cytochrome-c-t and Prostatic-Neoplasms

Abiraterone acetate (AA) is a selective inhibitor of CYP17 α-hydroxylase, which is crucial for androgen biosynthesis. Apigenin (Api) is a natural plant-derived flavonoid with potent antiproliferative and antimigration effects.. We aimed to investigate the possible role of Api in combination with the androgen receptor inhibitor AA in the treatment of androgen-sensitive human prostate cancer LNCaP cells.. The cells were either exposed to 10 μM AA, 25 μM Api, or in combination for 48 hours, then the viability rate was determined by the MTT test, whilst apoptosis and cell cycle phases were assessed by image-based cytometry. The expression of selected mRNA and proteins were evaluated by RT-qPCR and Western blot, respectively.. The combination of AA and Api significantly inhibited LNCaP as well as androgen-insensitive PC3 cell survival in a manner more marked than observed with either single treatment. Co-administration of Api with AA triggered apoptosis. This effect was demonstrated by Hoechst staining, and up-regulation of Bax, cytochrome c, caspase -3, and - 8 and down-regulation of Bcl-2 expression confirmed the effect. AA and Api each individually arrested the cell cycle in the G1 phase, with dual applications, leading to no further increase in the effect produced. The expression of NF-κB p105/p50 and the phosphorylation of AKT markedly decreased after apigenin treatment, with combination treatment leading to a favourable effect in terms of further augmenting the reduction.. The co-administration of Api with AA strongly enhanced the efficacy of AA therapy in the treatment of prostate cancer cells. These data suggested that the combination of AA and Api would be a potential chemotherapeutic strategy against prostate cancer.

Topics: Abiraterone Acetate; Androgens; Apigenin; Apoptosis; bcl-2-Associated X Protein; Caspases; Castration; Cytochromes c; Flavonoids; Humans; Male; NF-kappa B; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Proto-Oncogene Proteins c-akt; Receptors, Androgen; RNA, Messenger; Steroid 17-alpha-Hydroxylase

Brassica oleracea var. acephala is a good source of health-promoting biologically active compounds like phenolics, vitamins, and glucosinolates.. This in vitro research was conducted to evaluate the apoptotic, antioxidant, anti-inflammatory, and antiproliferative properties of ethanolic extract of Brassica oleracea var. acephala (EEBO) in PC3 prostate cancer cells. The LC-MS/MS technique was applied to identify the biomolecules of EEBO. The MTT assay was used to evaluate the cytotoxic effects of EEBO on PC3 cells. Moreover, qRT-PCR was used to examine the expression levels of Nrf2, NQO1, HO-1, NF-κB, TNF-α, IL-6, BAX, and BCL-2 in PC3 cell line. MMP was predicted by Rhodamine 123 staining, and release of cytochrome c was detected by an ELISA kit. Further, apoptosis was quantified by DNA fragmentation assay. The Western blotting method was used to detect the protein expression levels, and The DPPH assay was applied to determine the antioxidant effect of EEBO. The formula and structure of 19 biomolecules were predicted by LC-MS/MS. EEBO exhibited scavenging activity for DPPH. The MTT test showed EEBO reduced the viability of PC3 cells. The mRNA and protein levels of NRF2 pathway genes and BAX were increased, but those of the NF-κB pathway genes and BCL-2 were decreased in the EEBO-treated cells. Moreover, EEBO led to the diminution of MMP and enhanced the release of cytochrome c and DNA fragmentation, which resulted in apoptosis.. Molecular changes due to the anticancer impact of EEBO on PC3 were involved in the induction of Nrf2 antioxidant pathway and apoptosis and inhibition of inflammation.

Topics: Antioxidants; Apoptosis; bcl-2-Associated X Protein; Brassica; Chromatography, Liquid; Cytochromes c; Humans; Male; NF-E2-Related Factor 2; NF-kappa B; PC-3 Cells; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tandem Mass Spectrometry

Prostate cancer is the second leading cause of cancer-related death in men. Two classic cancer hallmarks are a metabolic switch from oxidative phosphorylation (OxPhos) to glycolysis, known as the Warburg effect, and resistance to cell death. Cytochrome

Topics: Acetylation; Animals; Apoptosis; Cardiolipins; Caspase 3; Cell Line, Tumor; Crystallography, X-Ray; Cytochromes c; Electron Transport Complex IV; Humans; Lysine; Male; Mice; Molecular Dynamics Simulation; Mutation; Oxidation-Reduction; Oxygen Consumption; Peroxidase; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Reactive Oxygen Species; Warburg Effect, Oncologic; Xenograft Model Antitumor Assays

The TLK1/Nek1 axis contributes to cell cycle arrest and implementation of the DDR to mediate survival upon DNA damage. However, when the damage is too severe, the cells typically are forced into apoptosis, and the contribution of TLKs in this process has not been investigated. In contrast, it is known that Nek1 may play a role by phosphorylating VDAC1 maintaining proper opening and closure of the channel and thus mitochondrial integrity. We now show that the activating phosphorylation of Nek1-T141 by TLK1 contributes to the phosphorylation and stability of VDAC1 and thereby to mitochondrial permeability and integrity. Treatment of three different cell lines model that overexpress Nek1-T141A mutant with doxorubicin showed exquisite sensitivity to the drug, with implementation of rapid accumulation of cells with subG1 DNA content (apoptotic) and other alterations in the cell cycle. In addition, these cells displayed reduced oxygen consumption under normal conditions and less reliance on mitochondria and more dependence on glycolysis for energy production. Consistent with greater apoptosis, upon treatment with low doses of doxorubicin, cells overexpressing Nek1-T141A displayed leakage of Cyt-C into the cytoplasmic fraction. This suggests that inhibiting the TLK1/Nek1/VDAC1 nexus could sensitize cancer cells to apoptotic killing in combination with an appropriate DNA damaging agent. We in fact have previously reported that Nek1 expression is elevated in advanced Prostate Cancer (PCa) and we now report that VDAC1 expression is elevated and correlated with disease stage, thereby making the TLK1/Nek1/VDAC1 nexus a very attractive target for PCa.

Topics: Antibiotics, Antineoplastic; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Damage; Doxorubicin; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Male; Mitochondria; NIMA-Related Kinase 1; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Voltage-Dependent Anion Channel 1

Natural products are a valuable source of anticancer agents, with many naturally derived compounds currently used in clinical and preclinical treatments. This study aims to investigate the antiproliferative activity and potential mechanism of action of the xanthoquinodin JBIR-99, isolated from fungi Parengyodontium album MEXU 30,054 and identified by single-crystal X-ray crystallography. Cytotoxicity of xanthoquinodin was evaluated in a panel of human cancer cells lines and CCD-112-CoN normal colon cells, using the sulforhodamine B assay. PC-3 prostate cancer cells were used in biochemical assays including cell cycle, mitochondrial transmembrane potential (MTP), reactive oxygen species (ROS) and caspase activity. Expression levels of apoptosis-pathway-related proteins were analyzed by Western blot. The in vivo toxicity of xanthoquinodin was determined using a zebrafish model. Xanthoquinodin showed cytotoxicity in all cancer cell lines but demonstrated relative selective potency against PC-3 cells with an IC

Topics: Apoptosis; Ascomycota; Cell Line, Tumor; Chromones; Crystallography, X-Ray; Cytochromes c; Humans; I-kappa B Kinase; Male; Membrane Potential, Mitochondrial; Molecular Conformation; Poly (ADP-Ribose) Polymerase-1; Prohibitins; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Repressor Proteins; S Phase Cell Cycle Checkpoints; Signal Transduction

Although African-American (AA) patients with prostate cancer tend to develop greater therapeutic resistance and faster prostate cancer recurrence compared with Caucasian-American (CA) men, the molecular mechanisms of this racial prostate cancer disparity remain undefined. In this study, we provide the first comprehensive evidence that cytochrome

Topics: Animals; Apoptosomes; Black or African American; Cell Line, Tumor; Cytochromes c; Humans; Male; Mice; Mice, SCID; Mitochondria; Mitochondrial Membranes; NF-kappa B; Nuclear Respiratory Factor 1; Oxidative Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc

Tumor cell mediated immune-suppression remains a question of interest in tumor biology. In this study, we focused on the metabolites that are released by prostate cancer cells (PCC), which could potentially attenuate T cell immunity.. Prostate cancer cells (PCC) media (PCM) was used to treat T cells, and its impact on T cell signaling was evaluated. The molecular mechanism was further verified in vivo using mouse models. The clinical significance was determined using IHC in human clinical specimens. Liquid chromatography mass spectroscopy (LC/MS-MS) was used to identify the metabolites that are released by PCC, which trigger T cells inactivation.. PCM inhibits T cells proliferation and impairs their ability to produce inflammatory cytokines. PCM decreases ATP production and increases ROS production in T cells by inhibiting complex III of the electron transport chain. We further show that SHP1 as the key molecule that is upregulated in T cells in response to PCM, inhibition of which reverses the phenotype induced by PCM. Using metabolomics analysis, we identified 1-pyrroline-5-carboxylate (P5C) as a vital molecule that is released by PCC. P5C is responsible for suppressing T cells signaling by increasing ROS and SHP1, and decreasing cytokines and ATP production. We confirmed these findings in vivo, which revealed changed proline dehydrogenase (PRODH) expression in tumor tissues, which in turn influences tumor growth and T cell infiltration.. Our study uncovered a key immunosuppressive axis, which is triggered by PRODH upregulation in PCa tissues, P5C secretion in media and subsequent SHP1-mediated impairment of T cell signaling and infiltration in PCa.

Topics: Adenosine Triphosphate; Animals; Cell Line, Tumor; Cell Proliferation; Chromatography, Liquid; Cytochromes c; Cytokines; Disease Models, Animal; Electron Transport Chain Complex Proteins; Heterografts; Humans; Lymphocyte Activation; Male; Metabolome; Metabolomics; Mice; Mitochondria; Prostatic Neoplasms; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Pyrroles; Reactive Oxygen Species; T-Lymphocytes; Tandem Mass Spectrometry

Norcantharidin, a modified pure compound from blister beetles, was previously demonstrated to induce apoptosis of cancer cells. This study investigated its anti-cancer activity in prostate cancer cells and the mechanisms involved.. Two human prostate cancer cell lines, 22Rv1 and Du145, were treated with norcantharidin at concentrations ranging from 3 to 30μg/ml. Cytotoxic effect of norcantharidin was determined by use of the 3-(4,5-dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) assay. The effects of apoptosis were evaluated by cell death assay, Caspase-3, -8, -9 activity and cytochrome c release. The apoptotic related protein expressions (Bcl-2 family and inhibitor of apoptosis proteins) were determined using western blotting.. An MTT assay revealed that norcantharidin induced cytotoxicity against both prostate cancer cells in dose- and time-dependent manners. Treatment with norcantharidin at 3μg/ml or higher significantly increased oligonucleosomal formation with concomitant appearance of PARP cleavage, implicating the induction of apoptosis. Norcantharidin intrinsically elevated cytosolic cytochrome c levels and activated caspase-3, -8, and -9. Extrinsically, it upregulated the expression of not only the death receptors Fas and DR5 in 22Rv1 cells, but also of RIP and TRADD adaptor proteins in Du145 cells. Mechanistically, norcantharidin increased ratios of pro-/anti-apoptotic proteins and decreased expression of IAP family member proteins, including cIAP1 and survivin, regardless of the distinct status of androgen receptor expression in both cells.. Norcantharidin exhibited cytotoxicity against 22Rv1 and Du145 prostate cancer cells by inducing both intrinsic and extrinsic apoptotic pathways and could thus potentially be a remedy for prostate cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Bridged Bicyclo Compounds, Heterocyclic; Caspases; Cell Line, Tumor; Cytochromes c; fas Receptor; Humans; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; TNF Receptor-Associated Death Domain Protein; Up-Regulation

Obacunone and obacunone glucoside (OG) are naturally occurring triterpenoids commonly found in citrus and other plants of the Rutaceae family. The current study reports the mechanism of cytotoxicity of citrus-derived obacunone and OG on human androgen-dependent prostate cancer LNCaP cells. Both limonoids exhibited time- and dose-dependent inhibition of cell proliferation, with more than 60% inhibition of cell viability at 100 μM, after 24 and 48 h. Analysis of fragmentation of DNA, activity of caspase-3, and cytosolic cytochrome-c in the cells treated with limonoids provided evidence for activation of programmed cell death by limonoids. Treatment of LNCaP cells with obacunone and OG resulted in dose-dependent changes in expression of proteins responsible for the induction of programmed cell death through the intrinsic pathway and down-regulation of Akt, a key molecule in cell signaling pathways. In addition, obacunone and OG also negatively regulated an inflammation-associated transcription factor, androgen receptor, and prostate-specific antigen, and activated proteins related to the cell cycle, confirming the ability of limonoids to induce cytotoxicity through multiple pathways. The results of this study provided, for the first time, an evidence of the cytotoxicity of obacunone and OG in androgen-dependent human prostate cancer cells.

Topics: Apoptosis; Benzoxepins; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Citrus paradisi; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Down-Regulation; Glucosides; Humans; Inflammation; Limonins; Male; Plant Extracts; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Signal Transduction; Triterpenes

Docetaxel (DTX) is widely used for the treatment of metastatic prostate and breast cancers. Despite the clinical success of DTX, drug-related cumulative toxicity restricts its clinical use in cancer therapy. Thus, there is an urgent need for new therapeutic options. Octreotide (OCT) is a synthetic somatostatin analog that induces apoptosis in different cancer cell lines in vitro. In this study, we investigated the possible synergistic apoptotic effects of DTX in combination with OCT in prostate and breast cancer cell lines.. The XTT cell viability assay was used to determine cytotoxicity. Apoptosis was evaluated by Cell Death Detection ELISA(Plus) Kit. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array. Levels of SSTR2 and SSTR5 proteins were determined by western blot analysis.. DTX and OCT combination induced apoptosis in both breast and prostate cancer cells in a concentration- and time-dependent manner. Moreover, combination treatment resulted in inhibition of anti-apoptotic proteins such as Bcl-2 and Bcl-xL and induction of pro-apoptotic proteins Bax, Cytochrome c and IAPs in all of the tested cancer cell lines. SSTR2 and SSTR5 protein levels were induced as compared to any agent alone.. These results indicate that this combination treatment is a significant inducer of apoptosis in a synergistic manner in breast and prostate cancer cells. This strong synergism helps to lower the dose of DTX in both types of cancers, thus letting DTX to be used for longer periods by delaying resistance development and lesser side effects.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cytochromes c; Docetaxel; Drug Synergism; Female; Humans; Inhibitor of Apoptosis Proteins; Male; Octreotide; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptors, Somatostatin; Taxoids

Galeterone (Gal) is a first-in-class multi-target oral small molecule that will soon enter pivotal phase III clinical trials in castration resistant prostate cancer (CRPC) patients. Gal disrupts androgen receptor (AR) signaling via inhibition of CYP17, AR antagonism and AR degradation. Resistance to current therapy is attributed to up-regulation of full-length AR (fAR), splice variants AR (AR-Vs) and AR mutations. The effects of gal and VNPT55 were analyzed on f-AR and AR-Vs (AR-V7/ARv567es) in LNCaP, CWR22Rv1 and DU145 (transfected with AR-Vs) human PC cells in vitro and CRPC tumor xenografts. Galeterone/VNPT55 decreased fAR/AR-V7 mRNA levels and implicates Mdm2/CHIP enhanced ubiquitination of posttranslational modified receptors, targeting them for proteasomal degradation. Gal and VNPT55 also induced significant apoptosis in PC cells via increased Bax/Bcl2 ratio, cytochrome-c release with concomitant cleavage of caspase 3 and PARP. More importantly, gal and VNPT55 exhibited strong in vivo anti-CRPC activities, with no apparent host toxicities. This study demonstrate that gal and VNPT55 utilize cell-based mechanisms to deplete both fAR and AR-Vs. Importantly, the preclinical activity profiles, including profound apoptotic induction and inhibition of CRPC xenografts suggest that these agents offer considerable promise as new therapeutics for patients with CRPC and those resistant to current therapy.

Topics: Androstadienes; Animals; Antineoplastic Agents; Apoptosis; Benzimidazoles; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Male; Mice; Mice, SCID; Neoplasm Transplantation; Phosphorylation; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Proteasome Endopeptidase Complex; Protein Isoforms; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-mdm2; Receptors, Androgen; RNA, Small Interfering

PACE4 is a proprotein convertase capable of processing numerous substrates involved in tumor growth, invasion, and metastasis. However, the precise role of PACE4 during prostate cancer cell apoptosis has not been reported.. In the present study, human prostate cancer cell lines DU145, LNCaP, and PC3 were transfected with PACE4 small interfering (si)RNA to investigate the underlying mechanisms of apoptosis.. We revealed that PACE4 siRNA exhibited antitumor activity by inducing apoptosis, as determined by Cell Counting Kit-8 (CCK-8), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT) assay, cell cycle analysis, Hoechst staining, caspase-3/7 activity, and western blot analysis. In addition, PACE4 siRNA significantly increased the ratio of Bax/Bcl-2, which led to the release of cytochrome c. Moreover, PACE4 siRNA also induced endoplasmic reticulum stress by increasing the expression of GRP78, GRP94, p-PERK, and p-eIF2α. The ratio of Bax/Bcl-2 and GRP78 were also increased in PACE4 gene knockdown prostate cancer cells compared with the control cells.. These data demonstrate that PACE4 siRNA may exert its antitumor activity through mitochondrial and endoplasmic reticulum stress signaling pathways, indicating it may be a novel therapeutic target for prostate cancer.

Topics: Apoptosis; Cell Line, Tumor; Cytochromes c; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; Mitochondria; Proprotein Convertases; Prostatic Neoplasms; RNA, Small Interfering; Serine Endopeptidases; Signal Transduction

Oxidative stress is one causative factor of the pathogenesis and aggressiveness of most of the cancer types, including prostate cancer (CaP). A moderate increase in reactive oxygen species (ROS) induces cell proliferation whereas excessive amounts of ROS promote apoptosis. In this study, we explored the pro-oxidant property of 3,9-dihydroxy-2-prenylcoumestan (psoralidin [pso]), a dietary agent, on CaP (PC-3 and C4-2B) cells. Pso greatly induced ROS generation (more than 20-fold) that resulted in the growth inhibition of CaP cells. Overexpression of anti-oxidant enzymes superoxide dismutase 1 (SOD1), SOD2, and catalase, or pretreatment with the pharmacological inhibitor N-acetylcysteine (NAC) significantly attenuated both pso-mediated ROS generation and pso-mediated growth inhibition in CaP cells. Furthermore, pso administration significantly inhibited the migratory and invasive property of CaP cells by decreasing the transcription of β-catenin, and slug, which promote epithelial-mesenchymal transition (EMT), and by concurrently inducing E-cadherin expression in CaP cells. Pso-induced ROS generation in CaP cells resulted in loss of mitochondrial membrane potential, cytochrome-c release, and activation of caspase-3 and -9 and poly (ADP-ribose) polymerase (PARP), which led to apoptosis. On the other hand, overexpression of anti-oxidants rescued pso-mediated effects on CaP cells. These findings suggest that increasing the threshold of intracellular ROS could prevent or treat CaP growth and metastasis.

Topics: Acetylcysteine; Antioxidants; Apoptosis; Benzofurans; beta Catenin; Cadherins; Caspase 3; Caspase 9; Catalase; Cell Line, Tumor; Cell Movement; Cell Proliferation; Coumarins; Cytochromes c; Enzyme Activation; Epithelial-Mesenchymal Transition; Humans; Male; Membrane Potential, Mitochondrial; Neoplasm Invasiveness; Oxidative Stress; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Reactive Oxygen Species; Snail Family Transcription Factors; Superoxide Dismutase; Superoxide Dismutase-1; Transcription Factors; Transcription, Genetic

In this study, a new apoptotic monoterpenoid indole alkaloid, subditine (1), and four known compounds were isolated from the bark of Nauclea subdita. Complete (1)H- and (13)C- NMR data of the new compound were reported. The structures of isolated compounds were elucidated with various spectroscopic methods such as 1D- and 2D- NMR, IR, UV and LCMS. All five compounds were screened for cytotoxic activities on LNCaP and PC-3 human prostate cancer cell-lines. Among the five compounds, the new alkaloid, subditine (1), demonstrated the most potent cell growth inhibition activity and selective against LNCaP with an IC50 of 12.24±0.19 µM and PC-3 with an IC50 of 13.97±0.32 µM, compared to RWPE human normal epithelial cell line (IC50 = 30.48±0.08 µM). Subditine (1) treatment induced apoptosis in LNCaP and PC-3 as evidenced by increased cell permeability, disruption of cytoskeletal structures and increased nuclear fragmentation. In addition, subditine (1) enhanced intracellular reactive oxygen species (ROS) production, as reflected by increased expression of glutathione reductase (GR) to scavenge damaging free radicals in both prostate cancer cell-lines. Excessive ROS could lead to disruption of mitochondrial membrane potential (MMP), release of cytochrome c and subsequent caspase 9, 3/7 activation. Further Western blot analyses showed subditine (1) induced down-regulation of Bcl-2 and Bcl-xl expression, whereas p53 was up-regulated in LNCaP (p53-wild-type), but not in PC-3 (p53-null). Overall, our data demonstrated that the new compound subditine (1) exerts anti-proliferative effect on LNCaP and PC-3 human prostate cancer cells through induction of apoptosis.

Topics: Alkaloids; Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Cell Nucleus; Cytochromes c; Cytoskeleton; DNA Fragmentation; Drug Screening Assays, Antitumor; Enzyme Activation; Gene Expression Regulation, Neoplastic; Glutathione Reductase; Humans; Indole Alkaloids; Indoles; Magnetic Resonance Spectroscopy; Male; Membrane Potential, Mitochondrial; Models, Biological; Plant Bark; Prostatic Neoplasms; Quinolizidines; Reactive Oxygen Species; Terpenes

Saikosaponin‑d (SSd), a triterpene saponin compound derived from Bupleurum radix, has been shown to have a cytotoxic effect on various cancer cell lines. However, its effect on prostate cancer cells has remained unexplored. The present study reports the apoptosis‑inducing effect of SSd on the DU145 human prostate carcinoma cell line. Treatment with SSd inhibited DU145 cell proliferation in a concentration‑dependent manner. Flow cytometric analysis showed that SSd inhibited the proliferation of DU145 cells by induction of apoptosis and cell cycle arrest at G0/G1 phase. Further mechanistic experiments demonstrated that SSd arrested the cell cycle at G0/G1 phase via upregulation of p53 and p21 and induced apoptosis by modulating B‑cell lymphoma 2 family proteins, dissipation of the mitochondrial membrane potential, release of cytochrome c into the cytosol and activation of caspase‑3. In conclusion the present study indicated that SSd induced apoptosis in DU145 cells by the intrinsic apoptotic pathway. Therefore, SSd may become a leading candidate drug for the therapy of prostate carcinoma.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; G1 Phase Cell Cycle Checkpoints; Humans; Male; Membrane Potential, Mitochondrial; Oleanolic Acid; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Saponins; Tumor Suppressor Protein p53; Up-Regulation

Gonadotropin-releasing hormone-I (GnRH-I) has attracted strong attention as a hormonal therapeutic tool, particularly for androgen-dependent prostate cancer patients. However, the androgen-independency of the cancer in advanced stages has spurred researchers to look for new medical treatments. In previous reports, we developed the GnRH-II antagonist Trp-1 to inhibit proliferation and stimulate the autophagic death of various prostate cancer cells, including androgen-independent cells. We further screened many GnRH-II antagonists to identify molecules with higher efficiency. Here, we investigated the effect of SN09-2 on the growth of PC3 prostate cancer cells. SN09-2 reduced the growth of prostate cancer cells but had no effect on cells derived from other tissues. Compared with Trp-1, SN09-2 conspicuously inhibited prostate cancer cell growth, even at low concentrations. SN09-2-induced PC3 cell growth inhibition was associated with decreased membrane potential in mitochondria where the antagonist was accumulated, and increased mitochondrial and cytosolic reactive oxygen species. SN09-2 induced lactate dehydrogenase release into the media and annexin V-staining on the PC3 cell surface, suggesting that the antagonist stimulated prostate cancer cell death by activating apoptotic signaling pathways. Furthermore, cytochrome c release from mitochondria to the cytosol and caspase-3 activation occurred in a concentration- and time-dependent manner. SN09-2 also inhibited the growth of PC3 cells xenotransplanted into nude mice. These results demonstrate that SN09-2 directly induces mitochondrial dysfunction and the consequent ROS generation, leading to not only growth inhibition but also apoptosis of prostate cancer cells.

Topics: Animals; Antinematodal Agents; Apoptosis; Cell Line, Tumor; Cytochromes c; Gonadotropin-Releasing Hormone; Humans; Lactate Dehydrogenases; Male; Mice; Mice, Nude; Oligopeptides; Prostatic Neoplasms; Signal Transduction

Kallikreins play a pivotal role in establishing prostate cancer.. In contrast to the classical Kunitz plant inhibitor SbTI, the recombinant kallikrein inhibitor (rBbKIm) led to prostate cancer cell death, whereas fibroblast viability was not affected.. rBbKIm shows selective cytotoxic effect and angiogenesis inhibition against prostate cancer cells.. New actions of rBbKIm may contribute to understanding the mechanisms of prostate cancer. Prostate cancer is the most common type of cancer, and kallikreins play an important role in the establishment of this disease. rBbKIm is the recombinant Bauhinia bauhinioides kallikreins inhibitor that was modified to include the RGD/RGE motifs of the inhibitor BrTI from Bauhinia rufa. This work reports the effects of rBbKIm on DU145 and PC3 prostate cancer cell lines. rBbKIm inhibited the cell viability of DU145 and PC3 cells but did not affect the viability of fibroblasts. rBbKIm caused an arrest of the PC3 cell cycle at the G0/G1 and G2/M phases but did not affect the DU145 cell cycle, although rBbKIm triggers apoptosis and cytochrome c release into the cytosol of both cell types. The differences in caspase activation were observed because rBbKIm treatment promoted activation of caspase-3 in DU145 cells, whereas caspase-9 but not caspase-3 was activated in PC3 cells. Because angiogenesis is important to the development of a tumor, the effect of rBbKIm in this process was also analyzed, and an inhibition of 49% was observed in in vitro endothelial cell capillary-like tube network formation. In summary, we demonstrated that different properties of the protease inhibitor rBbKIm may be explored for investigating the androgen-independent prostate cancer cell lines PC3 and DU145.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents, Phytogenic; Apoptosis; Calcium Signaling; Caspase 3; Caspase 9; Cell Adhesion; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Survival; Cytochromes c; Fibroblasts; Human Umbilical Vein Endothelial Cells; Humans; Hydrophobic and Hydrophilic Interactions; Kallikreins; Lipopolysaccharides; Male; Mitochondria; Plant Proteins; Prostatic Neoplasms; Recombinant Proteins; Trypsin Inhibitor, Kunitz Soybean

Cancer of the prostate gland is the most common invasive malignancy and the second leading cause of cancer-related death in human males. Many studies have shown that black tea reduces the risk of several types of cancer. We studied the effects of active extracts of black tea and the black tea polyphenols theaflavins (TFs), on the cellular proliferation and mitochondria of the human prostate cancer cell line PC-3. Our studies revealed that Yinghong black tea extracts (YBT), Assam black tea extracts (ABT) and TFs inhibited cell proliferation in a dose-dependent manner. We also showed that TFs, YBT and ABT affected the morphology of PC-3 cells and induced apoptosis or even necrosis in PC-3 cells. In addition, it was observed that the samples significantly caused loss of the mitochondrial membrane potential, release of cytochrome c from the intermembrane space into the cytosol, decrease of the ATP content and activation of caspase-3 compared with the control. Taken together, these findings suggest that black tea could act as an effective anti-proliferative agent in PC-3 cells, and TFs, YBT and ABT induced apoptosis of PC-3 cells through mitochondrial dysfunction.

Topics: Adenosine Triphosphate; Apoptosis; Biflavonoids; Camellia sinensis; Caspase 3; Catechin; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Humans; Male; Membrane Potential, Mitochondrial; Mitochondria; Necrosis; Polyphenols; Prostatic Neoplasms

Chronic inflammation promotes prostate cancer formation and progression. Furthermore, alterations in energy metabolism are a hallmark of prostate cancer cells. However, the actions of inflammatory factors on the energy metabolism of prostate epithelial cells have not been previously investigated. This is the first study to report on the effect of the inflammatory cytokine tumor necrosis factor alpha (TNFα) on the glycolytic and oxidative metabolism, and the mitochondrial function of widely used prostate epithelial cells.. Pre-malignant RWPE-1 and cancerous LNCaP and PC-3 cells were treated with low-dose TNFα. Glycolytic and oxidative metabolism was quantified by measuring extracellular acidification and oxygen consumption rates, respectively. ATP content and lactate export were measured by luminescence and fluorescence, respectively. Mitochondrial content and the expression of glucose transporter 1 (GLUT1), peroxisome proliferator-activated receptor co-activator 1 alpha (PGC-1α), and Cytochrome C were measured by flow cytometry.. Our data suggest that TNFα increases glycolysis, ATP production, and lactate export, while it reduces oxidative metabolism and mitochondrial function in prostate epithelial cells. The highly aggressive PC-3 cells tend to be less responsive to the actions of TNFα than the pre-malignant RWPE-1 and the non-aggressive LNCaP cells.. Cellular energetics, that is, glycolytic and oxidative metabolism is significantly influenced by low-level inflammation in prostate epithelial cells. In widely used prostate epithelial cell models, the micro-environmental inflammatory cytokine TNFα induces aerobic glycolysis while inhibiting oxidative metabolism. This supports the hypothesis that low-level inflammation can induce Warburg metabolism in prostate epithelial cells, which may promote cancer formation and progression.

Topics: Cell Line, Tumor; Cytochromes c; Disease Progression; Energy Metabolism; Epithelial Cells; Glucose Transporter Type 1; Glycolysis; Humans; Inflammation; Male; Mitochondria; Oxidative Stress; PPAR alpha; Precancerous Conditions; Prostate; Prostatic Neoplasms; Tumor Microenvironment; Tumor Necrosis Factor-alpha

Chemotherapy is one of the therapeutic strategies that has been used for the inhibition of cancer cell proliferation in several types of cancer, including prostate cancer. Although monoamine oxidase (MAO) inhibitors, phytoestrogen and antioxidants used in chemotherapy have been systematically studied, their effects on cancer cell growth remain to be fully understood. The purpose of this study was to investigate the effects of the MAO inhibitors, pargyline and tranylcypromine on cell survival in human prostate carcinoma (LNCaP-LN3) cells. After treating LNCaP-LN3 cells with pargyline or tranylcypromine, we examined cell proliferation, cell cycle pattern, apoptosis and the expression levels of apoptosis-related genes. The proliferation of cells exposed to pargyline decreased in a dose- and time-dependent manner, while tranylcypromine-treated cells showed the opposite results. Treatment with pargyline significantly induced cell cycle arrest at the G1 phase compared to the control and tranylcypromine-treated cells. In addition, pargyline induced an increase in the cell death rate by promoting apoptosis; however, tranylcypromine had no effect on LNCaP-LN3 cells. Based on our results, we suggest that pargyline is more powerful than tranylcypromine for the treatment of human prostate cancer.

Topics: Apoptosis; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; G1 Phase Cell Cycle Checkpoints; Humans; Male; Monoamine Oxidase Inhibitors; Pargyline; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Tranylcypromine

Norcantharidin (NCTD), a demethylated analog of cantharidin derived from blister beetles, has attracted considerable attentions in recent years due to their definitely toxic properties and the noteworthy advantages in stimulating bone marrow and increasing the peripheral leukocytes. Hence, it is worth studying the anti-tumor effect of NCTD on human prostate cancer cells DU145. It was found that after the treatment of NCTD with different concentrations (25-100 μM), the cell proliferation was significantly inhibited, which led to the appearance of micronucleus (MN). Moreover, the cells could be killed in a dose-/time-dependent manner along with the reduction of PCNA (proliferating cell nuclear antigen) expression, destruction of mitochondrial membrane potential (MMP), down-regulation of MnSOD, induction of ROS, depletion of ATP, and activation of AMPK (Adenosine 5'-monophosphate -activated protein kinase) . In addition, a remarkable release of cytochrome c was found in the cells exposed to 100 μM NCTD and exogenous SOD-PEG could eliminate the generation of NCTD-induced MN. In conclusion, our studies indicated that NCTD could induce the collapse of MMP and mitochondria dysfunction. Accumulation of intercellular ROS could eventually switch on the apoptotic pathway by causing DNA damage and depleting ATP.

Topics: Adenosine Triphosphate; AMP-Activated Protein Kinases; Antineoplastic Agents; Apoptosis; Bisbenzimidazole; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Humans; In Situ Nick-End Labeling; Male; Membrane Potential, Mitochondrial; Micronucleus Tests; Mitochondria; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Reactive Oxygen Species; Superoxide Dismutase

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti-neoplastic agents of natural product origin. We previously reported anti-proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC-3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase-3-dependent pathway that is activated due to dysregulated mitochondrial function. DPT-treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca (i)(2+) surge, increased mitochondrial membrane potential (MMP, ΔΨ(m)), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase-3 activation, which in turn induced apoptosis. The antioxidant N-acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca (i)(2+) surge, on the other hand the Ca(2+) chelator BAPTA inhibited the Ca( i)(2+) overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca(2+) flux. This suggested that both ROS and Ca( i)(2+) signaling play roles in the increased MMP via Ca (i)(2+)-dependent and/or -independent mechanisms, since ΔΨ(m) elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS- and Ca( i)(2+)-activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca(2+) flux homeostasis.

Topics: Apoptosis; bcl-2-Associated X Protein; Calcium; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Egtazic Acid; Enzyme Activation; G2 Phase Cell Cycle Checkpoints; Homeostasis; Humans; Intracellular Space; M Phase Cell Cycle Checkpoints; Male; Membrane Potential, Mitochondrial; Mitochondria; Models, Biological; Podophyllotoxin; Prostatic Neoplasms; Protein Transport; Reactive Oxygen Species

Cordycepin is the main functional component of Cordyceps militaris, which has been widely used in oriental traditional medicine. This compound has been shown to possess many pharmacological properties, such as enhancing the body's immune function, and anti-inflammatory, anti-aging and anticancer effects. In the present study, we investigated the apoptotic effects of cordycepin in human prostate carcinoma cells. We found that treatment with cordycepin significantly inhibited cell growth by inducing apoptosis in PC-3 cells. Apoptosis induction of PC-3 cells by cordycepin showed correlation with proteolytic activation of caspase-3 and -9, but not caspase-8, and concomitant degradation of poly (ADP-ribose) polymerases, collapse of the mitochondrial membrane potential (MMP). In addition, cordycepin treatment resulted in an increase of the Bax/Bcl-2 (or Bcl-xL) ratio, downregulation of inhibitor of apoptosis protein (IAP) family members, Bax conformational changes, and release of cytochrome c from the mitochondria to the cytosol. The cordycepin-induced apoptosis was also associated with the generation of intracellular reactive oxygen species (ROS). However, the quenching of ROS generation with antioxidant N-acetyl-L-cysteine conferred significant protection against cordycepin-elicited ROS generation, disruption of the MMP, modulation of Bcl-2 and IAP family proteins, caspase-3 and -9 activation and apoptosis. This indicates that the cellular ROS generation plays a pivotal role in the initiation of cordycepin-triggered apoptotic death. Collectively, our findings suggest that cordycepin is a potent inducer of apoptosis of prostate cancer cells via a mitochondrial-mediated intrinsic pathway and that this agent may be of value in the development of a potential therapeutic candidate for both the prevention and treatment of cancer.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; Deoxyadenosines; Down-Regulation; Enzyme Activation; Humans; Inhibitor of Apoptosis Proteins; Male; Membrane Potential, Mitochondrial; Mitochondria; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Vitexicarpin (3', 5-dihydroxy-3, 4', 6, 7-tetramethoxyflavone), a polymethoxyflavone isolated from Viticis Fructus (Vitex rotundifolia Linne fil.), has long been used as an anti-inflammatory herb in traditional Chinese medicine. It has also been reported that vitexicarpin can inhibit the growth of various cancer cells. However, there is no report elucidating its effect on human prostate carcinoma cells. The aim of the present study was to examine the apoptotic induction activity of vitexicarpin on PC-3 cells and molecular mechanisms involved. MTT studies showed that vitexicarpin dose-dependently inhibited growth of PC-3 cells with an IC50~28.8 μM. Hoechst 33258 staining further revealed that vitexicarpin induced apoptotic cell death. The effect of vitexicarpin on PC-3 cells apoptosis was tested using prodium iodide (PI)/Annexin V-FITC double staining and flow cytometry. The results indicated that vitexicarpin induction of apoptotic cell death in PC-3 cells was accompanied by cell cycle arrest in the G2/M phase. Furthermore, our study demonstrated that vitexicarpin induction of PC-3 cell apoptosis was associated with upregulation of the proapoptotic protein Bax, and downregulation of antiapoptotic protein Bcl-2, release of Cytochrome c from mitochondria and decrease in mitochondrial membrane potential. Our findings suggested that vitexicarpin may become a potential leading drug in the therapy of prostate carcinoma.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma; Cell Cycle Checkpoints; Cell Death; Cell Division; Cell Line, Tumor; Cytochromes c; Down-Regulation; Flavonoids; G2 Phase; Humans; Male; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mitochondria; Plant Extracts; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Up-Regulation

Prostate cancer is the second most common cause of mortality. Gallic acid (GA) is a natural polyphenol, and we tested its in-vitro cytotoxicity after 24 h in prostate cancer LNCaP cells.. GA autoxidation was measured fluorimetrically for H(2)O(2), and O(2)(•-) radicals by chemiluminescence. Intracellular reactive oxygen species (ROS) levels were detected with 2',7'-dichlorodihydrofluorescein diacetate. Cytotoxicity was evaluated by crystal-violet, while apoptosis and mitochondrial membrane potential were determined by flow cytometry. Cytochrome c release was detected by enzyme-linked immunosorbent assay, and caspase-8, -9 and -3 activities were measured calorimetrically.. GA autoxidation produced significant levels of H(2)O(2) and O2.-. Increased intracellular ROS levels with GA were reduced by N-acetyl-L-cysteine (NAC) and L-glutathione (GSH). Cells were protected against GA cytotoxicity when pretreated with increasing levels of superoxide dismutase/catalase mixture, NAC, or GSH for 3 h. The number of apoptotic cells increased with GA dose. GA caused mitochondrial potential loss, cytochrome c release, and activation of caspases 3, 8 and 9.. The ROS-dependent apoptotic mechanism of GA kills malignant cells effectively; it is likely that GA could be a good anticancer agent.

Topics: Acetylcysteine; Apoptosis; Caspases; Cell Line, Tumor; Cytochromes c; Gallic Acid; Glutathione; Humans; Male; Membrane Potential, Mitochondrial; Oxidation-Reduction; Prostatic Neoplasms; Reactive Oxygen Species

Diagnostic tissue biomarkers for prostate cancer (PC) include basal cell markers and α-methylacyl-coenzyme A-racemase (AMACR), often used in combination. Their sensitivity and specificity are not perfect and there is a need for additional diagnostic biomarkers for PC in cases that are difficult to diagnose on routine stained sections.. This study investigated the diagnostic accuracy of three novel tissue biomarkers for PC found through a search in the Human Protein Atlas database ( www.proteinatlas.com ): somatic cytochrome c (CYCS), intestinal cell kinase (ICK) and inhibitor of nuclear factor-κB kinase subunit beta (IKBKB), and compared the results with AMACR. A tissue microarray was constructed from 40 consecutive radical prostatectomy (RP) specimens including benign prostatic tissue, atrophy, high-grade prostatic intraepithelial neoplasia (HGPIN) and PC. Immunoreactivity was scored based on staining intensity and extent. Real-time polymerase chain reaction (PCR) was performed on malignant and benign frozen tissue samples from 32 RP specimens.. All four biomarkers showed a stronger expression in PC and HGPIN than in benign tissue (p < 0.001). The highest diagnostic accuracy for PC was achieved with ICK and AMACR at 97%. The area under the curve for CYCS, ICK, IKBKB and AMACR was 0.859, 0.997, 0.865 and 0.983, respectively. The presence of mRNA transcripts of the genes was confirmed by real-time PCR in benign and malignant prostatic tissue.. AMACR is an accurate diagnostic tissue marker for PC. However, in some PCs AMACR is false negative and a panel of CYCS, ICK and IKBKB may serve as ancillary diagnostic tool.

Topics: Biomarkers, Tumor; Biopsy; Cytochromes c; Humans; I-kappa B Kinase; Male; Prostate; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Racemases and Epimerases; Reproducibility of Results; RNA, Messenger; Sensitivity and Specificity; Tissue Array Analysis

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol, which is present in cruciferous vegetables and has been reported to possess anti-carcinogenic properties. In the present study, we examined whether DIM inhibits the development of prostate cancer using the transgenic adenocarcinoma mouse prostate (TRAMP) model. DIM feeding inhibited prostate carcinogenesis in TRAMP mice, reduced the number of cells expressing the SV40 large tumor antigen and proliferating cell nuclear antigen, and increased the number of terminal dUTP nick-end labeling-positive cells in the dorsolateral lobes of the prostate. Additionally, DIM feeding reduced the expression of cyclin A, cyclin-dependent kinase (CDK)2, CDK4, and Bcl-xL, and increased p27 and Bax expression. To assess the mechanisms by which DIM induces apoptosis, LNCaP and DU145 human prostate cancer cells were cultured with various concentrations of DIM. DIM induced a substantial reduction in the numbers of viable cells and induced apoptosis in LNCaP and DU145 cells. DIM increased the cleavage of caspase-9, -7, -3, and poly (ADP-ribose) polymerase (PARP). DIM increased mitochondrial membrane permeability and the translocation of cytochrome c and Smac/Diablo from the mitochondria. Additionally, DIM induced increases in the levels of cleaved caspase-8, truncated Bid, Fas, and Fas ligand, and the caspase-8 inhibitor Z-IETD-FMK was shown to mitigate DIM-induced apoptosis and the cleavage of caspase-3, PARP, and Bid. These results indicate that DIM inhibits prostate carcinogenesis via induction of apoptosis and inhibition of cell cycle progression. DIM induces apoptosis in prostate cancer cells via the mitochondria- and death receptor-mediated pathways.

Topics: Adenocarcinoma; Animals; Anticarcinogenic Agents; Antigens, Polyomavirus Transforming; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Carrier Proteins; Caspases; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin A; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Disease Models, Animal; Humans; Indoles; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondrial Membranes; Mitochondrial Proteins; Oligopeptides; Peptides; Permeability; Poly(ADP-ribose) Polymerases; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Protein Transport

A key player in prostate cancer development and progression is the androgen receptor (AR). Tumor-associated lipogenesis can protect cancer cells from carcinogenic- and therapeutic-associated treatments. Increased synthesis of fatty acids and cholesterol is regulated by androgens through induction of several genes in androgen-responsive cancer cells. Acetyl-CoA-carboxylase-α (ACCA) is a key enzyme in the regulation of fatty acids synthesis. Here we show that AR binds in vivo to intron regions of human ACCA gene. We also show that the level of ACCA protein in LNCaP depends on AR expression and that DHT treatment increases ACCA expression and fatty acid synthesis. Inhibition of ACCA by TOFA (5-tetradecyl-oxy-2-furoic acid) decreases fatty acid synthesis and induces caspase activation and cell death in most PCa cell lines. Our data suggest that TOFA can kill cells via the mitochondrial pathway since we found cytochrome c release after TOFA treatment in androgen sensitive cell lines. The results also imply that the pro-apoptotic effect of TOFA may be mediated via a decrease of neuropilin-1(NRP1) and Mcl-1expression. We have previously reported that Mcl-1 is under AR regulation and plays an important role in resistance to drug-induced apoptosis in prostate cancer cells, and NRP1 is known to regulate Mcl-1 expression. Here, we show for the first time that NRP1 expression is under AR control. Taken together, our data suggest that TOFA is a potent cell death inducing agent in prostate cancer cells.

Topics: Acetyl-CoA Carboxylase; Androgen Receptor Antagonists; Antineoplastic Agents; Apoptosis; Carcinoma; Cell Death; Cell Line, Tumor; Cytochromes c; Dihydrotestosterone; Fatty Acids; Furans; Genes, p53; Humans; Introns; Lipids; Male; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neuropilin-1; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptors, Androgen

Docetaxel and sodium selenite are well known for their anticancer properties. While resistance to docetaxel remains an obstacle in prostate cancer chemotherapy, sodium selenite, has been exploited as a new therapeutic approach. Currently, development of therapies affecting a multitude of cell targets, have been proposed as a strategy to overcome drug resistance. This association may reduce systemic toxicity counteracting a wide range of side effects. Here we report the effect of docetaxel and sodium selenite combination on the PC3 prostate cancer cell line, derived from bone metastasis. Therefore we evaluate cell growth, cell cycle progression, viability, mitochondria membrane potential, cytochrome C, Bax/Bcl2 ratio, caspase-3 expression and reactive oxygen species production. Our results suggest that sodium selenite and docetaxel combination have a synergistic effect on cell growth inhibition (67%) compared with docetaxel (22%) and sodium selenite (24%) alone. This combination also significantly induced cell death, mainly by late apoptosis vs necrosis, which is correlated with mitochondria membrane potential depletion. On the other hand, cytochrome C, Bax/Bcl2 ratio and caspase-3, known as proapoptotic factors, significantly increased in the presence of sodium selenite alone, but not in the presence of docetaxel in monotherapy or in combination with sodium selenite. These findings suggest that docetaxel and sodium selenite combination may be more effective on prostate cancer treatment than docetaxel alone warranting further evaluation of this combination in prostate cancer therapeutic approach.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Docetaxel; Drug Synergism; Humans; Male; Peroxides; Prostatic Neoplasms; Sodium Selenite; Taxoids

Phenethyl isothiocyanate (PEITC), one of many compounds found in cruciferous vegetables, has been reported as a potential anticancer agent. In earlier studies, PEITC was shown to inhibit cell growth and induction of apoptosis in many cancer cell lines. However, no report has shown whether PEITC can induce apoptosis in human prostate cancer cells. Herein, we aimed to determine whether PEITC has anticancer activity in DU 145 human prostate cancer cells. As a result, we found that PEITC induced a dose-dependent decrease in cell viability through induction of cell apoptosis and cell cycle arrest in the G(2)/M phase of DU 145 cells. PEITC induced morphological changes and DNA damage in DU 145 cells. The induction of G(2)/M phase arrest was mediated by the increase of p53 and WEE1 and it reduced the level of CDC25C protein. The induction of apoptosis was mediated by the activation of caspase-8-, caspase-9- and caspase-3-depedent pathways. Results also showed that PEITC caused mitochondrial dysfunction, increasing the release of cytochrome c and Endo G from mitochondria, and led cell apoptosis through a mitochondria-dependent signaling pathway. This study showed that PEITC might exhibit anticancer activity and become a potent agent for human prostate cancer cells in the future.

Topics: Anticarcinogenic Agents; Apoptosis; Blotting, Western; Caspases; Cell Division; Cell Proliferation; Comet Assay; Cytochromes c; DNA Damage; Flow Cytometry; Fluorescent Antibody Technique; G2 Phase; Humans; Isothiocyanates; Male; Membrane Potential, Mitochondrial; Mitochondria; Prostatic Neoplasms; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytosol; Humans; Male; Membrane Potential, Mitochondrial; Mitochondria; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Pyrans; Reactive Oxygen Species

We explored the mechanisms of apoptosis after Bcl-2 protein downmodulation in metastatic LNCaP-LN3 cells (LN3).. LNCaP, LNCaP-Pro5 (Pro5) and LN3 cells were cultured in 5% charcoal-stripped serum (CSS) or in R1881 (synthetic androgen) and bicalutamide (synthetic anti-androgen) and growth inhibition was assessed. Expression levels of androgen receptor (AR) and Bcl-2 were determined. LN3 cells were transfected with small interfering RNA Bcl-2 (siRNA Bcl-2) or control siRNA oligonucleotides. Rates of apoptosis and proliferation were obtained. Cytochrome c localization in treated and control cells was assessed +/- cyclosporine A (CsA). Caspases 9, 3, and poly (ADP-ribose) polymerase cleavage (PARP) were measured upon downmodulation of Bcl-2; and cell growth inhibition in vitro after Bcl-2 modulation combined with docetaxel chemotherapy was determined.. LN3 cells maintained growth under castrate conditions in vitro. AR protein amplification did not explain castrate-resistant LN3 cell growth. Bcl-2 protein levels in LN3 cells were significantly higher than in Pro5 cells, and were effectively downmodulated by siRNA Bcl-2. Subsequently increased apoptosis and decreased proliferation mediated by cytochrome c was noted and this was reversed by CsA. siRNA Bcl-2-transfected LN3 cells exhibited elevated levels of caspases 9, 3, and PARP cleavage. Exposure of LN3 cells to docetaxel led to increased apoptosis, and simultaneous downmodulation of Bcl-2 substantially enhanced this effect.. Downmodulation of Bcl-2 in metastatic castrate-resistant LNCaP-LN3 cells led to apoptosis via a cytochrome c-dependent pathway that was enhanced with docetaxel treatment.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Docetaxel; Down-Regulation; Humans; Male; Neoplasm Metastasis; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptors, Androgen; RNA, Small Interfering; Signal Transduction; Taxoids

The present study investigated the effects of the multikinase inhibitor sorafenib on androgen-independent cancer cells viability and intracellular signaling. Human androgen-independent PC-3 prostate cancer cells were treated with sorafenib. At concentration that suppresses extracellular signal-regulated kinase phosphorylation, sorafenib treatment reduced the mitochondrial transmembrane potential. Sorafenib also down-modulated the levels of myeloid cell leukemia 1, survivin and cellular inhibitor of apoptosis protein 2. Sorafenib induced caspase-3 cleavage and the mitochondrial release of cytochrome c. However, no nuclear translocation of apoptosis inducing factor was detected after treatment and the pan-caspase inhibitor Z-VAD-FMK had an obvious protective effect against the drug. In conclusion, sorafenib induces apoptosis through a caspase-dependent mechanism with down-regulated anti-apoptotic proteins in androgen-independent prostate cancer cells in vitro.

Topics: Apoptosis; Benzenesulfonates; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Extracellular Signal-Regulated MAP Kinases; Humans; Inhibitor of Apoptosis Proteins; Male; Microtubule-Associated Proteins; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Niacinamide; Phenylurea Compounds; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Pyridines; Sorafenib; Survivin

Teriflunomide (TFN) is an inhibitor of de novo pyrimidine synthesis and the active metabolite of leflunomide. Leflunomide is prescribed to patients worldwide as an immunomodulatory and anti-inflammatory disease-modifying prodrug. Leflunomide inhibited the growth of human prostate cancer xenographs in mice, and leflunomide or TFN promoted cytostasis and/or apoptosis in cultured cells. These findings suggest that TFN could be useful in prostate cancer chemoprevention. We investigated the possible mechanistic aspects of this tenet by characterizing the effects of TFN using premalignant PWR-1E and malignant DU-145 human prostate epithelial cells. TFN promoted a dose- and time-dependent cytostasis or apoptosis induction in these cells. The cytostatic effects of TFN, which were reversible but not by the presence of excess uridine in the culture medium, included diminished cellular uridine levels, an inhibition in oxygen consumption, a suppression of reactive oxygen species (ROS) generation, S-phase cell cycle arrest, and a conspicuous reduction in the size and number of the nucleoli in the nuclei of these cells. Conversely, TFN's apoptogenic effects were characteristic of catastrophic mitochondrial disruption (i.e., a dissipation of mitochondrial inner transmembrane potential, enhanced ROS production, mitochondrial cytochrome c release, and cytoplasmic vacuolization) and followed by DNA fragmentation. The respiration-deficient derivatives of the DU-145 cells, which are also uridine auxotrophs, were markedly resistant to the cytostatic and apoptotic effects of TFN, implicating de novo pyrimidine synthesis and mitochondrial bioenergetics as the primary targets for TFN in the respiration competent cells. These mechanistic findings advocate a role for TFN and mitochondrial bioenergetics in prostate cancer chemoprevention.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; Caspases; Cell Line, Transformed; Cytochromes c; Dose-Response Relationship, Drug; Epithelial Cells; Flow Cytometry; Humans; Immunoblotting; Isoxazoles; Leflunomide; Male; Membrane Potential, Mitochondrial; Mitochondria; Oxygen Consumption; Prostate; Prostatic Neoplasms; Reactive Oxygen Species; Time Factors; Tumor Cells, Cultured

Progression of prostate cancer is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. To investigate the effect of quercetin on insulin-like growth factor signaling and apoptosis in androgen independent prostate cancer cells (PC-3), IGF-IR, PI-3K, p-Akt, Akt, cyclin D1, Bad, cytochrome c, PARP, caspases-9 and 10 protein levels were assessed by western blot analysis. Mitochondrial membrane potency was detected by rhodamine-123 staining. Quercetin induced caspase-3 activity assay was performed for activation of apoptosis. Further, RT-PCR was also performed for Bad, IGF-I, II, IR, and IGFBP-3 mRNA expression. Quercetin significantly increases the proapoptotic mRNA levels of Bad, IGFBP-3 and protein levels of Bad, cytochrome C, cleaved caspase-9, caspase-10, cleaved PARP and caspase-3 activity in PC-3 cells. IGF-IRβ, PI3K, p-Akt, and cyclin D1 protein expression and mRNA levels of IGF-I, II and IGF-IR were decreased significantly. Further, treatment with PI3K inhibitor (LY294002) and quercetin showed decreased p-Akt levels. Apoptosis is confirmed by loss of mitochondrial membrane potential in quercetin treated PC-3 cells. This study suggests that quercetin decreases the survival of androgen independent prostate cancer cells by modulating the expression of insulin-like growth factors (IGF) system components, signaling molecules and induces apoptosis, which could be very useful for the androgen independent prostate cancer treatment.

Topics: Androgens; Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Cytochromes c; Humans; Male; Membrane Potentials; Microscopy, Fluorescence; Prostatic Neoplasms; Quercetin; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Somatomedins

beta-Elemene, a natural compound extracted from over 50 different Chinese medicinal herbs and plants, has been effective in the treatment of hyperplastic and proliferative disorders such as prostatic hypertrophy, hysteromyoma and neoplasms. Our previous studies have demonstrated that beta-elemene exhibits strong inhibitory activity in ovarian cancer cells. The aim of the present study was to assess the effect of beta-elemene on prostate cancer cells as well as other types of tumour cells and to determine whether the effect of beta-elemene on prostate cancer cell death was mediated through the induction of apoptosis.. The MTT assay was used to evaluate the ability of beta-elemene to inhibit cellular proliferation in cancer cells. Cellular apoptosis was assessed by annexin V binding, TUNEL and ELISA-based assays. Caspase activity was measured using a caspases assay kit. The protein levels of Bcl-2, caspases, cytochrome c and poly(ADP-ribose) polymerase (PARP) were analysed by Western blotting.. Here, we showed that beta-elemene had an antiproliferative effect on androgen-insensitive prostate carcinoma DU145 and PC-3 cells. Treatment with beta-elemene also inhibited the growth of brain, breast, cervical, colon and lung carcinoma cells. The effect of beta-elemene on cancer cells was dose dependent, with IC50 values ranging from 47 to 95 microg/ml (230-465 microm). TUNEL assay and flow cytometric analysis using annxin V/propidium iodide staining revealed that the percentage of apoptotic prostate cancer cells was increased by beta-elemene in a dose- and time-dependent manner. Moreover, beta-elemene exposure resulted in a decreased Bcl-2 protein level, increased cytochrome c release, and activated PARP and caspase-3, -7, -9, and -10 in prostate cancer cells.. Overall, these findings suggest that beta-elemene exerts broad-spectrum antitumour activity against many types of solid carcinoma and supports a proposal of beta-elemene as a new potentially therapeutic drug for castration-resistant prostate cancer and other solid tumours.

Topics: Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; Lung Neoplasms; Male; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes; Time Factors; Uterine Cervical Neoplasms

In this study, we investigated the functional role of early growth response-1 (Egr1 gene) in the regulation of radiation-induced clonogenic inhibition and apoptosis in p53 wild-type and mutant prostate cancer cells 22Rv1 and DU145, respectively. 22Rv1 cells were more sensitive to irradiation compared with DU145 cells, and the sensitivity was enhanced by overexpression of EGR-1 in both cells. Dominant-negative EGR-1 mutant (dnEGR-1) or repressor of EGR-1, NGFIA binding protein 1 (NAB1), increased radioresistance of these cells. Significant activation of caspases 3 and 9 and Bcl2-associated X (Bax) with increased poly(ADP-ribose) polymerase (PARP) cleavage and cytochrome c release was observed in radiation-exposed EGR-1 overexpressing cells. Gel shift analysis and chloramphenicol acetyl transferase (CAT) reporter assays indicate that EGR-1 transactivates the promoter of the Bax gene. Interaction of EGR-1 and Yes kinase-associated protein 1 (YAP-1) through the WW domain of YAP-1 enhances the transcriptional activity of EGR-1 on the Bax promoter as shown by chromatin immunoprecipitation and reporter assays. Irradiation of PC3 cell xenografts that were treated with adenoviral EGR-1 showed significant regression in tumor volume. These findings establish the radiation-induced pro-apoptotic action of EGR-1, in a p53-independent manner, by directly transactivating Bax, and prove that alters the B-cell CLL/lymphoma 2 (Bcl-2)/Bax ratio as one of the mechanisms resulting in significant activation of caspases, leading to cell death through the novel interaction of EGR-1 with YAP-1.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Chloramphenicol O-Acetyltransferase; Colony-Forming Units Assay; Cytochromes c; Early Growth Response Protein 1; Electrophoretic Mobility Shift Assay; Humans; Immunoprecipitation; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphoproteins; Poly(ADP-ribose) Polymerases; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Array Analysis; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factors; Transcriptional Activation; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured; Up-Regulation; X-Rays; YAP-Signaling Proteins

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.

Topics: Apoptosis; Cytochromes c; Humans; Male; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Phytotherapy; Plant Extracts; Prostatic Neoplasms; Serenoa; Tumor Cells, Cultured

It has been recently demonstrated that small gold compounds could have a potential anti-tumoral activity. Here, we report that aurothiomalate (ATM), a gold compound already used in clinical therapy for the treatment of rheumatoid arthritis, has a pro-apoptotic effect in aggressive prostate cancer (PC3U) cells. In contrast, treatment of human primary epithelial prostate cells (PrEC) with ATM did not cause apoptosis. We demonstrated that ATM is able to disrupt the PKCiota-Par6 complex in PC3U cells and that this disruption leads to the activation of ERK in a dose-dependent manner. Interestingly, we also showed that ERK acts upstream of the activation of caspase 3, leading to apoptosis. ATM treatment also causes activation of p38 and JNK MAP kinases. Moreover we could link ATM treatment to activation of the mitochondrial or so called intrinsic pathway, as we observed release of cytochrome c from mitochondria to cytoplasm, suggesting that the mitochondrial pathway is involved in the pro-apoptotic effect mediated by ATM. Taken together our data suggest that ATM could be a new promising drug for the treatment of advanced prostate cancer.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cytochromes c; Cytoplasm; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Gold Sodium Thiomalate; Humans; Male; Mitochondria; p38 Mitogen-Activated Protein Kinases; Prostatic Neoplasms

Gonadotropin-releasing hormone-I (GnRH-I) is known to directly regulate prostate cancer cell proliferation. However, the role of GnRH-II in prostate cancer is unclear. Here, we investigated the effect of the GnRH-II antagonist trptorelix-1 (Trp-1) on growth of PC3 prostate cancer cells. Trp-1 induced growth inhibition of PC3 cells in vitro and inhibited growth of PC3 cells xenografted into nude mice. FITC-N3, an FITC-conjugated Trp-1 analogue, was largely present in the mitochondria of prostate cancer cells, but not in other cells that are not derived from the prostate. Trp-1-induced PC3 growth inhibition was associated with decreased mitochondrial membrane potential and increased levels of mitochondrial and cytosolic reactive oxygen species (ROS). Growth inhibition was partially prevented by cotreating cells with N-acetyl cysteine, an antioxidant. Cytochrome c release and caspase-3 activation were not detected in Trp-1-treated cells. However, Trp-1 induced autophagosome formation, as seen by increased LysoTracker staining and recruitment of microtubule-associated protein 1 light chain 3 to these new lysosomal compartments. Trp-1-induced autophagy was accompanied by decreased AKT phosphorylation and increased c-Jun NH(2) terminal kinase phosphorylation, two events known to be linked to autophagy. Taken together, these data suggest that Trp-1 directly induces mitochondrial dysfunction and ROS increase, leading to autophagy of prostate cancer cells. GnRH-II antagonists may hold promise in the treatment of prostate cancer.

Topics: Animals; Autophagy; Caspase 3; Cell Growth Processes; Cell Line, Tumor; Cytochromes c; Female; Gonadotropin-Releasing Hormone; HeLa Cells; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Oligopeptides; Prostatic Neoplasms; Reactive Oxygen Species

We observed that treatment of prostate cancer cells for 24 h with magnolol, a phenolic component extracted from the root and stem bark of the oriental herb Magnolia officinalis, induced apoptotic cell death in a dose- and time-dependent manner. A sustained inhibition of the major survival signal, Akt, occurred in magnolol-treated cells. Treatment of PC-3 cells with an apoptosis-inducing concentration of magnolol (60 microM) resulted in a rapid decrease in the level of phosphorylated Akt leading to inhibition of its kinase activity. Magnolol treatment (60 microM) also caused a decrease in Ser((136)) phosphorylation of Bad (a proapoptotic protein), which is a downstream target of Akt. Protein interaction assay revealed that Bcl-xL, an anti-apoptotic protein, was associated with Bad during treatment with magnolol. We also observed that during treatment with magnolol, translocation of Bax to the mitochondrial membrane occurred and the translocation was accompanied by cytochrome c release, and cleavage of procaspase-8, -9, -3, and poly(ADP-ribose) polymerase (PARP). Similar results were observed in human colon cancer HCT116Bax(+/-) cell line, but not HCT116Bax(-/-) cell line. Interestingly, at similar concentrations (60 microM), magnolol treatment did not affect the viability of normal human prostate epithelial cell (PrEC) line. We also observed that apoptotic cell death by magnolol was associated with significant inhibition of pEGFR, pPI3K, and pAkt. These results suggest that one of the mechanisms of the apoptotic activity of magnolol involves its effect on epidermal growth factor receptor (EGFR)-mediated signaling transduction pathways.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; bcl-X Protein; Biphenyl Compounds; Cell Line, Tumor; Cytochromes c; Epidermal Growth Factor; ErbB Receptors; Humans; Lignans; Male; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction

Green tea and its major constituent epigallocatechin gallate (EGCG) are known for their chemopreventive effects including those against prostate cancer, which could be mediated by metal ions. Zn(2+) is an essential trace element that is required for human health and plays an important role in the normal function of the prostate gland. In the present study, the effect of EGCG on cell membrane and mitochondria of PC-3 (prostate carcinoma) cells in the presence and absence of Zn(2+) was studied. These studies revealed that EGCG, Zn(2+), or EGCG + Zn(2+) affected the morphology of PC-3 cells and induced apoptosis in PC-3 cells. It was observed that effects of treatment with EGCG, Zn(2+), or EGCG + Zn(2+)on mitochondria showed EGCG + Zn(2+) > Zn(2+) > EGCG, including cytochrome C release from the intermembrane space into the cytosol, inhibited the synthesis of ATP, loss of mitochondrial membrane potential, and activation of caspase-9. However, the order of effect on depressing membrane fluidity of PC-3 cells was EGCG > EGCG + Zn(2+) > Zn(2+). In summary, these findings suggest that EGCG, Zn(2+), and EGCG + Zn(2+) induce necrosis or apoptosis of PC-3 cells through mitochondria-mediated apoptotic pathway and free Zn(2+)-enhanced effects of EGCG on PC-3 cells due to its interactions with mitochondria.

Topics: Adenosine Triphosphate; Apoptosis; Caspase 9; Catechin; Cell Division; Cell Line, Tumor; Cytochromes c; Drug Synergism; Humans; Male; Membrane Fluidity; Membrane Potential, Mitochondrial; Mitochondria; Prostatic Neoplasms; Zinc

Ion channel modulators have been previously associated with cell proliferation and cell death in human cancer cell lines.. The effects of riluzole, an ion channel modulator, on cell proliferation, apoptosis and the apoptotic pathway in the LNCaP and C4-2 prostate cancer cell lines were investigated.. Riluzole inhibited DNA synthesis and increased apoptotic cells in both cell lines. The activities of caspase-3, -8 and -9 were significantly increased, and caspase inhibitors for caspase-3, -8 and -9 significantly rescued the cell viability of both carcinoma cell lines treated with riluzole. However, a change in mitochondrial membrane potential, release of cytochrome c and cleavage of Bid were not observed in the riluzole-treated cells. Riluzole significantly induced elevation of caspase-4 activity, fluorescence indicating cytosolic calcium, and morphological changes in endoplasmic reticulum (ER) as observed by transmission electron microscopy.. Riluzole induces inhibition of DNA synthesis and apoptotic cell death via ER stress in both the LNCaP and C4-2 prostate cancer cell lines.

Topics: Apoptosis; Caspase Inhibitors; Caspases; Cell Proliferation; Cytochromes c; Endoplasmic Reticulum; Excitatory Amino Acid Antagonists; Humans; Male; Membrane Potential, Mitochondrial; Prostatic Neoplasms; Riluzole; Signal Transduction; Tumor Cells, Cultured

To study the effect of hydrogen peroxide (H(2)O(2)) on radiation-induced apoptosis in human prostate cancer PC-3 cells.. At 4h before the irradiation, PC-3 cells were exposed to 10mM ammonium chloride (NH(4)Cl) concentrations. Subsequently, cells were exposed to 0.1mM H(2)O(2) just before the irradiations, which were administered with 10-MV X-rays at doses of 10 Gy.. The percentage of apoptotic cells at 48 h after X-irradiation alone, H(2)O(2) alone, and combined X-irradiation and H(2)O(2) was 1.85%, 4.85%, and 28.4%, respectively. With use of combined X-irradiation and H(2)O(2), production of reactive oxygen species (ROS) occurred 4h after the irradiation. This resulted in lysosomal rupturing, mitochondrial fragmentation, and the release of cytochrome c into the cytoplasm from the mitochondria. In contrast, when cells were exposed to NH(4)Cl before the X-irradiation and H(2)O(2) administration, apoptosis was almost completely suppressed, ROS production did not occur, lysosomal rupture and mitochondrial fragmentation were blocked, and cytochrome c was not released.. Hydrogen peroxide strongly enhanced lysosome-dependent radiation-induced apoptosis in human prostate cancer PC-3 cells. A combined use of X-rays and H(2)O(2) can also injure the mitochondrial cytoplasmic organelles and lead to the production of ROS that in and of itself might possibly induce apoptosis.

Topics: Ammonium Chloride; Apoptosis; Cell Line, Tumor; Cytochromes c; Humans; Hydrogen Peroxide; Lysosomes; Male; Mitochondria; Organelles; Oxidants; Oxidative Stress; Prostatic Neoplasms; Radiotherapy Dosage; Reactive Oxygen Species

Piceatannol (trans-3,4,3',5'-tetrahydroxystilbene) is a polyphenol that is found in grapes, red wine, Rheum undulatum, and the seeds of Euphorbia lagascae. It has been previously reported that piceatannol inhibits the proliferation of a variety of cancer cell types. In the present study, we assessed the effects of piceatannol on the growth of androgen-insensitive DU145 prostate cancer cells at concentrations of 1-10 micromol/L. Piceatannol reduced the viable numbers and increased the numbers of apoptotic DU145 cells in a dose-dependent manner. Western blot analysis revealed that piceatannol increased the protein levels of cleaved caspase-8, -9, -7, and -3 and cleaved poly(ADP-ribose) polymerase (PARP). Piceatannol increased mitochondrial membrane permeability and cytochrome c release from the mitochondria to the cytosol. Piceatannol induced an increase in the levels of truncated Bid, Bax, Bik, Bok, and Fas but caused a decrease in the levels of Mcl-1 and Bcl-xL. Caspase-8 and -9 inhibitors mitigated piceatannol-induced apoptosis. The caspase-8 inhibitor suppressed the piceatannol-induced cleavage of Bid, caspase-3, and PARP. These results indicate that piceatannol induces apoptosis via the activation of the death receptor and mitochondrial-dependent pathways in prostate cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Humans; Intracellular Signaling Peptides and Proteins; Male; Mitochondrial Membranes; Mitochondrial Proteins; Phytotherapy; Plant Extracts; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Signal Transduction; Stilbenes; Vitis

Prostate cancer is the leading cause of cancer-related death in men, incidences of which are increasing gradually in India. Protein kinase C (PKC), an enzyme, gets overexpressed in prostate cancer and results in a resistance to chemotherapy. Telomerase, a reverse transcriptase, is highly activated in prostate cancer cells. Both of these enzymes can be considered as potential molecular markers for prostate cancer. The present study investigates the effects of natural isothiocyanate phenethyl isothiocyanate (PEITC) in modulating the activities of PKC and telomerase in the androgen-independent human prostate adenocarcinoma (PC-3) cell line. We observed that PEITC downregulated the antiapoptotic isoforms (PKC alpha and epsilon) efficiently and zeta moderately. Basal level of PKC delta, a proapoptotic form, was very poor and its modulation was not significant. PEITC also inhibited the activity of telomerase. Studies were conducted to measure the degree of apoptotic cell death induced either by PEITC alone or in combination with adriamycin or etoposide. Apoptosis was evident from the release of mitochondrial cytochrome c, apoptotic index, and by the induction of caspases 3 and 8. PEITC exhibited remarkable efficacy in sensitizing PC-3 cells to undergo cell death by adriamycin and etoposide, which might prove to be of considerable value in synergistic therapy of cancer.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Caspases; Cell Line, Tumor; Cytochromes c; Down-Regulation; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; Humans; Isothiocyanates; Male; Prostatic Neoplasms; Protein Kinase C; Telomerase

The physical manifestations of aging reflect a loss of homeostasis that effects molecular, cellular and organ system functional capacity. As a sentinel homeostatic pathway, changes in apoptosis can have pathophysiological consequences in both aging and disease. To assess baseline global apoptosis balance, sera from 204 clinically normal subjects had levels of sFas (inhibitor of apoptosis), sFasL (stimulator of apoptosis), and total cytochrome c (released from cells during apoptosis) measured. Serum levels of sFas were significantly higher while sFasL and cytochrome c levels were lower in men compared to women. With increasing age there was a decrease in apoptotic markers (cytochrome c) and pro-apoptotic factors (sFasL) and an increase in anti-apoptotic factors (sFas) in circulation. The observed gender differences are consistent with the known differences between genders in mortality and morbidity. In a separate cohort, subjects with either breast (n = 66) or prostate cancer (n = 38) exhibited significantly elevated sFas with reduced sFasL and total cytochrome c regardless of age. These markers correlated with disease severity consistent with tumor subversion of apoptosis. The shift toward less global apoptosis with increasing age in normal subjects is consistent with increased incidence of diseases whose pathophysiology involves apoptosis dysregulation.

Topics: Adult; Aged; Aged, 80 and over; Aging; Apoptosis; Biomarkers; Body Mass Index; Breast Neoplasms; Cytochromes c; Fas Ligand Protein; fas Receptor; Female; Humans; Male; Middle Aged; Neoplasm Staging; Neoplasms; Prostatic Neoplasms; Sex Characteristics; Young Adult

Prostate cancer is a major cause of cancer mortality in American males. Once prostate cancer has metastasized, there is currently no curative therapy available. The development of effective agents is therefore a continuing effort to combat this disease. In the present study, the effects and potential mechanisms of NSC606985 (NSC), a water-soluble camptothecin analog, in prostate cancer cells were investigated.. Prostatic tumor cells, DU-145, LNCaP and PC-3, were used for the study. Cell proliferation, cell cycle, cell apoptosis and caspase 3/7 activity were determined in the presence or absence of NSC. The levels of Bax and Bak, and the release of cytochrome c from mitochondria were analyzed by Western blot.. Treatment with NSC at nanomolar concentrations produced a time- and dose-dependent decrease in viable cell numbers of multiple prostate cancer cells. In DU-145 cells, NSC produced a time-and dose-dependent induction of cell apoptosis and cell cycle arrest as evidenced by cell morphological changes, increases in S-phase and sub-G1 cell fractions, an elevation of caspase 3/7 activity, DNA fragmentation and apoptotic cells. NSC increased the levels of apoptotic proteins, Bax and Bak, and induced a release of cytochrome c from mitochondria to cytosol in DU-145 cells. Co-administration of Z-VAD-FMK, a pan-caspase inhibitor, blocked NSC-induced caspase 3/7 activity and cell apoptosis without affecting NSC-induced cell cycle arrest. In contrast, co-administration of a PKCdelta inhibitor, rottlerin, had no significant effect on NSC induction of caspase activity, and slightly potentiated NSC-induced cell death. Furthermore, like camptothecin, a mutation of topoisomerase 1 that prevents the binding of camptothecin to the enzyme completely abolished the NSC effect in DU-145 cells.. The data obtained suggest that NSC is able to decrease cell growth, induce cell apoptosis and cause growth arrest in prostatic tumor cells, which may involve an interaction with topoisomerase 1 and an activation of mitochondrial apoptotic pathway.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Camptothecin; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Flow Cytometry; Humans; Male; Prostatic Neoplasms

Development of effective agents for treatment of hormone-refractory prostate cancer (HRPC) has become a national medical priority. D-Allose is a monosaccharide (C-3 epimer of glucose) distributed rarely in nature; because of its scarcity and cost, the biological effect has hardly been studied. In the present study, we demonstrated the inhibitory action of D-allose on proliferation of human HRPC cell lines, DU145 and PC-3 in a dose- and time-dependent manner, while human normal prostate epithelial (NPE) cell line, PrEC showed no remarkable effect. In vitro treatment of D-allose resulted in the alteration of Bcl-2/Bax ratio in favor of apoptosis (programmed cell death, PCD) in both the HRPC cell lines, which was associated with the lowering of mitochondrial transmembrane potential (Deltapsi(m)) and the release of cytochrome C (cyt C), the cleavage of caspase 3 and poly (ADP-ribose) polymerase (PARP), and the elevation of calcium concentration in cytosol ([Ca(2+)](c)). D-Allose also induced G1 phase arrest of the cell cycle in DU145 cell line. This study for the first time suggested the antiproliferative effect of D-allose through induction of PCD in HRPC cell lines, which could be due to the modulation of mitochondria mediated intrinsic apoptotic pathway.

Topics: Androgens; Apoptosis; bcl-2-Associated X Protein; Calcium Signaling; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Drug Screening Assays, Antitumor; G1 Phase; Gene Expression Regulation, Neoplastic; Glucose; Humans; Male; Membrane Potential, Mitochondrial; Models, Biological; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; RNA, Messenger

Through cytotoxicity screening with naphthoquinone derivatives, a novel compound 6-(1-oxoallkyl)-5,8-dimethoxy-1,4-naphthoquinone-S17 (DMNQ-S17) showed its potency against human myeloid leukemia U937 cells. Thus, to elucidate the apoptotic mechanism of DMNQ-S17, this study was performed in myeloid leukemia U937 cells by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay, eletrophoretic mobility shift assay (EMSA), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay, 4',6-diamidino-2-phenylindole (DAPI) staining, and Western blotting. In the present study, DMNQ-S17 inhibited constitutive NF kappaB activation and its transcriptional activity in U937 cells. In addition, DMNQ-S17 induced apoptotic features such as apoptotic bodies, cell shrinkage and chromatin condensation in U937 cells. Consistently, flow cytometric analysis showed that DMNQ-S17 increased sub-G1 portion and TUNEL positive cells in a concentration-dependent manner. Furthermore, DMNQ-S17 effectively attenuated mitochondrial membrane potential, released cytochrome C, activated caspase-3 expression, and cleaved poly (ADP-ribose) polymerase (PARP). Reversely, caspase-3 and -9 inhibitors also blocked the DMNQ-S17 induced caspase-3 activation and PARP cleavage in U937 cells. Taken together, these findings suggest that DMNQ-S17 can be a potent anticancer candidate for myeloid leukemias by the suppression of NF-kappaB activation leading to the activation of caspase-3 in human myeloid leukemia U937 cells.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspase Inhibitors; Cytochromes c; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Fluorescent Dyes; Humans; In Situ Nick-End Labeling; Indoles; Leukemia, Myeloid; Lymphoma, Large B-Cell, Diffuse; Male; Membrane Potential, Mitochondrial; Naphthoquinones; NF-kappa B; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; U937 Cells

We examined the antiproliferation effect of Jaceosidin (4', 5, 7-trihydroxy-3', 6-dimethoxyflavone) isolated from the herb of Artemisia vestita Wall on several human cancer cell lines. Jaceosidin significantly reduced the proliferation of CAOV-3, SKOV-3, HeLa, and PC3 cells in a concentration-dependent manner. A time-dependent inhibition was also observed in CAOV-3 cells by Jaceosidin. By flow cytometric analysis, we found that Jaceosidin treatment resulted in an increased apoptosis in CAOV-3 cells. The cells treated with Jaceosidin exhibited a decreased mitochondrial membrane potential. Jaceosidin also increased the level of cleaved caspase-9 and induced the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP), while caspase-3 inhibitor Z-DEVD-FMK significantly reversed the proapoptotic effect of Jaceosidin in CAOV-3 cells. Moreover, Jaceosidin elevated the level of cytochrome c in cytosol. These findings suggest that the anticancer effect of Jaceosidin may be contributed by an induction of apoptosis involving cytochrome c release from mitochondria to cytosol.

Topics: Apoptosis; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Proliferation; Cytochromes c; Female; Flavonoids; HeLa Cells; Humans; Male; Membrane Potential, Mitochondrial; Mitochondria; Oligopeptides; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms

Saussurea lappa (SL) is a plant regularly utilized in traditional herbal medicine, and in vitro cell culture studies have demonstrated that SL has anti-ulcer, anti-inflammatory, and anti-tumor properties. In order to explore the possibility that SL exerts chemopreventive effects in androgen-independent prostate cancer, we attempted to determine whether the hexane extract of SL (HESL) induces apoptosis of DU145 cells, as well as the mechanisms underlying this effect. HESL substantially reduced the number of viable cells and induced apoptosis in DU145 cells in a dose-dependent manner. HESL-induced the cleavage of poly (ADP-ribose) polymerase (PARP) and caspases 8, 9, 7, and 3. HESL increased the protein levels of Bax, Bak, Bok, Bik, truncated Bid (t-Bid), and Bmf with a concomitant increase in the permeability of the mitochondrial membrane and in the release of cytochrome c from the mitochondria. The active fraction of HESL was isolated by column chromatography and the structure of the active compound dehydrocostus lactone (DHCL) was identified via (1)H NMR and (13)C NMR. DHCL promoted apoptosis with increased activation of caspases 8, 9, 7, 3, enhanced PARP cleavage, decreased Bcl-xL expression and increased levels of Bax, Bak, Bok, Bik, Bmf, and t-Bid. We have demonstrated that HESL and its active principle, DHCL, inhibit cell growth and induce apoptosis in DU145 cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspases; Cell Cycle; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Hexanes; Humans; Indicators and Reagents; Lactones; Male; Membrane Potentials; Mitochondrial Membranes; Plant Extracts; Plant Roots; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Saussurea; Sesquiterpenes; Solvents

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

Phytochemicals show promise as potential chemopreventive or chemotherapeutic agents against various cancers. Here we report the chemotherapeutic effects of berberine, a phytochemical, on human prostate cancer cells. The treatment of human prostate cancer cells (PC-3) with berberine induced dose-dependent apoptosis but this effect of berberine was not seen in non-neoplastic human prostate epithelial cells (PWR-1E). Berberine-induced apoptosis was associated with the disruption of the mitochondrial membrane potential, release of apoptogenic molecules (cytochrome c and Smac/DIABLO) from mitochondria and cleavage of caspase-9,-3 and PARP proteins. This effect of berberine on prostate cancer cells was initiated by the generation of reactive oxygen species (ROS) irrespective of their androgen responsiveness, and the generation of ROS was through the increased induction of xanthine oxidase. Treatment of cells with allopurinol, an inhibitor of xanthine oxidase, inhibited berberine-induced oxidative stress in cancer cells. Berberine-induced apoptosis was blocked in the presence of antioxidant, N-acetylcysteine, through the prevention of disruption of mitochondrial membrane potential and subsequently release of cytochrome c and Smac/DIABLO. In conclusion, the present study reveals that the berberine-mediated cell death of human prostate cancer cells is regulated by reactive oxygen species, and therefore suggests that berberine may be considered for further studies as a promising therapeutic candidate for prostate cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Berberine; Caspase 3; Caspase 9; Cell Line, Tumor; Collagen Type XI; Cytochromes c; Dose-Response Relationship, Drug; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Oxidative Stress; Prostatic Neoplasms; Reactive Oxygen Species

The aim of this study was to investigate the effect of quercetin, a flavonoid, on the apoptotic pathway in a human prostate cell line (LNCaP). We observed that treatment of cells for 24h with quercetin-induced cell death in a dose-dependent manner. A sustained inhibition of the major survival signal, Akt, occurred in quercetin-treated cells. Treatment of LNCaP cells with an apoptosis inducing concentration of quercetin (100 microM) resulted in a rapid decrease in the inhibitory Ser473 phosphorylation of Akt leading to inhibition of its kinase activity. Quercetin treatment (100 microM) also caused a decrease in Ser136 phosphorylation of Bad, which is a downstream target of Akt. Protein interaction assay revealed that during treatment with quercetin, Bcl-xL dissociated from Bax and then associated with Bad. Our results also show that quercetin decreases the Bcl-xL:Bax ratio and increases translocation and multimerization of Bax to the mitochondrial membrane. The translocation is accompanied by cytochrome c release, and procaspases-3, -8 and -9 cleavage and increased poly(ADP-ribose) polymerase (PARP) cleavage. Similar results were observed in human colon cancer HCT116Bax+/+ cell line, but not HCT116Bax-/- cell line. Interestingly, at similar concentrations (100 microM), quercetin treatment did not affect the viability or rate of apoptosis in normal human prostate epithelial cell line (PrEC) and rat prostate epithelial cell line (YPEN-1). Our results indicate that the apoptotic processes caused by quercetin are mediated by the dissociation of Bax from Bcl-xL and the activation of caspase families in human prostate cancer cells.

Topics: Annexin A5; Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; bcl-X Protein; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytosol; Humans; Male; Mitochondria; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Quercetin

p66Shc is shown to negatively regulate the life span in mice through reactive oxygen species (ROS) production. Recent reports, however, revealed that p66Shc protein level is significantly elevated in several human cancer tissues and growth-stimulated carcinoma cells, suggesting a mitogenic and carcinogenic role for p66Shc. In this communication, we demonstrate for the first time that p66Shc mediates androgenic growth signals in androgen-sensitive human prostate cancer cells through mitochondrial ROS production. Growth stimulation of prostate cancer cells with 5alpha-dihydrotestosterone (DHT) is accompanied by increased p66Shc level and ROS production, which is abolished by antioxidant treatments. However, antioxidant treatments do not affect the transcriptional activity of androgen receptor (AR) as observed by its inability to block DHT-induced prostate-specific antigen expression, an AR-dependent correlate of prostate cancer progression. Elevated expression of p66Shc by cDNA transfection increases the basal cell proliferation and, thus, reduces additional DHT-induced cell proliferation. Furthermore, DHT increases the translocation of p66Shc into mitochondria and its interaction with cytochrome c. Conversely, both redox-negative p66Shc mutant (W134F), which is deficient in cytochrome c interaction, and p66Shc small interfering RNA decrease DHT-induced cell proliferation. These results collectively reveal a novel role for p66Shc-ROS pathway in androgen-induced prostate cancer cell proliferation and, thus, may play a role in early prostate carcinogenesis.

Topics: Adaptor Proteins, Signal Transducing; Androgens; Cell Proliferation; Cytochromes c; Disease Progression; Humans; Male; Mitochondria; Models, Biological; Neoplasms, Hormone-Dependent; Oxidation-Reduction; Prostatic Neoplasms; Protein Binding; Reactive Oxygen Species; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Testosterone; Tumor Cells, Cultured

Bcl-2 overexpression is an important mechanism underlying the aggressive behavior of prostate cancer cells and their resistance to radio- or chemotherapy. HA14-1, a recently discovered organic Bcl-2 inhibitor, potently induces apoptosis in various human cancer cells. Sequential exposure of radioresistant LNCaP (wild-type (wt) p53), LNCaP/Bcl-2 (wt p53) and PC3 (mutant p53) prostate cancer cells to a minimally cytotoxic concentration of 10 microM HA14-1 for 1 h followed by 1-6 Gy gamma radiation, resulted in a highly synergistic (combination index <1.0) induction of cell death as determined by an apoptosis assay at 72 h, and a clonogenicity assay at 12 days, after the initial treatment. The reverse treatment sequence did not cause a synergistic induction of cell death. When compared to individual treatments, cell death induced by the combined treatment was associated with dramatically increased reactive oxygen species (ROS) generation, c-Jun N-terminal kinase (JNK) activation, Bcl-2 phosphorylation, cytochrome c release, caspase-3 activation and DNA fragmentation. Exposure to either 200 microg/ml of the antioxidant alpha-tocopherol or 10 microM JNK inhibitor SP600125 before the combined treatment resulted in decreased activation of JNK and caspase-3 as well as decreased DNA fragmentation. However, treatment with the pancaspase inhibitor carbobenzoxyl-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone before the combined treatment inhibited apoptosis without affecting JNK activation, and this inhibitory effect was enhanced in the presence of alpha-tocopherol or SP600125. Taken together, our results indicate that HA14-1 potently sensitizes radioresistant LNCaP and PC3 cells to gamma radiation, regardless of the status of p53. ROS and JNK are important early signals that trigger both caspase-dependent and -independent cell death pathways and contribute to the apoptotic synergy induced by the combined treatments.

Topics: Apoptosis; Benzopyrans; Caspases; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Gamma Rays; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinases; Nitriles; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured; Tumor Suppressor Protein p53

In this study, we evaluated, for the first time, the antiproliferative and cytotoxic effects induced by a combination of a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib, with other chemotherapeutic drugs including estrogen receptor-beta (ER-beta) antagonist (tamoxifen) and topoisomerase II inhibitor (etoposide) on some metastatic prostate cancer (PC) cell lines. Immunohistochemial analyses revealed that EGFR expression was enhanced in 38% of primary prostatic adenocarcinomas (Gleason scores 4-10) as compared to the corresponding normal tissues of the same prostate gland from 32 PC patients. The RT-PCR and Western blot data have also indicated the higher expression levels of EGFR and ER-beta transcripts and proteins in metastatic LNCaP, DU145 and PC3 cells relative to nonmalignant normal prostate cells. Moreover, the results from MTT and FACS analyses revealed that the drugs, alone or in combination at lower concentrations, inhibited the growth of 17beta-estradiol (E2) plus EGF and serum-stimulated androgen-responsive LNCaP-C33 and androgen-independent LNCaP-C81, DU145 and PC3 cells. Importantly, the combined gefitinib, tamoxifen and etoposide also caused a higher rate of apoptotic death of PC cells as compared to single agents. The cytotoxic effects induced by these drugs in PC3 cells appear to be mediated through the accumulation of cellular ceramide and activation of caspase cascades via a mitochondrial pathway. These findings indicate that the combined use of inhibitors of EGF-EGFR and E2-ER-beta signaling with etoposide, which act by increasing the cellular ceramide levels and caspase activity, represents a promising strategy for a more effective treatment of metastatic PC forms.

Topics: Adenocarcinoma; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Caspases; Cell Proliferation; Cells, Cultured; Ceramides; Cytochromes c; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Etoposide; Flow Cytometry; Gefitinib; Humans; Immunoenzyme Techniques; Male; Membrane Potential, Mitochondrial; Neoplasms, Hormone-Dependent; Prostate; Prostatic Neoplasms; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tamoxifen

The epidermal growth factor receptor (EGFR) and hedgehog cascades provide a critical role in prostate cancer progression and contribute to the resistance to clinical therapies and disease relapse. Therefore, we evaluated, for the first time, the antiproliferative and cytotoxic effects induced by a combination of selective inhibitors of EGFR tyrosine kinase and smoothened hedgehog signaling element, gefitinib and cyclopamine, with a current chemotherapeutic drug used in the clinics, docetaxel, on some metastatic prostate cancer cell lines. Immunohistochemical analyses revealed that sonic hedgehog (SHH) expression was enhanced in 39% of primary prostatic adenocarcinomas (Gleason scores 4-10) compared with the corresponding normal tissues of the same prostate gland from 32 prostate cancer patients. The confocal microscopy and Western blot analyses have also indicated the high expression levels of SHH and EGFR in metastatic LNCaP, DU145, and PC3 cells. Moreover, the results revealed that the drugs, alone or in combination, at lower concentrations inhibited the growth of EGF plus SHH-stimulated and serum-stimulated androgen-responsive LNCaP-C33 and androgen-independent LNCaP-C81, DU145, and PC3 cells. Importantly, the combined docetaxel, gefitinib, and cyclopamine also caused a higher rate of apoptotic death of prostate cancer cells compared with individual agents. The cytotoxic effects induced by these drugs in PC3 cells seem to be mediated in part through the cellular ceramide production and activation of caspase cascades via a mitochondrial pathway and the release of cytochrome c into the cytosol. Additionally, the combined agents were more effective at suppressing the invasiveness of PC3 cells through Matrigel in vitro than the single drugs. These findings indicate that the combined use of inhibitors of EGF-EGFR and hedgehog signaling with docetaxel could represent a more promising strategy for treatment in patients with metastatic and androgen-independent prostate cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Cell Movement; Cell Proliferation; Cytochromes c; Docetaxel; ErbB Receptors; Gefitinib; Hedgehog Proteins; Humans; Immunohistochemistry; Male; Membrane Potential, Mitochondrial; Neoplasm Invasiveness; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Quinazolines; Signal Transduction; Taxoids; Tumor Cells, Cultured; Veratrum Alkaloids

The mammalian lignan enterolactone is a major metabolite of plant-based lignans that has been shown to inhibit the growth and development of prostate cancer. However, little is known about the mechanistic basis for its anticancer activity. In this study, we report that enterolactone selectively suppresses the growth of LNCaP prostate cancer cells by triggering apoptosis. Mechanistic studies showed that enterolactone-induced apoptosis was characterized by a dose-dependent loss of mitochondrial membrane potential, release of cytochrome c and cleavage of procaspase-3 and poly(ADP-ribose)-polymerase (PARP). Caspase dependence was indicated by the ability of the pan-caspase inhibitor z-VAD-fmk to attenuate enterolactone-mediated apoptosis. Mechanistic studies suggested roles for Akt, GSK-3beta, MDM2, and p53 in enterolactone-dependent apoptosis. Our findings encourage further studies of enterolactone as a promising chemopreventive agent against prostate cancer.

Topics: 4-Butyrolactone; Apoptosis; Caspases; Cell Survival; Cytochromes c; Enzyme Activation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunoblotting; In Situ Nick-End Labeling; Lignans; Male; Membrane Potential, Mitochondrial; Mitochondria; Phytoestrogens; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Our study has shown that the Amaryllidaceae isocarbostyril narciclasine induces marked apoptosis-mediated cytotoxic effects in human cancer cells but not in normal fibroblasts by triggering the activation of the initiator caspases of the death receptor pathway (caspase-8 and caspase-10) at least in human MCF-7 breast and PC-3 prostate carcinoma cells. The formation of the Fas and death receptor 4 (DR4) death-inducing signaling complex was clearly evidenced in MCF-7 and PC-3 cancer cells. Caspase-8 was found to interact with Fas and DR4 receptors on narciclasine treatment. However, narciclasine-induced downstream apoptotic pathways in MCF-7 cells diverged from those in PC-3 cells, where caspase-8 directly activated effector caspases such as caspase-3 in the absence of any further release of mitochondrial proapoptotic effectors. In contrast, in MCF-7 cells, the apoptotic process was found to require an amplification step that is mitochondria-dependent, with Bid processing, release of cytochrome c, and caspase-9 activation. It is postulated that the high selectivity of narciclasine to cancer cells might be linked, at least in part, to this activation of the death receptor pathway. Normal human fibroblasts appear approximately 250-fold less sensitive to narciclasine, which does not induce apoptosis in these cells probably due to the absence of death receptor pathway activation.

Topics: Amaryllidaceae Alkaloids; Antineoplastic Agents, Phytogenic; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carcinoma; Caspases; Cytochromes c; DNA Fragmentation; Drug Resistance, Neoplasm; Enzyme Activation; fas Receptor; Female; Fibroblasts; Humans; Male; Mitochondria; Narcissus; Neoplasm Proteins; Phenanthridines; Prostatic Neoplasms; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor

B cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)-only proteins of the Bcl-2 family are important functional adaptors that link cell death signals to the activation of Bax and/or Bak. The BH3-only protein Nbk/Bik induces cell death via an entirely Bax-dependent/Bak-independent mechanism. In contrast, cell death induced by the short splice variant of Bcl-x depends on Bak but not Bax. This indicates that Bak is functional but fails to become activated by Nbk. Here, we show that binding of myeloid cell leukemia 1 (Mcl-1) to Bak persists after Nbk expression and inhibits Nbk-induced apoptosis in Bax-deficient cells. In contrast, the BH3-only protein Puma disrupts Mcl-1-Bak interaction and triggers cell death via both Bax and Bak. Targeted knockdown of Mcl-1 overcomes inhibition of Bak and allows for Bak activation by Nbk. Thus, Nbk is held in check by Mcl-1 that interferes with activation of Bak. The finding that different BH3-only proteins rely specifically on Bax, Bak, or both has important implications for the design of anticancer drugs targeting Bcl-2.

Topics: Adenoviridae; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Benzimidazoles; Carbocyanines; Cell Line; Cell Line, Tumor; Cell Membrane Permeability; Cytochromes c; Fluorescent Dyes; HCT116 Cells; Humans; Kidney; Male; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Models, Biological; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Transgenes

Apoptin, a chicken anemia virus-derived protein, selectively induces apoptosis in transformed but not in normal cells, thus making it a promising candidate as a novel anticancer therapeutic. The mechanism of apoptin-induced apoptosis is largely unknown. Here, we report that contrary to previous assumptions, Bcl-2 and Bcl-xL inhibit apoptin-induced cell death in several tumor cell lines. In contrast, deficiency of Bax conferred resistance, whereas Bax expression sensitized cells to apoptin-induced death. Cell death induction by apoptin was associated with cytochrome c release from mitochondria as well as with caspase-3 and -7 activation. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad spectrum caspase inhibitor, was highly protective against apoptin-induced cell death. Apoptosis induced by apoptin required Apaf-1, as immortalized Apaf-1-deficient fibroblasts as well as tumor cells devoid of Apaf-1 were strongly protected. Thus, our data indicate that apoptin-induced apoptosis is not only Bcl-2- and caspase dependent, but also engages an Apaf-1 apoptosome-mediated mitochondrial death pathway.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-X Protein; Breast Neoplasms; Capsid Proteins; Caspase 3; Caspase 7; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochromes c; Enzyme Activation; Female; Fibroblasts; Humans; Intracellular Signaling Peptides and Proteins; Lymphoma, B-Cell; Male; Mitochondria; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Tumor Cells, Cultured

Berberine, a naturally occurring isoquinoline alkaloid, has been shown to possess anti-inflammatory and antitumor properties in some in vitro systems. Here, we report that in vitro treatment of androgen-insensitive (DU145 and PC-3) and androgen-sensitive (LNCaP) prostate cancer cells with berberine inhibited cell proliferation and induced cell death in a dose-dependent (10-100 micromol/L) and time-dependent (24-72 hours) manner. Treatment of nonneoplastic human prostate epithelial cells (PWR-1E) with berberine under identical conditions did not significantly affect their viability. The berberine-induced inhibition of proliferation of DU145, PC-3, and LNCaP cells was associated with G1-phase arrest, which in DU145 cells was associated with inhibition of expression of cyclins D1, D2, and E and cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 proteins, increased expression of the Cdk inhibitory proteins (Cip1/p21 and Kip1/p27), and enhanced binding of Cdk inhibitors to Cdk. Berberine also significantly (P < 0.05-0.001) enhanced apoptosis of DU145 and LNCaP cells with induction of a higher ratio of Bax/Bcl-2 proteins, disruption of mitochondrial membrane potential, and activation of caspase-9, caspase-3, and poly(ADP-ribose) polymerase. Pretreatment with the pan-caspase inhibitor z-VAD-fmk partially, but significantly, blocked the berberine-induced apoptosis, as also confirmed by the comet assay analysis of DNA fragmentation, suggesting that berberine-induced apoptosis of human prostate cancer cells is mediated primarily through the caspase-dependent pathway. The effectiveness of berberine in checking the growth of androgen-insensitive, as well as androgen-sensitive, prostate cancer cells without affecting the growth of normal prostate epithelial cells indicates that it may be a promising candidate for prostate cancer therapy.

Topics: Androgens; Antineoplastic Agents, Phytogenic; Apoptosis; Berberine; Carcinoma; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Activation; Epithelial Cells; G1 Phase; Humans; Male; Membrane Potentials; Mitochondria; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2

Isoliquiritigenin (ISL), a simple chalcone derivative, 4,2',4'-trihydroxychalcone, found in licorice, shallot and bean sprouts, has been reported to have chemoprotective effects. To examine the effects of ISL on the growth of prostate cancer cells, we cultured MAT-LyLu (MLL) rat and DU145 human prostate cancer cells with various concentrations (0-20 micromol/L) of ISL. Treatment of the cells with increasing concentrations of ISL led to dose-dependent decreases in the viable cell numbers in both DU145 and MLL cells (P<.05). Hoechst 33258 dye staining of condensed nuclei and annexin V binding to surface phosphatidylserine revealed increased numbers of apoptotic cells after ISL treatment. Western blot analysis revealed that ISL increased the levels of membrane-bound Fas ligand (FasL), Fas, cleaved casapse-8, truncated Bid (tBid), Bax and Bad in DU145 cells (P<.05). Isoliquiritigenin increased the percentage of cells with depolarized mitochondrial membranes, in a concentration-dependent manner (P<.05). Isoliquiritigenin induced the release of cytochrome c and Smac/Diablo from the mitochondria into the cytoplasm (P<.05). Isoliquiritigenin dose-dependently increased the levels of cleaved caspase-9, caspase-7, caspase-3 and poly(ADP-ribose) polymerase (P<.05). The present results indicate that ISL inhibits prostate cancer cell growth by the induction of apoptosis, which is mediated through mitochondrial events, which are associated with an evident disruption of the mitochondrial membrane potential, and the release of cytochrome c and Smac/Diablo, and the activation of caspase-9.

Topics: Animals; Apoptosis; Blotting, Western; Caspases; Cell Division; Cell Line, Tumor; Chalcone; Chalcones; Cytochromes c; Enzyme Activation; Fas Ligand Protein; fas Receptor; Flow Cytometry; Humans; Male; Membrane Glycoproteins; Membrane Potentials; Mitochondrial Membranes; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Necrosis Factors

Although the antitumor effect of alpha-tocopheryl succinate (vitamin E succinate) has been well demonstrated, its underlying mechanism remains elusive. This study provides evidence that inhibition of Bcl-xL/Bcl-2 function represents a major pathway whereby alpha-tocopheryl succinate mediates apoptosis induction in prostate cancer cells. In vitro data indicate that alpha-tocopheryl succinate was able to disrupt the binding of Bak BH3 peptide to Bcl-xL and Bcl-2 with IC50 of 26 microm, in line with its potency in antiproliferation. Treatment of PC-3 cells with this agent led to reduced association of Bcl-2 and Bcl-xL with Bak, leading to caspase-dependent apoptosis. Moreover, overexpression of Bcl-xL protected LNCaP cells from the apoptosis induction. This mechanistic finding provided a basis to develop potent Bcl-xL/Bcl-2 inhibitors. Docking of alpha-tocopheryl succinate into the Bak peptide-binding site indicates that it adopted a unique hairpin-shaped conformation for protein interactions. We rationalized that the hemisuccinate and the two proximal isopranyl units of the side chain played a crucial role in ligand anchoring and protein-ligand complex stabilization, respectively. However, exposure of the distal isopranyl unit to a polar environment might diminish the binding affinity of alpha-tocopheryl succinate. This premise was corroborated by a structure-activity analysis of a series of derivatives with truncated side chains and/or altered carboxyl terminus. This computer model predicted that the removal of the distal isopranyl unit from the side chain would improve binding affinity, leading to two agents with significantly higher potency in inhibiting Bak peptide binding and in suppressing prostate cancer cell proliferation.

Topics: Apoptosis; bcl-X Protein; Cell Proliferation; Cytochromes c; Humans; Male; Models, Molecular; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Tocopherols; Tumor Cells, Cultured; Vitamin E

Prostate cancer is one of the malignant tumors which exhibit resistance to anticancer drugs, at least in part due to enhanced antiapoptotic mechanisms. Therefore, the understanding of such mechanisms should improve the design of chemotherapy against prostate cancer. Galectin-3 (Gal-3), a multifunctional oncogenic protein involved in the regulation of tumor proliferation, angiogenesis, and apoptosis has shown antiapoptotic effects in certain cell types. Here, we show that the expression of exogenous Gal-3 in human prostate cancer LNCaP cells, which do not express Gal-3 constitutively, inhibits anticancer drug-induced apoptosis by stabilizing the mitochondria. Thus, Gal-3-negative cells showed 66.31% apoptosis after treatment with 50 micromol/L cis-diammine-dichloroplatinum for 48 hours, whereas two clones of Gal-3-expressing cells show only 2.92% and 1.42% apoptotic cells. Similarly, Gal-3-negative cells showed 43.8% apoptosis after treatment with 300 micromol/L etoposide for 48 hours, whereas only 15.38% and 14.51% of Gal-3-expressing LNCaP cells were apoptotic. The expression of Gal-3 stimulated the phosphorylation of Ser(112) of Bcl-2-associated death (Bad) protein and down-regulated Bad expression after treatment with cis-diammine-dichloroplatinum. Gal-3 also inhibited mitochondrial depolarization and damage after translocation from the nuclei to the cytoplasm, resulting in inhibition of cytochrome c release and caspase-3 activation. These findings indicate that Gal-3 inhibits anticancer drug-induced apoptosis through regulation of Bad protein and suppression of the mitochondrial apoptosis pathway. Therefore, targeting Gal-3 could improve the efficacy of anticancer drug chemotherapy in prostate cancer.

Topics: Apoptosis; bcl-Associated Death Protein; Caspase 3; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Nucleus; Cisplatin; Cytochromes c; Enzyme Activation; Etoposide; Galectin 3; Humans; Male; Mitochondria; Prostatic Neoplasms; Transfection

Advanced prostate cancer is not curable by current treatment strategies indicating a significant need for new chemotherapeutic options. Highly substituted ansa-titanocene compounds have shown promising cytotoxic activity in a range of cancers. The objectives of this study are to examine the effects of these titanocene compounds on prostate cancer cells. Prostate cell lines were treated with three novel titanocene compounds and compared to titanocene dichloride and cisplatin. Percent apoptosis, viability and cell cycle were assessed using propidium iodide DNA incorporation with flow cytometry. Cytochrome C was assessed by western blotting of mitochondrial and cytoplasmic fractions. Apoptosis Inducing Factor was assessed by confocal microscopy. These novel compounds induced more apoptosis compared to cisplatin in a dose dependent manner. Compound Y had the most significant effect on cell cycle and apoptosis. Despite the release of cytochrome C from the mitochondrial fraction there was no inhibition of apoptosis with the pan caspase inhibitor, ZVAD-FMK. AIF was shown to translocate from the cytosol to the nucleus mediating a caspase independent cell death. Bcl-2 over expressing PC-3 cells, which were resistant to cisplatin induced apoptosis, underwent apoptosis following treatment with all the titanocene compounds. This study demonstrates possible mechanisms by which these novel titanocene compounds can mediate their apoptotic effect in vitro. The fact that they can induce more apoptosis than cisplatin in advanced cancer cell lines would confer an advantage over cisplatin. They represent exciting new agents with future potential for the treatment of advanced prostate cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Cysteine Proteinase Inhibitors; Cytochromes c; Humans; Male; Molecular Structure; Organometallic Compounds; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Transfection

Prostate cancer is the second leading cancer diagnosed in elderly males in the Western world. Epidemiologic studies suggest that dietary modifications could be an effective approach in reducing various cancers, including prostate cancer, and accordingly cancer-preventive efficacy of dietary nutrients has gained increased attention in recent years. We have recently shown that grape seed extract (GSE) inhibits growth and induces apoptotic death of advanced human prostate cancer DU145 cells in culture and xenograft. Because prostate cancer is initially an androgen-dependent malignancy, here we used LNCaP human prostate cancer cells as a model to assess GSE efficacy and associated mechanisms. GSE treatment of cells led to their detachment within 12 hours, as occurs in anoikis, and caused a significant decrease in live cells mostly due to their apoptotic death. GSE-induced anoikis and apoptosis were accompanied by a strong decrease in focal adhesion kinase levels, but an increase in caspase-3, caspase-9, and poly(ADP-ribose) polymerase cleavage; however, GSE caused both caspase-dependent and caspase-independent apoptosis as evidenced by cytochrome c and apoptosis-inducing factor release into cytosol. Additional studies revealed that GSE causes DNA damage-induced activation of ataxia telangiectasia mutated kinase and Chk2, as well as p53 Ser(15) phosphorylation and its translocation to mitochondria, suggesting this to be an additional mechanism for apoptosis induction. GSE-induced apoptosis, cell growth inhibition, and cell death were attenuated by pretreatment with N-acetylcysteine and involved reactive oxygen species generation. Together, these results show GSE effects in LNCaP cells and suggest additional in vivo efficacy studies in prostate cancer animal models.

Topics: Acetylcysteine; Anoikis; Anticarcinogenic Agents; Apoptosis; Apoptosis Inducing Factor; Ataxia Telangiectasia Mutated Proteins; Caspases; Cell Cycle Proteins; Checkpoint Kinase 2; Cytochromes c; DNA-Binding Proteins; Focal Adhesion Kinase 1; Histones; Humans; Male; Plant Extracts; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The present study reports a novel response to sulforaphane, a highly promising anticancer constituent of several edible cruciferous vegetables, in PC-3 and LNCaP human prostate cancer cells involving induction of autophagy. Exposure of PC-3 and LNCaP cells to sulforaphane resulted in several specific features characteristic of autophagy, including appearance of membranous vacuoles in the cytoplasm as revealed by transmission electron microscopy and formation of acidic vesicular organelles as revealed by fluorescence microscopy following staining with the lysosomotropic agent acridine orange. The sulforaphane-induced autophagy was associated with up-regulation, processing, and recruitment to autophagosomes of microtubule-associated protein 1 light chain 3 (LC3), which is a mammalian homologue of the yeast autophagy regulating protein Apg8/Aut7p. Treatment of cells with a specific inhibitor of autophagy (3-methyladenine) attenuated localization of LC3 to autophagosomes but exacerbated cytosolic release of cytochrome c as well as apoptotic cell death as revealed by analysis of subdiploid fraction and cytoplasmic histone-associated DNA fragmentation. In conclusion, the present study indicates that induction of autophagy represents a defense mechanism against sulforaphane-induced apoptosis in human prostate cancer cells. To the best of our knowledge, the present study is the first published report to convincingly document induction of autophagy by an isothiocyanate class of dietary chemopreventive agent.

Topics: Anticarcinogenic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Cytochromes c; Humans; Isothiocyanates; Male; Microtubule-Associated Proteins; Prostatic Neoplasms; Sulfoxides; Thiocyanates

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in prostate cancer cells through DR4 and DR5 death receptors, but not in normal prostate cells, which do not express these receptors. Therefore, TRAIL has excellent potential to be a selective prostate cancer therapeutic agent with minimal toxic side effects. However, prostate cancer cells, as many other cancer types, develop resistance to TRAIL, and the underlying molecular mechanisms require further investigation. We hypothesize that selenium may sensitize TRAIL-resistant cells to undergo caspase-mediated apoptosis and increase therapeutic efficacy. Here, we report that TRAIL signaling in LNCaP prostate cancer cells stalled at downstream of caspase-8 and BID cleavage, as indicated by the lack of Bax translocation into mitochondria, and no subsequent activation of the caspase-9 cascade. Selenite induced a rapid generation of superoxide and p53 Ser(15) phosphorylation and increased Bax abundance and translocation into the mitochondria. Selenite and TRAIL combined treatment led to synergistic increases of Bax abundance and translocation into mitochondria, loss of mitochondrial membrane potential, cytochrome c release, and cleavage activation of caspase-9 and caspase-3. Inactivating p53 with a dominant-negative mutant abolished apoptosis without affecting superoxide generation, whereas a superoxide dismutase mimetic agent blocked p53 activation, Bax translocation to mitochondria, cytochrome c release, and apoptosis induced by selenite/TRAIL. In support of Bax as a crucial target for cross-talk between selenite and TRAIL pathways, introduction of Bax into p53 mutant DU145 cells enabled selenite to sensitize these cells for TRAIL-induced apoptosis. Taken together, the results indicate that selenite induces a rapid superoxide burst and p53 activation, leading to Bax up-regulation and translocation into mitochondria, which restores the cross-talk with stalled TRAIL signaling for a synergistic caspase-9/3 cascade-mediated apoptosis execution.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Caspases; Cytochromes c; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Humans; Male; Membrane Glycoproteins; Membrane Potentials; Mitochondrial Membranes; Prostatic Neoplasms; Reactive Oxygen Species; Sodium Selenite; Superoxides; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

A proteomic analysis was pursued to identify new signaling effectors of transforming growth factor beta1 (TGF-beta1) that serve as potential intracellular effectors of its apoptotic action in human prostate cancer cells. The androgen-sensitive and TGF-beta-responsive human prostate cancer cells, LNCaP T beta RII, were used as in vitro model. In response to TGF-beta, significant posttranslational changes in two proteins temporally preceded apoptotic cell death. TGF-beta mediated the nuclear export of prohibitin, a protein involved in androgen-regulated prostate growth, to the cytosol in the LNCaP T beta RII cells. Cofilin, a protein involved in actin depolymerization, cell motility, and apoptosis, was found to undergo mitochondrial translocation in response to TGF-beta before cytochrome c release. Loss-of-function approaches (small interfering RNA) to silence prohibitin expression revealed a modest decrease in the apoptotic response to TGF-beta and a significant suppression in TGF-beta-induced cell migration. Silencing Smad4 showed that the cellular localization changes associated with prohibitin and cofilin action in response to TGF-beta are independent of Smad4 intracellular signaling.

Topics: Cell Line, Tumor; Cell Survival; Cofilin 1; Cytochromes c; Dihydrotestosterone; Humans; Hydrogen-Ion Concentration; Male; Prohibitins; Prostatic Neoplasms; Proteome; Repressor Proteins; Signal Transduction; Transforming Growth Factor beta

The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.

Topics: Carrier Proteins; Caspase 2; Caspase 8; Caspase 9; Cell Death; Cell Line, Tumor; CRADD Signaling Adaptor Protein; Cytochromes c; Death Domain Receptor Signaling Adaptor Proteins; Enzyme Activation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Mitochondria; Prostatic Neoplasms; RNA, Small Interfering; Transfection

Ceramide analogs are potential chemotherapeutic agents. We report that a ceramide analog induces apoptosis in human prostate cancer cells. The ceramide analog induced cell death through an apoptotic mechanism, which was demonstrated by DNA fragmentation, the cleavage of poly ADP ribose polymerase (PARP), and a loss of membrane asymmetry. Treating the cells with ceramide analog resulted in the release of various proapoptotic mitochondrial proteins including cytochrome c and Smac/DIBLO into the cytosol, and a decrease in the mitochondrial membrane potential. In addition, the ceramide analog decreased the phospho-Akt and phospho-Bad levels. The expression of the antiapoptotic Bcl-2 decreased slightly with increasing Bax to Bcl-2 ratio. These results suggest that the ceramide analog induces apoptosis by regulating multiple signaling pathways that involve the mitochondrial pathway.

Topics: Apoptosis; Blotting, Western; Cell Proliferation; Ceramides; Cytochromes c; DNA Fragmentation; Genes, bcl-2; Humans; Male; Membrane Potentials; Mitochondria; Neoplasm Proteins; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Signal Transduction; Subcellular Fractions

TNFalpha-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in prostate cancer cells. However, some prostate cancer cells, such as LNCaP are resistant to TRAIL. In addition to the involvement of several pathways in the TRAIL-resistance of LNCaP, it has been shown that mitochondrial response to TRIAL is low in these cells. Therefore, in this study, using in vitro cell free and reconstitution models, we have demonstrated that mitochondria from these cells are capable of responding to apoptotic stimuli. Furthermore, experiments to determine the influence of cytochrome c on apoptotic response noted that incubation of cytosol with exogenous cytochrome c induced truncation of Bid. We have demonstrated that truncation of Bid by exogenous cytochrome c is mediated through the activation of caspases-9 and -3. Incubation of cytosol with recombinant caspases-9 and -3 in the absence or presence of inhibitors showed that activation of caspase-9, leading to the activation of caspase-3 was necessary for the truncation of Bid. Published results indicate that in apoptotic cells cytochrome c is released from the mitochondria in two installments, an early small amount and a late larger amount. Our results suggest that the initial release of cytochrome generates tBid that is capable of translocation into the mitochondria causing further release of cytochrome c. Thus, in addition to providing functional explanation for the biphasic release of cytochrome c from mitochondria, we demonstrate the presence of a feedback amplification of mitochondrial apoptotic signal.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Fractionation; Cell Line, Tumor; Cell-Free System; Cysteine Proteinase Inhibitors; Cytochromes c; Cytosol; Drug Resistance, Neoplasm; Humans; Male; Membrane Glycoproteins; Mitochondria; Models, Biological; Prostatic Neoplasms; Protein Transport; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Hypoxia occurs in association with cancer development, the result being a more aggressive and metastatic cancer phenotype. Hypoxia, which activates hypoxia-inducible factor-1 alpha (HIF-1alpha), is associated with a number of cellular changes including increased apoptotic resistance. The authors hypothesized that HIF-1alpha is central to the cell's ability to resist apoptosis induced during the hypoxia selection process.. PWR-1E, LNCaP, LNCaP-HOF, PC-3, and DU-145 cells were cultured in normoxic and hypoxic conditions. Apoptosis was assessed by propidium iodide DNA staining. Cleavage of specific substrates was used to assess caspase activity and Western blotting was used to assess mitochondrial release of cytochrome c and second mitochondria-derived activator caspase (SMAC)/Diablo. A dominant negative HIF-1alpha construct was transfected into the PC-3 and LNCaP cells to block HIF-1alpha activity.. PC-3 and DU-145 were resistant to apoptosis induced by exposure to hypoxia, but the PWR-1E and LNCaP cells were susceptible. This induction of apoptosis in the LNCaP cells was caspase dependent but independent of cytochrome c release. Blocking the activity of HIF-1alpha had no effect on increased apoptotic susceptibility in the PC-3 cells. LNCaP-HOF cells, which were resistant to hypoxia-induced apoptosis, showed no increase in HIF-1alpha expression or activity.. Apoptotic resistance is already established in cells that survive a hypoxic insult and whereas increased HIF-1alpha activity may be essential for the development of a more aggressive cancer phenotype, it may not be responsible for the initial selection of an apoptotic resistance phenotype.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Caspases; Cell Hypoxia; Cell Nucleus; Cytochromes c; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intracellular Signaling Peptides and Proteins; Male; Mitochondrial Proteins; Prostatic Neoplasms; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Sulforaphane, a constituent of many edible cruciferous vegetables, including broccoli, effectively suppresses proliferation of cancer cells in culture and in vivo by causing apoptosis induction, but the sequence of events leading to cell death is poorly defined. Here, we show that multidomain proapoptotic Bcl-2 family members Bax and Bak play a critical role in apoptosis induction by sulforaphane. This conclusion is based on the following observations: (a) sulforaphane treatment caused a dose- and time-dependent increase in the protein levels of both Bax and Bak and conformational change and mitochondrial translocation of Bax in SV40-transformed mouse embryonic fibroblasts (MEF) derived from wild-type mice to trigger cytosolic release of apoptogenic molecules (cytochrome c and Smac/DIABLO), activation of caspase-9 and caspase-3, and ultimately cell death; (b) MEFs derived from Bax or Bak knockout mice resisted cell death by sulforaphane, and (c) MEFs derived from Bax and Bak double knockout mice exhibited even greater protection against sulforaphane-induced cytochrome c release, caspase activation, and apoptosis compared with wild-type or single knockout cells. Interestingly, sulforaphane treatment also caused a dose- and time-dependent increase in the protein level of Apaf-1 in wild-type, Bax-/-, and Bak-/- MEFs but not in double knockout, suggesting that Bax and Bak might regulate sulforaphane-mediated induction of Apaf-1 protein. A marked decline in the protein level of X-linked inhibitor of apoptosis on treatment with sulforaphane was also observed. Thus, it is reasonable to postulate that sulforaphane-induced apoptosis is amplified by a decrease in X-linked inhibitor of apoptosis level, which functions to block cell death by inhibiting activities of caspases. In conclusion, the results of the present study indicate that Bax and Bak proteins play a critical role in initiation of cell death by sulforaphane.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; Apoptosis Regulatory Proteins; Apoptotic Protease-Activating Factor 1; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Carrier Proteins; Caspase 3; Caspase 9; Caspases; Cell Proliferation; Cells, Cultured; Chemoprevention; Complement Membrane Attack Complex; Complement System Proteins; Cytochromes c; Enzyme Activation; Epithelial Cells; Fibroblasts; Glycoproteins; Isothiocyanates; Male; Membrane Proteins; Mice; Mice, Knockout; Mitochondria; Mitochondrial Proteins; Prostate; Prostatic Neoplasms; Protein Conformation; Protein Transport; Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfoxides; Thiocyanates

We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.

Topics: Acetylcysteine; Animals; Antioxidants; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 8; Caspases; Cell Line, Tumor; Cells, Cultured; Cytochromes c; Enzyme Activation; Humans; Isothiocyanates; Male; Membrane Potentials; Mice; Mice, Knockout; Mitochondria; Models, Biological; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Sulfoxides; Thiocyanates

Anticancer agents that selectively kill tumor cells and spare normal tissues are urgently needed. Here, we engineered a cell-permeable peptidomimetic, shepherdin, modeled on the binding interface between the molecular chaperone Hsp90 and the antiapoptotic and mitotic regulator, survivin. Shepherdin makes extensive contacts with the ATP pocket of Hsp90, destabilizes its client proteins, and induces massive death of tumor cells by apoptotic and nonapoptotic mechanisms. Conversely, shepherdin does not reduce the viability of normal cells, and does not affect colony formation of purified hematopoietic progenitors. Systemic administration of shepherdin in vivo is well tolerated, and inhibits human tumor growth in mice without toxicity. Shepherdin could provide a potent and selective anticancer agent in humans.

Topics: Adenosine Triphosphate; Animals; Antennapedia Homeodomain Protein; Antimetabolites, Antineoplastic; Apoptosis; Benzoquinones; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Design; Enzyme Inhibitors; Female; Fibroblasts; Gene Products, tat; HeLa Cells; Homeodomain Proteins; HSP90 Heat-Shock Proteins; Humans; Inhibitor of Apoptosis Proteins; Lactams, Macrocyclic; Male; Mice; Mice, SCID; Microtubule-Associated Proteins; Models, Molecular; Molecular Mimicry; Neoplasm Proteins; Nuclear Proteins; Peptide Fragments; Prostatic Neoplasms; Protein Binding; Protein Conformation; Proto-Oncogene Proteins c-bcl-2; Rifabutin; Stem Cells; Survivin; Telomerase; Transcription Factors; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Caspases; Cell Proliferation; Collagen Type XI; Cytochromes c; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Ligands; Male; Membrane Glycoproteins; Prostatic Neoplasms; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/APO-2L), a member of the tumor necrosis factor (TNF) gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively kill tumor cells. Although microenvironments of solid tumors (hypoxia, nutrient deprivation, and low pH) often affect the effectiveness of chemotherapy, few studies have been reported on the relationship between tumor microenvironments and TRAIL. In this study, we investigated whether low extracellular pH affects TRAIL-induced apoptotic death. When human prostate carcinoma DU145 cells were treated with 200 ng/ml His-tagged TRAIL for 4 h, the survival was approximately 10% at pH 6.3-6.6 and 61.3% at pH 7.4. Similar results were observed in human colorectal carcinoma CX-1 cell line. The TRAIL-mediated activation of caspase, cytochrome c release, and poly (ADP-ribose) polymerase (PARP) cleavage was promoted at low extracellular pH. Immunoprecipitation followed by western blot analysis shows that low extracellular pH enhances the association of truncated Bid with Bax during treatment with TRAIL. Western blot analysis also shows that the low extracellular pH-enhanced TRAIL cytotoxicity does not involve modulation of the levels of TRAIL receptors (DR4, DR5, and DcR2), FLIP, inhibitor of apoptosis (IAP), and Bcl-2. Overexpression of Bcl-2 effectively prevented low extracellular pH-augmented TRAIL cytotoxicity. Taken together, we propose that TRAIL-mediated cytotoxicity is greatly enhanced in low pH environments by promoting caspase activation.

Topics: Adenocarcinoma; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carcinoma; Carrier Proteins; Caspases; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cytochromes c; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Male; Membrane Glycoproteins; Mitochondria; Models, Biological; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Curcumin (Diferuloylmethane) is a major chemical component of turmeric (curcuma longa) and is used as a spice to give a specific flavor and yellow color in Asian food. Curcumin exhibits growth inhibitory effects in a broad range of tumors as well as in TPA-induced skin tumors in mice. This study was undertaken to investigate the radiosensitizing effects of curcumin in p53 mutant prostate cancer cell line PC-3. Compared to cells that were irradiated alone (SF(2)=0.635; D(0)=231 cGy), curcumin at 2 and 4 microM concentrations in combination with radiation showed significant enhancement to radiation-induced clonogenic inhibition (SF(2)=0.224: D(0)=97 cGy and SF(2)=0.080: D(0)=38 cGy) and apoptosis. It has been reported that curcumin inhibits TNF-alpha-induced NFkappaB activity that is essential for Bcl-2 protein induction. In PC-3 cells, radiation upregulated TNF-alpha protein leading to an increase in NFkappaB activity resulting in the induction of Bcl-2 protein. However, curcumin in combination with radiation treated showed inhibition of TNF-alpha-mediated NFkappaB activity resulting in bcl-2 protein downregulation. Bax protein levels remained constant in these cells after radiation or curcumin plus radiation treatments. However, the downregulation of Bcl-2 and no changes in Bax protein levels in curcumin plus radiation-treated PC-3 cells, together, altered the Bcl2 : Bax ratio and this caused the enhanced radiosensitization effect. In addition, significant activation of cytochrome c and caspase-9 and -3 were observed in curcumin plus radiation treatments. Together, these mechanisms strongly suggest that the natural compound curcumin is a potent radiosesitizer, and it acts by overcoming the effects of radiation-induced prosurvival gene expression in prostate cancer.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Combined Modality Therapy; Curcumin; Cyclin D1; Cytochromes c; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Gene Expression Regulation, Neoplastic; Humans; Male; NF-kappa B; Prostatic Neoplasms; Radiation Tolerance; Radiation-Sensitizing Agents; Radiation, Ionizing; Tumor Necrosis Factor-alpha

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a highly promising candidate for the treatment of cancer because it elicits cell death in the majority of tumor cells while sparing most normal cells. Some cancers, however, display resistance to TRAIL, suggesting that treatment with TRAIL alone may be insufficient for cancer therapy. In the present study, we explored whether the apoptotic responsiveness of PC-3 prostate cancer cells to TRAIL could be enhanced by targeting the novel protein kinase C (PKC) isoform eta. Transfection of PC-3 cells with second-generation chimeric antisense oligonucleotides against PKCeta caused a time- and dose-dependent knockdown of PKCeta, as revealed by real-time RT-PCR and Western blot analyses. Knockdown of PKCeta resulted in a marked amplification of TRAIL's cytotoxic activity. Cell killing could be substantially prevented by the pan-caspase inhibitor z-VAD-fmk. In addition, PKCeta knockdown and administration of TRAIL significantly synergized in activation of caspase-3 and internucleosomal DNA fragmentation. Knockdown of PKCeta augmented TRAIL-induced dissipation of the mitochondrial transmembrane potential and release of cytochrome c from mitochondria into the cytosol, indicating that PKCeta acts upstream of mitochondria. We conclude that PKCeta represents a considerable resistance factor with respect to TRAIL and a promising target to exploit the therapeutic potential of TRAIL.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Down-Regulation; Humans; Male; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Oligonucleotides, Antisense; Prostatic Neoplasms; Protein Kinase C; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

E7389, a macrocyclic ketone analog of the marine natural product halichondrin B, currently is undergoing clinical trials for cancer. This fully synthetic agent exerts its highly potent in vitro and in vivo anticancer effects via tubulin-based antimitotic mechanisms, which are similar or identical to those of parental halichondrin B. In an attempt to understand the impressive potency of E7389 in animal models of human cancer, its ability to induce apoptosis following prolonged mitotic blockage was evaluated. Treatment of U937 human histiocytic lymphoma cells with E7389 led to time-dependent collection of cells in the G2-M phase of the cell cycle, beginning as early as 2 h and becoming maximal by 12 h. Increased numbers of hypodiploid events were seen beginning at 12 h, suggesting initiation of apoptosis after prolonged E7389-induced mitotic blockage. The identity of hypodiploid events as apoptotic cells under these conditions was confirmed by two additional morphologic criteria: green to orange/yellow shifts on acridine orange/ethidium bromide staining, and cell surface annexin V binding as assessed by flow cytometry. Several biochemical correlates of apoptosis also were seen following E7389 treatment, including phosphorylation of the antiapoptotic protein Bcl-2, cytochrome c release from mitochondria, proteolytic activation of caspase-3 and -9, and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). In LNCaP human prostate cancer cells, treatment with E7389 also led to generation of hypodiploid cells, activation of caspase-3 and -9, and appearance of cleaved PARP, indicating that E7389 can activate cellular apoptosis pathways under anchorage-independent and -dependent cell culture conditions. These results show that prolonged mitotic blockage by E7389 can lead to apoptotic cell death of human cancer cells in vitro and can provide a mechanistic basis for the significant in vivo anticancer efficacy of E7389.

Topics: Apoptosis; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Membrane; Cell Polarity; Cytochromes c; Diploidy; Enzyme Activation; Ethers, Cyclic; Furans; G2 Phase; Humans; Ketones; Male; Mitochondria; Mitosis; Phosphorylation; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; U937 Cells

Prostate cancer is the most common malignancy and the second leading cause of male death in Western countries. Prostate cancer mortality results from metastases to the bones and lymph nodes and progression from androgen-dependent to androgen-independent disease. Although androgen ablation was found to be effective in treating androgen-dependent prostate cancer, no effective life-prolonging therapy is available for androgen-independent cancer. Epidemiological studies have shown a strong correlation between consumption of cruciferous vegetables and a lower risk of prostate cancer. These vegetables contain glucosinolates, which during metabolism give rise to several breakdown products, mainly indole-3-carbinol (I3C), which may be condensed to polymeric products, especially 3,3'-diindolylmethane (DIM). It was previously shown that these indole derivatives have significant inhibitory effects in several human cancer cell lines, which are exerted through induction of apoptosis. We have previously reported that I3C and DIM induce apoptosis in prostate cancer cell lines through p53-, bax-, bcl-2- and fasL-independent pathways. The objective of this study was examination of the apoptotic pathways that may be involved in the effect of DIM in the androgen-independent prostate cancer cell line, PC3, in vitro. Our results suggest that DIM induces apoptosis in PC3 cells, through the mitochondrial pathway, which involves the translocation of cytochrome c from the mitochondria to the cytosol and the activation of initiator caspase, 9, and effector caspases, 3 and 6, leading to poly ADP-ribose polymerase (PARP) cleavage and induction of apoptosis. Our findings may lead to the development of new therapeutic strategies for the treatment of androgen-independent prostate cancer.

Topics: Androgens; Anticarcinogenic Agents; Apoptosis; Cytochromes c; Cytosol; Diet; Humans; Indoles; Male; Mitochondria; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Tumor Cells, Cultured; Vegetables

Irofulven (hydroxymethylacylfulvene) is a novel antitumor drug, which acts by alkylating cellular macromolecular targets. The drug is a potent inducer of apoptosis in various types of tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to irofulven involving mitochondrial dysfunction and leading to death of prostate tumor LNCaP-Pro5 cells. Irofulven caused early (2-5 hours) translocation of the proapoptotic Bax from cytosol to mitochondria followed by the dissipation of mitochondrial membrane potential and cytochrome c release at 4 to 12 hours. These effects preceded caspase activation and during the first 6 hours were not affected by caspase inhibitors. Processing of caspase-9 initiated the caspase cascade at approximately 6 hours and progressed over time. The activation of the caspase cascade provided a positive feedback loop that enhanced Bcl-2-independent translocation and cytochrome c release. General and specific caspase inhibitors abrogated irofulven-induced apoptotic DNA fragmentation with the following order of potency: pan-caspase > or = caspase-9 > caspase-8/6 > caspase-2 > caspase-3/7 > caspase-1/4. Abrogation of caspase-mediated DNA fragmentation failed to salvage irofulven-treated cells from growth inhibition and loss of viability, demonstrating a substantial contribution of a caspase-independent cell death. Monobromobimane, an inhibitor of alternative caspase-independent apoptotic pathway that is mediated by mitochondrial permeability transition, antagonized both apoptosis, measured as phosphatidylserine externalization, and cytotoxicity of irofulven. Collectively, the results indicate that irofulven-induced signaling is integrated at the level of mitochondrial dysfunction. The induction of both caspase-dependent and caspase-independent death pathways is consistent with pleiotropic effects of irofulven, which include targeting of cellular DNA and proteins.

Topics: Apoptosis; bcl-2-Associated X Protein; Bridged Bicyclo Compounds; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Enzyme Inhibitors; Humans; Male; Mitochondria; Molecular Structure; Prostatic Neoplasms; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes; Signal Transduction

Lack of life-prolonging therapies has provided much of the impetus for seeking complementary and alternative management/treatment options by prostate cancer (CaP) patients. Among these, the use of dietary supplements and botanical products has been showing a sustained increase in recent years, owing in part to some encouraging pre-clinical and clinical data shown in a limited number of herbal products. Notably, however, the majority of herbal and dietary supplement products have not been rigorously studied with regard to their efficacy. In vitro mechanistic experiments are considered essential preludes and requisites to more lengthy and costly animal and human studies, in that they may provide relevant insights and scientific basis for effects some of these products purportedly might demonstrate. In vitro studies in our laboratory have shown that a polyherbal supplement, Equiguard, exhibits anti-tumor activity against hormone dependent LNCaP cells cultured in both androgen-proficient (FBS) and -deficient (CS-FBS) conditions. Clinically relevant anti-prostate cancer effects of Equiguard are vividly illustrated by growth suppression and down regulated expression of prostate specific genes, respectively, androgen receptor (AR) and prostate specific antigen (PSA). However, the mechanistic bases contributing to these effects have not been well characterized. This communication describes experiments aimed at further understanding growth arrest elicited by Equiguard in LNCaP cells cultured in FBS and CS-FBS conditions. We have focused on aspects of cell cycle control and induction of apoptosis. Regulation of cell cycle progression by Equiguard was analyzed by examining changes in the expression of Rb and cyclins D/E. Using Western blot analysis, we showed that treatment caused inhibition of Rb phosphorylation, which was accompanied by the reduction of cyclins D/E expression, in both culture conditions. Moreover, cells treated with Equiguard and cultured with FBS-supplemented media showed up-regulation of cyclin-dependent kinase inhibitor Kip1/p27. These results support the interpretation that suppression of Rb phosphorylation mediated the observed growth arrest induced by Equiguard under androgen-proficient condition. In contrast, Equiguard-treated cells cultured in CS-FBS had lowered expression of the Kip1/p27, suggesting that different control mechanisms, possibly evoked by changes in cellular microenvironments, contributed to growth suppression by Equiguard.

Topics: Apoptosis; Cell Cycle; Cell Proliferation; Cytochromes c; Down-Regulation; Drugs, Chinese Herbal; Humans; Male; Prostatic Neoplasms; Retinoblastoma Protein; Tumor Cells, Cultured

Antiandrogens such as Casodex (Bicalutamide) are designed to treat advance stage prostate cancer by interfering with androgen receptor-mediated cell survival and by initiating cell death. Treatment of androgen sensitive, non-metastatic LNCaP human prostate cancer cells with 0-100 microM Casodex or 0-10 ng/ml TNF-alpha induces cell death in 20-60% of the cells by 48 h in a dose-dependent manner. In cells treated with TNF-alpha, this is accompanied by the loss of mitochondrial membrane potential (DeltaPsim) and cell adhesion. In contrast, cells treated with Casodex display loss of cell adhesion, but sustained mitochondrial dehydrogenase activity. Overexpression of Bcl-2 in LNCaP cells attenuates the induction of cell death by TNF-alpha but not Casodex, suggesting that mitochondria depolarization is not required for the induction of cell death by Casodex. While both TNF-alpha and Casodex-induced release of cytochrome c in LNCaP cell is predominantely associated with the translocation and cleavage of Bax, our data also suggest that Casodex induces cell death by acting on components downstream of decline of DeltaPsim and upstream of cytochrome c release. Furthermore, while induction of both caspase-3 and caspase-8 activities are observed in TNF-alpha and Casodex-treated cells, a novel cleavage product of procaspase-8 is seen in Casodex-treated cells. Taken together, these data support the hypothesis that Casodex induces cell death by a pathway that is independent of changes in DeltaPsim and Bcl-2 actions and results in an extended lag phase of cell survival that may promote the induction of an invasive phenotype after treatment.

Topics: Androgen Antagonists; Anilides; bcl-2-Associated X Protein; Carcinoma; Caspases; Cell Adhesion; Cell Death; Cell Nucleus; Cell Survival; Cytochromes c; Humans; Male; Membrane Potentials; Mitochondria; Nitriles; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tosyl Compounds; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The role of Bax in the release of cytochrome c from mitochondria and the induction of apoptosis has been demonstrated in many systems. Using immunocytochemical staining, we observed that photodynamic therapy (PDT) with the photosensitiser Pc 4 induced Bax translocation from the cytosol to mitochondria, and the release of cytochrome c from mitochondria as early signalling for the intrinsic pathway of apoptosis in human breast cancer MCF-7c3 cells. To test the role of Bax in apoptosis, MCF-7c3 cells were treated with Bax antisense oligonucleotides, which resulted in as much as a 50% inhibition of PDT-induced apoptosis. In the second approach, Bax-negative human prostate cancer DU-145 cells were studied. Following PDT, the hallmarks of apoptosis, including the release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, caspase activation, and chromatin condensation and fragmentation, were completely blocked in these cells. Restoration of Bax expression in DU-145 cells restored apoptosis, indicating that the resistance of DU-145 cells to PDT-induced apoptosis is due to the lack of Bax rather than to another defect in the apoptotic machinery. However, despite the inhibition of apoptosis, the Bax-negative DU-145 cells were as photosensitive as Bax-replete MCF-7c3 cells, as determined by clonogenic assay. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to Bax activation.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Death; Cytochromes c; Female; Immunohistochemistry; Indoles; Male; Mitochondria; Photochemotherapy; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured