Effect of MTRR gene on apoptosis and autophagy pathways in multi-resistant epithelial ovarian cancer / 中华妇产科杂志

ABSTRACT

Objective To explore the effect of down-regulated methionine synthase reductase (MTRR) gene on the apoptosis and autophagy pathway, and offer a possible approach for the MTRR to reverse the multi-resistant ovarian cancer. Methods (1) The experiment was divided into 3 groups, SKOV3/DDP-MTRRi (down-regulated MTRR group), SKOV3/DDP-NC (negative control group), and SKOV3/DDP (blank control group). Different concentration of cisplatin (0, 1, 2, and 4 μg/ml) treated on 3 groups cells. The apoptosis rate was measured by flow cytometry (FCM). Autophagy was detected by immunofluorescence. Autophagy microtubule associated protein light chain 3β(LC3B) and p62 were detected by western blot. The formation of autophagosome of cells was observed by transmission electron microscope. (2) Detection of autophagy and apoptosis of SKOV3/DDP-MTRRi induced by rapamycin. The experiment was divided into 4 groups included rapamycin group (5 nmol/L rapamycin), rapamycin+cisplatin group (5 nmol/L rapamycin+4μg/ml cisplatin), cisplatin group (4μg/ml cisplatin) and blank control group. LC3B and p62 protein were detected by western blot. The survival rate cells were detected by methyl thiazolyl tetrazolium (MTT) method. The apoptosis rate was measured by FCM. (3) The 3 groups cells (SKOV3/DDP, SKOV3/DDP-NC and SKOV3/DDP-MTRRi) induced by a certain concentration of cisplatin (4 μg/ml) after 48 hours, then detecting the protein expression of caspase, Bcl-2 family in apoptosis pathway and the key proteins in phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) autophagy pathways by western blot, getting the time when the proteins′expression changed. Results (1) The 3 groups cells (SKOV3/DDP, SKOV3/DDP-NC, and SKOV3/DDP-MTRRi) induced by a certain concentration of cisplatin (4 μg/ml) after 48 hours, apoptosis and autophagy of 3 groups of cells were gradually increased with the increased concentration of cisplatin. The apoptosis rate of SKOV3/DDP-MTRRi cells [(26.2 ± 1.4)%] were significantly increased compared with the SKOV3/DDP-NC cells or SKOV3/DDP cells [(14.8 ± 2.4)%, (14.2 ± 2.4)%;all P0.05), but cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, and cleaved PARP were significantly increased in SKOV3/DDP-MTRRi cells (P<0.05). For the autophagy pathway, the expression of phosphorylated Akt (p-Akt) and phosphorylated mammalian target of rapamycin (p-mTOR) were significantly increased (P<0.05), but Akt and mTOR had no significant variation. The expression of phosphatase and tensin homologue deleted on chromosome ten (PTEN) was significantly decreased (P<0.05). Conclusions MTRR silencing significantly increase cisplatin-induced apoptosis and reduce the autophagy induced by cisplatin in SKOV3/DDP cells. Down-regulation of MTRR enhanced the chemosensitivity of cisplatin-resistant ovarian cancer cells may be by activating caspase and Bcl-2 apoptosis family and inhibiting the PI3K/Akt autophagy pathway.