Genistein (GEN) has been previously reported to induce apoptosis through changing bcl-x(l) expression and autophagy on cancer cells, the mechanism of which remains unclear. In this study we investigated the effect of the differentiation of Bcl-xl distribution induced by GEN on cell death by examining the activation of autophagy and apoptosis in A549 cell.
CCK-8 assay indicated the resistance to radiation, the subcellular distribution of bcl-xl was observed by Immunofluorescence, cell DNA damage were analyzed by neutral Comet assays the interaction of Beclin1/Bcl-xl was detected by Co-Immunoprecipitation, western blot measured the expression of cell autophagy and apoptosis related protein, flow cytometry analyzed cell autophagy and apoptosis level, The expression of cytoplasmic Bcl-xl protein was analyzed using immunohistochemistry in NSCLC patient tumor tissue.
We initially showed that the differentiation of cytoplasmic Bcl-xl in GEN A549 cells. By examining both cytoplasmic and nuclear of Bcl-xl in H1975, A549 and 293T cell, we found that the Bcl-xl cytoplasmic expression and cell death caused by radiation were negatively related. Furthermore, the expression of Bcl-xl cytoplasmic immunohistochemistry levels in tumor tissue, scored for intensity as 0, 1 + , 2 + , 3 + , were 26.7, 43.3, 26.7, and 3.3% in human non-small lung cancer, respectively, clearly showing the high expression of Bcl-xl cytoplasmic have a low overall response rate (ORR) of radiotherapy. Interestingly, we found that the treatment of combining GEN with radiation inhibited the cytoplasmic Bcl-xl and increased the Autophagy associated cell death by promoting the dissociation of Bcl-xl/beclin1 complex. In addition, the combinatorial treatment significantly inhibited the DNA damage repair and contributed to oligomerization of Bax and activation of cleaved caspase-3 and PARP.
Our data suggest that the autophagy and apoptosis promote cell death induced by radiation when the distribution of cytoplasmic Bcl-xl is inhibited by GEN. Based on this evidence, we hereby report that crosstalk between apoptosis and autophagy by Inhibition of cytoplasmic Bcl-xl will support the application of using GEN on radiation therapy.
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All authors have declared no conflicts of interest.