The effect of the PI3K/AKT pathway inhibition on leiomyosarcoma cells

Abstract

9002 Background: Leiomyosarcomas (LMS) are one of the most common histological types of soft tissue sarcomas and are usually resistant to standard chemotherapy. Novel agents that can cause apoptosis must be explored. The PI3K/AKT pathway is an important survival pathway and is upregulated in many tumor cells. Recent publications have shown that the AKT pathway is an important survival pathway for a uterine LMS cell line, SKLMS-1. In this study we examined the effects of LY294002, a specific PI 3-kinase inhibitor on the survival of the SKLMS-1 cell line. Methods: Various doses of LY294002 were added to growing SKLMS-1 cells for 24, 48 and 72 hours. Cells were harvested at these time points and counted. Cell cycle distribution was analyzed by propidium iodide staining. Apoptosis was analyzed by both TUNEL assay and cleavage of PARP by western blotting. Cell lysates were analyzed for cyclin D3, cyclin A, and p27 by western blot. Results: At all doses (10, 20 and 50 μM) LY294002 caused G0/G1 growth arrest at 24hours in a dose-dependent manner. Cells that were treated for 48 and 72 hours were killed as demonstrated by decreased cell counts, TUNEL assay and PARP cleavage. Apoptosis occurred in both a time and dose dependent manner. At a dose of 10 μM, a 5-fold and 14-fold decrease in cell number was found at 48 and 72 hours respectively, compared to cells treated with the control DMSO. At a dose of 50 μM, cell count revealed more than a 12-fold decrease in cell numbers for both 48 and 72 hours. Analysis of the cell cycle distribution correlated with the cell counts. Results showed that LY294002 arrested the cells early in G0/G1 allowing them to transverse through one cell cycle, and then become trapped in G0 and ultimately die through apoptosis. Expression of cyclin A and cyclin D3 was reduced in the treated cells. Interestingly, p27 levels did not increase with treatment. Conclusions: The PI3K inhibitor, LY294002 caused apoptosis in a both dose and time dependent manner. At 24 hours cells were arrested in G0/G1. At higher doses and at 48 and 72 hours, cells were killed by apoptosis. These preclinical data may provide an opportunity for the development of novel therapies that target the P13K/AKT pathway No significant financial relationships to disclose.