Generation and Validation of Monoclonal Antibodies Against the Maltose Binding Protein.

The maltose binding protein (MBP) is a commonly used protein tag. Two monoclonal antibodies (mAbs) were generated against the MBP by immunizing mice with purified 6xHis-tagged MBP (6xHis-MBP). A nontoxic adjuvant cocktail of poly(I:C) and anti-CD40 mAb was used.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Caspase-8 activation is necessary but not sufficient for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in the prostatic carcinoma cell line LNCaP.

Background: The differential sensitivity of tumor cells to TRAIL-induced apoptosis may be mediated by different intracellular inhibitors of apoptosis, and only a few reports have described the pathway(s) that are activated in response to TRAIL in prostate cells.

Methods: LNCaP was transfected with a dominant-negative form of FADD (FADD-DN) and cells were selected in the presence of hygromycin. Cell viability was estimated by calcein assay.