[Effect of MTRR gene on apoptosis and autophagy pathways in multiresistant epithelial ovarian cancer].
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+ 4 μg/ml cisplatin), cisplatin group(4 μg/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 P<0.05]at 2 μg/ml cisplatin. Immunofluorescence tests revealed that the aggregates of LC3B in SKOV3/DDP-MTRRi cells were more than that of SKOV3/DDP-NC cells and SKOV3/DDP cells. The expression of LC3B of SKOV3/DDP-MTRRi cells was lower than those of SKOV3/DDP-NC cells and SKOV3/DDP cells(P<0.05). The expression of p62 of SKOV3/DDP-MTRRi cells was higher than those of SKOV3/DDP-NC cells and SKOV3/DDP cells(P<0.05). The structure of chloroplast was integrity and autophagosome was dispersing in plastids of SKOV3/DDP-NC cells and SKOV3/DDP cells. Organelles disappear and vacuoles increased obviously in SKOV3/DDP-MTRRi cells, no autophagosome was observed.(2)The expression of LC3B of rapamycin + cisplatin group was higher than those of other 3 group cells(1.72±0.08, 1.43±0.04, 1.37±0.11, and 1.11±0.09; P<0.05). The expression of p62 of rapamycin + cisplatin group was significant decreased(0.58 ± 0.10, 0.94 ± 0.12, 1.21 ± 0.11, and 1.57 ± 0.10; P<0.05). The survival rate of rapamycin + cisplatin group was higher than that of cisplatin group[(0.78±0.03)% vs(0.62±0.03)%; P=0.018], the apoptosis rate was significant decreased in rapamycin+ cisplatin group[(59.0 ± 3.9)% vs(40.4 ± 3.0)%, P=0.019].(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, the expression of Bax in 3 groups cell were not evidently changed(P= 0.661). The expression of Bcl-2 was significantly decreased in SKOV3/DDP-MTRRi cells(P=0.030). The expression of caspase-3, caspase-7, caspase-9, and poly(ADP-ribose)polymerase(PARP)were not evidently changed(P>0.05), 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 cisplatinresistant ovarian cancer cells may be by activating caspase and Bcl-2 apoptosis family and inhibiting the PI3K/Akt autophagy pathway.
Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, and Key Laboratory of High-Incidence-Tumor Prevention and Treatment, Ministry of Education, Nanning 530021, China.
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D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D000077216:Carcinoma, Ovarian Epithelial; D053148:Caspase 3; D053453:Caspase 9; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002945:Cisplatin / Q000494:pharmacology*; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D005260:Female; D005287:Ferredoxin-NADP Reductase; D006801:Humans; D009375:Neoplasms, Glandular and Epithelial; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt
D000970:Antineoplastic Agents; C111241:methionine synthase reductase; D005287:Ferredoxin-NADP Reductase; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D053148:Caspase 3; D053453:Caspase 9; D002945:Cisplatin