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.

It has been suggested in many studies that combined treatment with chemotherapeutic agents and apoptosisinducing 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-a 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-a 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.

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.

The human prostatic carcinoma cell line LNCaP is sensitive to TNF-a treatment and expresses wild-type p53. To analyse the possible role of p53 in TNF-amediated apoptosis, we generated a derivative of LNCaP, LN-56, expressing a dominant-negative element of p53, GSE56. P53 inactivation in LN-56 was associated with an increased resistance to apoptosis induced by TNF-a. Surface expression of TNF-a receptors was unchanged in LN-56 compared to LNCaP. TNF-a treatment resulted in accumulation of p53 in LNCaP and upregulation of p21/WAF1. Activation of caspase-7 and PARP proteolysis were delayed in LN-56 under TNF-a treatment. TNF-a-induced apoptosis in LNCaP cells was accompanied by caspase-dependent proteolysis of p21/WAF1 and Rb, which was signi®-cantly attenuated in LN-56. Cytochrome c release was induced by TNF-a treatment in both cell lines, but caspase-9 was not activated. LNCaP and LN-56 were injected s.c. in nude mice and tumors were identi®ed in all LN-56, but not LNCaP, bearing mice indicating that p53 plays an important role in growth control of prostatic neoplasms. Interestingly, accumulation of p53 in TNF-a-treated LNCaP cells was decreased in the presence of the caspase inhibitor Z-VAD-FMK, suggesting a new role of activated caspases in acceleration of p53 response. In summary, these results indicate that p53 is involved in TNF-a-mediated apoptosis in LNCaP.

We and others have previously described that the androgen-responsive human prostatic carcinoma cell line LNCaP is resistant to TRAIL and that TRAIL-mediated apoptosis in LNCaP is PI3K/Akt-dependent. In this study, we found that LNCaP remained resistant to treatment with TRAIL after androgen deprivation even in the presence of the PI3K/Akt pathway inhibitor wortmannin. This resistance was determined by failure to form the TRAIL-DISC and by decreased TRAIL-R1 and TRAIL-R2 levels after androgen deprivation; the capacity of TRAIL to induce DISC formation was completely restored in the presence of DHT. TRAIL and wortmannin together accelerated processing of caspase-8 on the DISC and apparently the release of caspase-8 from the DISC into the cytoplasm. Surprisingly, we found that wortmannin decreased the total amount of TRAIL-R1, but not TRAIL-R2, in the cells as well as the amount of TRAIL-R1 precipitated by TRAIL. Our data suggest that TRAIL-DISC formation and sensitivity to TRAIL treatment are androgen-dependent in LNCaP.