JS-K, a glutathione/glutathione S-transferase-activated nitric oxide releasing prodrug inhibits androgen receptor and WNT-signaling in prostate cancer cells.

BACKGROUND: Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC). METHODS: Prostate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay. RESULTS: The NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway. CONCLUSIONS: Our results suggest that small molecules able to inhibit WNT- and AR-signaling via NO-release represent a promising platform for the development of new compounds for the treatment of CRPC.

Down-regulation of CK2 activity results in a decrease in the level of cdc25C phosphatase in different prostate cancer cell lines.

Protein kinase CK2 is implicated in the regulation of the cell cycle. In addition to a variety of functions, CK2 has anti-apoptotic properties. So far the role of CK2 linking both pathways in the cell is not clear. Some years ago we found that CK2 phosphorylates cdc25C, one member of the cdc25 family of proteins. In this study, we showed that inhibition of CK2 activity by three different inhibitors led to a down-regulation of the level of cdc25C. Inhibition of CK2 activity by transfecting the dominant-negative CK2α subunit also resulted in a down-regulation of the level of cdc25C whereas inhibition of CK2α' had no effect on the cdc25C level. In both cases, we observed apoptosis by PARP cleavage as well as by an increase in γH2AX phosphorylation. These results show that down-regulation of the level of cdc25C is not a prerequisite for the induction of apoptosis.

p53 is dispensable for the induction of apoptosis after inhibition of protein kinase CK2.

BACKGROUND: Protein kinase CK2 is a ubiquitously expressed heterotetramer consisting of two catalytic alpha/alpha' and two regulatory beta subunits. Expression of CK2 is highly elevated in tumor cells where it protects cells from apoptosis. A variety of different compounds were tested as inhibitors of protein kinase CK2 in order to find new therapy strategies. To analyze the role of p53 in the response to CK2 inhibition we used one of the most specific CK2 inhibitors available, TBB, in different prostate cancer cell lines. METHODS: We treated prostate cancer cells with the CK2 inhibitor TBB and determined its effect on CK2 activity by an in vitro phosphorylation assay and its effect on viability by an MTT assay. Furthermore, we analyzed changes in the expression of p53 and PARP cleavage by Western Blot analysis. RESULTS: Inhibition of CK2 by TBB led to a decrease in cell viability and apoptosis in two cell lines which express wild-type p53 whereas two other cell lines expressing mutant or no p53 failed to show signs of apoptosis. Moreover, cell lines expressing wild-type p53 showed an increase of the amount of p53 and of its transactivation efficiency. However, down-regulation of p53 by RNAi showed that p53 is not necessary for the induction of apoptosis. CONCLUSIONS: Wild-type p53 is not necessary for the induction of apoptosis by TBB in prostate cancer cells.

DMAT, an inhibitor of protein kinase CK2 induces reactive oxygen species and DNA double strand breaks.

Elevated levels of protein kinase CK2 were found in tumour cells compared to normal cells. Thus, inhibition of CK2 kinase activity seems to be an attractive method to stop growth of cancer cells. Two drugs, namely tetrabromobenzotriazole, TBB, and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, DMAT were found to specifically inhibit CK2 and to induce apoptosis in tumour cells. The aim of the present study was the elucidation of the mode of action of these two inhibitors in addition to the inhibition of CK2 activity. The decrease in CK2 kinase activity induced by both inhibitors was accompanied by a reduction in cell viability and induction of apoptosis. Most interestingly, we detected that DMAT in contrast to TBB induced reactive oxygen species, ROS and DNA-double-strand-breaks (DSBs). Thus, in addition to inhibition of CK2 one has to consider ROS and DSBs contributing to the induction of apoptosis by DMAT.

Inhibition of CK2 activity provokes different responses in hormone-sensitive and hormone-refractory prostate cancer cells.

Protein kinase CK2 seems to play an essential role in cellular growth regulation as well as in apoptosis. By using a pair of prostate carcinoma cell lines which are either hormone-sensitive (LNCaP cells) or hormone-refractory (PC-3 cells) we analysed the contribution of protein kinase CK2 to their different growth behaviour as well as to apoptosis. We found the same amount of CK2 subunits in both cell lines although the enzymatic activity of CK2 was much higher in the hormone-refractory cells. These results for the first time show a correlation between the specific activity of protein kinase CK2 and specific growth properties of prostate cancer cells. The antiproliferative flavonoid apigenin led to an inhibition of the CK2 activity in both types of cells but only the hormone-sensitive LNCaP cells responded with apoptosis. Thus, these results demonstrate that a high CK2 activity is dispensable for growth and not necessary for a protection against apoptosis in hormone-refractory prostate cancer cells.

CK2 inhibition induces apoptosis via the ER stress response.

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase consisting of two catalytic α/α' and two regulatory ß subunits. Expression of CK2 is highly elevated in tumor cells where it protects cells from apoptosis. Accordingly inhibition of CK2 is known to induce programmed cell death, making it a promising target for cancer therapy. In the present study we investigated apoptosis induction by the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in prostate tumor cells. In contrast to PC-3 cells LNCaP cells respond to CK2 inhibition with apoptosis. Most interestingly we found the mitochondrial pathway induced in LNCaP as well as in PC-3 cells as monitored by down-regulation of bcl-2 and subsequent cytochrome c release. In both cell lines activation of caspase 9 was not detected. Instead, an activation of the endoplasmic reticulum (ER) stress response in LNCaP cells after treatment with the CK2 inhibitor TBB was found. We show that this ER stress response led to an up-regulation of the death receptor DR5 and subsequent apoptosis in LNCaP cells.

Inability of NCoR/SMRT to repress androgen receptor transcriptional activity in prostate cancer cell lines.

The molecular mechanisms leading to castration-resistant prostate cancer (CRPC) are poorly understood. Among several mechanisms leading to CRPC growth a dysregulation of androgen receptor (AR) co-regulators (i.e. up-regulation of co-activators or down-regulation of co-repressors) is discussed. There are numerous reports demonstrating an increased expression of co-activators during prostate cancer progression. On the contrary, the impact of co-repressors on tumor growth and development is less clear. In this study we compared the effects of two known co-repressors, NCoR and SMRT, on AR transcriptional activity in prostate cancer (PCa) cell lines and compared them to that in COS-1 cells. Interestingly, we found that NCoR/SMRT overexpression did not repress AR-dependent gene expression in the PCa cell lines, but rather activated it. This finding is probably due to an impaired AR-co-repressor interaction in the prostate cancer cell lines. In conclusion, we provide evidence that up-regulation of NCoR or SMRT may increase transcriptional activity of the AR in a cell type-specific context.