Effects of sirolimus and starved culture on autophagy of human A431 cells / 中华皮肤科杂志

ABSTRACT

Objective To evaluate effects of sirolimus(a classic autophagy inducer)and starved culture on autophagy of a human cutaneous squamous cell carcinoma cell line A431. Methods Cultured A431 cells and HeLa (a human cervical carcinoma cell line)cells were both classified into 5 groups to be treated with DMEM alone(control group), 0.1%dimethyl sulfoxide alone(DMSO group), 20 nmol/L sirolimus(20?nmol/L sirolimus group), 80 nmol/L sirolimus(80?nmol/L sirolimus group), and Earle′s balanced salt solution(EBSS group)respectively. After 4?hour treatment, Western blot analysis was performed to measure the expressions of autophagy?related markers microtubule?associated protein 1 light chain 3A/3B (LC3A/B) and recombinant gamma?aminobutyric acid receptor associated protein(GABARAP), and acridine orange staining to determine autophagy levels in these cells. Results As Western blot analysis showed, the ratio of LC3A/B?Ⅱto LC3A/B?Ⅰin A431 cells was similar between the control group and DMSO group(P > 0.05), but significantly higher in the 20?nmol/L sirolimus group, 80?nmol/L sirolimus group and EBSS group than in the control group(all P < 0.05). Western blot results from HeLa cells were similar to those from A431 cells. Bivariate correlation analysis revealed that the protein expression of GABARAP was positively correlated with that of LC3A/B ?Ⅰ in both HeLa cells(r = 0.869, 95% CI 0.807 - 0.999, P = 0.051)and A431 cells(r = 0.837, 95% CI -0.173 - 0.989, P = 0.037), but negatively correlated with that of LC3A/B?Ⅱ in both HeLa cells(r = -0.742, 95% CI -0.982 - 0.406, P = 0.042)and A431 cells(r = - 0.684, 95% CI -0.977 - 0.500, P = 0.047). Acridine orange staining showed that the percentages of autophagosome?positive A431 cells and HeLa cells were significantly increased in both the 80?nmol/L sirolimus group(23.750% ± 0.260% and 33.307% ± 0.715% respectively)and EBSS group(32.450% ± 0.488% and 66.097% ± 1.141% respectively) compared with the control group(15.987% ± 0.242% and 14.117% ± 0.295%, respectively, all P < 0.05). Conclusion The classic autophagy inducer sirolimus and starved culture can upregulate the autophagy level of A431 cells, and GABARAP may be highly correlated with LC3A/B.