The effect of cytoplasmic Ca2+ on cisplatin-induced autophagy in ovarian carcinoma SKOV3 cells and its mechanism
10.3969/j.issn.1007-3969.2016.04.005
- VernacularTitle:Ca2+在顺铂诱导人卵巢癌SKOV3细胞自噬反应中的作用
- Author:
Linchuan ZENG
;
Huimin DENG
;
Jun CHEN
;
Minghan DOU
;
Ye XU
- Publication Type:Journal Article
- Keywords:
Cisplatin;
Ca2+;
ER Stress;
Autophagy
- From:
China Oncology
2016;26(4):313-319
- CountryChina
- Language:Chinese
-
Abstract:
Background and purpose:Ca2+plays a very important role in the maintenance of cell biological functions. The storage, release and uptake capacity of Ca2+ is controlled by endoplasmic reticulum (ER). Ca2+ homeo-stasis is essential for cellular energy metabolism and proper protein folding. This study aimed to investigate the effect of cytoplasmic Ca2+ on cisplatin induced ER stress-mediated autophagy in ovarian carcinoma SKOV3 and its underlying mechanism.Methods:The ovarian cancer SKOV3 was used as a study object. The experiment consisted of three parts:① To explore the possible relationship between cisplatin-induced ER stress and autophagy, SKOV3 cells were treated with cisplatin for 0, 6, 12 and 24 h, respectively;② To explore the possible relationship between ER stress induced Ca2+ effux and autophagy, SKOV3 cells were treated with cisplatin for 0, 9 and 12 h, respectively, and TG was used as a positive control;③ To explore the effects of blocking calcium effux on autophagy, SKOV3 cells were divided into control group, cisplatin group, TG group, BAPTA-AM group, cisplatin combined with BAPTA-AM group and TG com-bined with BAPTA-AM group. Western blot was used to detect the protein levels of GRP78 and LC3. Fluo-4 calcium lfuorescent probe was used to examine cytoplasmic Ca2+ levels. Confocal microscopy was used to detect LC3 level by immunolfurescence staining.Results:Compared to control group (0.679±0.011), GRP78 was signiifcantly accumulated at 6, 12 and 24 h after cisplatin treatment and reached the maximum value at 6 h (1.393±0.004,P=0.000). Similarly, compared to control group (0.038±0.000), LC3 puncta were clearly seen after cisplatin treatment and reached the maxi-mum value at 12 h (0.072±0.002,P=0.000). Using confocal microscopy, we found that cisplatin and TG increased LC3 punctate accumulation and cytoplasmic Ca2+ levels in a time-dependent manner. Immunolfuorescent method showed that treatment with cisplatin combined with BAPTA-AM or TG combined with BAPTA-AM increased LC3 punctate accumulation induced by cisplatin or TG. The results of Western blot showed that cisplatin combined with BAPTA-AM (0.071±0.001) or TG combined with BAPTA-AM (0.065±0.001) signiifcantly increased LC3Ⅱ/LC3Ⅰ ratio induced by cisplatin (0.039±0.000,P=0.000) or TG (0.035±0.001,P=0.000).Conclusion:Cisplatin induces intracellular ER stress and autophagy in SKOV3 cells, accompanied by increased cytoplasmic Ca2+ levels. Chelating cytoplasmic Ca2+enhanc-es cisplatin-induced autophagy.
