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. The amplified TRAP products are separated on 10 % native PAGE and detected by SYBR Green I dye.

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 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.

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. RESULTS: 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. CONCLUSIONS: 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.

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. 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%.

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. 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.

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. These results demonstrate for the first time that physiologically achievable concentrations of genistein enhance telomerase reverse transcriptase transcriptional activity in prostate cancer cells via the activation of STAT3. Consequently, these concentrations of genistein will augment the growth of prostate cancer cells that could be detrimental to individuals with prostate cancer and therefore, caution should be exercised when genistein is considered for chemotherapeutic purposes.

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. Here, we show that IP6 represses telomerase activity in mouse and human prostate cancer cells dose-dependently. In addition, IP6 prevents the translocation of TERT to the nucleus. Since phosphorylation of TERT by Akt and/or PKCalpha is necessary for nuclear translocation, we examined phosphorylation of Akt and PKCalpha after IP6 treatments. Our results show that IP6 inhibits phosphorylation of Akt and PKCalpha. These results show for the first time that IP6 represses telomerase activity in prostate cancer cells by posttranslational modification of TERT via the deactivation of Akt and PKCalpha.

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. We demonstrated that hTERT shRNA down-regulated and hTERT overexpression up-regulated the expression and transcriptional activity of a key cell cycle regulator, cyclin D1, in human prostate epithelial cell lines, DU-145 and BPH-1. Based on these observations, we propose that in addition to its well-defined function in telomere length regulation, hTERT has a novel role in the modulation of cyclin D1 expression.

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. 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. CONCLUSIONS: Mahanine inhibits growth and induces apoptosis in both androgen-responsive, LNCaP and androgen-independent, PC3 cells by targeting cell survival pathway.

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. Binding study showed that calmodulin has two types of binding sites for phenoxazines. One is high affinity binding site (Kd value 0.07-0.46 microM) and the other, a low affinity binding site (Kd value 0.7-34.5 microM). The change in secondary structure of calmodulin upon binding to phenoxazines was studied by circular dichroism (CD) method, which showed that the percentage of helicity decreased with an extensive change in tertiary structure of calmodulin. Kinetic analysis of the phenoxazine-calmodulin interaction showed that phenoxazines competitively inhibited the activation of phosphodiesterase without affecting Vmax. Thus, these studies showed a good correlation between the ability of phenoxazines to block the activation of phosphodiesterase and their ability to bind to the activator.

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. Here, we show that genistein-induced inhibition in cell proliferation is associated with a reduction in telomerase activity. Using reverse transcriptase-PCR and hTERT promoter activity assays, we showed that genistein decreased hTERT expression and transcriptional activity dose-dependently. Using various deleted hTERT promoter constructs, we defined that the hTERT core promoter is enough to observe the genistein-induced repression of hTERT transcriptional activity. Because c-Myc is involved in transcriptional regulation of hTERT, c-Myc expression was examined. A dose-dependent decrease in c-Myc message and proteins was observed with genistein treatment. These results indicate that genistein represses hTERT transcriptional activity via the down-regulation of c-Myc expression. However, genistein-induced repression of hTERT transcriptional activity was not blocked by the mutation of c-Myc at the hTERT promoter, suggesting that additional factors are involved in genistein-dependent repression of telomerase activity. Interestingly, we observed that genistein down-regulates the activation of Akt thereby phosphorylation of hTERT and inhibits its translocation to the nucleus. These results show for the first time that genistein represses telomerase activity in prostate cancer cells not only by repressing hTERT transcriptional activity via c-Myc but also by posttranslational modification of hTERT via Akt.