Non-radioactive assay methods for the assessment of telomerase activity and telomere length.

The Telomeric repeat amplification protocol (TRAP) is a highly sensitive PCR-based assay and prove to be an important tool for understanding the role of telomerase in cancer and various tissues that harbors telomerase positive stem cells. This assay measures telomerase activity where the amount of target is dependent upon the activity of the enzyme. This protocol consists of two steps: first, telomeric repeats are added to the substrate by telomerase present in the cell and second, the extended products are amplified by Taq-DNA polymerase.

A naturally derived small molecule disrupts ligand-dependent and ligand-independent androgen receptor signaling in human prostate cancer cells.

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 restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

Background: 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.

Fluorescent epigenetic small molecule induces expression of the tumor suppressor ras-association domain family 1A and inhibits human prostate xenograft.

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.

Non-radioactive assay methods for the assessment of telomerase activity and telomere length.

The telomeric repeat amplification protocol (TRAP) assay is a highly sensitive PCR based assay and is an important tool for understanding the role of telomerase in cancer. This assay measures an enzymatic activity where the amount of target is dependent upon the activity of the enzyme. This protocol consists of two steps: first, telomeric repeats are added to the substrate by the enzyme and second, the extended products will be amplified by Taq-DNA polymerase.

Mahanine reverses an epigenetically silenced tumor suppressor gene RASSF1A in human prostate cancer cells.

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.

Physiologically achievable concentrations of genistein enhance telomerase activity in prostate cancer cells via the activation of STAT3.

Telomerase contributes to the infinite replicative potential of cancer cells by conferring proliferation and survival through the regulation of growth factors and apoptotic proteins. Although it is generally known that the phytoestrogen, genistein, has telomerase-repressing and anti-proliferative effects on various cancer cells at pharmacological concentrations, we report here that physiologically achievable concentrations of genistein enhance telomerase activity, the proliferation of human prostate cancer cells and tumor growth in the transgenic adenocarcinoma mouse prostate model. In determining the mechanism for enhanced telomerase activity, we observed that physiological concentrations of genistein activated signal transducers and activators of transcription 3 (STAT3) both in vitro and in vivo and increased STAT3 binding to the telomerase reverse transcriptase promoter in human prostate cancer cells.

Inositol hexaphosphate represses telomerase activity and translocates TERT from the nucleus in mouse and human prostate cancer cells via the deactivation of Akt and PKCalpha.

Inositol hexaphosphate (IP6) has anti-proliferative effects on a variety of cancer cells, including prostate cancer. However, the molecular mechanism of anti-proliferative effects of IP6 is not entirely understood. Since the activation of telomerase is crucial for cells to gain immortality and proliferation ability, we examined the role of IP6 in the regulation of telomerase activity in prostate cancer cells.

Telomerase reverse transcriptase regulates the expression of a key cell cycle regulator, cyclin D1.

Cells require a mechanism to maintain telomere stability in order to overcome replicative senescence and telomerase catalyzes the synthesis and extension of telomeric DNA, therefore, be a rate-limiting step in cellular immortality and oncogenesis. However, some studies have raised questions about whether the stabilization of chromosome ends entirely explains the ability of telomerase to promote tumorigenesis. To elucidate non-canonical functions of human telomerase reverse transcriptase (hTERT), we used loss-of-function and gain-of-function studies.

Mahanine inhibits growth and induces apoptosis in prostate cancer cells through the deactivation of Akt and activation of caspases.

Background: 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.

Methods: 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.

Results: Mahanine inhibited growth of PC3 and LNCaP prostate cancer cells in a dose and time-dependent manner.

Inhibition of calmodulin-dependent cyclic AMP phosphodiesterase by phenoxazines.

Phenoxazine derivatives were examined for their ability to inhibit the calmodulin-mediated activation of phosphodiesterase, which is based on the hydrolysis of cAMP to AMP by phosphodiesterase in the presence or absence of inhibitor, followed by quantitative analysis by HPLC method. Anticalmodulin activity of phenoxazines with respect to substitution at C-2 position follows the order: 2-trifluoromethyl>2-chloro>unsubstituted phenoxazines. The interaction of phenoxazines with calmodulin using fluorescence spectroscopy has been performed.

Genistein represses telomerase activity via both transcriptional and posttranslational mechanisms in human prostate cancer cells.

Genistein, the most abundant isoflavone present in soybean has antiproliferative effects on a variety of cancer cells, including prostate cancer. However, the molecular mechanism of antiproliferative effects of genistein is not entirely understood. Because the activation of telomerase is crucial for cells to gain immortality and proliferation ability, we examined the role of genistein in the regulation of telomerase activity in prostate cancer cells.