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Effects of ethanol on autophagy and the role of P62 in ethanol-induced autophagy in pheochromocytoma cells
DONG Yue-juan SHI Zhen-yu LIU Shi-meng ZHANG Wei-juan HUANGFU Chao-shen DENG Jin-bo LIU Bin*
Acta Anatomica Sinica ›› 2013, Vol. 44 ›› Issue (6) : 772-777.
Effects of ethanol on autophagy and the role of P62 in ethanol-induced autophagy in pheochromocytoma cells
Objective To utilize pheochromocytoma (PC12) cells to examine the effect of ethanol on autophagy and the role of P62 in ethanol-induced autophagy. Methods The inhibition effect of ethanol on proliferation of PC12 cells was examined by MTT assay. Indirect immunofluorescence was used to detect location of LC3, a biomarker of autophagy, and P62. The LC3 fluorescence intensity in cellular cytosol was measured using a high content screening imaging system. The ultrastructural morphology of autophagic vacuoles and autophagy-lysosome was observed under the transmission electron microscope. Protein expression of P62 was detected with Western blotting analysis. Results Compared to the control in serumfree medium after 24hours, ethanol at the concentrations of 100mmol/L, 200mmol/L, 400mmol/L and 800mmol/L decreased cell proliferation of PC12 cells to 91.97% (P<0.05)，72.63%(P<001)，58.23%(P<0.01) and 50.82%(P<0.01) respectively, indicating a dose-dependent inhibitory effect of ethanol on cell proliferation.Indirect immunofluorescence staining and the high content screening imaging system showed that ethanol increased LC3 fluorescence intensity and the co-localization of LC3 with P62 in PC12 cellular cytosol. The transmission electron microscope showed the ultrastructural morphology of autophagic vacuoles and autophagy-lysosome in PC12 cells incubated for 2hours in the presence of 200mmol/L ethanol. Compared with the control in serum-free medium, the protein expression of P62 increased significantly in different ethanol concentration treatment groups (P<0.01). The crest-time appeared at 2hours after the PC12 cells were treated with 200mmol/L ethanol. Conclusion Ethanol may induce autophagy of PC12 cells. P62 may be involved in the autophagy against ethanol-induced cytotoxicity.
Ethanol / Autophagy / LC3 / P62 / PC12 cell / Indirect immunofluorescence / Transmission electron microscopy
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