Regulation Of Akt Phosphorylation By A Cdk Inhibitor Purvalanol A In Breast Cancer Cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Akt is a serine/threonine kinase and an essential component in the PI3K-Akt-mTOR axis. Multiple cell signals stimulate the activation of Akt through Akt activating kinases. The Akt is activated through the phosphorylation in serine and threonine residues. The activated Akt increases in cancer cells and plays promoting functions in proliferation, survival, invasion, and drug/radiation resistance. Several Akt inhibitors have been tested for cancer treatment. Purvalanol A is such a CDK inhibitor. In synchronized cells, purvalanol A induced a reversible cell cycle arrest in the G1 and G2 phases. Purvalanol A treated MCF7 cells induced apoptosis via activating the polyamine catabolic pathway. In the study of the cell cycle and radiation resistance of breast cancer cells, purvalanol A was found to inhibit phosphorylated Akt. MCF7 cells were treated with 20µM of purvalanol A one hour before the radiation. The protein was extracted at 15, 60, and 300 minutes after the radiation at 6Gy. Western analyses were performed with antibody against phosph-Akt(Ser473). Compared to control cells, purvalanol A treated cells showed reduced phosph-Akt(Ser473) by 59 to 88% depending on the specific time point. To clarify if the reduction of phosphorylated Akt in purvalanol A treated cells is a result of radiation, the effect of purvalanol A on the expression of phosphorylated Akt was examined in cells without radiation. Two breast cancer cell lines, MCF7 and MDA-MB231 cells, and one prostate cancer cell line, PC3 cells were treated with purvalanol A for 2 hours at doses of 0.5,1,5,10, and 20µM. Expression of phosph-Akt(Ser473) and phosph-Akt(Thr308) was investigated by Western blot analyses. The result from three cell lines showed that purvalanol A regulated the expression of phosph-Akt(Ser473) and phosph-Akt(Thr308) in a dose-dependent way. At a low dose of 1µM, purvalanol A increased the phosph-Akt(Ser473) in MCF7 cells by 34% and phosph-Akt(Thr308) by 50%, respectively. In MDA-MB231 cells treated with 1µM of purvalanol A, phosph-Akt(Ser473) increased by 35% and phosph-Akt(Thr308) increased by 1.6 fold. In PC3 cells, treatment with 1µM of purvalanol A led to an 87% increase in phosph-Akt(Ser473). However, the same treatment also reduced phosph-Akt(Thr308) by 13%. At a high dose of 20µM, purvalanol A treatment significantly reduced phosphorylated Akt. The phosph-Akt(Ser473) was reduced by 80% in MDA-MB231 and by 84% in MCF7 cells, respectively. The phosph-Akt(Thr308) was reduced by 91% in MDA-MB231 and by 97% in MCF7 cells, respectively. In PC3 cells treatment with 20µM of purvalanol A reduced phosph-Akt(Ser473) by 50% and phosph-Akt(Thr308) by 90%. The results suggest that purvalanol A could be a regulator of Akt activation and a multiple targeting effector in cancer treatment. Citation Format: Hong Yin. Regulation of AKT phosphorylation by a CDK inhibitor purvalanol A in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1326. doi:10.1158/1538-7445.AM2014-1326