Rebecca A. Glover

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-α 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-α 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.

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 β, γ, and δ 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-β and –γ 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.

Bisindolylmaleimide IX Facilitates Tumor Necrosis Factor Receptor Family-mediated Cell Death and Acts as an Inhibitor of Transcription

Bisindolylmaleimides (Bis) were originally described as protein kinase C inhibitors. Several studies have shown that Bis potentiate tumor necrosis factor (TNF) receptor family-mediated apoptosis in lymphoid and dendritic cells, but the inhibition of protein kinase C cannot account for these effects (Zhou, T., Song, L., Yang, P., Wang, Z., Lui, D., and Jope, R. S. (1999) Nat. Med. 5, 42–48). We investigated the effect of four Bis derivatives (I, II, VIII, and IX) in human prostatic carcinoma cell lines and found that Bis IX was the most potent inducer of apoptosis under simultaneous treatment with TNF-α, agonistic anti-Fas monoclonal antibody, and TNF-related apoptosis-inducing ligand (TRAIL). Bis IX synergistically induced caspase activity in combination with apoptosis-inducing ligands and converted the phenotype of cell lines from apoptosis-resistant to -sensitive. Bis IX induced p53 accumulation in LNCaP (lymph node carcinoma of prostate), which expresses wild-type p53 that was not accompanied by the induction of p53-responsive genes, p21/WAF1, and Mdm2. Moreover, the induction of p21/WAF1 and Mdm2 by doxorubicin was abrogated by simultaneous treatment with Bis IX. These effects apparently result from general inhibition of transcription by Bis IX. We have shown by Northern blot analysis that the transcription activity of the hygromycin gene after transient transfection of pcDNA3.1-Hygro plasmid in 293 and HeLa cells was inhibited by Bis IX in a dose-dependent manner. Moreover, DNA binding activity of Bis IX was prevented by actinomycin D, suggesting that actinomycin D and Bis IX have similar mechanisms of interaction with DNA. In addition, we found that actinomycin D and Bis IX induced caspase activity to the same extent during TRAIL-mediated apoptosis. In summary, these results suggest that Bis IX potentiates TNF receptor family-mediated cell death in part as an inhibitor of transcription.

TRAIL-DISC Formation Is Androgen-Dependent in the Human Prostatic Carcinoma Cell Line 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.

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 down-regulation 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.

Multiple effects of N-a-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.

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.