Abstract 98: TET suppresses protein translation, differentially effects polysome loading of cell cycle regulatory proteins and inhibits cell growth and proliferation of prostate cancer cells
Authors: Jaiswal Praveen K. , Koul Sweaty , Dong Qin , Koul Hari 
Published: July 2017

Introduction: Translational control is a critical component in development and progression of cancer and could be explored to develop antineoplastic therapies. Results from our laboratory have shown that TET (a derivative of Tetrandrine) inhibits cell growth and promotes apoptosis of prostate cancer (PCa) cells. However, the underlying molecular mechanisms are not clearly understood. We observed that TET inhibited 4EBP phosphorylation, suggesting that it might inhibit translational initiation. In the present study we evaluated the effects of TET on Translation in general and cell cycle related proteins in particular.

Methods: Androgen dependent PCa cells LNCaP and Enz resistant CRPCa cells 22Rv1 were maintained in supplemented RPMI as recommended. Where indicated cells were treated with TET (0 or 20µM) for various time points (2h, 8h, 18h and 24h). At the end of the experimental periods cells were pulsed with cycloheximide for 10 minutes in RPMI media. Cytoplasmic RNA was collected by lysing cells in polysome lysis buffer. Cell lysates were centrifuged and the resulting cytosolic supernatant was separated by density (a 10% to 50% sucrose) gradient and centrifugation. Fractions were collected using a TELEDYNE ISCO Density Gradient Fractionation System with continuous monitoring of untranslated mRNAs. Polysome (P)/Monosome (M) ratio were measured. The total RNA in each fraction was extracted using TRIzol LS (Invitrogen) and qRT PCR was done for p21, p27, Cyclin D1 and c-Myc gene for each fraction. Cell viability was measured by MTT assay at various time points.

Results: TET treatment resulted in decreased protein synthesis. TET treatment resulted in decrease of RNA associated with Polysomes. There were a significant shift in P/M ratio from 0.82 (control) to 0.48 (24 h. TET) [TET 2hrs (0.75), 8h (0.69) and 18hrs (0.52)] in LNCaP and P/M ratio form 0.91 (control) to 0.32 (8 h TET) [TET 2h (0.78) and 4h (0.53)] in 22Rv1 cells. The decrease of the P/M ratio suggests that TET inhibits translation initiation in prostate cancer cells independent of the androgen dependence. While there was generalized decrease in translation and specific decrease in mRNA for Cyclin D1, c-Myc associated with polysomes following TET treatment. In quite contrast to these findings, we observed an increase in mRNA for p21 and p27 in polysomal fractions of TET treated cells as compare to control at 24 h in LNCaP cells. These data suggest differential effects of TET treatment on polysome loading of cell cycle regulatory proteins. Moreover, TET inhibited growth and proliferation of LNCaP and 22Rv1 cells in dose as well as in time dependent fashion.

Conclusions: TET targets translational machinery and differentially effects translation of cell cycle regulatory genes by effecting their polysome loading and effects cell growth and proliferation of androgen dependent and castrate resistant PCa cells.

Citation Format: Praveen K. Jaiswal, Sweaty Koul, Qin Dong, Hari Koul. TET suppresses protein translation, differentially effects polysome loading of cell cycle regulatory proteins and inhibits cell growth and proliferation of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 98. doi:10.1158/1538-7445.AM2017-98