Abstract 4384: TET inhibits phosphorylation of eIF4E-binding protein, in prostate cancer cells
Authors: Koul Sweaty , Dong Qin , Slepenkov Sergey , Koul Hari K. 
Published: August 2015

Introduction and Objectives: All eukaryotic cellular mRNAs are blocked at their 5-prime ends with the 7-methyl-guanosine cap structure. EIF4E is a eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs. The EIF4E polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. Tetrandrine (TET) is a bis-benzylisoquinoline alkaloid, isolated from the root of Stefenia tatrandra. Previous studies have shown that TET has anti-inflammatory, immunologic and antiallergenic effects. We have shown that TET targets AR signaling in PCa cells and induces expression of p21. However the molecular mechanism by which TET exerts its effects are not completely understood. In the present study we investigated the effects of TET in prostate cancer cells in culture (PC-3 and LNCaP cells).

Methods: LNCaP, C-4 and PC3 cells were grown in supplemented RPMI media and DMEM/F12 Media respectively, and maintained at 37C in a 5%CO2 incubator (as described previously). Protein levels were measured by Western Blot and by immuno-fluorescence assays. In vitro translation system from HeLa cell lysates was used to measure protein synthesis in vitro. Protein Synthesis in vivo was measured by a reduction in the rate of firefly luciferase production following introduction of luciferase mRNA into cells by nucleofection. mRNA expression measured in RTPCR assays. Promoter activities were measured by measuring promoter driven luciferase expression.

Results: TET inhibited protein synthesis in dose-dependent way with IC50 value of 20 μM as measured by a reduction in the rate of firefly luciferase production following introduction of luciferase mRNA into cells by nucleofection. The inhibition of protein synthesis was 88% at 40 μM. Interestingly TET did not inhibit protein synthesis in in vitro translation system from HeLa cell lysates. This suggested that inhibition by TET occurs in signaling pathways regulating protein synthesis that are operative in intact cells but not cell-free lysates. It is well known that un-phosphorylated 4E-BP1 binds with high affinity to cap-binding protein eIF4E, thus preventing recruitment of 48S translation initiation complex to capped mRNA and inhibiting protein synthesis. By western blotting, we demonstrated that tetrandrine inhibited phosphorylation of translational repressor 4E-BP1 at Ser65, the substrate of multiple upstream signaling cascades.

Conclusions: Our results suggest that TET is a novel agent to target EIF4E and that Tet inhibits EIF4. Current research is aimed at determining the involvement of other upstream signaling pathways that might explain inhibition of 4E-BP1 phosphorylation following TET treatment.Financial support : (VA-1BX001258; NCI RO1-161880; Carroll W. Feist Endowed Chair Funds (Koul H), Funds from, FWCC, the Dean School SOM and Chancellor- LSUHSC-Shreveport.

Citation Format: Sweaty Koul, Qin Dong, Sergey Slepenkov, Hari K. Koul. TET inhibits phosphorylation of eIF4E-binding protein, in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4384. doi:10.1158/1538-7445.AM2015-4384