purvalanol-a and Prostatic-Neoplasms

Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors that induce cell cycle arrest and apoptosis in various cancer cells. We further hypothesized that co-treatment of CDK inhibitors with rapamycin, an mTOR inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer regard to androgen receptor (AR) status due to inhibition of proliferative pathway, PI3K/AKT/mTOR, and induction of cell death mechanisms. Androgen responsive (AR+), PTEN(-/-) LNCaP and androgen independent (AR-), PTEN(+/-) DU145 prostate cancer cells were exposed to purvalanol (20 µM) and roscovitine (30 µM) with or without rapamycin for 24 h. Cell viability assay, immunoblotting, flow cytometry and fluorescence microscopy was used to define the effect of CDK inhibitors with or without rapamycin on proliferative pathway and cell death mechanisms in LNCaP and DU145 prostate cancer cells. Co-treatment of rapamycin modulated CDK inhibitors-induced cytotoxicity and apoptosis that CDK inhibitors were more potent to induce cell death in AR (+) LNCaP cells than AR (-) DU145 cells. CDK inhibitors in the presence or absence of rapamycin induced cell death via modulating upstream PI3K/AKT/mTOR signaling pathway in LNCaP cells, exclusively only treatment of purvalanol have strong potential to inhibit both upstream and downstream targets of mTOR in LNCaP and DU145 cells. However, co-treatment of rapamycin with CDK inhibitors protects DU145 cells from apoptosis via induction of autophagy mechanism. We confirmed that purvalanol and roscovitine were strong apoptotic and autophagy inducers that based on regulation of PI3K/AKT/mTOR signaling pathway. Co-treatment of rapamycin with purvalanol and roscovitine exerted different effects on cell survival and death mechanisms in LNCaP and DU145 cell due to their AR receptor status. Our studies show that co-treatment of rapamycin with CDK inhibitors inhibit prostate cancer cell viability more effectively than either agent alone, in part, by targeting the mTOR signaling cascade in AR (+) LNCaP cells. In this point, mTOR is a fine-tuning player in purvalanol and roscovitine-induced apoptosis and autophagy via regulation of PI3K/AKT and the downstream targets, which related with cell proliferation.

Topics: Apoptosis; Autophagy; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Humans; Male; Prostatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Purines; Receptors, Androgen; Roscovitine; Signal Transduction; TOR Serine-Threonine Kinases

Androgen signaling is critical in prostate cancer development and progression. The co-existence of hormone responsive and irresponsive cells due to functional androgen receptor (AR) in prostate gland is the major obstacle in prostate cancer therapy models. Targeting aberrant cell cycle by novel cell cycle blocking agents is a promising strategy to treat various types of malignancies. Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors able to activate apoptotic cell death by inducing cell cycle arrest at G1/S and G2/M phases in cancer cells. Polyamines are unique cationic amine derivatives involved in the regulation of cell proliferation. Although the elevated intracellular level of polyamines (putrescine, spermidine and spermine) is typical for prostate gland, abnormal regulation of polyamine metabolism might result in rapid cell proliferation and, thus in prostate cancer progression. Therefore, treatment with drug-induced depletion of intracellular polyamine levels through the activated polyamine catabolism is critical to achieve successful strategies for prostate cancer. In this study we aimed to investigate the apoptotic efficiency of CDK inhibitors in three prostate cancer cell lines (LNCaP, DU145 and PC3), showing different AR expression profile. We found that both purvalanol and roscovitine were able to induce apoptosis at moderate cytotoxic concentrations by decreasing mitochondria membrane potential. The apoptotic effect of both CDK inhibitors was due to activation of caspases by modulating Bcl-2 family members. The efficiency of drugs was quite similar on the three prostate cell lines used in this study. However, DU145 cells were found the least sensitive against CDK inhibitors while purvalanol was more potent than roscovitine. Similarly to classical chemotherapeutic agents, both drugs could up-regulate polyamine catabolic enzymes (SSAT, SMO and PAO) in cell type dependent manner. Transient silencing of SSAT and/or inhibition of PAO/ SMO with MDL72527 prevented CDK inhibitors- induced apoptotic cell death in DU145 and PC3 cells. Although roscovitine was less effective in DU145 cells, pre-treatment with α-difluoromethylornithine (DFMO), an inhibitor of ODC, enhanced the roscovitine-induced apoptotic cell death through the cleavage of caspase-9 and caspase-3. Therefore, we conclude that polyamine catabolism might have essential role in the cellular responses against CDK inhibitors in different androgen-responsive or irresponsiv

Topics: Apoptosis; Caspases; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Male; Mitochondria; Polyamines; Prostatic Neoplasms; Protein Kinase Inhibitors; Purines; Receptors, Androgen; Roscovitine