P53 and the proteasome regulate androgen receptor activity.

Mutual regulation of expression between p53 and AR has been reported. To further investigate the role of p53 in the regulation of AR expression, an ARE-Luciferase vector was inserted into LNCaP and into LNCaP-sip53 transfectants, and AR activity was quantitatively estimated after treatment with proteasome inhibitors. LNCaP expresses a mutated form of AR. Therefore, to investigate whether p53 can modulate the expression of wild-type (wt) of AR, we transfected PC3-wtAR with a p53 vector together with ARE-Luc and showed that p53 expression decreased DHT-dependent activity of wtAR. Since proteasomes also participate in AR transcriptional activity, we investigated the role of p53 in proteasome-dependent inhibition of AR activity. More than 80% of AR activity was inhibited by 3 µM of lactacystin in LNCaP whereas no inhibition was noted in LN-sip53. We also found that lactacystin decreased AR-DNA binding 3-fold in LNCaP but no binding decrease was observed in LN-sip53. Taken together, our data show that the inhibitory effects of proteasome inhibitors are dependent on p53 status, at least in prostate cancer. Therefore, the role of p53 during treatment with proteasome inhibitors in different tumors should be further investigated.

Inhibition of p53 expression modifies the specificity of chromatin binding by the androgen receptor.

The androgen receptor (AR) is known to play a critical role in prostate cancer (PC). p53 likely also plays a role given that p53 mutations are commonly found in advanced PC, and loss of wild-type protein function contributes to the phenotype of castration-resistant prostate cancer (CRPC). Nevertheless, the extent of the contribution of p53 dysfunction to PC remains unclear. Here we analyze the effects of p53 inhibition in PC cells and show that it has significant consequences for both the interaction between AR, and chromatin and the proliferative capacity of these cells. Inhibition of p53 expression enabled LNCaP cells to proliferate independently of androgens. Moreover, it modified the genome-wide binding pattern of AR. ChIP-sequnce analyis (ChIP-seq) revealed that fewer AR-binding sites were present in the context of p53 inhibition, suggesting that wild-type p53 is required for stable binding of AR to certain chromatin regions. Further analysis revealed that a lower AR occupancy was accompanied by a reduction in FoxA1 binding at regulatory regions of AR-dependent genes. Our study also identifies a pool of genes that may be transcriptionally regulated by AR only in the absence of p53, and that may contribute to the CRPC phenotype. Overall, our results point to p53 playing an important role in regulating AR activity across the genome.

Caspase-2-Based Regulation of the Androgen Receptor and Cell Cycle in the Prostate Cancer Cell Line LNCaP.

Caspase-2 can induce apoptosis in response to extrinsic and intrinsic signals. Unlike other caspases, this protein is not expressed solely in nonnuclear compartments; a subpopulation is constitutively localized in the nucleus. As one of the most evolutionarily conserved caspases, caspase-2 may have roles in multiple cellular processes. However, its contribution to nonapoptotic processes remains a mystery. In this study, we show that caspase-2 activity is important for proliferation by cells of the androgen-dependent prostate cancer cell line LNCaP. LNCaP cells expressing either a dominant-negative (dn) form of caspase or an siRNA against caspase-2 had lower androgen receptor (AR)-dependent proliferative responses than control cells, and application of the siRNA resulted in downregulation of the expression of both AR-dependent prostate-specific antigen (PSA) and AR-dependent reporter luciferase. Also, caspase-2 formed complexes with the cell cycle regulatory proteins cyclin D3, CDK4, and p21/Cip1, and caspase-2 regulated AR transactivation by inhibiting the repressive function of cyclin D3. Taken together, these results reveal, for the first time, that caspase-2 is involved in cell cycle promotion and AR activation. Given that prostate cancer cells depend on AR activity in order to survive, the fact that our data indicate that caspase-2 positively regulates AR activity suggests that caspase-2 has potential as a target in the treatment of prostate cancer.

TOFA (5-tetradecyl-oxy-2-furoic acid) reduces fatty acid synthesis, inhibits expression of AR, neuropilin-1 and Mcl-1 and kills prostate cancer cells independent of p53 status.

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.

KN-93 inhibits androgen receptor activity and induces cell death irrespective of p53 and Akt status in prostate cancer.

It has been suggested that the downregulation of AR expression should be considered the principal strategy for the treatment of hormone-refractory prostate cancer. We have previously shown that inhibition of AR induced PI3K-independent activation of Akt that was mediated by CaMKII. In this study, we found that the CaMKII inhibitor KN-93 has a broader effect on apoptosis than just inhibition of CaMKII: first, KN-93 inhibits AR activity and induces cell death in PCa cells after androgen deprivation when many other drugs fail to kill prostate cancer cells; second, KN-93 inhibits expression of the anti-apoptotic protein Mcl-1 and induces expression of the pro-apoptotic protein PUMA; third, KN-93-mediated cell death is p53-independent; and fourth, KN-93 induces the generation of ROS. The ROS induction allows KN-93 to circumvent the activation of Akt, which occurs in prostate cancer cells under androgen deprivation, since Akt could not inhibit ROS-mediated apoptosis. KN-93 also synergistically induces cell death in combination with low doses of doxorubicin and converts the phenotype of prostate cancer cells from TRAIL-resistant to -sensitive. These data suggest that KN-93 could be used for novel therapeutic approaches when hormonal therapy has failed.

Mechanisms of prostate cancer cell survival after inhibition of AR expression.

Recent reports have shown that the AR is the key determinant of the molecular changes required for driving prostate cancer cells from an androgen-dependent to an androgen-independent or androgen depletion-independent (ADI) state. Several recent publications suggest that down-regulation of AR expression should therefore be considered the principal strategy for the treatment of ADI prostate cancer. However, no valid data is available about how androgen-dependent prostate cancer cells respond to apoptosis-inducing drugs after knocking down AR expression and whether prostate cancer cells escape apoptosis after inhibition of AR expression. This review will focus on mechanisms of prostate cancer cell survival after inhibition of AR activity mediated either by androgen depletion or by targeting the expression of AR by siRNA. We have shown that knocking down AR expression by siRNA induced PI3K-independent activation of Akt, which was mediated by calcium/calmodulin-dependent kinase II (CaMKII). We also showed that the expression of CaMKII genes is under AR control: active AR in the presence of androgens inhibits CaMKII gene expression whereas inhibition of AR activity results in an elevated level of kinase activity and in enhanced expression of CaMKII genes. This in turn activates the anti-apoptotic PI3K/Akt pathways. CaMKII also express anti-apoptotic activity that is independent from the Akt pathway. This may therefore be an important mechanism by which prostate cancer cells escape apoptosis after androgen depletion or knocking down AR expression. In addition, we have found that there is another way to escape cell death after AR inhibition: DNA damaging agents cannot fully activate p53 in the absence of AR and as a result p53 down stream targets, for example, microRNA-34, cannot be activated and induce apoptosis. This implies that there may be a need for re-evaluation of the therapeutic approaches to human prostate cancer.

Functional genetic screening reveals the role of mitochondrial cytochrome b as a mediator of FAS-induced apoptosis.

Functional selection of genetic suppressor elements (GSEs), engineered gene fragments that interfere with the function of a particular gene product, was used to identify regulators of FAS-induced apoptosis. Chicken DF-1 cells expressing human FAS receptor and susceptible to FAS-induced apoptosis were infected with a GSE library consisting of randomly fragmented normalized chicken cDNAs in a replication-competent avian retroviral vector. Virus-producing cells were subjected to several rounds of selection using FAS agonistic antibodies, resulting in isolation of a set of GSEs conferring resistance to FAS-induced apoptosis. Surprisingly, one of the isolated GSEs encoded a 42 amino acid-long polypeptide derived from the C-terminal half of cytochrome b (Cyt b) encoded by the mitochondrial genome. Subsequent experiments showed that caspase 8-dependent cleavage of mitochondrial Cyt b and translocation of its C-terminal half into the cytoplasm occurred during FAS-induced apoptosis in both chicken and human cells. Ectopic cytoplasmic expression of either full-length Cyt b or its C-terminal half in several human cell lines induced apoptosis, which could be suppressed by the isolated GSE, but not by Bcl2 over-expression or Apaf-1 or cytochrome c knock-down. These results reveal a cytochrome c-independent branch of FAS-induced apoptosis involving cleavage and cytoplasmic release of mitochondrial Cyt b.

Prostate cancer-specific monoclonal antibody 5D4 significantly enhances the cytotoxicity of doxorubicin-loaded liposomes against target cells in vitro.

Long-circulating liposomes loaded with doxorubicin (Dox) were additionally modified with the prostate cell-specific monoclonal antibody 5D4 (mAb 5D4). The resultant Dox-loaded 5D4-immunoliposomes specifically recognized prostate cancer cell lines of several different types expressing the mAb 5D4 antigen, PSMA, and significantly enhanced cytotoxicity toward these cells compared with the non-targeted Dox-liposomes in vitro while no increased toxicity was observed toward non-prostate (lung) cancer cell line.

MicroRNA-34 mediates AR-dependent p53-induced apoptosis in prostate cancer.

We investigated whether knocking down AR expression effects apoptosis after treatment with different apoptosis-inducing agents. We found that siRNA AR (si-AR) significantly decreased apoptosis induced by topoisomerase inhibitors doxorubicin (DOX) and camptothecin (Campt). It is known that DNA double-strand break inducing agents leads to activation (phosphorylation) of p53 that in turn regulates the expression of a variety of apoptosis-related genes including microRNA(miR)-34a and 34b/c. We found that DOX induced five phosphorylation sites of p53 (Ser15, 20, 37, 46 and 392); all of these sites were inhibited by si-AR. Subsequently we identified three kinases, SPAK, MDC1 and CaMKII that are under AR control and two of them, MDC1 and CaMKII, apparently participate in p53 upstream events that resulted in p53 inhibition. Using qPCR we showed that the level of miR-34a increased by 3-fold after DOX, but no increase was found with si-AR. MiR-34c expression increased 27 fold after DOX and only by 2.7 times with si-AR. It appears that AR-dependent inhibition of p53 resulted in suppression of miR-34a and -34c expression. Importantly, DOX did not induce miR-34 in LNCaP grown in an androgen free medium or in AR-negative prostate cancer cell lines, DU145 and PC3. To directly investigate the role of miR-34 in DOX-mediated apoptosis, we transfected cells with anti-miR-34 oligonucleotides or with miR-34. We found that inhibition of individual miR-34, either 34a or 34c, or forced overexpression of miR-34a or miR-34c did not modulate DOX-mediated apoptosis. Only simultaneous inhibition or forced overexpression of both miR-34 resulted in modulation of DOX-mediated apoptosis. Taken together, our data indicate that cooperation between miR-34a and 34c plays an important role in AR-dependent p53-mediated apoptosis in prostate cancer.

Unique resistance of breast carcinoma cell line T47D to TRAIL but not anti-Fas is linked to p43cFLIP(L).

The majority of breast cancer cell lines are resistant to tumor necrosis factor -related apoptosis inducing ligand (TRAIL) induced apoptosis. TRAIL and Fas receptor death-inducing signaling complex (DISCs) formation are similar and involve ligand-dependent recruitment of FADD and caspase-8. We have found that the breast carcinoma cell line T47D is an unusual example of selective sensitivity to anti-Fas mAb treatment but resistant to TRAIL. Therefore, a detailed comparison of these two signaling pathways in one cell line should provide insight into the mechanism of TRAIL resistance. We observed that only anti-Fas mAb induces caspase activation and cell death in T47D. Further, FADD and caspase-8 interact with both TRAIL-R1 and TRAIL-R2, and that the amount of caspase-8 recruited by Fas-, TRAIL-R1 and TRAIL-R2 are the same. cFLIP(S) and cFLIP(R )isoforms block death receptor-induced apoptosis by inhibiting caspase-8 activation at the DISC; the role of cFLIP(L )at the DISC is still controversial. It has been suggested that the presence of the cleaved form of FLIP(L)-p43 at the DISC prevents caspase-8 cleavage. We found that both TRAIL and anti-Fas mAb-induced DISCs contain the cleaved form of p43 cFLIP(L) and its amount at the Fas DISC was higher compared to the TRAIL DISC. We also found that inhibition of cFLIP(L) expression in T47D cells decreased Fas-mediated caspase-8 activation and activation of effector caspases. We propose that in T47D p43 cFLIP(L) in the Fas-DISC may promote caspase-8 activation. The mechanism by which different amounts of p43cFLIP(L) regulates caspase-8 activation remains to be investigated.

Chemotherapeutic agents up-regulate the cytomegalovirus promoter: implications for bioluminescence imaging of tumor response to therapy.

Bioluminescence imaging is widely used to evaluate tumor growth and response to therapy in living animals. In cells expressing luciferase under the control of a constitutive promoter, light output in part depends on viable cell number, so that changes in bioluminescence intensity may be correlated with changes in viable tumor mass over time. We have found that treatment of cancer cell lines expressing luciferase under control of the cytomegalovirus (CMV) promoter with staurosporine, doxorubicin, and paclitaxel results in a transient increase in bioluminescence, which is positively correlated with apoptosis and inversely correlated with cell viability. In contrast, similar treatment of cell lines expressing luciferase under control of the SV40 promoter did not exhibit this result. We found that low doses of staurosporine induced bioluminescence in CMV- but not SV40-driven luciferase cell lines, whereas high doses elicited induction in both, indicating promoter-dependent and promoter-independent mechanisms of bioluminescence induction. The promoter-dependent increase in bioluminescence intensity from CMV-driven luciferase is a result of induction of luciferase mRNA and protein expression. We extended these findings in vivo; doxorubicin treatment resulted in a transient induction in bioluminescence when normalized to tumor volume in CMV- but not SV40-driven luciferase-expressing xenografts. We found that inhibition of the p38 mitogen-activated protein kinase pathway blocked bioluminescence induction by doxorubicin, paclitaxel, and staurosporine in CMV-driven luciferase-expressing cells. These findings have important implications when using bioluminescence to monitor the efficacy of anticancer therapy and underscore the complex regulation of the CMV promoter, which is widely used for high-level protein expression in mammalian cells.

Calcium/calmodulin-dependent kinase II plays an important role in prostate cancer cell survival.

It has recently been shown that the androgen receptor (AR) is the main factor that required for prostate cancer cells survival. We show that knocking down AR expression by siRNA induces PI3K-independent activation of Akt, which was mediated by calcium/calmodulin-dependent kinase II (CaMKII). We further show, for the first time, that prostate cancer cells express beta,gamma and delta CaMKII genes, and the expression of these genes is under the control of AR activity: active AR in the presence of androgens inhibits CaMKII gene expression whereas inhibition of AR activity results in elevated level of kinase activity and in enhanced expression of CaMKII-beta and -gamma genes. Overexpression of CaMKII genes results in resistance to apoptosis induced by KN-93, a CaMKII inhibitor, or wortmanninn, a PI3K/Akt inhibitor, in combination with doxorubicin, thapsigargin and TRAIL. Moreover, overexpression of CaMKII increases secretion of prostate specific antigen and promotes cell growth of LNCaP in steroid-free condition. Our data show that there is cross-talk between AR- and CaMKII-mediated pathways. The results of this study suggest that CaMKII is an important player in prostate cancer cells ability to escape apoptosis under androgen ablation and facilitate the progression of prostate cancer cells to an androgen independent state.

TSA-induced cell death in prostate cancer cell lines is caspase-2 dependent and involves the PIDDosome.

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.

Mechanisms of cell death induced by histone deacetylase inhibitors in androgen receptor-positive prostate cancer cells.

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.

Androgen regulates apoptosis induced by TNFR family ligands via multiple signaling pathways in LNCaP.

It has been suggested in many studies that combined treatment with chemotherapeutic agents and apoptosis-inducing ligands belonging to TNFR family is a more effective strategy for cancer treatment. However, the role of androgen regulation of TNFR family-induced apoptosis in prostate cancer is poorly understood. In this study, we investigated the dose-dependent effects of androgen on TNF-alpha and TRAIL-mediated apoptosis in LNCaP. To investigate the interaction between the androgen receptor (AR) and the caspase-2 gene, chromatin immunoprecipitation analysis was used, and we are the first to identify that AR interacts in vivo with an androgen-responsive elements in intron 8 of caspase-2 gene. We have found that DHT inhibited apoptosis in dose-dependent manner. There is a direct, androgen-dependent correlation between the levels of activated Akt and caspase activation after treatment with TNF-alpha and TRAIL. We have also found that there are at least two different regulatory mechanisms of p53 expression by androgen: at the gene and protein levels. At the same time, the level of AR was found to be higher in LNCaP-si-p53 compared to LNCaP-mock cells. These data indicate that there is a mutual regulation of expression between p53 and AR. Our study suggests that androgen-dependent outcome of apoptotic treatment can occur, at least in part, via the caspase-2, Akt and p53-mediated pathways.

p53 is a suppressor of inflammatory response in mice.

Chronic inflammation is known to promote cancer, suggesting that negative regulation of inflammation is likely to be tumor suppressive. We found that p53 is a general inhibitor of inflammation that acts as an antagonist of nuclear factor kappaB (NFkappaB). We first observed striking similarities in global gene expression profiles in human prostate cancer cells LNCaP transduced with p53 inhibitory genetic element or treated with TNF, suggesting that p53 inhibits transcription of TNF-inducible genes that are largely regulated by NFkappaB. Consistently, ectopically expressed p53 acts as an inhibitor of transcription of NFkappaB-dependent promoters. Furthermore, suppression of inflammatory response by p53 was observed in vivo in mice by comparing wild-type and p53 null animals at molecular (inhibition of transcription of genes encoding cytokines and chemokines, reducing accumulation of reactive oxygen species and protein oxidation products), cellular (activation of macrophages and neutrophil clearance) and organismal (high levels of metabolic markers of inflammation in tissues of p53-deficient mice and their hypersensitivity to LPS) levels. These observations indicate that p53, acting through suppression of NFkappaB, plays the role of a general "buffer" of innate immune response in vivo that is well consistent with its tumor suppressor function and frequent constitutive activation of NFkappaB in tumors.

Trichostatin A (TSA) sensitizes the human prostatic cancer cell line DU145 to death receptor ligands treatment.

The human prostatic carcinoma cell line DU145 has previously been found to be resistant to treatment with TNF-family ligands. However, TRAIL, TNF-alpha and anti-Fas antibodies (Ab) treatment in combination with the histone deacetylase inhibitor Trichostatin A (TSA) converted the phenotype of DU145 from resistant to sensitive. TSA induced 15% cell death but simultaneous treatment with TRAIL, TNF-alpha and anti-Fas Ab resulted in 55%, 70% and 40% cell death, respectively. Simultaneous treatment did not increase the level of TSA-induced histone acetylation, but induced the release of acetylated histones from chromatin into the cytosol. This release was caspase dependent since it was abrogated by Z-VAD-fmk. In addition, treatment with TSA induced caspase-9 activation and resulted in the release of cytochrome c and Smac/DIABLO from mitochondria. To further investigate the role of caspase-9 in TSA-mediated apoptosis we used two different approaches: (1) cells were pretreated with the caspase-9 inhibitor Z-LEHD-fmk, and (2) cells were transfected with a dominant-negative form of caspase-9. Both approaches gave similar results: cells became resistant to treatment with TSA. These data indicate that TSA mediates its effect via the mitochondrial pathway. This was confirmed by examining DU145 overexpressing Bcl-2. These transfectants were resistant to TSA treatment. Taken together, our data shows that only simultaneous treatment with TNF-family ligands and TSA in DU145 resulted in caspase activity sufficient to induce apoptosis. The combination of TSA and TNF-family ligands could potentially be the basis for the treatment of prostate cancer.

Tumor necrosis factor-related apoptosis-inducing ligand-mediated activation of mitochondria-associated nuclear factor-kappaB in prostatic carcinoma cell lines.

It has been suggested that some nuclear transcription factors may participate in the regulation of mitochondrial functions through transcriptional control of mitochondrial DNA. Very little is known about the response of transcription factors within mitochondria to the activation of death receptors. Recent publications indicate that nuclear factor-kappaB (NF-kappaB) is localized in mitochondria of mammalian cells. Because of the critical role of mitochondria in the execution of many apoptotic pathways, we suggest that NF-kappaB-dependent mechanisms operating at the level of mitochondria contribute to its role in regulating death receptor signaling. We have found NF-kappaB p65 and p50 subunits with DNA binding activity in the mitochondria of prostatic carcinoma cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) affects DNA binding activity of mitochondria-associated NF-kappaB but does not change the amount of p65 in mitochondria, which suggests activation of mitochondrial NF-kappaB without additional translocation of NF-kappaB subunits to mitochondria. We have also shown that TRAIL decreases mitochondrial genome encoded mRNA levels and inhibition of NF-kappaB prevents this decrease. TRAIL effects on mitochondrial NF-kappaB-DNA binding and mitochondrial genome encoded mRNA levels also depend on Bcl-2 overexpression. In addition, transcription factor activator protein-1 with DNA binding activity is also found in mitochondria of prostatic carcinoma cells and TRAIL treatment affects this binding. In summary, NF-kappaB is found in mitochondria of prostatic carcinoma cells, where it is thought to regulate mitochondria genome encoded mRNA levels in response to TRAIL treatment.

Multiple effects of N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) on apoptotic pathways in human prostatic carcinoma cell lines.

TPCK is widely used as an inhibitor of chymotrypsin-like proteases but has recently been identified as an inhibitor of the PDK1/Akt pathway. In this study, we show that TPCK inhibits TRAIL-induced caspase activity but potentiates wortmannin-dependent caspase activity in prostatic carcinoma cell lines. The inhibitory activity of TPCK was found to be death ligand-specific since TPCK inhibits TRAIL-mediated caspase activity but does not affect Fas-induced caspase activity. Our data also show that impaired TRAIL-DISC formation in the presence of TPCK is responsible for caspase inhibition. Further, TPCK induces p53 expression and inhibits the PDK1/Akt pathway resulting in BAD dephosphorylation, and the release of cytochrome c and Smac/DIABLO from mitochondria. TPCK also selectively decreases the levels of androgen receptor and caspase-2 whereas it does not change the levels of other proteins (caspases-3, -7, -8, -9; heat shock proteins 27, 70, 90). Finally, TPCK-induced degradation of caspase-2 is protected by Bcl-2 overexpression, apparently by an adapter protein since direct interaction between caspase-2 and Bcl-2 was not detected. Together, these features suggest that TPCK could be used as a therapeutic agent for treatment of those tumor cells that are resistant to ligand-induced treatment because of aberrant signaling pathways downstream of the DISC.

Death receptor-induced cell death in prostate cancer.

Prostate cancer mortality results from metastasis and is often coupled with progression from androgen-dependent to androgen-independent growth. Unfortunately, no effective treatment for metastatic prostate cancer increasing patient survival is available. The absence of effective therapies reflects in part a lack of knowledge about the molecular mechanisms involved in the development and progression of this disease. Apoptosis, or programmed cell death, is a cell suicide mechanism that enables multicellular organisms to regulate cell number in tissues. Inhibition of apoptosis appears to be a critical pathophysiological factor contributing to the development and progression of prostate cancer. Understanding the mechanism(s) of apoptosis inhibition may be the basis for developing more effective therapeutic approaches. Our understanding of apoptosis in prostate cancer is relatively limited when compared to other malignancies, in particular, hematopoietic tumors. Thus, a clear need for a better understanding of apoptosis in this malignancy remains. In this review we have focused on what is known about apoptosis in prostate cancer and, more specifically, the receptor/ligand-mediated pathways of apoptosis as potential therapeutic targets.