mtt-formazan and Prostatic-Neoplasms

Trichosanthin produced in the root tube of Trichosanthes kirilowii shows anti-tumor activity on a series of cancer cells including Hela, MCF-7, HL-60. But there is little information about its effect on the carcinogenesis of prostate cancer.. This work was designed to study the role of trichosanthin on prostate cancer cells PC3.. Trichosanthin was expressed in BL21 strain and purified by affinity chromatography. MTT assay was designed to determine the effect of trichosanthin on growth of PC3 cells at doses of 10, 20, 40, 60, 80, and 120 μg/ml. Then the effect of 50 μg/ml rTCS alone or combined with 2 μM IL-2 on PC3 cell proliferation was analyzed. And the mechanism of rTCS was studied by western blot. After that the in vivo effect of rTCS combined with IL-2 was explored in mice bearing PC3 xenograft tumor.. Trichosanthin was successfully expressed in BL21 and purified by 100 mM imidazole. It was shown to inhibit proliferation of PC3 cells in a dose-dependent manner with IC50 50.6 μg/ml. When combined with cytokine IL-2, a significant synergic effect was obtained. The inhibition rate on PC3 was around 50 % in combination group while only 35.5 % in single rTCS group at 50 μg/ml. Further, the expression of full length caspase-8 and Bcl-2 decreased significantly while cleaved caspase-8 and Bax were up-regulated, which suggest that caspase-8-mediated apoptosis pathway may be activated by rTCS in PC3 cells. Moreover, our data demonstrated that tumor volume and tumor weight were significantly reduced in rTCS-treated or rTCS/IL-2-treated nude mice bearing PC3 xenograft tumor compared with control. And significant difference was also found between rTCS and rTCS/IL-2 group.. This study demonstrates that rTCS is a potential agent with high in vitro and in vivo anti-tumor activity on PC3 cells. And rTCS combined with IL-2 is a promising strategy in treating patients with prostate cancer in future.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Female; Formazans; Male; Mice; Prostatic Neoplasms; Recombinant Proteins; Reproducibility of Results; Tetrazolium Salts; Time Factors; Trichosanthin; Tumor Burden

Alternol is an original compound purified from the fermentation products of Alternaria alternata var. monosporus, a microorganism from the bark of the yew tree. It has been reported that Alternol can inhibit proliferation of mouse leukemia cells and human gastric carcinoma cells, the aim of this study was to investigate the effects of Alternol on prostate cancer cells in comparison to prostate cells.. The MTT assay was utilized to assess cell viability. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Protein expression levels were examined by Western blotting.. Alternol treatment resulted in a significant decrease in the viability of prostate cancer cells but had lesser effects on prostate cells. Alternol inhibited AMP-activated protein kinase (AMPK) phosphorylation in prostate cancer C4-2 cells but stimulated AMPK phosphorylation in prostate RWPE-1 cells. Inhibition of p27 phosphorylation was observed in C4-2 cells whereas a promotion of p27 phosphorylation was seen in RWPE-1 cells. Alternol treatment resulted in a profound increase in the LC3II/LC3I protein ratio in RWPE-1 cells but not in C4-2 cells. A dose-dependent down-regulation of Bcl-2 protein was detected in C4-2 cells but not in RWPE-1 cells. Pretreatment of cells with Compound C (AMPK inhibitor) before Alternol treatment abolished the selective antitumor effect of Alternol.. These results reveal for the first time that Alternol exerts a selective antitumor effect on prostate cancer cells when compared with RWPE-1 prostate epithelial cells. In addition, the AMPK signaling pathway is responsible for the selective antitumor effects of Alternol.

Topics: AMP-Activated Protein Kinases; Autophagy; Blotting, Western; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p27; Drug Interactions; Flow Cytometry; Formazans; Heterocyclic Compounds, 4 or More Rings; Humans; Male; Phosphorylation; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; Pyrimidines; Signal Transduction; Tetrazolium Salts

The present paper is focused on zinc(ii) treatment effects on prostatic cell lines PC-3 (tumour) and PNT1A (non-tumour). Oxidative status of cells was monitored by evaluation of expression of metallothionein (MT) isoforms 1A and 2A at the mRNA and protein level, glutathione (oxidised and reduced), and intracellular zinc(ii) after exposition to zinc(ii) treatment at concentrations of 0-150 μM using electrochemical methods, western blotting and fluorescent microscopy. A novel real-time impedance-based growth monitoring system was compared with widely used end-point MTT assay. Impedance-based IC(50) for zinc(ii) is 55.5 and 150.8 μM for PC-3 and PNT1A, respectively. MTT-determined IC(50) are >1.3-fold higher. Impedance-based viability correlates with viable count (r > 0.92; p < 0.03), not with MTT. Two-fold lower intracellular zinc(ii) in the tumour PC-3 cell line was found. After zinc(ii) treatment >2.6-fold increase of intracellular zinc(ii) was observed in non-tumour PNT1A and in tumour PC-3 cells. In PC-3 cells, free and bound zinc(ii) levels were enhanced more markedly as compared to PNT1A. PNT1A produced 4.2-fold less MT compared to PC3. PNT1A cells showed a 4.8-fold increase trend (r = 0.94; p = 0.005); PC-3 did show a significant trend at MT1 and MT2 protein levels (r = 0.93; p = 0.02) with nearly ten-fold increase after 100 μM zinc(ii) treatment. In terms of redox state, PNT1A had a predominance of reduced GSH forms (GSH : GSSG ratio > 1), when exposed to zinc(ii) compared to PC3, where predominance of oxidised forms remains at all concentrations. IC(50) differs significantly when determined by MTT and real-time impedance-based assays due to dependence of impedance on cell morphology and adhesion. When real-time growth monitoring, precise electrochemical methods and fluorescent microscopy are performed together, accurate information for metal fluxes, their buffering by thiol compounds and monitoring of the redox state become a powerful tool for understanding the role of oxidative stress in carcinogenesis.

Topics: Blotting, Western; Cell Growth Processes; Cell Line, Tumor; Cell Survival; Dielectric Spectroscopy; Fluorescent Dyes; Formazans; Glutathione; Humans; Linear Models; Male; Metallothionein; Microscopy, Fluorescence; Oxidation-Reduction; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA; Tetrazolium Salts; Zinc Sulfate

The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) and the androgen receptor (AR) play important roles in tumor development and progression in prostate carcinogenesis. Among many functions, PTEN negatively regulates the cytoplasmic phosphatidylinositol-3-kinase/AKT anti-apoptotic pathway; and nuclear PTEN affects the cell cycle by also negatively regulating the MAPK pathway via cyclin D. Decreased PTEN expression is correlated with prostate cancer progression. Over-expression of AR and upregulation of AR transcriptional activity are often observed in the later stages of prostate cancer. Recent studies indicate that PTEN regulates AR activity and stability. However, the mechanism of how AR regulates PTEN has never been studied. Furthermore, resveratrol, a phytoalexin enriched in red grapes, strawberries and peanuts, has been shown to inhibit AR transcriptional activity in prostate cancer cells. In this study, we use prostate cancer cell lines to test the hypothesis that resveratrol inhibits cellular proliferation in both AR-dependent and -independent mechanisms. We show that resveratrol inhibits AR transcriptional activity in both androgen-dependent and -independent prostate cancer cells. Additionally, resveratrol stimulates PTEN expression through AR inhibition. In contrast, resveratrol directly binds epidermal growth factor receptor (EGFR) rapidly inhibiting EGFR phosphorylation, resulting in decreased AKT phosphorylation, in an AR-independent manner. Thus, resveratrol may act as potential adjunctive treatment for late-stage hormone refractory prostate cancer. More importantly, for the first time, our study demonstrates the mechanism by which AR regulates PTEN expression at the transcription level, indicating the direct link between a nuclear receptor and the PI3K/AKT pathway.

Topics: Androgen Antagonists; Androgens; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; ErbB Receptors; Formazans; Gene Expression Regulation, Neoplastic; Humans; Male; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptors, Androgen; Resveratrol; Signal Transduction; Stilbenes; Tetrazolium Salts; Time Factors

Taxanes are first line drugs for treating prostate cancer recurrence after the failure of anti-androgen therapy. There is a need to make taxanes more effective since they only provide palliative benefit. Exploiting endoplasmic reticulum (ER) stress death signaling to enhance drug efficacy has not been delineated. Human PC-3 cells were used as a model of hormone refractory prostate cancer. Thapsigargin and methylseleninic acid (MSA) were examined as sensitizers. Thapsigargin is a classic ER stress inducer. The activity of MSA in inducing ER stress has recently been studied by our group. The efficacy of single drug and the various combinations was evaluated by measuring apoptosis with a cell death ELISA kit. Thapsigargin increased the cell killing potency of paclitaxel or docetaxel by 10- to 12-fold, while MSA caused a 5- to 8-fold increase. Since thapsigargin is not used clinically because of its toxicity, the follow-up experiments were done with MSA. To test the hypothesis that a threshold level of ER stress is crucial to chemotherapeutic sensitization, three different approaches designed to dampen the severity of ER stress induced by MSA were examined. Lowering ER stress consistently attenuated the efficacy of MSA/taxane. GADD153 is a pro-apoptotic transcription factor which is upregulated during ER stress. Knocking down GADD153 by siRNA also reduced the cell killing effect of MSA/taxane. Both the intrinsic and extrinsic apoptotic pathways were involved in the sensitization mechanism. Our study supports the idea that marshalling ER stress apoptotic response is conducive to chemotherapeutic sensitization.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Death; Cell Line, Tumor; Docetaxel; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme-Linked Immunosorbent Assay; Formazans; Heat-Shock Proteins; Humans; Male; Molecular Chaperones; Organoselenium Compounds; Paclitaxel; Prostatic Neoplasms; RNA, Small Interfering; Taxoids; Tetrazolium Salts; Thapsigargin; Time Factors; Transcription Factor CHOP; Transfection

Survivin, a member of inhibitor of apoptosis family protein, has become an attractive therapeutic target in cancer due to its selective expression in tumor cells and its important roles for tumor cell viability. Here, we show that vector-based small interfering RNAs (siRNAs) silenced survivin expression in prostate cancer cells, resulting in significantly reduced cell proliferation and enhanced apoptosis, and increased the sensitivity of prostate cancer cells (PC-3) to the apoptosis- inducing agent, platinol. Furthermore, PC-3 cells transfected with the siRNA-expressing vector showed lower tumor formation in nude mice xenografts in vivo. These results demonstrated that inhibition of survivin expression by siRNA attenuated the malignant phenotypes of prostate cancer cells, and may provide a novel approach for gene therapy of androgen-independent prostate cancer.

Topics: Androgens; Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Feasibility Studies; Formazans; Gene Silencing; Genetic Therapy; Humans; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Nude; Microtubule-Associated Proteins; Prostatic Neoplasms; RNA Interference; RNA, Messenger; RNA, Small Interfering; Survivin; Tetrazolium Salts; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

The standard hormonal therapy with currently available antiandrogens and the leutinizing hormone releasing hormone (LHRH) analogs is not effective in the hormone-refractory stage of prostate cancer due to changes in androgen receptor (AR) signaling axis. In this refractory stage, AR continues to play a significant role in the growth of cancer cells even though the cancer cells are no longer dependent on the level of circulating androgens.. A series of 11beta-Delta(9)-19 nortestosterone compounds were designed through structure-based rationale and tested for their binding affinity against AR and glucocorticoid receptor (GR) using fluorescence polarization assays, their agonistic ability to induce AR dependent transcription using PSA-driven report gene assays, and their growth inhibitory affects against a series of AR positive (LAPC4, LNCap, and CWR22R) and negative human prostate cancer cell lines (PC3) using MTT cell proliferation assays.. This study proposes the design of novel bifunctional antiandrogens based on the conjugation of 11beta and/or 7alpha-Delta(9)-19 nortestosterone class of steroidal compounds to the synthetic ligand for FK506-binding proteins. As a critical step towards the development of bifunctional antiandrogens, highly potent and AR-specific lead compounds were identified using in vitro data. The lead compounds identified in this study possessed low binding affinity for GR, indicating the absence of undesirable antiglucocorticoid activity.. The results of this study validate our drug discovery rationale based on the structural biology of AR and pave the pay for future development of bifunctional compounds in order to block AR function in hormone refractory stage of prostate cancer.

Topics: Androgen Antagonists; Androgen Receptor Antagonists; Cell Growth Processes; Cell Line, Tumor; Drug Design; Fluorescence Polarization; Formazans; Humans; Male; Nandrolone; Neoplasms, Hormone-Dependent; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Tetrazolium Salts; Transcription, Genetic

Curcumin (diferuloylmethane) is the major active component of turmeric and is being actively investigated for its anti-cancer properties. To better understand the biological mechanisms of the chemopreventive potential of curcumin in prostate cancer, we have evaluated curcumin regulated transcriptome in prostate cancer cells. Hierarchical clustering methods and functional classification of the Curcumin-Gene Expression Response (Cu-GER) showed temporal co-regulation of genes involved in oxidative stress response and growth signaling pathways. Interestingly, C4-2B, androgen independent metastatic prostate cancer cells exhibited attenuated Cu-GER response in comparison to parental androgen dependent and less aggressive LNCaP cells. Androgen Receptor (AR) regulated genes which play critical roles in normal growth and differentiation of the prostate gland, as well as in prostate cancer, were also a part of the Cu-GER. Of note, curcumin downregulated transcript encoded by the potentially causal TMPRSS2-ERG gene fusion, a common oncogenic alteration noted in 50-70% of prostate cancer patients. Further more, expression of EGFR and ERBB2 receptor were found to be downregulated in curcumin treated LNCaP and C4-2B cells. This report for the first time establishes novel features of Cu-GER in prostate cancer cells of varying tumorigenic phenotypes and provides potentially novel read-outs for assessing effectiveness of curcumin in prostate cancer and likely in other cancers. Importantly, new gene-networks identified here further delineate molecular mechanism(s) of action of curcumin in prostate cancer cells.

Topics: Androgens; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Curcumin; Dose-Response Relationship, Drug; Formazans; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Heme Oxygenase (Decyclizing); Humans; Male; Models, Biological; Neoplasms, Hormone-Dependent; Oxidative Stress; Prostatic Neoplasms; Tetrazolium Salts; Time Factors

Prostate cancer has its highest incidence in the USA and is becoming a major concern in Asian countries. Bufadienolides are extracts of toxic glands from toads and are used as anticancer agents, mainly on leukemia cells. In the present study, the antiproliferative and apoptotic mechanisms of bufalin and cinobufagin on prostate cancer cells were investigated. Proliferation of LNCaP, DU145, and PC3 cells was measured by 3-(4,5-dimethylthiazol-2-yle)-2,5-diphenyltetrazolium bromide assay and the doubling time (tD) was calculated. Bufalin and cinobufagin caused changes in the tD of three prostate cancer cell lines, which were more significant than that of human mesangial cells. In addition, bufadienolides induced prostate cancer cell apoptosis more significantly than that in breast epithelial cell lines. After treatment, the caspase-3 activity and protein expression of caspase-3, -8, and -9 were elevated. The expression of other apoptotic modulators, including mitochondrial Bax and cytosolic cytochrome c, were also increased. However, expression of p53 was only enhanced in LNCaP cells. Downregulation of p53 by antisense TP53 restored the cell viability suppressed by bufalienolides. Furthermore, the increased expression of Fas was more significant in DU145 and PC3 cells with mutant p53 than in LNCaP cells. Transfection of Fas small interfering RNA restored cell viability in the bufadienolide-treated cells. These results suggest that bufalin and cinobufagin suppress cell proliferation and cause apoptosis in prostate cancer cells via a sequence of apoptotic modulators, including Bax, cytochrome c, and caspases. The upstream mediators might be p53 and Fas in androgen-dependent LNCaP cells and Fas in androgen-independent DU145 and PC3 cells.

Topics: Androgens; Apoptosis; bcl-2-Associated X Protein; Bufanolides; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; fas Receptor; Fluorescent Antibody Technique, Indirect; Formazans; Gene Expression Regulation, Neoplastic; Humans; Male; Prostatic Neoplasms; RNA, Messenger; Signal Transduction; Tetrazolium Salts; Tumor Suppressor Protein p53

Development of chemoresistance in androgen-refractory prostate cancer cells is partly due to constitutive activation of Rel/NF-kappaB transcription factors that regulate several cell survival and anti-apoptotic genes. In this study we examined whether betulinic acid (BetA), a pentacyclic triterpene from the bark of white birch, is effective in inhibiting NF-kappaB expression in androgen-refractory human prostate cancer cells exhibiting high constitutive NF-kappaB expression. Treatment of PC-3 cells with BetA inhibited DNA binding and reduced nuclear levels of the NF-kappaB/p65. BetA-mediated NF-kappaB inhibition involved decreased IKK activity and phosphorylation of IkappaBalpha at serine 32/36 followed by its degradation. Reporter assays revealed that NF-kappaB inhibition by BetA is transcriptionally active. These effects were found to correlate with a shift in Bax/Bcl-2 ratio and cleavage of poly(ADP)ribose polymerase more towards apoptosis. BetA also inhibited TNFalpha-induced activation of NF-kappaB via the IkappaBalpha pathway, thereby sensitizing the cells to TNFalpha-induced apoptosis. Our studies demonstrate that BetA effectively inhibits constitutive NF-kappaB activation and supports the rationale for targeting NF-kappaB through combination protocols with BetA in androgen-refractory prostate cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Betulinic Acid; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Formazans; Genes, Reporter; Humans; Luciferases; Male; NF-kappa B; Pentacyclic Triterpenes; Prostatic Neoplasms; Tetrazolium Salts; Time Factors; Transfection; Triterpenes; Tumor Necrosis Factor-alpha

Considering the role of aberrant beta-catenin signaling in tumorigenesis, we investigated the mechanism by which the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist troglitazone facilitated beta-catenin down-regulation. We demonstrate that troglitazone and its more potent PPARgamma-inactive analogs Delta2TG and STG28 mediated the proteasomal degradation of beta-catenin in prostate cancer cells by up-regulating the expression of beta-transducin repeat-containing protein (beta-TrCP), an F-box component of the Skp1-Cul1-F-box protein E3 ubiquitin ligase. Evidence indicates that although small interfering RNA-mediated beta-TrCP knockdown protected cells against STG28-facilitated beta-catenin ablation, ectopic beta-TrCP expression enhanced the degradation. The involvement of beta-TrCP in beta-catenin degradation was also corroborated by the pull-down analysis and the concurrent down-regulation of known beta-TrCP substrates examined, including Wee1, Ikappabetaalpha, cdc25A, and nuclear factor-kappaB/p105. Furthermore, glycogen synthase kinase-3beta represented a key regulator in the effect of these thiazolidinedione derivatives on beta-catenin proteolysis even though these agents increased its phosphorylation level. It is noteworthy that this drug-induced beta-TrCP up-regulation was accompanied by the concomitant down-regulation of Skp2 and Fbw7, thereby affecting many of the target proteins of these two F-box proteins (such as p27 and cyclin E). As a consequence, the ability of troglitazone to target these F-box proteins provides a molecular basis to account for its reported effect on modulating the expression of aforementioned cell-cycle regulatory proteins. Despite this complicated mode of pharmacological actions, normal prostate epithelial cells, relative to LNCaP cells, were less susceptible to the effects of STG28 on modulating the expression of beta-catenin and beta-TrCP, suggesting the translation potential of using STG28 as a scaffold to develop more potent chemopreventive agents.

Topics: beta Catenin; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cullin Proteins; F-Box Proteins; Formazans; Glutathione Transferase; Humans; Male; PPAR gamma; Prostatic Neoplasms; Recombinant Fusion Proteins; RNA Interference; S-Phase Kinase-Associated Proteins; Tetrazolium Salts; Thiazolidinediones; Ubiquitin-Protein Ligases

We compared the effects of chelerythrine (CHE) and sanguinarine (SA) on human prostate cancer cell lines (LNCaP and DU-145) and primary culture of human gingival fibroblasts. CHE and SA treatment of cell lines for 24 h resulted in (1) inhibition of cell viability in a dose-dependent manner in all tested cells (as evaluated by MTT test and bromodeoxyuridine incorporation assay); (2) dose-dependent increase in DNA damage in all tested cells (as evaluated by DNA comet assay); (3) changes in apoptosis (assessed by western blot analysis and TUNEL assay); and (4) significant induction of cyclin kinase inhibitors p21(Waf1/Cip1) and p27(Kip1) in prostate cancer cells (identified by western blot analysis). Our study demonstrates that CHE had significant cytotoxic effect, independent of p53 and androgen status, on human prostate cancer cell lines. Normal gingival fibroblasts and DU-145 cells were more sensitive to the treatment with both alkaloids than were LNCaP cells. CHE and SA may be prospective natural molecules for use in the treatment of prostate cancer owing to their involvement in apoptosis and cell cycle regulation.

Topics: Alkaloids; Antineoplastic Agents; Apoptosis; Benzophenanthridines; Blotting, Western; Bromodeoxyuridine; Cell Cycle; Cell Line, Tumor; Cell Survival; Comet Assay; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; DNA Damage; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Fibroblasts; Formazans; Gingiva; Humans; In Situ Nick-End Labeling; Isoquinolines; Male; Prostatic Neoplasms; Tetrazolium Salts

Advanced glycation end products (AGE) are produced with normal aging. Recently, some reports indicated that the interaction between AGE and the cognate receptor (RAGE) has a role in cancer dependent.. We investigated RAGE and amphoterin mRNA expression in prostate cancer cell lines (DU145, PC-3, and LNCaP cells), hormone-refractory prostate cancer tissues, and paired untreated primary prostate cancer and normal prostate (including benign prostatic hypertrophy (BPH)) tissues using real-time quantitative PCR. Moreover, to confirm the AGE-RAGE interaction in prostate cancer, DU145 cells stimulated with AGE-bovine serum albumin (AGE-BSA) were examined by in vitro matrigel assay, cell viability assay, MTT assay, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot.. DU145 cells, a hormone-independent prostate cancer cell line, showed the highest RAGE mRNA expression. Amphoterin mRNA was expressed in all three cell lines. In prostate tissues, untreated prostate cancer tissue and hormone-refractory prostate cancer tissue showed higher RAGE and amphoterin mRNA expression than normal prostate tissue. The AGE-RAGE interaction induced the invasion and growth in DU145 cells stimulated with AGE-BSA.. The AGE-RAGE interaction is important in prostate cancer development, and inhibition of this interaction has potential as a new molecular target for cancer therapy or prevention.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Formazans; Gene Expression Regulation, Neoplastic; Glycation End Products, Advanced; HMGB1 Protein; Humans; Ligands; Male; Neoplasm Invasiveness; Prostatic Hyperplasia; Prostatic Neoplasms; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts

GnRH analogs have antiproliferative and/or apoptotic effects on prostate cancer cells. Also, neurotrophin receptors TrkA and p75 have been reported in normal prostate suggesting a role in the gland growth control. In prostate cancer, TrkA receptors seem to be overexpressed and p75 receptors show a decreased expression. These changes in neurotrophin receptors may be related with unbalanced growth in malignant cells. In the present study we investigate the effects of GnRH analogs (leuprolide and cetrorelix) on the expression of TrkA and p75 neurotrophin receptors in primary cultures of human prostate cancer cells.. Tissue was obtained from radical prostatectomies due to prostate adenocarcinoma. Cells were isolated after sequential enzyme digestion and cultured in defined media. Nerve growth factor (NGF) receptors in untreated cultures were estimated by immunofluorescence. Cultures were treated with leuprolide (agonist) or cetrorelix (antagonist) and expression of TrkA and p75 receptors were evaluated by semi quantitative RT-PCR (polymerase chain reaction) and western blot. Cell proliferation was estimated by MTT method and apoptosis through COMET assay.. Both leuprolide and cetrorelix induced a significant increase in p75 receptor gene and protein expression at a concentration that induce apoptosis and decrease proliferation. TrkA receptors showed no changes in presence of GnRH analogs.. GnRH analogs, leuprolide, and cetrorelix, change the ratio between neurotrophin receptors TrkA and p75 by increasing gene and protein expression of p75 receptor. Considering that TrkA receptor is related with proliferation and p75 with apoptosis, we suggest that our findings may explain, in part, the effect of GnRH analogs on prostate cancer growth.

Topics: Adenocarcinoma; Antineoplastic Agents, Hormonal; Apoptosis; Blotting, Western; Cell Proliferation; Comet Assay; Epithelial Cells; Formazans; Gonadotropin-Releasing Hormone; Humans; Leuprolide; Male; Prostatic Neoplasms; Receptor, Nerve Growth Factor; Receptor, trkA; Receptors, Nerve Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts

Once prostate cancer has metastasized, current treatment methods are generally ineffective. Due to the reported anti-tumor properties of specific nutrients, we investigated the effect of a unique formulation (NS) of lysine, proline, arginine, ascorbic acid, and epigallocatechin gallate on human prostate cancer cell lines: PC-3, DU145 (androgen insensitive) and LNCaP (androgen sensitive), by measuring cell proliferation, MMP expression, and invasion potential. Cell lines DU145, PC-3, and LNCaP were treated at near confluence with NS at various concentrations. Cell proliferation was measured by MTT assay after 24 hours, MMP expression was measured by gelatinase zymography in condition media, and invasion activity was measured by Matrigel. The nutrient mixture did not significantly inhibit PC-3 cell proliferation at 50 microg/ml, but showed significant antiproliferative effect at 500 ug/ml. When treated with NS, proliferation of LNCaP cells was inhibited by 80% of control at 100 microg/ml. NS showed dose-dependent inhibition of DU145 cell proliferation with 47% reduction at 1000 microg/ml. NS showed a dose-dependent inhibition of both MMP-2 and MMP-9 expression by PC-3 cells and MMP-9 expression by PMA-treated (200 ng/ml) DU145 cells. Neither MMP-2 nor MMP-9 gelatinolytic activity was detected in LNCaP cell culture. Invasion of DU145 and LNCaP cells through Matrigel was completely inhibited at 500 microg/ml and PC-3 at 1000 microg/ml. Inhibition of MMP expression and invasion suggests the mixture of nutrients studied is a potent, natural anticancer agent for the treatment of prostate cancer.

Topics: Antioxidants; Arginine; Ascorbic Acid; Catechin; Cell Line, Tumor; Cell Proliferation; Chemistry, Pharmaceutical; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Drug Synergism; Formazans; Humans; Laminin; Lysine; Male; Matrix Metalloproteinases; Neoplasm Invasiveness; Proline; Prostatic Neoplasms; Proteoglycans; Tetrazolium Salts

7-Hydroxystaurosporine (UCN-01), a non-selective inhibitor of protein kinase C (PKC), and phorbol ester (PMA), a PKC activator, are undergoing clinical evaluations. We investigated the effects of UCN-01 and PMA on a panel of prostate cancer cell lines. While PMA induced p21WAF1/CIP1 and arrest growth of LNCaP cancer cells (IC50 = 0.5-1 nM), aggressive cancer cell lines (DU145, PC3, and PC3M) were resistant to PMA (IC50 >5000 nM). Low concentrations (25-50 nM) of UCN-01 abrogated PMA-induced p21 and growth arrest in LNCaP cells. These low doses of UCN-01 however did not inhibit proliferation of any prostate cancer cell line. PMA-sensitive LNCaP cells were resistant to clinically relevant concentrations of UCN-01 (IC50 = 1.2 microM), but UCN-01 inhibited growth of DU145 and PC3/3M with an IC50 of 200-400 nM. For comparison, PMA-sensitive HL60 leukemia cells were sensitive to UCN-01 due to rapid apoptosis caused by UCN-01. In PMA-resistant prostate cancer cells, UCN-01 downregulated cyclin D1, induced p21, caused morphological differentiation, and G1-phase arrest leading to slow cell death without caspase activation. Importantly, normal prostate epithelial cells (PrEC) were very sensitive to both PMA (IC50 = 0.2 nM) and UCN-01. In PrEC, UCN-01 downregulated cyclin D1 and arrest growth with an IC50 less than 100 nM. We conclude that loss of sensitivity to either UCN-01 or PMA accompanies progression of prostate cancer.

Topics: Alkaloids; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA, Neoplasm; Down-Regulation; Drug Resistance, Neoplasm; Formazans; Humans; Immunoblotting; Male; Prostatic Neoplasms; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate; Tetrazolium Salts; Transfection; Tumor Cells, Cultured

Enzymatically inactive procathepsin D secreted from cancer cells has been confirmed to play a role in breast cancer development. We focused on prostate cancer and the role of activation peptide in mitogenic activity.. Synthetic peptides and monoclonal antibodies raised against individual fragments of activation peptide were employed. Cell proliferation was measured by MTT (3-[4,5-dimethylthiatol-2-yl]-2,5-diphenyl tetrazolium bromide) assay or by in vivo growth in nude mice.. We demonstrated that the growth factor activity of activation peptide is localized in amino-acid region 27-44. In addition, both anti-activation peptide and anti-27-44 peptide antibodies administered in vivo inhibited the growth of human prostate tumors in mice.. Based on these data, we hypothesize that the interaction of procathepsin D activation peptide with an unknown receptor is mediated by amino-acid sequence 27-44. This interaction leads in certain types of tumor to a proliferation and higher motility. Blocking of this interaction by antibodies or antagonists might be a valuable tool in prostate cancer inhibition.

Topics: Animals; Antibodies, Monoclonal; Cathepsin D; Cell Division; Chromatography, Affinity; Enzyme Precursors; Enzyme-Linked Immunosorbent Assay; Formazans; Gelatin; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Microspheres; Peptide Fragments; Prostate; Prostatic Neoplasms; Tetrazolium Salts; Tumor Cells, Cultured

The strongly metastatic MAT-LyLu and the weakly metastatic AT-2 rat prostatic cancer cell lines have been shown to express voltage-gated ion channels differentially. In the present study, the possible contribution of voltage-gated ion channel activity to the proliferation of these cell lines was investigated, in a comparative approach.. Several voltage-gated ion channel modulators were tested for their effects on proliferation over 54 hr, using an in vitro assay. The modes of action of the chemicals were monitored by electrophysiological (patch-clamp) recording.. The voltage-gated K(+) channel blockers 4-aminopyridine (4-AP; 2 mM), margatoxin (5 nM), charybdotoxin (4.5 nM), and verapamil (50 microM) inhibited the K(+) channels of both cell lines by between 38-65% and reduced the proliferation of the AT-2 cell line, in a dose-dependent manner, by 8-51%. However, only 4-AP reduced proliferation of the MAT-LyLu cell line. Tetrodotoxin (6 microM) blocked completely the voltage-gated Na(+) channel expressed selectively in the MAT-LyLu cell line, but had no effect on the proliferation of either cell line. On the other hand, the presumed Na(+) channel "opener" veratridine (10-50 microM) reduced significantly, in a dose-dependent manner, the proliferation of both cell lines by up to approximately 30%.. We conclude that the mechanism(s) controlling the proliferation of the weakly metastatic AT-2 cells involves voltage-gated K(+) channels. In contrast, the proliferation of strongly metastatic MAT-LyLu cells is much less dependent upon voltage-gated K(+) channel activity.

Topics: 4-Aminopyridine; Animals; Calcium Channel Blockers; Cell Division; Charybdotoxin; Dose-Response Relationship, Drug; Formazans; Ion Channel Gating; Male; Neurotoxins; Patch-Clamp Techniques; Potassium Channels; Prostatic Neoplasms; Rats; Scorpion Venoms; Sodium Channels; Tetrazolium Salts; Tetrodotoxin; Tumor Cells, Cultured; Verapamil; Veratridine

The ADAMs are a multi-functional gene family, some of which have been shown to play a role in diverse biological processes such as fertilization, myogenesis, neurogenesis and the activation of growth factors/immune regulators such as TNF-alpha. So-named because they possess both A Disintegrin And Metalloprotease domain, the ADAMs have potential implications for the metastasis of human tumour cells via cell adhesion and protease activities. However, no studies have yet comprehensively examined the expression or regulation of ADAMs in solid tumours. Therefore, the aim of this study was to examine the expression of the ADAMs in human prostate cancer cell lines and to examine their possible regulation by androgen, a primary hormonal regulator of prostate cancer cell proliferation and metastasis. Applying RT-PCR, ADAM-9, -10, -11, -15 and -17 mRNA expression was found in the androgen-dependent prostate cancer cell lines, LNCaP and ALVA-41 and the androgen-independent cell lines, DU-145 and PC-3. Northern blotting of LNCaP cell total RNA revealed transcripts for ADAM-9 (3.8 kb), ADAM-10 (4.4, 3.2 and 0.54 kb), ADAM-15 (3 kb) and ADAM-17 (4 and 2.6 kb). ADAM-11 transcript was not detected by Northern blotting possibly due to low levels of ADAM-11 mRNA expression. This is the first report of ADAM expression in prostate cancer cell lines. Since androgens are implicated in prostate cancer cell growth and maintenance, the regulation of ADAMs by dihydrotestosterone (DHT) was investigated in the androgen-dependent cell line LNCaP. It was shown by quantitative RT-PCR using continuous fluorescence monitoring that ADAM-10 mRNA expression was regulated in a bell shaped, dose-dependent manner by DHT. Maximum stimulation was observed at 1.0 nM DHT (5-fold significant increase). For ADAM-9 mRNA, a significant upregulation was found at 1.0 and 10 nM (1.5-1.7-fold increase). In contrast, ADAM-17 mRNA, was significantly inhibited at 0.1 and 1.0 nM (1.7-fold decrease). This is the first report, to our knowledge, illustrating hormonal regulation of ADAM mRNA. The novel data described here also provide a strong stimulus to the development of specific quantitative and functional assays for particular ADAMs. These assays, which are not yet available, are required to enable subsequent investigation, both in vitro and in vivo, of the specific roles of each ADAM in prostate cancer cell proliferation, cell motility and invasion.

Topics: Blotting, Northern; Cell Division; Dihydrotestosterone; Dose-Response Relationship, Drug; Formazans; Humans; Male; Membrane Glycoproteins; Metalloendopeptidases; Prostate-Specific Antigen; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Tumor Cells, Cultured