mahanine and Prostatic-Neoplasms

Continued reliance on androgen receptor (AR) signaling is a hallmark of prostate cancer, including the development of castration-resistant prostate cancer (CRPC), making it an attractive therapeutic target for prostate cancer treatment. Mahanine is a novel carbazole alkaloid derived from the leaves of Murraya koenigii, commonly known as the curry leaf plant, which grows widely across East-Asia. We show here that mahanine possesses the ability to inhibit ligand-dependent and -independent AR transactivation, leading to a prominent decline in AR target gene expression. Mahanine treatment causes a time- and dose-dependent decline in AR protein levels, including truncated AR splice variants, in a panel of androgen-responsive and -independent prostate cancer cells. The decrease in AR levels induced by mahanine occurs posttranslationally by proteasomal degradation, without any change in the AR gene expression. Mahanine treatment induces an outward movement of the AR from the nucleus to the cytoplasm, leading to an initial increase in cytoplasmic AR levels, followed by a gradual decline in the AR levels in both cellular compartments. Ligand-induced AR phosphorylation at Ser-81, a phospho-site associated with prostate cancer cell growth and AR transactivity, is greatly diminished in the presence of mahanine. The decline in AR phosphorylation at Ser-81 by mahanine occurs via the inactivation of mitotic kinase CDK1. Collectively, our data demonstrate that mahanine strongly disrupts AR signaling and inhibits the growth of androgen-dependent and -independent prostate cancer cells, thereby implicating a therapeutic role of mahanine in prostate cancer treatment.

Topics: Androgens; Blotting, Western; Carbazoles; CDC2 Protein Kinase; Cytoplasm; Dihydrotestosterone; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Ligands; Male; Microscopy, Fluorescence; Molecular Structure; Murraya; Phosphorylation; Plant Leaves; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Protein Transport; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Serine; Signal Transduction; Transcriptional Activation

Hypermethylation of the promoter of the tumor suppressor gene RASSF1A silences its expression and has been found to be associated with advanced grade prostatic tumors. The DNA methyltransferase (DNMT) family of enzymes are known to be involved in the epigenetic silencing of gene expression, including RASSF1A, and are often overexpressed in prostate cancer. The present study demonstrates how mahanine, a plant-derived carbazole alkaloid, restores RASSF1A expression by down-regulating specific members of the DNMT family of proteins in prostate cancer cells.. Using methylation-specific PCR we establish that mahanine restores the expression of RASSF1A by inducing the demethylation of its promoter in prostate cancer cells. Furthermore, we show that mahanine treatment induces the degradation of DNMT1 and DNMT3B, but not DNMT3A, via the ubiquitin-proteasome pathway; an effect which is rescued in the presence of a proteasome inhibitor, MG132. The inactivation of Akt by wortmannin, a PI3K inhibitor, results in a similar down-regulation in the levels DNMT1 and DNMT3B. Mahanine treatment results in a decline in phospho-Akt levels and a disruption in the interaction of Akt with DNMT1 and DNMT3B. Conversely, the exogenous expression of constitutively active Akt inhibits the ability of mahanine to down-regulate these DNMTs, suggesting that the degradation of DNMT1 and DNMT3B by mahanine occurs via Akt inactivation.. Taken together, we show that mahanine treatment induces the proteasomal degradation of DNMT1 and DNMT3B via the inactivation of Akt, which facilitates the demethylation of the RASSF1A promoter and restores its expression in prostate cancer cells. Therefore, mahanine could be a potential therapeutic agent for advanced prostate cancer in men when RASSF1A expression is silenced.

Topics: Androstadienes; Antineoplastic Agents; Carbazoles; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3B; Down-Regulation; Epigenesis, Genetic; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Male; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Promoter Regions, Genetic; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins c-akt; Tumor Suppressor Proteins; Ubiquitination; Wortmannin

Epigenetic silencing of Ras-association domain family 1A (RASSF1A) protein in cancer cells results in a disruption of cell cycle control, genetic instability, enhanced cell motility, and apoptotic resistance. Ectopic expression of RASSF1A reverses this tumorigenic phenotype. Thus, small molecules with the ability to restore RASSF1A expression may represent a new class of therapeutic agents. Recently, we designed and synthesized a fluorescent carbazole analogue of mahanine (alkaloid from Murraya koenigii) that restored RASSF1A mRNA expression. Our fluorescent lead compound up-regulated RASSF1A in vitro, potently inhibited human prostate cancer cell proliferation, and fluoresced at a visible wavelength, allowing for the observation of intracellular distribution. The small molecule lead was not acutely toxic up to 550 mg/kg, and dosing at 10 mg/kg reduced human xenograft tumor volume by about 40%.

Topics: Animals; Carbazoles; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Dose-Response Relationship, Drug; Fluorescence; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Chemical; Molecular Structure; Murraya; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

It is becoming clear that frequent epigenetic silencing of tumor suppressor genes could be responsible for the development of cancer in various organs. Several recent reports suggest that suppression of RASSF1A is associated with the advanced grade and stage of prostate cancer and many other cancers. In this investigation, we demonstrated that, mahanine, a plant derived carbazole alkaloid, induced RASSF1A expression in both androgen-responsive (LNCaP) and androgen-negative (PC3) prostate cancer cells by inhibiting DNA methyltransferase (DNMT) activity. Mahanine-induced expression of RASSF1A in turn significantly reduced cyclin D1 but not other cyclins. To understand the inverse relationship between RASSF1A and cyclin D1, we observed that mahanine treatment down-regulates cyclin D1 and addition of RASSF1A siRNA prevented this inhibition. This study show for the first time that mahanine can reverse an epigenetically silenced gene, RASSF1A in prostate cancer cells by inhibiting DNMT activity that in turn down-regulates a key cell cycle regulator, cyclin D1. Mahanine therefore, promises an encouraging therapeutic choice for advanced prostatic cancer.

Topics: Alkaloids; Carbazoles; Cell Line, Tumor; Cyclin D1; DNA (Cytosine-5-)-Methyltransferases; Dose-Response Relationship, Drug; Down-Regulation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Male; Prostatic Neoplasms; Tumor Suppressor Proteins

The present study was undertaken to evaluate anti-proliferative and -apoptotic activities of mahanine, a plant derived carbazole alkaloid, in prostate cancer cells and to determine its molecular mechanism by which it induces apoptotic cell death.. The growth inhibitory and apoptotic inductive effect of mahanine on prostate cancer cells were examined by measuring cell proliferation and BrdU labeling, caspase activity, DNA fragmentation, and Western blot analyses.. Mahanine inhibited growth of PC3 and LNCaP prostate cancer cells in a dose and time-dependent manner. Mechanistically, mahanine inhibited cell-survival pathway by dephosphorylation of PIP3 dependent kinase 1 (PDK1) thereby deactivation of Akt and downregulation of Bcl-xL. In addition, mahanine activated caspase pathway (caspases 9 and 3) and eventually cleavage of DNA repair enzyme, PARP resulting DNA fragmentation and apoptosis.. Mahanine inhibits growth and induces apoptosis in both androgen-responsive, LNCaP and androgen-independent, PC3 cells by targeting cell survival pathway.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Apoptosis; bcl-X Protein; Carbazoles; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Proliferation; DNA Fragmentation; DNA, Neoplasm; Dose-Response Relationship, Drug; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Male; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Time Factors; Toxoids