Androgens down-regulate myosin light chain kinase in human prostate cancer cells

Androgens play a major role in the growth and survival of primary prostate tumors. The molecular mechanisms involved in prostate cancer progression are not fully understood but genes that are regulated by androgens clearly influence this process. We searched for new androgen-regulated genes using the Affymetrix GeneChip Human Genome U95 Set in the androgen-sensitive LNCaP prostate cancer cell line. Analysis of gene expression profiles revealed that myosin light chain kinase (MLCK) mRNA levels were markedly down-regulated by the synthetic androgen R1881. The microarray data were confirmed by ribonuclease protection assays. RNA and protein analyses revealed that LNCaP cells express both long (non-muscle) and short (smooth muscle) isoforms, and that both isoforms are down-regulated by androgens. Taken together, these data identify MLCK as a novel downstream target of the androgen signalling pathway in prostate cells.     

Androgen receptor action in hormone-dependent and recurrent prostate cancer

The importance of androgens and androgen receptors (AR) in primary prostate cancer is well established. Metastatic disease is usually treated with some form of androgen ablation, which is effective for a limited amount of time. The role of AR in prostate cancers that recur despite androgen ablation therapy is less certain. Most of these tumors express prostate specific antigen (PSA), an androgen-regulated gene; moreover, AR is generally highly expressed in recurrent prostate cancer. We propose that AR continues to play a role in many of these tumors and that it is not only the levels of AR, ligands, and co-regulators, but also the changes in cell signaling that induce AR action in recurrent prostate cancer. These pathways are, therefore, potential therapeutic targets.     

Antioxidants Abrogate Alpha-Tocopherylquinone-Mediated Down-Regulation of the Androgen Receptor in Androgen-Responsive Prostate Cancer Cells

Tocopherylquinone (TQ), the oxidation product of alpha-tocopherol (AT), is a bioactive molecule with distinct properties from AT. In this study, AT and TQ are investigated for their comparative effects on growth and androgenic activity in prostate cancer cells. TQ potently inhibited the growth of androgen-responsive prostate cancer cell lines (e.g., LAPC4 and LNCaP cells), whereas the growth of androgen-independent prostate cancer cells (e.g., DU145 cells) was not affected by TQ. Due to the growth inhibitory effects induced by TQ on androgen-responsive cells, the anti-androgenic properties of TQ were examined. TQ inhibited the androgen-induced activation of an androgen-responsive reporter and inhibited the release of prostate specific antigen from LNCaP cells. TQ pretreatment was also found to inhibit AR activation as measured using the Multifunctional Androgen Receptor Screening assay. Furthermore, TQ decreased androgen-responsive gene expression, including TM4SF1, KLK2, and PSA over 5-fold, whereas AT did not affect the expression of androgen-responsive genes. Of importance, the antiandrogenic effects of TQ on prostate cancer cells were found to result from androgen receptor protein down-regulation produced by TQ that was not observed with AT treatment. Moreover, none of the androgenic endpoints assessed were affected by AT. The down-regulation of androgen receptor protein by TQ was abrogated by co-treatment with antioxidants. Overall, the biological actions of TQ were found to be distinct from AT, where TQ was found to be a potent inhibitor of cell growth and androgenic activity in androgen-responsive prostate cancer cells.     

Quantum dots-based multiplexed immunohistochemistry of protein expression in human prostate cancer cells

Semiconductor quantum dots (QDs) are bright fluorescent nanoparticles that have been successfully used for the detection of biomarker expression in cells. The objective of the present study is to use this technology in a multiplexing manner to determine at a single cell level the expression of a cell-specific bio-marker, prostate-specific antigen (PSA) expressed by human prostate cancer LNCaP and ARCaP cell lines. Here we compared the sensitivity of immunohistochemistry (IHC) and QD-based detection of AR and PSA expression in these cell lines. Further, we conducted multiplexing QD-based detection of PSA and androgen receptor (AR) expression in LNCaP cells subjecting to androgen (R1881) stimulation. The involvement of AR in PSA regulation in LNCaP cells, at a single cell level, was confirmed by the co-incubation of LNCaP cells in the presence of both R1881 and its receptor antagonist, bicalutamide (Casodex). We showed here the superior quality of QDs, in comparison to IHC, for the detection of AR and PSA in cultured LNCaP and ARCaP cells. Multiplexing QDs technique can be used to detect simultaneously AR and PSA expression induced by R1881 which promoted AR translocation from its cytosolic to the nuclear compartment. We observed AR antagonist, bicalutamide, inhibited AR nuclear translocation and PSA, but not AR expression in LNCaP cells.