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Prompt Report
silencing-mediated increase in autophagy and apoptosis of human gastric cancer BGC-823 cells after doxorubicin treatment
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Abstract:
Objective: To investigate the effect of high-mobility group box 1 ( HMGB1 ) gene silencing on chemotherapeutic agent Doxorubicin (DOX)-induced autophagy and apoptosis in human gastric cancer BGC-823 cells. Methods: Cell viability, microtubule light chain 3 (LC3-Ⅰ, LC3-Ⅱ) protein content and autophagic vesicle formation in DOX-induced BGC-823 cells were assessed by MTT assay, Western blotting and confocal microscopy, respectively. BGC-823 cells were transfected with pSuper-shHMGBl (a shRNA targeting the HMGB1 gene) and pSuper-shNC (a control vector) respectively and subjected to G418 selection. BGC-823 cells stably expressing pSuper-shHMGBl or pSuper-shNC were treated with DOX. After treatment, autophagic vesicle formation was assessed by confocal microscopy. Cell viability and cell apoptosis were determined by the MTT assay and flow cytometry respectively. HMGB1, LC3, Bcl-2, Bcl-XL, Mcl-1 and cleaved caspase-3 proteins were assessed by Western blotting. Results: Treatment with DOX increased the protein content of LC3-Ⅱ and the formation of autophagic vesicles in BGC-823 cells ( P <0.01). As compared to the control, HMGB1 silencing inhibited DOX-induced autophagy ( P <0.01), promoted apoptosis (\[46.12±3.15\]% vs \[12.37±2.84\]%, P <0.05) and up-regulated the expression of cleaved caspase-3 in BGC-823 cells. Furthermore, antiapoptotic Mcl-1 protein degradation was increased in BGC-823 cells after induction of autophagy, however, Bcl-2 and Bcl-XL protein levels did not change significantly. HMGB1 gene silencing significantly attenuated the degradation of Mcl-1 protein. Conclusion: Autophagy can be induced by Doxorubicin in BGC-823 cells, which may contribute to DOX resistance. Knockdown of HMGB1 attenuates the DOX-induced autophagy and promotes apoptosis in gastric cancer cells.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No. 81101558), the Natural Science Foundation of Fujian Province (No. 2012J01364), and the Fundation for the Doctoral Program from the Ministry of Education of China (No. 20113518120002)
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