INVESTIGATION OF THE IMPACT OF THE PI3K/AKT/mTOR PATHWAY INHIBITOR ON THE RADIO SENSITIVITY IN BREAST CANCER STEM CELL LINE BY PHOSPHORILATION ASSAY
Project no. 1127 · Status: Kapanmış · 2018
Summary
Breast cancer is a complex type of cancer and is ranked first among women worldwide and second among cancer-related deaths worldwide. Breast cancer is a heterogeneous disease that is divided into subtypes according to clinical features, exposed tumor markers and histological type. Ductal and lobular carcinomas are the most common histological types. Each subtype has a different prognosis and treatment response. Many risk factors have been identified in the formation of breast cancer. Mutations play an important role in the development of breast cancer. The abnormal activation of the PI3K/Akt/mTOR pathway due to mutations is frequently observed in breast cancer. Studies have shown that inhibition of this pathway helps prevent breast cancer development. Surgical intervention, chemotherapy and radiotherapy in breast cancer treatments are usually successful, but resistance to treatment or recurrence is usually seen. Stem cells are potentially responsible for the resistance and reccurence, cells that have potency regenerating cancer cell bulk. For this reason, current research has focused on the elucidation of the mechanisms by which cancer cells, as well as cancer stem cells, are eliminated. In this study , the cytotoxic, apoptotic activity of PI3K/Akt/mTOR inhibitor PKI-402 was investigated in different breast cancer subtypes and breast cancer stem cells in comparison with normal cells. For this purpose, cytoxicity, apoptosis, ?-H2AX assay, clonogenic survival assay and phosphorylation assay were performed. PKI-402 cytotoxicity was found for these cells at a dose of 109 nM in the MCF-7 cell line, 2.44 µM in the MDA-MB-231 cell line, 5.941 µM in the BCSC cell line and 142 nM in the MCF-10A cell line at 48 hours. Advanced experiments were continued with these doses and it was found that when this IC50 dose was combined with radiation, apoptosis was induced in the MCF-7, BCSC and MCF-10A cell lines and not in the MDA-MB-231 cell line. As a result of the ?-H2AX , MCF-7, MDA-MB-231 cell lines showed a significant percentage of ?-H2AX between controls and between groups (IR and PKI-402 + IR). The percentage of ?-H2AX in the MCF-10A and BCSC cell lines was not significantly assessed. The survival capacity after the treatments was assessed by clonogenic assay. A significant change in survival percentages difference between MCF-7 and BCSC cell lines was observed between IR and PKI-402 + IR groups and control. As a result of the phosphorylation assay, it was observed that some of the important 16 proteins repressing the PI3K/Akt/mTOR pathway inhibition in MCF-7, MDA-MB-231 and BCSC cell lines were observed to suppress the phosphorylation of only 4E-BP1(Thr37/46) in normal mammary epithelial cell line MCF-10A . Based on all these results, we think PKI-402 should be confirmed in further experiments, considering that it enhances radiation sensitivity.