Ionizing radiation enhances the therapeutic potential of TRAIL in prostate cancer in vitro and in vivo: Intracellular mechanisms

BACKGROUND: We assessed the influence of sequential treatment of ionizing radiation followed by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on intracellular mechanisms of apoptosis of prostate tumor cells in vitro and in vivo. METHODS: Prostate normal and cancer cells were exposed to irradiation and TRAIL. Four- to 6-week-old athymic nude mice were injected s.c. with PC-3 tumor cells. Tumor bearing mice were exposed to irradiation and TRAIL, either alone or in combination (TRAIL after 24 hr of irradiation), and tumor growth, apoptosis, and survival of mice were examined. Expressions of death receptors, Bcl-2 family members, and caspase were measured by Western blotting, ELISA, and ribonuclease protection assay; tumor cellularity was assessed by H&E staining; inhibition of p53 was performed by RNA interference (RNAi) technology, and apoptosis was measured by annexin V/propidium iodide staining, and terminal deoxynucleotidyltransferase-mediated nick end labeling assay. RESULTS: Irradiation significantly augmented TRAIL-induced apoptosis in prostate cancer cells through upregulation of DR5, Bax, and Bak, and induction of caspase activation. Dominant negative FADD and p53 siRNA inhibited the synergistic interaction between irradiation and TRAIL. The pretreatment of cells with irradiation followed by TRAIL significantly enhanced more apoptosis than single agent alone or concurrent treatment. Furthermore, irradiation sensitized TRAIL-resistant LNCaP cells to undergo apoptosis. The sequential treatment of xenografted mice with irradiation followed by TRAIL-induced apoptosis through activation of caspase-3, induction of Bax and Bak, and inhibition of Bcl-2, and completely eradicated the established tumors with enhanced survival of nude mice. CONCLUSION: The sequential treatment with irradiation followed by TRAIL can be used as a viable option to enhance the therapeutic potential of TRAIL in prostate cancer.     

Dichloroacetate (DCA) sensitizes both wild-type and over expressing Bcl-2 prostate cancer cells in vitro to radiation

BACKGROUND: Bcl-2 protects cells from apoptosis and provides a survival advantage to cells over-expressing this oncogene. In addition, over expression of Bcl-2 renders cell resistant to radiation therapy. Recently, dichloroacetate (DCA) was proven to potentiate the apoptotic machinery by interacting with Bcl-2. In this study, we investigated whether treating human prostate cancer cells with DCA could modulate Bcl-2 expression and if the modulation in Bcl-2 expression could render the Bcl-2 over expressing cells more susceptible to cytotoxicity effects of radiation. METHODS: PC-3-Bcl-2 and PC-3-Neo human prostate cancer cells treated with DCA in addition to irradiation were analyzed in vitro for changes in proliferation, clonogenic survival, apoptosis, cell cycle phase distribution, mitochondrial membrane potential, and expression of Bcl-2, Bcl-xL, Bax, or Bak proteins. RESULTS: DCA alone produced significant cytotoxic effects and was associated with G1 cell cycle arrest. Furthermore, DCA was associated with an increased rate of apoptosis. The combination of DCA with irradiation sensitized both cell lines to radiation's killing effects. Treatment of PC-3-Bcl-2 or PC-3-Neo with DCA and irradiation resulted in marked changes in various members of the Bcl-2 family. In addition, DCA therapy resulted in a significant change in mitochondria membrane potential, thus supporting the notion that DCAs effect is on the mitochondria. CONCLUSIONS: This is the first study to demonstrate DCA can effectively sensitize wild-type and over expressing Bcl-2 human prostate cancer cells to radiation by modulating the expression of key members of the Bcl-2 family. Together, these findings warrant further evaluation of the combination of DCA and irradiation.     

Antitumor activity and downregulation of pro-angiogenic molecules in human prostate cancer cells by a novel thiazolidione compound

BACKGROUND: Current treatments for prostate cancer are effective in many patients with locally advanced disease, but many of these patients eventually have recurrence. It is therefore important to develop alternative therapeutic agents with improved efficacy and tolerability. We recently identified a synthetic thiazolidin compound, 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidione (DBPT), that induces apoptosis in human colon cancer cells, independent of p53 and P-glycoprotein status. Here, we investigated the antitumor properties and mechanisms of action of this compound in human prostate cancer cell lines. METHODS: The effect of DBPT on cell-cycle progression and apoptosis in LNCaP and DU145 cells was examined by flow cytometry and Western blotting. The effect of DBPT on pro-angiogenic molecules was analyzed by Western blotting and by an enzyme-linked immunosorbent assay. RESULTS: DBPT inhibited the growth of LNCaP and DU145 cells with 50% inhibitory concentrations ranging from 1.6 to 5.9 microM. Treating LNCaP and DU145 cells with DBPT led to a time-dependent cell-cycle arrest in the G(2)/M phase and increased levels of G(2)/M checkpoint proteins, such as cyclin B1, cdc25C, phosphorylated histone H(3), and MPM-2. DBPT induced the phosphorylation of Bcl-xL and Bim, and induced apoptosis, as evidenced by cleavage of caspase and poly (ADP-ribose) polymerase. DBPT also effectively induced apoptosis in Bcl-2-overexpressing DU145 cells. Furthermore, DBPT decreased hypoxia-inducible factor 1 alpha and vascular endothelial growth factor expression in LNCaP cells under both normoxia and hypoxia. CONCLUSIONS: DBPT can suppress proliferation, induce apoptosis, and down regulate pro-angiogenic molecules in prostate cancer cells, and might be useful in treating prostate cancer.     

Antisense Bcl-2 sensitizes prostate cancer cells to radiation

BACKGROUND: Bcl-2 is anti-apoptotic and overexpression is associated with prostate tumor aggressiveness. We hypothesized that Bcl-2 has a role in prostate cancer radiation (RT) response. The relationship of Bcl-2 expression in four prostate cancer cell lines, and the effect of modulating expression with a Bcl-2 antisense oligonucleotide (G3139, Genasense, oblimersen sodium, Genta Incorporated), to RT was examined. METHODS: The four cell lines studied were LNCaP (wild type-p53), PC3 (p53 null), Bcl-2 stably transfected LNCaP (LNCaP-BST), and Bcl-2 stably transfected PC3 (PC3-BST) cells. Cells were treated with antisense (AS) Bcl-2 alone or with RT (2-6 Gy). Following RT, cells were processed at 3-6 hr for Western blots, 18 hr for Annexin V staining and flow cytometric analysis, 24 hr for caspases 3+7 quantification by fluorometric assay, and immediately for clonogenic survival. RESULTS: AS caused a significant reduction in Bcl-2 expression in all cell lines. P53 expression was elevated following RT treatment in LNCaP and LNCaP-BST cells. P21 was increased by RT treatment in all cell lines. AS caused a significant increase in caspase 3+7 activity over the mismatch (MM) controls in all cell lines. When AS was combined with RT, caspase 3+7 activity was further increased significantly over all other groups in all cell lines. Moreover, AS+RT resulted in significantly reduced clonogenic survival over MM+RT, which was dampened in the Bcl-2 overexpressing lines. CONCLUSIONS: To our knowledge, these data demonstrate for the first time that a Bcl-2 specific AS oligonucleotide sensitizes prostate cancer cells to RT. p53 is not required for this effect.     

Regulation of Bcl-2 during androgen-unresponsive progression of prostate cancer

The progression of prostate cancer from androgen-responsive to an androgen-unresponsive state remains the greatest obstacle in the treatment of this disease. Androgen-unresponsive prostate cancer is highly resistant to chemotherapy and radiation treatment that kill cells by the induction of apoptosis. Elucidating the molecular mechanisms of apoptosis regulation in prostate cancer can be useful in the development of new strategies for effective therapy of androgen-unresponsive cancer. We analyzed the Bcl-2 family of apoptosis regulators using various passages of the LNCaP prostate cancer cell line, which serve as an in vitro model for the progression of prostate cancer from androgen-responsive to androgen-unresponsive. In our model, progressively higher passages of LNCaP cells represent the progression to androgen-unresponsiveness. We examined the basal mRNA expression of the Bcl-2 family of apoptosis regulators. Under normal growth conditions, both androgen-responsive and androgen-unresponsive LNCaP cells express the Bcl-2 family of genes at similar levels. Western blot analysis showed the presence of Bcl-2 protein in androgen-responsive cells but not in androgen-unresponsive cells. Both androgen-responsive and androgen-unresponsive cells expressed Bax protein at similar levels. When exposed to oxidative stress, androgen-responsive cells underwent apoptosis but androgen-unresponsive cells exhibited resistance suggesting that the progression to androgen-unresponsiveness was associated with altered regulation of apoptosis. Treatment with paclitaxel or sodium butyrate induced apoptosis in both androgen-responsive and androgen-unresponsive cells suggesting that the apoptotic machinery is still intact in androgen-unresponsive LNCaP cells.     

The leader sequence triggers and enhances several functions of clusterin and is instrumental in the progression of human prostate cancer in vivo and in vitro

OBJECTIVE: To investigate the role of the leader sequence (which during clusterin biosynthesis facilitates its proper post-translational processing and secretion) in the functional activities of clusterin, a ubiquitous secretory glycoprotein with many biological functions, reported to be pro-apoptotic and anti-apoptotic in target cells, but for which the dual mechanism remains unclear. MATERIALS AND METHODS: We designed an expression vector starting from the second in-frame ATG on the full-length human clusterin cDNA that was capable of driving the expression of both the full-length and the truncated isoforms of clusterin. We established stable expression clones of the androgen-dependent prostate cancer line LNCaP expressing clusterin with and without the leader sequence. This induced expression provided an opportunity to evaluate both the in vivo and in vitro actions of clusterin expression. RESULTS: The LNCaP cells expressing clusterin with the leader sequence resisted apoptosis induced by tumour necrosis factor (TNF)-alpha, but clones with no leader sequence were highly susceptible to TNF-alpha-induced apoptosis. Furthermore, in the absence of the leader sequence, the expressed clusterin had a molecular weight consistent with that of the predicted holoprotein (40 kDa), suggesting a compromised post-translational processing with diffuse distribution throughout the cytoplasm. However, cells transfected with the full-length vector expressed clusterin of 60 and 35 kDa variants, and located exclusively in the Golgi apparatus. In vivo, only the overexpression of the full-length clusterin is anti-apoptotic and stimulates the proliferation of tumour. CONCLUSION: The leader sequence is important in determining the functions of clusterin, which include anti-apoptotic and anti-necrotic properties. The lack of the leader sequence allowed the incompletely processed clusterin to induce apoptosis in target cells; without the leader sequence, clusterin functions differently. Thus, the leader sequence is a trigger for many functions of clusterin in the progression of human prostate cancer cells.     

ABL-N诱导前列腺癌细胞凋亡及其机制

目的 探讨旋覆花内酯(ABL)-N诱导前列腺癌细胞凋亡的作用及其机制.方法 0～40μmol/L ABL-N分别处理前列腺痛细胞后,利用噻唑蓝(MTF)检测对细胞增长的抑制作用;流式细胞学、TUNEL染色等方法检测其诱导凋亡的作用,并检测Capase活性,Western blot测定bax、bel-2水平变化.结果 ABL-N明显抑制前列腺癌细胞PC3、LNCaP及DU145的生长,并呈剂量依赖性.40 μmol/L ABL-N作用24 h后,(73.34±4.41)%的PrEC细胞存活,而3种前列腺癌细胞分别为(11.92±2.31)%、(12.55±1.94)%、(13.28±2.26)%.膜联蛋白/碘化丙锭(Annexin V/PI)及TUNEL染色表明ABL-N呈剂量依赖性诱导PC3凋亡.ABL-N可激活Caspase活性,尤其Caspase-3,20μmol/L ABL-N作用24 h后其活性是对照组的4.23倍;bax/bcl-2比率随浓度增加明显增高.结论 ABL-N可通过Caspase途径及bax/bcl-2蛋白途径,诱导PC3等前列腺癌细胞凋亡,抑制前列腺癌细胞增殖.

Abstract：

Objective To explore the ABL-N-induced apoptosis of human prostate cancer cells and the mechansim. Methods After administration of 0-40 μmol/L ABL-N for 24 h, the effects of ABL-N on the induction of apoptosis in human prostate cancer cells PC3 were measured by methyl thiazol tetrazolium ( MTT) colorimetry, Annexin V/propidium iodide staining and TUNEL staining. The levels of bax and bcl-2 were tested by Western blotting. Caspase activity was assayed. Results ABL-N treatment to PC3,LNCaP, and DU145 cells resulted in a dose-dependent inhibition of cell growth without any substantial effect on normal human prostate epithelial PrEc cells. About (73. 34 ±4. 41)% of PrEC cells were viable following a 24-h exposure to 40 μmol/L ABL-N, whereas only (11. 92 ± 2. 31) % of PC3, (12. 55 ±1. 94) % of LNcap, and (13. 28 ± 2. 26) % of DU145 cells survived under similar conditions of ABL-N treatment. ABL-N treatment resulted in a dose-dependent induction of apoptosis of PC3 cells. Furthermore,ABL-N induced the activation of Caspases, especially Caspase 3. The Caspase-3 activity of PC3 cells treated with ABL-N (20 μmol/L) (0. 95) was significantly increased by about 4. 2-fold of the untreated cells (0. 24) at 24 h. The ratio of bax/bcl-2 was also increased significantly. Conclusion ABL-N induces apoptosis though the activation of Caspase 3 and pro- and anti-apoptotic Bcl-2 family proteins in prostate cancer cells.     

Overexpression of the cytoprotective protein clusterin decreases radiosensitivity in the human LNCaP prostate tumour model

OBJECTIVE: To evaluate the effect of clusterin overexpression on radiation-induced tumour growth rates and apoptosis in human prostate LNCaP cells, as prostate cancer cells are relatively resistant to radiation-induced apoptosis and local recurrences are common, but overexpression of the anti-apoptotic protein clusterin can accelerate progression to androgen-independence and to confer a chemoresistant phenotype in various prostate cancer models. MATERIALS AND METHODS: Western blot analysis and immunohistochemistry were used to compare clusterin expression levels in parental (P) and clusterin-transfected (T) LNCaP cells in vitro and in vivo. The effects of radiation on clusterin-expression in both parental LNCaP/P and clusterin-transfected LNCaP/T tumours were analysed by Northern blot analysis. The cellular response to radiation was determined up to 3 weeks after irradiation using tetrazolium and re-growth assays, and cell-cycle analysis by flow cytometry. RESULTS: Clusterin mRNA expression increased from undetectable to low levels in LNCaP/P tumours after radiation and more than three-fold in LNCaP/T tumours. Clusterin overexpression decreased the radiosensitivity in a time-dependent manner, reducing the extent of growth arrest and apoptosis by up to 54%. Re-growth assays showed that the improved survival rates of LNCaP/T cells after radiation did not change after 3 days, remaining constant over 3 weeks. CONCLUSIONS: These results identify clusterin as a promoter of cell survival that may help mediate resistance to radiation-induced apoptosis. Furthermore, clusterin overexpression seems to provide an extended protection against radiation-induced cell cycle arrest and apoptosis.     

Interleukin-6 protects LNCaP cells from apoptosis induced by androgen deprivation through the Stat3 pathway

BACKGROUND: Elevated expression of interleukin-6 (IL-6) is implicated in the progression of hormone refractory prostate cancer. Previous studies demonstrated that IL-6 promotes androgen-independent growth of prostate cancer cells. In this study, the effect of IL-6 on apoptosis induced by androgen deprivation was investigated. METHODS: The effect of IL-6 on apoptosis induced by androgen deprivation in LNCaP cells was examined by cell death ELISA and Western blot using cleaved poly (ADP-ribose) polymerase (PARP) and caspase-9, as well as Bcl-xL and phosphorylated Bad. The Stat3 in IL-6-mediated anti-apoptosis in prostate cancer cells was examined using either dominant-negative or constitutively activated Stat3 mutants. RESULTS: Overexpression of IL-6 renders androgen sensitive LNCaP human prostate cancer cells more resistant to apoptosis induced by androgen deprivation. LNCaP cells undergo apoptosis after 72 hr of androgen deprivation, an outcome is largely absent in clones overexpressing IL-6 as measured by cell death ELISA and chromatin degradation assays. IL-6 over-expressing cells resulted in a significant decrease in the expression of cleaved PARP and cleaved caspase-9 as well as an increase in the expression of Bcl-xL and phosphorylated Bad. Addition of IL-6 antibody completely abolished the anti-apoptotic activity of IL-6. This protective effect of IL-6 was reversed by the expression of a dominant-negative Stat3 mutant, Stat3F. Furthermore, ectopic expression of a constitutively active Stat3 antagonized androgen deprivation-induced cell death of LNCaP cells. CONCLUSION: These results indicate that IL-6 protects androgen sensitive LNCaP cells from apoptosis induced by androgen deprivation, and Stat3 activation play an important role in IL-6-mediated anti-apoptosis in prostate cancer cells.     

Protection of androgen-dependent human prostate cancer cells from oxidative stress-induced DNA damage by overexpression of clusterin and its modulation by androgen

BACKGROUND: Recent studies reported that oxidative stress is one of the major factors associated with the progression of prostate cancer through the accumulation of DNA damage. In the present study, we investigated the effect of oxidative stress on cell injury using androgen-dependent human prostate cancer LNCaP cells overexpressing clusterin, which has been shown to play crucial roles in the acquisition of resistance to several apoptotic stimuli. METHODS: We introduced clusterin cDNA into LNCaP cells which do not express a detectable level of clusterin expression, and generated a clusterin-overexpressing cell line (LNCaP/Cl) and a control vector only-transfected cell line (LNCaP/Co). The effects of hydrogen peroxide (H2O2) treatment on the LNCaP sublines with and without the addition of dihydrotestosterone (DHT) were analyzed using the in vitro mitogenic assay and lipid peroxidation assay, and morphological changes in the LNCaP sublines after H2O2 treatment were examined by staining with Hoechst 33258. The degrees of DNA damage induced by H2O2 into the LNCaP sublines were evaluated by the measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) level. RESULTS: H2O2-induced apoptosis in LNCaP/Cl was significantly suppressed compared with that in LNCaP/Co through the inhibition of membrane damage; however, the measurement of 8-OHdG level demonstrated that DNA damage was more intensively accumulated in LNCaP/Cl cells than LNCaP/Co cells. Furthermore, DHT suppressed the incidence of apoptotic cell death and enhanced the formation of 8-OHdG in both LNCaP/Cl and LNCaP/Co cells after H2O2 treatment in a dose-dependent manner. CONCLUSIONS: These findings suggest that clusterin may contribute to conferring resistance to oxidative stress-mediated cellular injury on prostate cancer cells, especially in the presence of androgen.     

The role of survivin and Bcl-2 in zinc-induced apoptosis in prostate cancer cells

ObjectivesTo study the effects of zinc treatment on the gene expression levels of survivin and Bcl-2 in prostate cancer cells.Materials and methodsThe effects of zinc exposure on apoptosis were assessed using two human prostate cancer cell lines, LNCaP and PC-3. Zinc-induced apoptosis was measured by Annexin V staining. The direct effect of zinc on the expression levels of zinc transporters (ZnT-1 and ZnT-4) and apoptosis-related genes (Bax, Bcl-2, and survivin) was determined by RT-PCR analysis.ResultsWhen LNCaP and PC-3 cells were exposed to various concentrations of zinc sulfate for 48 hors, their growth was inhibited in a dose-dependent manner. The levels of zinc in both cell lines treated with zinc sulfate for 24 hours were higher than in untreated cells. Exposure to zinc induced apoptosis and necrosis in LNCaP and PC-3 cells. Apoptosis became more extensive as the treatment time with zinc increased. There was a significant increase in the gene expression levels of ZnT-1 and ZnT-4 in both cell lines treated with zinc sulfate compared with untreated cells. The expression of Bax mRNA was up-regulated, while the expression of Bcl-2 and survivin were decreased in both cell lines following zinc treatment.ConclusionsExposure to zinc sulfate in human prostate cancer cells increased intracellular levels of zinc, which resulted in increased apoptosis. The apoptogenic effect of elevated concentration of zinc could be due either to increased expression of zinc transporters and increased levels of Bax or decreased Bcl-2 and survivin expression.     

Apigenin drives the production of reactive oxygen species and initiates a mitochondrial mediated cell death pathway in prostate epithelial cells

BACKGROUND: Phytoestrogens may reduce tumorigenesis in prostate cancer. We screened five phytoestrogens for their effect on cell growth and apoptosis in PWR-1E, LNCaP, PC-3, and DU145 prostate epithelial cells in vitro. METHODS: We assessed cell number, proliferation, and apoptosis using crystal violet assays, flow cytometric analysis, and TUNEL. Focusing specifically on apigenin we assessed the ability of calpain, serine protease, caspase, estrogen receptor, and ceramide synthase inhibitors to block apigenin induced apoptosis. We also analyzed caspase 3, 7, 8, 9, Bcl-2, Bax, Bid, and cytochrome C by Western analysis, and mitochondrial permeability and reactive oxygen species production by flow cytometry using mitosensor(TM) and DCFH-DA, respectively. RESULTS: Apigenin and silybinin significantly reduced cell number, with apigenin inducing apoptosis in PWR-1E, LNCaP, PC-3, and DU145 cells. The PC-3 and DU145 cells were less susceptible to apigenin induced apoptosis then LNCaP and PWR-1E cells. The induction of apoptosis by apigenin was caspase dependent. Apigenin generated reactive oxygen species, a loss of mitochondrial Bcl-2 expression, mitochondrial permeability, cytochrome C release, and the cleavage of caspase 3, 7, 8, and 9 and the concomitant cleavage of the inhibitor of apoptosis protein, cIAP-2. The overexpression of Bcl-2 in LNCaP B10 cells reduced the apoptotic effects of apigenin. CONCLUSIONS: Apigenin induces cell death in prostate epithelial cells using a mitochondrial mediated cell death pathway. Bcl-2 has a role in inhibiting apigenin induced cell death in prostate epithelial cells.     

Androgen-mediated resistance to apoptosis

BACKGROUND: Previous studies demonstrate that androgen is capable of exerting a protective effect in the androgen-sensitive human prostate cancer cell line LNCaP. Limited studies, however, have addressed the underlying mechanisms involved, in particular the effects of androgen on both pro- and anti-apoptotic gene expression. METHODS: We investigated the effects of androgen on apoptotic sensitivity and the expression of the caspases and specific members of the Bcl-2 family in the LNCaP cell line. The effects of androgen on NF-kappaB activation were also investigated by using a gel mobility shift assay. RESULTS: 5alpha-Dihydrotestosterone (5-alphaDHT) conferred resistance to radiation (5 Gy) and etoposide-induced apoptosis in the LNCaP cell line. This finding was associated with a time-dependent decrease in the expression of the caspases and pro-apoptotic Bcl-2 family members. 5-alphaDHT did not confer protection against apoptosis in the LNCaP line transfected with the IkappaB super repressor of NF-kappaB, nor in the androgen insensitive PC-3 and DU-145 cell lines. CONCLUSION: The ability of 5-alphaDHT to raise the apoptotic threshold in the LNCaP cell line by altering specific pro-apoptotic gene expression suggests that androgen may serve as a general survival signal against diverse pathways that ultimately signal for apoptosis. We hypothesize that NF-kappaB serves as an important mediator in androgen survival signaling.     

Use of antisense oligonucleotides targeting the cytoprotective gene, clusterin, to enhance androgen- and chemo-sensitivity in prostate cancer

The discovery and targeting of genes mediating androgen-independence may lead to the development of novel therapies that delay progression of hormone refractory prostate cancer (HRPC). Clusterin is a stress-associated cell survival gene that increases after androgen ablation. Here, we review clusterins functional role in apoptosis and the use of antisense oligonucleotides (ASOs) against clusterin to enhance apoptosis in prostate cancer models. Immunostaining of tissue microarrays constructed from untreated and post-hormone treated radical prostatectomy specimens confirm that clusterin is highly expressed in virtually all HRPC cells, 80% of prostate cancer cells after neoadjuvant hormone therapy, but is low or absent (<20%) in untreated specimens. Overexpression of clusterin in LNCaP cells confers resistance to both androgen ablation and chemotherapy. Clusterin ASOs reduced clusterin levels in a dose-dependent and sequence-specific manner. Adjuvant treatment with murine clusterin ASOs after castration of mice bearing Shionogi tumors decreased clusterin levels, accelerated apoptotic tumor regression, and significantly delayed the recurrence of androgen-independent tumors. A human clusterin ASO targeting the translation initiation site and incorporating MOE-gapmer backbone (OGX-011) synergistically enhanced the cytotoxic effects of paclitaxel in human xenografts of prostate, renal cell, bladder, and lung cancer. Clusterin, is an anti-apoptosis protein upregulated in an adaptive cell survival manner by androgen ablation and chemotherapy that confers resistance to various cell death triggers. Suppression of clusterin levels using ASOs enhances cell death following treatment with androgen ablation, radiation, and chemotherapy.     

Pro-apoptotic and immunomodulatory activity of a mycobacterial cell wall-DNA complex towards LNCaP prostate cancer cells.

BACKGROUND: We have isolated a mycobacterial cell wall-DNA complex (MCC) possessing anti-cancer activity against bladder cancer cells. The anti-cancer activity of MCC appears to be due to two effects: a direct interaction with bladder cancer cells resulting in the induction of apoptosis and an indirect effect via the stimulation of monocytes and macrophages cytokine synthesis. In this study, the direct effect of MCC towards LNCaP cancer cells was evaluated. METHODS: Inhibition of proliferation, cell cycle arrest and induction of apoptosis were evaluated in vitro using LNCaP cells treated with MCC. The synthesis of IL-12, GM-CSF, and TNF-alpha by LNCaP cells in response to MCC was also determined. Experiments were performed to gain insight into the mechanism of action of MCC towards LNCaP cells. RESULTS: MCC caused a dose-dependent inhibition of the proliferation of LNCaP cells that was associated with cell cycle arrest at the G0/G1 phase. MCC-induced apoptosis of LNCaP cells was consistent with a mitochondrial pathway involving mitochondrial disruption, release of cytochrome c, and an increase in Bax protein levels leading to caspase-3 and -7 activation and cleavage of poly (ADP-ribose) polymerase and nuclear mitotic apparatus protein. Surprisingly, MCC also directly induced the synthesis of IL-12 and GM-CSF, but not TNF-alpha, by LNCaP cells. CONCLUSIONS: MCC possesses the ability to directly induce apoptosis of LNCaP cells and to trigger the synthesis of IL-12 and GM-CSF by these cells, suggesting a potential role of MCC for the treatment of prostate cancer.     

Nitrogen containing bisphosphonates induce apoptosis and inhibit the mevalonate pathway,impairing Ras membrane localization in prostate cancer cells

PURPOSE: Metastasis to bone is an important cause of morbidity in advanced prostate cancer. Despite the typically sclerotic nature of prostatic bone metastases osteolysis has a significant role in the pathogenesis of this disease. The nitrogen containing bisphosphonates (N-BPs), such as pamidronate and zoledronic acid, have greatly enhanced potency for inhibiting bone resorption and inducing apoptosis in osteoclasts. We investigated the effects of N-BPs on prostate cancer cells. MATERIALS AND METHODS: Cell viability was determined with an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeyhoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) dye reduction assay. Cell cycle analysis, DNA fragmentation and caspase 3 activity were assessed using flow cytometry. Ras, Bcl-2 and Bax were quantified by Western blotting. RESULTS: Pamidronate and zoledronic acid decreased cell viability in the 3 human cell lines DU145, PC3 and LNCaP. These effects were associated with changes in cell cycle distribution, induction of DNA fragmentation and a decrease in the Bcl-2-to-Bax ratio, which are features of apoptotic cell death. Pre-incubation with caspase inhibitors attenuated the effects of zoledronic acid and caspase 3 activity was demonstrated in treated DU145 cells. Zoledronic acid induced loss of cell viability in DU145 cells was prevented by co-treatment with farnesol, suggesting that N-BPs cause inhibition of the mevalonate pathway and Ras prenylation. A decrease in active, membrane bound Ras in zoledronic acid treated DU145 cells was shown by Western blot analysis. CONCLUSIONS: N-BPs induce apoptosis in prostate cancer via a caspase dependent mechanism. They have effects on protein prenylation via inhibition of the mevalonate pathway and impair membrane localization of Ras in prostate cancer cells.     

Bcl-2 antagonizes the combined apoptotic effect of transforming growth factor-beta and dihydrotestosterone in prostate cancer cells

BACKGROUND: We previously demonstrated that dihydrotestosterone (DHT) enhances transforming growth factor-beta (TGF-beta) -induced apoptosis in human prostate cancer cells (Endocrinology 2001;142:2419-2426). METHODS: In this study, the ability of the apoptosis suppressor bcl-2 to directly antagonize the combined apoptotic effect of TGF-beta and DHT in the androgen-sensitive LNCaP TbetaRII prostate cancer cells was examined. The previously cloned TGF-RbetaII receptor LNCaP cells, responsive to both TGF-beta and androgens, were engineered to overexpress the bcl-2 oncoprotein and the profile of apoptosis induction was analyzed in response to TGF-beta alone (5.0 ng/ml) or in combination with DHT (1 nM). RESULTS: Biological characterization of cloned LNCaP TbetaRII hygromycin/bcl-2 transfectants demonstrated that bcl-2 overexpression resulted in a significant inhibition of the combined TGF-beta and DHT apoptotic effect in prostate cancer cells (P < 0.01). Furthermore, molecular analysis indicated that this antagonistic effect of bcl-2 on apoptosis was due to partial suppression of TGF-beta and DHT-mediated induction of caspase-1 expression and activation in LNCaP TbetaRII-hygro/bcl-2 transfectants. These results support a potential bcl-2 interference with the TGF-beta and androgen apoptotic signaling in prostate cancer cells by means of an antagonistic effect on caspase-1 activation. CONCLUSION: This evidence may have mechanistic significance in understanding the contribution of bcl-2 overexpression in the development of androgen-independent prostate cancer by means of conferring resistance to TGF-beta-mediated apoptosis.     

苦参碱对雄激素依赖性前列腺癌细胞的影响

目的：探讨苦参碱（Matrine）对雄激素依赖性前列腺癌细胞株（LNCaP）凋亡及前列腺特异抗原（prostate specific antigen,PSA）表达的影响。方法：分别用0.5g／L、1.0g／L、1.5g／L、2.0g／L浓度的苦参碱作用于LNCaP细胞12h、24h、36h后MTT法检测细胞生长活性;24h后流式细胞仪测定细胞凋亡的变化;24h后Western印迹法检测细胞内Bel-2和Bax的表达;12h、24h、36h后化学发光法检测LNCaP细胞培养液中PSA的变化。结果：苦参碱能抑制LNCaP细胞的生长,呈剂量与时间依赖性,不同浓度苦参碱组之间与不同作用时间组之间的差异均有统计学意义（P〈0.05）。苦参碱诱导LNCaP细胞凋亡,各浓度组凋亡细胞比例均显著高于对照组,差异有统计学意义（P〈0.05）;LNCaP细胞内Bcl-2含量呈浓度依赖性下降,Bax含量呈浓度依赖性升高（P〈0.01）;LNCaP细胞培养液中PSA的表达显著下降（P均〈0.05）。结论：苦参碱能显著抑制LNCaP细胞的体外生长,诱导其凋亡,并抑制PSA的表达。