Apoptosis In Prostate Carcinoma Cells Research Articles

LncRNA BRE-AS1 interacts with miR-145-5p to regulate cancer cell proliferation and apoptosis in prostate carcinoma and has early diagnostic values.

Long non-coding RNA (LncRNA) BRE-AS1 has recently proven to be a tumor suppressor in lung cancer. The present study aimed to investigate the involvement of lncRNA BRE-AS1 in prostate carcinoma (PC). In the present study we found that plasma BRE-AS1 and miR-145-5p were both down-regulated in PC patients than in healthy controls. Down-regulation of BRE-AS1 and miR-145-5p effectively distinguished early-stage PC patients from healthy controls. A significant and positive correlation between BRE-AS1 and miR-145–5p was only found in PC patients. BRE-AS1 overexpression mediated miR-145-5p up-regulation in PC cells, while miR-145-5p overexpression did not significantly affect BRE-AS1. Overexpression of BRE-AS1 and miR-145-5p led to inhibited proliferation and promoted apoptosis of PC cells. miR-145-5p inhibitor attenuated the effects of BRE-AS1 overexpression on cancer cell behaviors. Therefore, lncRNA BRE-AS1 may regulate cancer cell proliferation and apoptosis in PC by interacting with miR-145-5p.

Eupalitin induces apoptosis in prostate carcinoma cells through ROS generation and increase of caspase-3 activity.

Prostate cancer is the second most common malignancy in the human reproductive system. Eupalitin is one of the O-methylated flavonol-exhibited enhanced cancer chemopreventive agents. The current study highlights the structural determination of eupalitin and aims to explore the antitumor activity of eupalitin in human prostate cancer cell (PC3) and its underlying mechanism. Eupalitin structure was determined by using FTIR, (1)H NMR, and (13)C NMR. PC3 cells were treated with increasing concentrations of eupalitin, followed by analysis of the cell viability with an MTT assay. The results demonstrated that eupalitin markedly inhibited the proliferation of PC3 cells in a concentration-dependent manner. The results from fluorescent microscopic analysis of nuclear condensation and intracellular ROS generation determined that eupalitin significantly induced ROS level lead to nuclear apoptosis. Cell cycle analysis revealed that eupalitin-induced cell cycle progression as a percentage of cells in G0/G1 phase decreased whereas S phase increased. Caspase-3 immunofluorescence analysis confirms the efficacy of eupalitin-inducing apoptotic pathway and cell death. Thus, our study is helpful in understanding the mechanism underlying these effects in prostate cancer and it may provide novel molecular targets for prostate cancer therapy.

15,16-Dihydrotanshinone I, a Compound ofSalvia miltiorrhizaBunge, Induces Apoptosis through Inducing Endoplasmic Reticular Stress in Human Prostate Carcinoma Cells

5,16-dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhiza Bunge (tanshen root) and was found to be the most effective compound of tanshen extracts against breast cancer cells in our previous studies. However, whether DHTS can induce apoptosis through an endoplasmic reticular (ER) stress pathway was examined herein. In this study, we found that DHTS significantly inhibited the proliferation of human prostate DU145 carcinoma cells and induced apoptosis. DHTS was able to induce ER stress as evidenced by the upregulation of glucose regulation protein 78 (GRP78/Bip) and CAAT/enhancer binding protein homologous protein/growth arrest- and DNA damage-inducible gene 153 (CHOP/GADD153), as well as increases in phosphorylated eukaryotic initiation factor 2α (eIF2α), c-jun N-terminal kinase (JNK), and X-box-binding protein 1 (XBP1) mRNA splicing forms. DHTS treatment also caused significant accumulation of polyubiquitinated proteins and hypoxia-inducible factor (HIF)-1α, indicating that DHTS might be a proteasome inhibitor that is known to induce ER stress or enhance apoptosis caused by the classic ER stress-dependent mechanism. Moreover, DHTS-induced apoptosis was reversed by salubrinal, an ER stress inhibitor. Results suggest that DHTS can induce apoptosis of prostate carcinoma cells via induction of ER stress and/or inhibition of proteasome activity, and may have therapeutic potential for prostate cancer patients.

Inositol Hexaphosphate Suppresses Growth and Induces Apoptosis in Prostate Carcinoma Cells in Culture and Nude Mouse Xenograft: PI3K-Akt Pathway as Potential Target

Constitutive activation of phosphoinositide 3-kinase (PI3K)-Akt pathway transmits growth-regulatory signals that play a central role in promoting survival, proliferation, and angiogenesis in human prostate cancer cells. Here, we assessed the efficacy of inositol hexaphosphate (IP6) against invasive human prostate cancer PC-3 and C4-2B cells and regulation of PI3K-Akt pathway. IP6 treatment of cells suppressed proliferation, induced apoptosis along with caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, and inhibited constitutive activation of Akt and its upstream regulators PI3K, phosphoinositide-dependent kinase-1 and integrin-linked kinase-1 (ILK1). Downstream of Akt, IP6 inhibited the phosphorylation of glycogen synthase kinase-3alpha/beta at Ser(21/9) and consequently reduced cyclin D1 expression. Efficacy studies employing PC-3 tumor xenograft growth in nude mice showed that 2% (w/v) IP6 feeding in drinking water inhibits tumor growth and weight by 52% to 59% (P < 0.001). Immunohistochemical analysis of xenografts showed that IP6 significantly reduces the expression of molecules associated with cell survival/proliferation (ILK1, phosphorylated Akt, cyclin D1, and proliferating cell nuclear antigen) and angiogenesis (platelet endothelial cell adhesion molecule-1 or CD31, vascular endothelial growth factor, endothelial nitric oxide synthase, and hypoxia-inducible factor-1alpha) together with an increase in apoptotic markers (cleaved caspase-3 and PARP). These findings suggest that, by targeting the PI3K-ILK1-Akt pathway, IP6 suppresses cell survival, proliferation, and angiogenesis but induces death in prostate cancer cells, which might have translational potential in preventing and controlling the growth of advanced and aggressive prostate cancer for which conventional chemotherapy is not effective.

Role of Protein Kinase CK2 in the Regulation of Tumor Necrosis Factor–Related Apoptosis Inducing Ligand–Induced Apoptosis in Prostate Cancer Cells

Protein kinase CK2 (formerly casein kinase 2 or II) is a ubiquitous and highly conserved protein Ser/Thr kinase that plays diverse roles such as in cell proliferation and apoptosis. With respect to the latter, we originally showed that elevated CK2 could suppress various types of apoptosis in prostate cancer cells; however, the downstream pathways that respond to CK2 for mediating the suppression of apoptosis have not been fully elucidated. Here, we report studies on the role of CK2 in influencing activities associated with tumor necrosis factor-related ligand (TRAIL/Apo2-L)-mediated apoptosis in prostate carcinoma cells. To that end, we show that both androgen-insensitive (PC-3) and androgen-sensitive (ALVA-41) prostate cancer cells are sensitized to TRAIL by chemical inhibition of CK2 using its specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). Furthermore, we have shown that overexpression of CK2alpha using pcDNA6-CK2alpha protected prostatic cancer cells from TRAIL-mediated apoptosis by affecting various activities associated with this process. Thus, overexpression of CK2 resulted in the suppression of TRAIL-induced apoptosis via its effects on the activation of caspases, DNA fragmentation, and downstream cleavage of lamin A. In addition, the overexpression of CK2 blocked the mitochondrial apoptosis machinery engaged by TRAIL. These findings define the important role of CK2 in TRAIL signaling in androgen-sensitive and -insensitive prostatic carcinoma cells. Our data support the potential usefulness of anticancer strategies that may involve the combination of TRAIL and down-regulation of CK2.

Effects of IKK inhibitor PS1145 on NF-κB function, proliferation, apoptosis and invasion activity in prostate carcinoma cells

A key antiapoptotic transcription factor, nuclear factor kappa-B (NF-kappaB), is known to be critically important for tumor cell growth, angiogenesis and development of metastatic lesions. We and others showed previously that NF-kappaB transcription factor was constitutively activated in androgen-independent prostate carcinoma (PC) cell lines due to the upregulated activity of inhibitor of NF-kappaB kinases (IKK). In this work, using luciferase assay, electrophoretic mobility shift assay and Northern blot analysis of expression of endogenous kappaB-responsive genes, we demonstrate that a novel highly specific small-molecule IKK inhibitor, PS1145, efficiently inhibited both basal and induced NF-kappaB activity in PC cells. We found that PS1145 induced caspase 3/7-dependent apoptosis in PC cells and significantly sensitized PC cells to apoptosis induced by tumor necrosis factor alpha. We also showed that PS1145 inhibited PC cell proliferation. Effects of PS1145 on proliferation and apoptosis correlated with inhibition of interleukin (IL)-6, cyclin D1, D2, inhibitor of apoptosis (IAP)-1 and IAP-2 gene expression and decreased IL-6 protein level. In addition, we found that incubation with PS1145 inhibited the invasion activity of highly invasive PC3-S cells in invasion chamber assay in a dose-dependent manner. Overall, this study provides the framework for development of a novel therapeutic approach targeting NF-kappaB transcription factor to treat advanced PC.

Nitroxide tempo, a small molecule, induces apoptosis in prostate carcinoma cells and suppresses tumor growth in athymic mice

In previous studies, nitroxide tempo (2, 2, 6, 6-tetramethyl-piperidine-1-oxyl), a small molecule, induced cell death in cancer cells. The current study examined the antineoplastic properties of tempo in the human hormone-dependent/hormone-independent prostate carcinoma models (LNCaP, DU-145, and PC-3). The apoptotic effects of tempo were examined by the flow cytometric analysis of cells labeled with fluorescein isothiocyanate-conjugated annexin-V, and by electron microscopy. Enzymatic assays were performed to measure the activities of 2 cysteine proteases, i.e., caspase-9 and caspase-3, in tempo-treated cells. The effects of tempo on cell proliferation and on cell cycle distribution profiles were measured by the flow cytometric assay using immunofluorescent staining of incorporated 5'-bromo-2'-deoxyuridine (BrdU) coupled with 7-amino-actinomycin D (7-AAD) staining of total DNA. The number of proliferating cells was also determined independently by enzyme-linked immunosorbent assay using chemiluminescent detection of incorporated BrdU. Subcutaneous growth of human prostate carcinoma in athymic mice was monitored after intratumoral administration of tempo into tumor-bearing mice. In addition, cell viability assays were performed to compare the cytotoxic effect of a combination of doxorubicin or mitoxantrone and tempo with single agents. Tempo treatment of prostate carcinoma cells caused a significant increase in the number of apoptotic cells compared with control groups (tempo, 2.5 mM, 24 hours: DU-145, approximately 3.4-fold; PC-3, approximately 6-7-fold; tempo 1 mM, 24 hours: LNCaP, approximately 12-fold). Tempo-induced loss of cell viability was blocked partially or completely after pretreatment of cells with actinomycin-D or cycloheximide, suggesting a de novo macromolecule synthesis-dependent mechanism of cell death. Electron microscopy revealed aggregation and marginalization of chromatin in the nuclei of a large number of tempo-treated LNCaP cells. Tempo treatment of LNCaP cells resulted in enhanced activities of caspase-9 (tempo, 5 mM, 15 hours: approximately 2-fold) and caspase-3 (tempo, 2.5 mM, 24 hours: approximately 12-fold). Tempo treatment also led to an enhanced number of cells in G2/M phase of the cell cycle (tempo, 5.0 mM, 24 hours: DU-145, approximately 1.6-fold; PC-3, approximately 1.5-fold; LNCaP, approximately 5.3-fold), and decreased BrdU incorporation indicative of a decline in the number of proliferating cells (tempo, 2.5 mM, 24 or 48 hours; DU-145, approximately 2-3-fold; PC-3, approximately 1.2-fold; LNCaP, approximately 5-10-fold). Administration of tempo into LNCaP tumor-bearing mice resulted in a significant inhibition of tumor growth (percent initial tumor volume [Day 30, n = 4]: vehicle, 845.35 +/- 272.83; tempo, 9.72 +/- 9.72; tempo vs. vehicle, P < 0.02). In hormone-refractory prostate carcinoma cells, a combination of relatively low doses of tempo and doxorubicin or mitoxantrone caused enhanced cytotoxicity as compared with single agents. These data demonstrated that nitroxide tempo induced apoptosis and activated a caspase-mediated signaling pathway in prostate carcinoma cells. Tempo treatment also caused cell cycle arrest in G2/M phase and decreased the number of proliferating cells (S phase). Tempo treatment of tumor-bearing mice led to inhibition of tumor growth, suggesting that tempo is a novel member of the small-molecule family of antineoplastic agents.

The 70kilodalton heat shock protein is an inhibitor of apoptosis in prostate cancer

The 70 kD heat shock protein (HSP70) plays essential cellular roles in mediating intra-cellular protein folding and protecting cells from proteotoxic stress. This study has examined the role of HSP70 in the expression of apoptosis in prostate carcinoma cells. Apoptosis was negatively correlated with HSP70 expression in PC-3 cells heat shocked in vivo. Further experiments carried out on an in vitro reconstituted system with isolated nuclei and cytoplasm from PC-3 cells showed that purified HSP70 directly inhibits apoptosis in a dose-dependant manner. Therefore, the potential role of depletion of intracellular HSP70 was examined as a means of inducing apoptosis in PC-3 cancer cells. Depletion of HSP70 by two independent strategies, either with anti-sense oligonucleotides directed against HSP70 mRNA or with the bioflavinoid drug quercetin, led to apoptosis in the absence of stress. In addition, quercetin pre-treatment synergistically enhanced apoptosis in combination with heat shock. Thus, HSP70 plays a physiological role in tumour cells as an inhibitor of apoptosis occurring both spontaneously and after stress and is a potential target for apoptosis-based cancer therapy.

Neuropeptides bombesin and calcitonin inhibit apoptosis-related elemental changes in prostate carcinoma cell lines.

Etoposide-induced apoptosis in prostate carcinoma cells is associated with changes in the elemental content of the cells. The authors previously reported that calcitonin and bombesin inhibited etoposide-induced apoptosis in these cells. In the current study, the authors investigated whether these neuropeptides block the etoposide-induced changes in elemental content. Cells from the PC-3 and Du 145 prostate carcinoma cell lines were grown either on solid substrates or on thin plastic films on titanium electron microscopy grids, and they were exposed to etoposide for 48 hours in the absence or presence of calcitonin and bombesin. After the exposure, the cells were frozen and freeze dried, and their elemental content was analyzed by energy-dispersive X-ray microanalysis in both in the scanning electron microscope and the scanning transmission electron microscope. Etoposide treatment consistently induced an increase in the cellular Na concentration and a decrease in the cellular K concentration, resulting in a marked increase of the Na/K ratio and also an increase in the phosphorus:sulphur (P/S) ratio. Both bombesin and calcitonin inhibited the etoposide-induced changes in the cellular Na/K ratio, and calcitonin, but not bombesin, inhibited the changes in the P/S ratio. No significant elemental changes were found with bombesin or calcitonin alone. The neuropeptides bombesin and calcitonin, which inhibited etoposide-induced apoptosis, also inhibited the etoposide-induced elemental changes in prostate carcinoma cells. This important fact strengthens the link between apoptosis and changes in the intracellular elemental content. This correlation provides an objective basis for the study of neuropeptide target points and may be helpful for alternative therapeutic protocols using neuropeptide inhibitors in the treatment of patients with advanced prostatic carcinoma.

Induction of apoptosis in prostate carcinoma cells by BH3 peptides which inhibit Bak/Bcl-2 interactions.

Interactions between proteins of the Bcl-2 family play an important role in the regulation of apoptosis. Anti-apoptotic family members can heterodimerize with pro-apoptotic family members and antagonize their function, thus protecting against death. In cells protected from death by overexpression of Bcl-2 much of the Bax is present in Bax/Bcl-2 hetero-multimers and its death signal is blocked as it cannot homodimerize. This led us to use the Bcl-2/Bax heterodimer as a target for new compounds which may provide a therapy particularly suited to tumour cells for which resistance to conventional therapy is associated with elevated expression of Bcl-2. We assessed whether apoptosis could be induced in prostate tumour cells by blocking this heterodimerization with synthetic peptide sequences derived from the BH3 domain of pro-apoptotic Bcl-2 family members. Prostate cells were found to undergo up to 40% apoptosis 48 h following the introduction of synthetic peptides from the BH3 domains of Bax and Bak. The caspase inhibitor z-VAD.fmk provided protection against apoptosis mediated by these peptides. Immunoprecipitation studies revealed that introduction of peptides derived from the BH3 regions of Bak and Bax into cells blocked Bak/Bcl-2 heterodimerization. These data suggest that by blocking the dimerization through which Bcl-2 would normally inhibit apoptosis the apoptotic pathway driven by Bak was re-opened. © 2001 Cancer Research Campaign http://www.bjcancer.com

Thiol-mediated apoptosis in prostate carcinoma cells.

Glutathione (GSH) maintains an optimum cellular redox potential. Chemical depletion, physical efflux from the cell, or intracellular redistribution of this thiol antioxidant is associated with the onset of apoptosis. The aim of this study was to determine the effects of a thiol-depleting agent, diethylmaleate (DEM), on androgen sensitive and insensitive prostate carcinoma cells. LNCaP and PC-3 cell lines were induced to undergo apoptosis by DEM and diamide. Apoptosis was quantified by annexin V binding and propidium iodide incorporation using flow cytometry and was confirmed by DNA gel electrophoresis. Intracellular GSH was quantified using a thiol quantitation kit and the generation of reactive oxygen intermediates was measured using dihydrorhodamine 123. Western blot assessed caspase-3, caspase-8, Bcl-2, and Bcl-XL protein expression. Mitochondrial permeability was measured using DiOC6 and stabilized using bongkrekic acid. DEM and diamide induced apoptosis in both androgen sensitive and insensitive cells. Apoptosis was also induced in an LNCaP transfectant cell line overexpressing Bcl-2. Apoptosis was caspase-3 dependent and caspase-8 independent. Bongkrekic acid partially prevented the effects of DEM on mitochondrial permeability but was unable to prevent the induction of apoptosis. Decreased Bcl-2 and Bci-XL protein expression was observed at the time of initial caspase-3 activation. This study demonstrates that thiol depletion can be used as an effective means of activating caspase-3 in both androgen sensitive and insensitive prostate carcinoma cells. Direct activation of this effector caspase may serve as a useful strategy for inducing apoptosis in prostate carcinoma cells.