Overexpression of BCL-X(L) underlies the molecular basis for resistance to staurosporine-induced apoptosis in PC-3 cells

We have reported previously that among human prostate cancer cell lines LNCaP but not PC-3 cells undergo apoptosis after treatment with the protein kinase inhibitor staurosporine (STS). We have now further investigated this model to uncover the molecular mechanism causing resistance to STS-induced apoptosis in PC-3 cells. S-100 lysates of both cell lines showed biochemical changes typical of apoptosis after the addition of cytochrome c and dATP, suggesting that the postmitochondrial phase of apoptosis was intact. Upon addition of STS, the proapoptotic molecules Bax and Bad became predominantly mitochondrial in both cell lines. This, in turn, was followed by loss of mitochondrial transmembrane potential, translocation of cytochrome c to the cytosol, activation of caspase-9, -3, and -7, and cleavage of the apoptotic targets, DNA fragmentation factor and poly(ADP-ribose) polymerase, in LNCaP but not in PC-3 cells. Components of the mitochondrial permeability transition pore, adenine nucleotide transporter and voltage-dependent anion channel, were normally expressed in the correct subcellular fraction of both cell lines. Overexpression of the proapoptotic proteins Bax and Bad, fused to a green fluorescent protein but not of green fluorescent protein alone, induced apoptosis in >80% of PC-3 cells. These experiments suggested that a factor protecting the mitochondria of PC-3 cells mediates resistance to STS-induced apoptosis. A wide search among the antiapoptotic Bcl-2 family members was performed, and Bcl-X(L) was found to be overexpressed in PC-3 cells. Experiments down-regulating Bcl-X(L) expression by using the tyrosine kinase inhibitor genistein, sodium butyrate, or an antisense Bcl-X(L) oligonucleotide restored sensitivity to apoptosis in PC-3 cells. Thus, Bcl-X(L) overexpression is one of the mediators of resistance to STS-induced apoptosis in the prostate cancer cell line PC-3.     

Phellinus linteus sensitises apoptosis induced by doxorubicin in prostate cancer

It has been demonstrated that the Phellinus linteus (PL) mushroom, which mainly consists of polysaccharides, possesses antitumour activity. The mechanisms of PL against malignant growth remain unknown. The anticancer drug doxorubicin (Dox) has been shown to induce apoptosis via initiating a caspase cascade. In this investigation, we tested the effect of PL on Dox-induced apoptosis in prostate cancer LNCaP cells. We showed that PL or Dox, at relatively low doses, does not induce apoptosis in the cells. However, combination treatment with low doses of PL and Dox results in a synergistic effect on the induction of apoptosis. In this apoptotic process, caspases 8, 3 and BID are cleaved, and the addition of caspase inhibitor z-VADfmk completely blocks apoptosis. In addition, JNK is activated in response to PL or the combination treatment in LNCaP cells. The suppression of JNK partially inhibits the induction of apoptosis elicited by the co-treatment. These findings indicate that PL has a synergistic effect with Dox to activate caspases in prostate cancer LNCaP cells. Our study also suggests that PL has therapeutic potential to augment the magnitude of apoptosis induced by antiprostate cancer drugs.     

雷公藤红素对人前列腺癌细胞凋亡及SENP1基因表达的影响

目的：研究雷公藤红素对人前列腺癌（PCa）细胞生长、凋亡及SUMO特异蛋白酶1（SUMO-specificproteases 1,SENP1）基因表达的影响。方法：对前列腺癌细胞PC-3和LNCaP进行雷公藤红素处理,通过测定细胞生长曲线,荧光染色,荧光显微镜观察,流式细胞仪分析和实时定量PCR,检测雷公藤红素对前列腺癌细胞生长、凋亡及SENP1 mRNA表达水平的影响。结果：雷公藤红素能够显著抑制PCa细胞的生长,并且抑制作用呈剂量依赖性;雷公藤红素能够诱导PCa细胞凋亡,1μmol/L雷公藤红素处理24h诱导14.8%的PC-3细胞和23.2%的LNCaP细胞发生凋亡或死亡;雷公藤红素还能够降低PCa细胞中SENP1 mRNA水平,尤其是PC-3细胞。结论：雷公藤红素能够显著抑制PCa细胞的生长并诱导细胞凋亡,表明雷公藤红素具有抗前列腺癌作用,雷公藤红素能够降低PCa细胞中SENP1 mRNA水平,揭示雷公藤红素可能通过SENP1相关信号通路达到抗前列腺癌作用。     

Finasteride induces apoptosis via Bcl-2, Bcl-xL, Bax and caspase-3 proteins in LNCaP human prostate cancer cell line

We investigated the effects of finasteride, a 5alpha-reductase inhibitor, on cell death machinery through the induction of apoptosis in an in vitro model for prostate cancer. Finasteride treatment of the LNCaP hormone-dependent human prostate cancer cell line caused the loss of cell viability and accelerated apoptosis in a concentration-dependent manner. The contents of immunoreactive procaspase-3 were examined by immunoblot analysis and the results suggest that the apoptosis induced by finasteride involves the increase of caspase-3 activity. Early cell changes that occur during apoptosis are associated with mitochondrial changes mediated by members of the Bcl-2 family of proteins. Therefore, Bcl-2, Bcl-xL and Bax were evaluated by the Western blot analysis. The immunoreactivity for pro-apoptotic Bax was markedly increased whereas antiapoptotic Bcl-2 and Bcl-xL expression was significantly reduced after incubation of cells with finasteride. These findings suggest that finasteride induces apoptosis in LNCaP cells via proteins of the Bcl-2 and caspase family.     

Bcl-2 and Bcl-x(L) differentially protect human prostate cancer cells from induction of apoptosis by melanoma differentiation associated gene-7, mda-7/IL-24

Subtraction hybridization identified melanoma differentiation associated gene-7, mda-7, in the context of terminally differentiated human melanoma cells. Based on its structure, cytokine-like properties and proposed mode of action, mda-7 has now been classified as IL-24. When expressed by means of a replication-incompetent adenovirus, Ad.mda-7 induces apoptosis in a broad range of cancer cells, without inducing harmful effects in normal fibroblast or epithelial cells. These unique properties of mda-7/IL-24 suggest that this gene will prove beneficial for cancer gene therapy. We now demonstrate that Ad.mda-7 decreases viability by induction of apoptosis in hormone-responsive (LNCaP) and hormone-independent (DU-145 and PC-3) human prostate carcinomas, without altering growth or survival in early-passage normal human prostate epithelial cells (HuPEC). Ad.mda-7 causes G(2)/M arrest and apoptosis in LNCaP (p53-wildtype), DU-145 (p53 mutant, Bax-negative) and PC-3 (p53-negative) prostate carcinomas, but not in HuPEC. Apoptosis induction correlated with changes in the ratio of pro- to antiapoptotic Bcl-2 protein family members. A potential functional role for changes in bcl-2 family gene expression in Ad.mda-7-induced apoptosis was suggested by the finding that forced overexpression of bcl-x(L) or bcl-2 differentially diminished the apoptotic effect of Ad.mda-7 in prostate carcinomas. These results confirm that induction of apoptosis by the mda-7/IL-24 gene in prostate cancer cells is Bax- and p53-independent and is mediated by mitochondrial pathways involving bcl-2 family gene members. The mda-7/IL-24 gene represents a new class of cancer-specific apoptosis-inducing genes with obvious potential for the targeted gene-based therapy of human prostate cancer.     

Suppression of galectin-3 expression enhances apoptosis and chemosensitivity in liver fluke-associated cholangiocarcinoma

Cholangiocarcinoma (CCA) is a fatal disease with high resistance to anticancer drugs. This is probably in part due to enhanced resistance to apoptosis. We have previously shown that galectin-3 (Gal-3), a β-galactoside-binding lectin, is highly expressed in CCA tissues. In this study, we demonstrated further that Gal-3 plays a direct role in anti-apoptosis regardless of the apoptotic insults. The anti-apoptotic activity and chemoresistance of CCA cells were related to Gal-3 expression level. Suppression of Gal-3 expression with siRNA stimulated apoptosis. siGal-3-K626 transiently depleted Gal-3 expression to the baseline and dramatically induced apoptosis, while siGal-3-K402 suppressed Gal-3 expression by 50% and provoked cell apoptosis, but only under apoptotic insults (hypoxic conditions or short UV radiation). These actions were reversed in Gal-3 overexpressing CCA cells. The correlation between the degree of anti-apoptotic activity and the level of endogenous Gal-3 was demonstrated. Suppression of Gal-3 expression in CCA cells with siGal-3-K402 significantly enhanced apoptosis induced by cisplatin or 5-fluorouracil by approximately 10 times, whereas overexpression of Gal-3 led to an increased resistance to drugs. In summary, the present study showed that the cellular level of Gal-3 might contribute to the anti-apoptotic activity and chemoresistance of CCA cells. Hence, Gal-3 expression level in cancer cells or tissues may be a marker for predicting chemotherapeutic response, and Gal-3 may be a specific gene-targeting therapy option for treating CCA. ( Cancer Sci 2009; 00: 000–000)     

Apoptotic signaling in bufalin‐ and cinobufagin‐treated androgen‐dependent and ‐independent human prostate cancer cells

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. (Cancer Sci 2008; 99: 2467–2476)     

CD29 and CD7 mediate galectin-3-induced type II T-cell apoptosis

Galectin (Gal)-3, a M(r) 31000 member of the beta-galactoside-binding protein family, is a multifunctional protein implicated in a variety of biological functions, including tumor cell adhesion, proliferation, differentiation, angiogenesis, apoptosis, cancer progression, and metastasis. Here, we report that secreted extracellular Gal-3 can signal apoptosis of human T leukemia cell lines, human peripheral blood mononuclear cells, and activated mouse T cells after binding to cell surface glycoconjugate receptors through carbohydrate-dependent interactions because the apoptotic effect was found to be inhibited by lactose, specific sugar inhibitor, and to be dose dependent. However, the apoptosis sensitivity to Gal-3 varied among the different cell lines tested. We report that Gal-3-null Jurkat, CEM, and MOLT-4 cells were significantly more sensitive to exogenous Gal-3 than SKW6.4 and H9 cells, which express Gal-3, suggesting a cross-talk between the antiapoptotic activity of intracellular Gal-3 and proapoptotic activity of extracellular Gal-3. Furthermore, Gal-3-transfected CEM cells were found to be more resistant to C(2)-ceramide-induced apoptosis than the control CEM cells. Identification of Gal-3 cell surface receptors revealed that Gal-3 binding to CD7 and CD29 (beta(1) integrin) induced apoptosis. Gal-3 binding to its cell surface receptors results in activation of mitochondrial apoptosis events including cytochrome c release and caspase-3 activation, but not caspase-8 activation. Taken together, these results suggest that the induction of T-cell apoptosis by secreted Gal-3 may play a role in the immune escape mechanism during tumor progression through the induction of apoptosis to cancer-infiltrating T cells. The induction of T-cell apoptosis by secreted Gal-3 is dependent in part on the presence or absence of cytoplasmic Gal-3, providing a new insight for the immune escape mechanism of cancer cells.     

Ethanolic extract of Brazilian green propolis sensitizes prostate cancer cells to TRAIL-induced apoptosis

Prostate cancer represents an ideal disease for chemopreventive intervention. Propolis possesses immuno-modulatory, anti-tumour and chemopreventive properties. The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is an important endogenous anti-cancer agent that induces apoptosis selectively in tumour cells. However, some cancer cells are resistant to TRAIL-mediated apoptosis. Naturally occurring phenolic and polyphenolic compounds sensitize TRAIL-resistant cancer cells and augment the apoptotic activity of TRAIL. The ethanolic extract of Brazilian green propolis (EEP) is rich in phenolic components. Our in vitro results indicate the potential targets in the TRAIL-induced apoptotic pathway for the cancer chemopreventive activity of Brazilian propolis. We examined the cytotoxic and apoptotic effects of Brazilian EEP and its bioactive components in combination with TRAIL on LNCaP prostate cancer cells. The chemical composition of Brazilian green propolis was determined by high performance liquid chromatography-diode array detection. The cytotoxicity was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl-tetrazolium and lactate dehydrogenase assays. Apoptosis was detected using annexin V-FITC by flow cytometry and fluorescence microscopy. The mitochondrial membrane potential (?Ψm) was evaluated using DePsipher staining by fluorescence microscopy. Flow cytometry was used to analyse death receptor (TRAIL-R1 and TRAIL-R2) expression in LNCaP cells. The inhibition of nuclear factor-κB (NF-κB) (p65) activation in cancer cells was confirmed by the ELISA-based TransAM NF-κB kit. The LNCaP cells were shown to be resistant to TRAIL-induced apoptosis. Our study demonstrates that EEP sensitizes TRAIL-resistant prostate cancer cells. The main phenolic components detected in Brazilian green propolis are artepillin C, quercetin, kaempferol and p-coumaric acid. Brazilian propolis and its bioactive components markedly augmented TRAIL-mediated apoptosis and cytotoxicity in prostate cancer cells. Brazilian EEP enhanced the expression of TRAIL-R2 and the activity of NF-κB in LNCaP cells. The co-treatment of prostate cancer cells with 100 ng/ml TRAIL and 50 μg/ml EEP increased the percentage of apoptotic cells to 65.8 ± 1.2% and caused a significant disruption of ?Ψm in LNCaP cells. We show that Brazilian EEP helped cells overcome TRAIL resistance by engaging both intrinsic and extrinsic apoptotic pathways and regulating NF-κB activity. The data demonstrate the important role of Brazilian green propolis and its bioactive compounds in prostate cancer chemoprevention through the enhancement of TRAIL-mediated apoptosis.     

TRAIL (APO-2L) induces apoptosis in human prostate cancer cells that is inhibitable by Bcl-2.

To determine if TRAIL-induced apoptosis in human prostate tumor cells was suppressed by bcl-2, we compared the levels of apoptosis induced by recombinant human TRAIL in pairs of isogenic cell lines that do or do not express bcl-2. Three human prostate tumor cell lines (PC3, DU145 and LNCaP) and their bcl-2-expressing counterparts were tested for their susceptibility to TRAIL. Cells were exposed to TRAIL in the presence of cycloheximide which acted as a sensitizer. Apoptosis was induced rapidly in PC3 and DU145 neo-control transfected cells, whereas induction in LNCaP required 24 h. All three cell line variants expressing bcl-2 were resistant to the apoptotic effects of TRAIL. Caspase 3 and 8 activation was also detected in the neo control cells after treatment with TRAIL, demonstrating the rapid activation of the caspase cascade similar to that seen with other death receptors. Bcl-2 overexpression in these cells blocked activation of these caspases, suggesting that bcl-2 expression of human cancer cells may be a critical factor in the therapeutic efficacy of TRAIL.     

Soy isoflavones augment the effect of TRAIL-mediated apoptotic death in prostate cancer cells

Prostate cancer represents an ideal disease for chemopreventive intervention. Genistein, daidzein and equol, the predominant soy isoflavones, have been reported to lower the risk of prostate cancer. Isoflavones exert their chemopreventive properties by affecting apoptosis signalling pathways in cancer cells. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous anticancer agent that induces apoptosis selectively in tumour cells. Soluble or expressed in immune cells, TRAIL molecules play an important role in immune surveillance and defense mechanisms against tumour cells. However, various types of cancer cells are resistant to TRAIL-mediated apoptosis. We examined the cytotoxic and apoptotic effects of genistein, daidzein and equol in combination with TRAIL in LNCaP cells. Cytotoxicity was measured by MTT and LDH assays. Apoptosis was analyzed by flow cytometry and fluorescence microscopy using Annexin V-FITC. Mitochondrial membrane potential (ΔΨm) was evaluated by fluorescence microscopy using DePsipher staining. Flow cytometry detected the expression of death receptor TRAIL-R1 (DR4) and TRAIL-R2 (DR5) on cell surfaces. The soy isoflavones sensitized TRAIL-resistant prostate cancer cells to apoptotic death. The isoflavones did not alter death receptor expression, but significantly augmented TRAIL-induced disruption of ΔΨm in the LNCaP cells. We showed for the first time that the chemopreventive effects of soy foods on prostate cancer are associated with isoflavone-induced support of TRAIL-mediated apoptotic death.     

Why anti-Bcl-2 clinical trials fail: a solution

The alteration in expression of B cell lymphoma-2 (Bcl-2) family of protein members in cancer is involved mainly in the regulation of apoptosis. Bcl-2 family proteins are currently used as major targets in the development of methods to improve treatment outcomes for cancer patients that underwent clinical trials. Although many agents have been developed for targeting Bcl-2 in the past decade, some previous attempts to target Bcl-2 have not resulted in beneficial clinical outcome for reasons unknown. Here, we propose that this was due in part for not considering the cellular level of a different antiapoptotic protein, i.e., galectin-3 (Gal-3). Gal-3 is a member of the β-galactoside binding protein family and a multifunctional oncogenic protein which regulates cell growth, cell adhesion, cell proliferation, angiogenesis, and apoptosis. Gal-3 is the sole protein that contains the NWGR anti-death motif of the Bcl-2 family and inhibits cell apoptosis induced by chemotherapeutic agents through phosphorylation, translocation and regulation of survival signaling pathways. It is now established that Gal-3 is a candidate target protein to suppress antiapoptotic activity and anticancer drug resistance. In this review, we describe the role and relevance of Gal-3 and Bcl-2 protein family in the regulation of apoptosis and propose a novel combination therapy modality. Combination therapy that targets Gal-3 could be essential for improvement of the efficacy of Bcl-2 targeting therapy in cancers and should be studied in future clinical trials. Otherwise, not considering Gal-3 cellular level could lead to trial failure.     

Celastrol, a triterpene extracted from the Chinese "Thunder of God Vine," is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice

Interest in the use of traditional medicines for cancer prevention and treatment is increasing. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as novel anticancer drugs. Celastrol, an active compound extracted from the root bark of the Chinese medicine "Thunder of God Vine" (Tripterygium wilfordii Hook F.), was used for years as a natural remedy for inflammatory conditions. Although Celastrol has been shown to induce leukemia cell apoptosis, the molecular target involved has not been identified. Furthermore, whether Celastrol has antitumor activity in vivo has never been conclusively shown. Here, we report, for the first time, that Celastrol potently and preferentially inhibits the chymotrypsin-like activity of a purified 20S proteasome (IC(50) = 2.5 micromol/L) and human prostate cancer cellular 26S proteasome (at 1-5 micromol/L). Inhibition of the proteasome activity by Celastrol in PC-3 (androgen receptor- or AR-negative) or LNCaP (AR-positive) cells results in the accumulation of ubiquitinated proteins and three natural proteasome substrates (IkappaB-alpha, Bax, and p27), accompanied by suppression of AR protein expression (in LNCaP cells) and induction of apoptosis. Treatment of PC-3 tumor-bearing nude mice with Celastrol (1-3 mg/kg/d, i.p., 1-31 days) resulted in significant inhibition (65-93%) of the tumor growth. Multiple assays using the animal tumor tissue samples from both early and end time points showed in vivo inhibition of the proteasomal activity and induction of apoptosis after Celastrol treatment. Our results show that Celastrol is a natural proteasome inhibitor that has a great potential for cancer prevention and treatment.     

Heparin/heparan sulfate interacting protein plays a role in apoptosis induced by anticancer drugs

Heparin/heparan sulfate interacting protein (HIP, also known as ribosome protein L29) is involved in cell-cell and cell-extracellular matrix interactions and influences cell proliferation, migration and differentiation. In the present study, we investigated the role of HIP in anticancer drug-induced apoptosis. Both colon cancer HCT-116 and HT-29 cells showed dose-dependent down-regulation of HIP expression when treated with sodium butyrate. The down-regulation was negatively correlated with the percentage of apoptotic cells (R = -0.955, P = 0.03 and R = -0.792, P = 0.06 for HCT-116 and HT-29 cells, respectively). The correlation between HIP expression and apoptosis in HCT-116 cells was also evident in the differential expression of HIP in the floating and adherent cell populations. Most apoptotic cells were distributed in the floating population. HIP expression in this population was approximately 30% lower than adherent and untreated control cells. HIP expression in HCT-116 cells was also significantly decreased in parallel with apoptosis after treatment with 50 micro M camptothecin and 20 micro M 5-fluorouracil. This indicates that the down-regulation of HIP may be a general phenomenon in anticancer drug-induced apoptosis. The down-regulation of HIP occurred in the early phase of apoptosis, in parallel with the activation of caspase-3 and the externalization of phosphatidylserine. The functional significance of HIP in apoptosis was shown by knocking down the expression of HIP using small interfering RNA. A 50% reduction in HIP expression was sufficient to increase the percentage of apoptotic cells (from 11 to 20%) and increase the sensitivity of the cells to apoptosis induced by 1 mM butyrate by 60%. These results indicate that HIP may play an important role in anticancer drug-induced apoptosis.     

PrLZ protects prostate cancer cells from apoptosis induced by androgen deprivation via the activation of Stat3/Bcl-2 pathway

PrLZ/PC-1 is a newly identified, prostate-specific and androgen-inducible gene. Our previous study showed that PrLZ can enhance the proliferation and invasive capability of LNCaP cells, contributing to the development of prostate cancer. However, its potential role in androgen-independent processes remains elusive. In this study, we showed that PrLZ enhanced in vitro growth and colony formation of prostate cancer cells on androgen deprivation as well as tumorigenicity in castrated nude mice. In addition, PrLZ stabilized mitochondrial transmembrane potential, prevented release of cytochrome c from mitochondria to cytoplasm, and inhibited intrinsic apoptosis induced by androgen depletion. Mechanistically, PrLZ elevated the phosphorylation of Akt and Stat3 and upregulated Bcl-2 expression. Our data indicate that PrLZ protects prostate cancer cells from apoptosis and promotes tumor progression following androgen deprivation. In summary, we propose that PrLZ is a novel antiapoptotic gene that is specifically activated in prostate cancer cells escaping androgen deprivation may offer an appealing therapeutic target to prevent or treat advanced prostate malignancy.     

Allyl isothiocyanate,a constituent of cruciferous vegetables,inhibits growth of PC-3 human prostate cancer xenografts in vivo

We have shown previously that allyl isothiocyanate (AITC), a constituent of cruciferous vegetables, significantly inhibits survival of PC-3 and LNCaP human prostate cancer cells in culture, whereas proliferation of a normal prostate epithelial cell line is minimally affected by AITC even at concentrations that are highly cytotoxic to the prostate cancer cells. The present studies were designed to test the hypothesis that AITC administration may retard growth of human prostate cancer xenografts in vivo. Bolus i.p. injection of 10 micromol AITC, three times per week (Monday, Wednesday and Friday) beginning the day of tumor cell implantation, significantly inhibited the growth of PC-3 xenograft (P < 0.05 by two-way ANOVA). For example, 26 days after tumor cell implantation, the average tumor volume in control mice (1025 +/- 205 mm3) was approximately 1.7-fold higher compared with AITC-treated mice. Histological analysis of tumors excised at the termination of the experiment revealed a statistically significant increase in number of apoptotic bodies with a concomitant decrease in cells undergoing mitosis in the tumors of AITC-treated mice compared with that of control mice. Western blot analysis indicated an approximately 70% reduction in the levels of anti-apoptotic protein Bcl-2 in the tumor lysate of AITC-treated mice compared with that of control mice. Moreover, the tumors from AITC-treated mice, but not control mice, exhibited cleavage of BID, which is known to promote apoptosis. Statistically significant reduction in the expression of several proteins that regulate G2/M progression, including cyclin B1, cell division cycle (Cdc)25B and Cdc25C (44, 45 and 90% reduction, respectively, compared with control), was also observed in the tumors of AITC-treated mice relative to control tumors. In conclusion, the results of the present study indicate that AITC administration inhibits growth of PC-3 xenografts in vivo by inducing apoptosis and reducing mitotic activity.     

Inhibition of the phosphatidylinositol 3'-kinase pathway promotes autocrine Fas-induced death of phosphatase and tensin homologue-deficient prostate cancer cells

Rationally designed therapeutics that target the phosphatidylinositol 3'-kinase (PI3K) cell survival pathway are currently in preclinical and clinical development for cancer therapy. Drugs targeting the PI3K pathway aim to inhibit proliferation, promote apoptosis, and enhance chemosensitivity and radiosensitivity of cancer cells. The phosphatase and tensin homologue (PTEN) phosphatidylinositol 3'-phosphatase is a key negative regulator of the PI3K pathway. Inactivation of the PTEN tumor suppressor results in constitutive activation of the PI3K pathway and is found in approximately 50% of advanced prostate cancers, which correlates with a high Gleason score and poor prognosis. Inhibition of the PI3K pathway leads to apoptosis of prostate cancer cells; however, the precise mechanism by which this occurs is unknown. Here we report that apoptotic cell death of PTEN-deficient LNCaP and PC3 prostate cancer cells induced by the PI3K inhibitor LY294002 can be abrogated by disrupting Fas/Fas ligand (FasL) interactions with recombinant Fas:Fc fusion protein or FasL neutralizing antibody (Nok-1), or by expressing dominant-negative Fas-associated death domain. Furthermore, we find that apoptosis induced by expression of wild-type PTEN, driven by a tetracycline-inducible expression system in LNCaP cells, can be inhibited by blocking Fas/FasL interaction using Fas:Fc or Nok-1. These data show that apoptosis induced by blockade of the PI3K pathway in prostate tumor cells is mediated by an autocrine Fas/FasL apoptotic mechanism and the Fas apoptotic pathway is both necessary and sufficient to mediate apoptosis by PI3K inhibition.