sirolimus and perillyl-alcohol

Topics: Autophagy; Cell Death; Chloroquine; Glioblastoma; Glioma; Humans; Lysosomes; Monoterpenes; Rolipram; Sirolimus; Temozolomide; TOR Serine-Threonine Kinases

Translation is mediated partly by regulation of free eukaryotic initiation factor 4E (eIF4E) levels through PI3K-Akt-mTOR signaling. Cancer cells treated with the plant-derived perillyl alcohol (POH) or the mechanistic target of rapamycin (mTOR) inhibitor rapamycin dephosphorylate eIF4E-binding protein (4E-BP1) and attenuate cap-dependent translation. We previously showed in cancer cell lines with elevated eIF4E that POH and rapamycin regulate telomerase activity through this pathway. Here, immortalized Chinese hamster ovary (CHO) control cells and CHO cells with forced eIF4E expression (rb4E) were used to elucidate eIF4E's role in telomerase regulation by POH and rapamycin. Despite 5-fold higher eIF4E amounts in rb4E, telomerase activity, telomerase reverse transcriptase (TERT) mRNA, and TERT protein were nearly equivalent in control and rb4E cells. In control cells, telomerase activity, TERT mRNA and protein levels were unaffected by either compound. In contrast, telomerase activity and TERT protein were both attenuated by either agent in rb4E cells, but without corresponding TERT mRNA decreases indicating a translational/post-translational process. S6K, Akt, and 4E-BP1 were modulated by mTOR mediators only in the presence of increased eIF4E. Thus, eIF4E-overexpression in rb4E cells enables inhibitory effects of POH and rapamycin on telomerase and TERT protein. Importantly, eIF4E-overexpression modifies cellular protein synthetic processes and gene regulation.

Topics: Animals; Antineoplastic Agents; CHO Cells; Cricetinae; Cricetulus; Enzyme Activation; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Enzymologic; Monoterpenes; Oncogene Protein v-akt; Phosphorylation; Ribosomal Protein S6 Kinases; Sirolimus; Telomerase; Transfection; Up-Regulation

We previously demonstrated in prostate cancer cells that a phytochemical-perillyl alcohol-and the mechanistic target of rapamycin (mTOR) inhibitor rapamycin rapidly attenuated telomerase activity. Protein levels of the telomerase catalytic subunit reverse transcriptase (hTERT) were diminished in the absence of an effect on hTERT mRNA, supporting an effect on 4E-BP1 phosphorylation and reduced initiation of protein translation. The decline in hTERT protein did not coincide wholly, however, with loss of telomerase activity suggesting a further level of regulation. We hypothesized that a hTERT-mTOR-S6K (S6 kinase)-Hsp90 (Heat shock protein 90)-Akt complex previously detected in activated NK cells was present in DU145 prostate cancer cells. Furthermore, we postulated that both perillyl alcohol and rapamycin disrupted this complex to control telomerase activity post-translationally. Antibodies directed against either RAPTOR, a binding partner of mTOR, or mTOR itself co-immunoprecipitated Hsp90, hTERT, and S6K confirming a similar TERT complex in prostate cancer cells. Perillyl alcohol or rapamycin caused rapid dissociation of the captured hTERT-mTOR-RAPTOR complex, establishing an additional mechanism by which these agents decrease telomerase activity. These findings provide convincing evidence for mTOR-mediated regulation of hTERT in DU145 cells.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Cell Line; Down-Regulation; HSP90 Heat-Shock Proteins; Humans; Immunoprecipitation; Monoterpenes; Multiprotein Complexes; Protein Binding; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases; Sirolimus; Telomerase; TOR Serine-Threonine Kinases

Isoprenoids are recognized for their ability to suppress carcinogenic processes in vivo and in vitro. We previously established that the isoprenoid, perillyl alcohol, acted mechanistically on translation of specific proteins through modulation of mechanistic target of rapamycin (mTOR) signaling. Telomerase-the enzyme responsible for immortalizing cells through the addition of telomeric repeats-is de-repressed early in an aspiring cancer cell. Here the effects of biologically-relevant concentrations and short incubations (1-16 h) of perillyl alcohol or the mTOR inhibitor, rapamycin, on telomerase activity were examined in prostate cancer cell lines. A rapid suppression of telomerase activity was observed (from ∼65% to >95%) determined by real-time quantitative telomerase repeat amplification protocol and confirmed by polyacrylamide gel-analysis. Using real-time reverse transcriptase-PCR, we demonstrated that human telomerase reverse transcriptase (hTERT) mRNA levels were unaltered. Western blot analysis revealed that hTERT protein levels decreased in response to perillyl alcohol or rapamycin. This decrease was partially blocked by pretreatment with a proteasome inhibitor MG-132, indicating that proteasomal degradation contributed to the loss of hTERT protein. No change in hTERT phosphorylation at Ser824 was observed, indicating the absence of cellular hTERT protein redistribution. These findings provide evidence for a unique link between nutrient- and macrolide-mediated regulation of mTOR and hTERT, a key enzyme that regulates DNA structure and stability.

Topics: Blotting, Western; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Male; Monoterpenes; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Telomerase; TOR Serine-Threonine Kinases