G Gucluler1, O Piskin, Y Baran. 1. Izmir Institute of Technology, Department of Molecular Biology and Genetics, Urla, Izmir, Turkey.
Abstract
PURPOSE: Sphingolipids are important signaling molecules mediating cell survival, proliferation, cell cycle regulation and apoptosis. Ceramide is the most vital sphingolipid which induces growth arrest, senescence, and apoptosis. In this study, we aimed to determine the roles of sphingosine kinase-1 (SK-1) and glucosylceramide synthase (GCS) genes in paclitaxel, doxorubicin, tamoxifen, cyclophosphamide and docetaxel induced apoptosis in human MCF-7 breast cancer cells. METHODS: IC50 values (drug concentration inhibiting cell growth by 50%) of the anticancer agents were calculated using XTT cell proliferation assay. Changes in mitochondrial membrane potential (MMP) were determined using JC-1 assay kit. Changes in the mRNA levels of SK-1 and GCS genes were measured by using RT-PCR technique. RESULTS: The results demonstrated significant decrease in cellular proliferation and increase in loss of MMP in a dose-dependent manner. Paclitaxel, doxorubicin, tamoxifen, cyclophosphamide and docetaxel application downregulated SK-1 expression while paclitaxel, tamoxifen, cyclophosphamide and docetaxel but not doxorubicin downregulated GCS comparing to untreated control cells. CONCLUSION: These results show for the first time that these agents induce apoptosis in MCF-7 cells by downregulating the antiapoptotic SK-1 and GCS genes that may result in accumulation of apoptotic ceramides.
PURPOSE:Sphingolipids are important signaling molecules mediating cell survival, proliferation, cell cycle regulation and apoptosis. Ceramide is the most vital sphingolipid which induces growth arrest, senescence, and apoptosis. In this study, we aimed to determine the roles of sphingosine kinase-1 (SK-1) and glucosylceramide synthase (GCS) genes in paclitaxel, doxorubicin, tamoxifen, cyclophosphamide and docetaxel induced apoptosis in human MCF-7 breast cancer cells. METHODS: IC50 values (drug concentration inhibiting cell growth by 50%) of the anticancer agents were calculated using XTT cell proliferation assay. Changes in mitochondrial membrane potential (MMP) were determined using JC-1 assay kit. Changes in the mRNA levels of SK-1 and GCS genes were measured by using RT-PCR technique. RESULTS: The results demonstrated significant decrease in cellular proliferation and increase in loss of MMP in a dose-dependent manner. Paclitaxel, doxorubicin, tamoxifen, cyclophosphamide and docetaxel application downregulated SK-1 expression while paclitaxel, tamoxifen, cyclophosphamide and docetaxel but not doxorubicin downregulated GCS comparing to untreated control cells. CONCLUSION: These results show for the first time that these agents induce apoptosis in MCF-7 cells by downregulating the antiapoptotic SK-1 and GCS genes that may result in accumulation of apoptotic ceramides.