splice variant of the Kruppel-like factor 6 (KLF6) tumor suppressor gene, known as KLF6-SV1, in tumors from men after prostatectomy predicted markedly poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that KLF6-SV1 levels were specifically upregulated in hormone-refractory metastatic PCa. In 2 complementary mouse models of metastatic PCa, KLF6-SV1-overexpressing PCa cells were shown by in vivo and ex vivo bioluminescent imaging to metastasize more rapidly and to disseminate to lymph nodes, bone, and brain more often. Interestingly, while KLF6-SV1 overexpression increased metastasis, it did not affect localized tumor growth. KLF6-SV1 inhibition using RNAi induced spontaneous apoptosis in cultured PCa cell lines and suppressed tumor growth in mice. Together, these findings demonstrate that KLF6-SV1 expression levels in PCa tumors at the time of diagnosis can predict the metastatic behavior of the tumor; thus, KLF-SV1 may represent a novel therapeutic target.

Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model

The AKT/mammalian target of rapamycin (AKT/mTOR) and ERK MAPK signaling pathways have been shown to cooperate in prostate cancer progression and the transition to androgen-independent disease. We have now tested the effects of combinatorial inhibition of these pathways on prostate tumorigenicity by performing preclinical studies using a genetically engineered mouse model of prostate cancer. We report here that combination therapy using rapamycin, an inhibitor of mTOR, and PD0325901, an inhibitor of MAPK kinase 1 (MEK; the kinase directly upstream of ERK), inhibited cell growth in cultured prostate cancer cell lines and tumor growth particularly for androgen-independent prostate tumors in the mouse model. We further showed that such inhibition leads to inhibition of proliferation and upregulated expression of the apoptotic regulator Bcl-2-interacting mediator of cell death (Bim). Furthermore, analyses of human prostate cancer tissue microarrays demonstrated that AKT/mTOR and ERK MAPK signaling pathways are often coordinately deregulated during prostate cancer progression in humans. We therefore propose that combination therapy targeting AKT/mTOR and ERK MAPK signaling pathways may be an effective treatment for patients with advanced prostate cancer, in particular those with hormone-refractory disease.

The transcription factor Egr1 regulates the HIF‐1α gene during hypoxia

Using oligonucleotide expression microarrays we have examined the modulation of gene expression in the DU145 prostate cancer cell line. Our findings confirm that the Egr1 transcription factor is rapidly and transiently upregulated by hypoxia. Furthermore, we have demonstrated that HIF-1alpha mRNA is also transiently upregulated, as is its target gene VEGF. To elucidate the mechanism of the transcriptional upregulation of the HIF-1alpha gene, we have shown that Egr1 is able to directly bind to the HIF-1alpha promoter using chromatin immunoprecipitation. We also provide evidence that the binding of Egr1 is necessary for the trans-activation of the HIF-1alpha promoter. These studies highlight the importance for the Egr1 transcription factor in the hypoxic response in cultured prostate cancer cell lines, and indicate that the response of Egr1 is upstream of HIF-1 in these cells. These studies are the first demonstration that the HIF-1alpha transcription factor is targeted directly by Egr1 in hypoxia.

Inhibitory effects of nordihydroguaiaretic acid (NDGA) on the IGF‐1 receptor and androgen dependent growth of LAPC‐4 prostate cancer cells

Nordihydroguaiaretic acid (NDGA) is an inhibitor of the IGF-1 receptor (IGF-1R) in breast and other cancers, and concomitantly inhibits tumor growth both in cultured cells and animals. The current study evaluates the effect of NDGA on the androgen-stimulated growth of human prostate cancer cells. LAPC-4 prostate cancer cells in tissue culture were androgen starved for 3 days, 1 nM dihydrotestosterone (DHT) and other androgens were then added for up to 7 days, and cell proliferation measured. IGF-1R protein expression was measured by Western blot, and IGF-1R mRNA expression by quantitative PCR. IGF-1R receptor kinase activation was measured by ELISA. After 7 days, LAPC-4 growth was doubled by 1 nM DHT. NDGA had a rapid effect to inhibit IGF-1R autophosphorylation induced by IGF-1. DHT increased the expression of IGF-1R protein and mRNA levels. Maximal IGF-1R protein levels were observed 3 days after the addition of androgen. In addition, NDGA, at 10 microM or less, inhibited DHT-induced proliferation in both cells grown in plates and cells grown in soft agar. Androgen receptor (AR) studies by FRET revealed that NDGA had no conformational effects on the AR in response to ligand. NDGA blocks the DHT-induced growth of LAPC-4 prostate cancer cells by several mechanisms including rapid inhibition of the IGF-1R kinase, and a dose-dependent inhibition of androgen stimulation of IGF-1R expression. Clinical studies of this agent will determine its efficacy in the setting of androgen-dependent prostate cancer.

Aerobic Fitness Levels in African American Men, Serum IGF-1, and Growth of Prostate Cancer Cells

Prostate cancer affects blacks disproportionately and its evolution may be affected by a sedentary lifestyle. The growth of prostate tumors may be modulated by androgens and insulin-like growth factors. PURPOSE: To test the hypothesis that the serum of males having low aerobic capacity contains concentrations of substances which promote the growth of prostate cancer cells. METHODS: Subjects were healthy young adult African-American males. A sedentary low fitness group (N=8) and active high fitness group (N=9) were identified by a physical activity questionnaire and verified by measurement of peak oxygen uptake (VO2peak, [<38 vs· > 40 ml · kg−1· min−1]). Serum sample from each subject was incubated separately with cultures of androgen dependent LNCaP prostate cancer cells. The serum concentration of testosterone, insulin-like growth factor (IGF-1) and the relative cell growth were measured. REULTS: The significance of intergroup differences was evaluated by a two-tailed t-test at P<0.05. VO2peak was found to be greater and body mass index tended to be lower in the high fitness than in the low fitness group (P<0.05). Intergroup differences in age, height, resting heart rate, blood pressure, body weight and in peak heart rate were not significant (P>0.05). IGF-1 concentration was higher in the serum from low fitness group compared to that from the high fitness group. The relative growth of androgen dependent LNCaP prostate cancer cells was significantly higher when cultured in medium supplemented with serum from individuals in the low fitness group than cultures grown in medium supplemented with serum from individuals in high fitness group. There was no significant intergroup difference in serum testosterone level. CONCLUSION: It is concluded that a lower growth rate of prostate cancer cells may be associated with lower serum concentration of IGF-1 in physically active males with a higher aerobic capacity than in sedentary males with a lower aerobic capacity. These findings suggest that a regimen of physical activity with increases in aerobic capacity inhibits the growth of prostate cancer cells. It is concluded that a sedentary lifestyle may promote the growth of prostate cancer cells.

The Heterochromatin Protein 1 Family is Regulated in Prostate Development and Cancer

The HP1 family of evolutionarily conserved proteins regulates heterochromatin packaging, in addition to a less defined role in the regulation of euchromatic genes. To examine the possible role of HP1 proteins in fetal prostate development and prostate cancer the protein expression of HP1alpha, beta and gamma was evaluated in human archival tissue. Tissue sections from human prostate cancer and fetal prostate were examined using antibodies against HP1 isoforms to evaluate HP1 modulation in cancer and development. Western blot analysis of HP1 proteins was also performed in extracts of cultured prostate cancer cells. HP1alpha, beta and gamma are differentially regulated in various cellular compartments in prostate development. HP1alpha is not expressed at 14 or 24 weeks of prostate development but it is expressed in adult prostate tissue. HP1beta is highly expressed at 14 and 24 weeks, and it appears predominantly in epithelial cells compared to HP1gamma, which is expressed at equal levels in epithelial and stromal cells. All 3 HP1 isoforms show altered expression in prostate cancer compared to that in normal adult prostate tissue. HP1 proteins are tightly regulated during prostate development. In the adult prostate HP1alpha, beta and gamma antibodies detect high levels of HP1 antigen in a contiguous layer of epithelial cells. However, the detection of HP1 in prostate cancer ranges from undetectable to inconsistent staining of noncontiguous epithelial cells.

Apoptotic Activity and Mechanism of 2-Cyano-3,12-Dioxoolean-1,9-Dien-28-Oic-Acid and Related Synthetic Triterpenoids in Prostate Cancer

Synthetic triterpenoids 2-cyano-3, 12-dioxooleana-1, 9-(11)-dien-28-oic acid (CDDO) and CDDO-Me (CDDO-methyl ester) have entered clinical trials for cancer. We determined that CDDO analogues at submicromolar concentrations induce apoptosis of cultured prostate cancer cell lines, LNCaP, ALVA31, Du145, PC3, and PPC1, with lethal dose 50% approximately 1 micromol/L for CDDO-Me and an imidazole analogue (CDDO-Im). These compounds induced apoptosis of prostate cancer cells as characterized by cleavage of caspase-3, caspase-7, caspase-8, caspase-9, caspase-10, BID, and poly(ADP)ribose polymerase and by dependence on caspase activity. Moreover, triterpenoid-induced cell death was abolished by caspase-8-targeting small interfering (si) RNA. To explore the mechanism(s) involved in caspase-8 activation, we examined cell surface expression of death receptor (DR)4 and DR5 after triterpenoid treatment. Cell surface DR4 and DR5 expression was significantly up-regulated by CDDO or CDDO-Im but not by CDDO-Me. DR4 and DR5 knockdown with siRNA significantly inhibited apoptosis induced by CDDO and CDDO-Im but had no effect on CDDO-Me-induced killing, suggesting that CDDO and CDDO-Im induce apoptosis by a different mechanism than CDDO-Me. In addition to activating the caspase-8-dependent extrinsic apoptosis pathway, we observed that Bcl-X(L) overexpression inhibited triterpenoid-mediated killing of prostate cancer cell line Du145, suggesting that the intrinsic pathway (via mitochondria) also participates in triterpenoid-mediated killing. In vivo antitumor activity of CDDO-Me was shown using a Du145 tumor xenograft model in nude rats. Altogether, these findings suggest CDDO and related synthetic triterpenoids should be further evaluated as potential novel therapeutics for hormone refractory prostate cancers.

Short Communication: Nanoparticle Thermotherapy and External Beam Radiation Therapy for Human Prostate Cancer Cells

Nanoparticle thermotherapy (NPTT) uses monoclonal antibody-linked iron oxide magnetic nanoparticles (bioprobes) for the tumor-specific thermotherapy of cancer by hysteretic heating of the magnetic component of the probes through an externally applied alternating magnetic field (AMF). The present study investigated the effect of NPTT on a human prostate cancer cell line, DU145. The concept of total heat dose (THD) as a measure for NPTT was validated on a cellular level and THD was correlated to cell death in vitro. The study, furthermore, explored the potential enhancement of the NPTT effect through added external beam radiation therapy (EBRT), because both forms of treatment have a different, and potentially complementary, mechanism of causing cell death. Using carbodiimide, (111)In-DOTA-ChL6 was conjugated to dextran iron oxide 20-nm particles with polyethylene glycol COOH groups on the surface and purified as (111)In-bioprobes. NPTT and EBRT were applied alone and combined to cells labeled with the bioprobes. Cell response was monitored by measuring lactate dehydrogenase (LDH), a product of cytolysis, in the medium. This distinct focus on the response to NPTT was possible, since we found in previous studies that the LDH assay was relatively insensitive to the response of cells (without bioprobes) to EBRT in the dose levels given here. NPTT showed a significantly increased cell death at a total calculated heat dose of 14.51 and 29.02 J/g cells (50% and 100% AMF duty, 350 Oe, 136 kHz, 12 cycles, 20 minutes total), compared with AMF exposure in the absence of bioprobes. Adding EBRT to NPTT did not increase cell death, as measured by LDH. However, EBRT given to cells labeled with bioprobes caused significant cell death at radiation doses of 10 Gy and higher. In human prostate cancer cell cultures, NPTT applied as a single modality caused cell death that correlated with THD estimation; complete cell death occurred at 14.51 J/g cells. Consequently, enhancement of the NPTT effect through the addition of EBRT could not be addressed. Interestingly, EBRT induced cell death on bioprobe-labeled cells at EBRT levels that did not show cell death in the absence of bioprobes; this phenomenon is worth investigating further.

Differential effects of resveratrol on androgen-responsive LNCaP human prostate cancer cells in vitro and in vivo

Resveratrol is a phytochemical that has been under consideration for use as a prostate cancer chemopreventive agent. However, the efficacy, as well as the mechanisms of action of resveratrol on prostate cancer prevention, remains largely unknown. This study seeks to address these questions and examine the cancer preventive effects of resveratrol using complementary human LNCaP prostate cancer cell culture and xenograft models. In cultured LNCaP cells, we found that resveratrol inhibited cell growth. The growth inhibitory effects of resveratrol appeared to be through modulation of both androgen- and estrogen-mediated events. Global gene expression analysis using microarrays identified androgen-responsive genes as a group of genes universally affected by resveratrol in LNCaP cells in vitro. The effect of resveratrol on expression of these genes appeared to be through inhibition of both androgen- and estrogen-mediated transcription. In a xenograft model, resveratrol delayed LNCaP tumor growth and inhibited expression of a marker for steroid hormone responses. However, exposure to resveratrol also led to increased angiogenesis and inhibition of apoptosis in the xenograft. In summary, resveratrol may act through modulation of steroid hormone-dependent pathways to inhibit prostate cancer cell growth in both culture and xenografts, but exposure in vivo may be of concern.

Transcription Factor Signal Transducer and Activator of Transcription 5 Promotes Growth of Human Prostate Cancer Cells In vivo

Signal transducer and activator of transcription 5a/b (Stat5a/b) is the key mediator of prolactin effects in prostate cancer cells via activation of Janus-activated kinase 2. Prolactin is a locally produced growth factor in human prostate cancer. Prolactin protein expression and constitutive activation of Stat5a/b are associated with high histologic grade of clinical prostate cancer. Moreover, activation of Stat5a/b in primary prostate cancer predicts early disease recurrence. Here, we inhibited Stat5a/b by several different methodologic approaches. Our goal was to establish a proof of principle that Stat5a/b is critical for prostate cancer cell viability in vitro and for prostate tumor growth in vivo. We inhibited Stat5a/b protein expression by antisense oligonucleotides or RNA interference and transcriptional activity of Stat5a/b by adenoviral expression of a dominant-negative mutant of Stat5a/b in prostate cancer cells in culture. Moreover, Stat5a/b activity was suppressed in human prostate cancer xenograft tumors in nude mice. Stat5a/b regulation of Bcl-X(L) and cyclin D1 protein levels was shown by antisense suppression of Stat5a/b protein expression followed by Western blotting. We show here that inhibition of Stat5a/b by antisense oligonucleotides, RNA interference, or adenoviral expression of dominant-negative Stat5a/b effectively kills prostate cancer cells. Moreover, we show that Stat5a/b is critical for human prostate cancer xenograft growth in nude mice. The effects of Stat5a/b on the viability of prostate cancer cells involve Stat5a/b regulation of Bcl-X(L) and cyclin D1 protein levels but not the expression or activation of Stat3. This work establishes Stat5a/b as a therapeutic target protein for prostate cancer. Pharmacologic inhibition of Stat5a/b in prostate cancer can be achieved by small-molecule inhibitors of transactivation, dimerization, or DNA binding of Stat5a/b.

Effect of bioflavonoids on the proliferation of DU145 human prostate cancer cells

Flavonoids are ubiquitously occurring and widely consumed secondary metabolites of plants and are therefore an integral part of the human diet. Apigenin, luteolin, Myricetin and Rutin are flavonoids that have various pharmacological activities including inhibitory effects on the growth and proliferation of tumor cells. We have investigated the effects of these flavonoids alone and in combination on the proliferation of androgen‐independent human prostate (DU145) cancer cells in culture. Our results indicate that apigenin, luteolin, myricetin and rutin inhibited cell proliferation and decreased the viability of DU145 cells after 96 hours of treatment. To determine whether the decreased viability and cell proliferation was due to cell death by apoptosis or necrosis, we performed western blot analysis using antibodies raised against the pro‐apoptotic protein Bad and the anti‐apoptotic proteins Bcl2 and BclxL, We observed a dose‐dependent and time dependent increase in the pro‐apoptotic Bad gene and a dose dependent and time dependent decrease in BcL2 and BclxL when cells were treated with flavonoids for 72 hours. DNA fragmentation studies and expression of matrix metalloproteinase (MMP) – 2 using identical doses and time points confirmed the pro‐apoptotic effect of flavonoids on the proliferation of DU145 cells.