Zhou Liqiang, Li Shihao, Liu Li, Zhou Qi, Yuan Yiwu, Xin Lin
Department of Gastrointestinal Surgery, Second Affiliated Hospital of Nanchang University, Nanchang 330006, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2020 Jan 30;40(1):27-33. doi: 10.12122/j.issn.1673-4254.2020.01.05.
To investigate the molecular mechanism of recombinant methioninase (rMETase) in promoting apoptosis of gastric cancer cells.
Gastric cancer SGC-7901 cells were treated with rMETase (final concentration of 1.25 and 2.50 mmmol/L) for 72 h, and the changes in the cell viability were detected using CCK-8 method and the cell morphology changes were observed under an inverted microscope. Plate colony formation assay was used to evaluate colony formation ability of the cells, and flow cytometry was performed to analyze the changes in cell apoptosis and cell cycles. Glucose and lactate levels in the culture medium were determined using a colorimetric method and ATP concentration was detected using a fluorescence microplate reader; Western blotting was used to assess the effect of rMETase on PI3K/Akt pathway, glucose transporter-1 (GLUT-1), glycolysis- related proteins and apoptotic proteins in SGC-7901 cells.
rMETase significantly inhibited the proliferation and clonal formation, promoted cell apoptosis, and induced cell cycle arrest in S phase in SGC-7901 cells ( < 0.05). With the increase of rMETase concentration, the cells showed obviously decreased glucose intake accompanied by decreased glycolysis and ATP concentration ( < 0.001). The results of Western blotting showed that the expressions of PI3K, p-Akt/t-Akt, GLUT-1, and the key glycolytic enzymes HK2, PFKM, LDHA, antiapoptosis protein Bcl-2 were all downregulated and the pro-apoptotic proteins Bax and caspase-3 were up-regulated in response to rMETase treatment in SGC-7901 cells ( < 0.01).
rMETase can inhibit aerobic glycolysis, induce apoptosis and inhibit the proliferation of SGC-7901 cells by inhibiting the activity of PI3K/Akt/GLUT-1 pathway, suggesting its potential as a therapeutic agent for gastric cancer.
探讨重组蛋氨酸酶（rMETase）促进胃癌细胞凋亡的分子机制。
用rMETase（终浓度为1.25和2.50 mmol/L）处理胃癌SGC-7901细胞72 h，采用CCK-8法检测细胞活力变化，倒置显微镜下观察细胞形态变化。平板克隆形成实验用于评估细胞的克隆形成能力，流式细胞术分析细胞凋亡和细胞周期的变化。采用比色法测定培养基中葡萄糖和乳酸水平，荧光酶标仪检测ATP浓度；蛋白质免疫印迹法评估rMETase对SGC-7901细胞中PI3K/Akt通路、葡萄糖转运蛋白-1（GLUT-1）、糖酵解相关蛋白和凋亡蛋白的影响。
rMETase显著抑制SGC-7901细胞的增殖和克隆形成，促进细胞凋亡，并诱导细胞周期阻滞于S期（<0.05）。随着rMETase浓度的增加，细胞葡萄糖摄取明显减少，同时糖酵解和ATP浓度降低（<0.001）。蛋白质免疫印迹结果显示，rMETase处理后，SGC-7901细胞中PI3K、p-Akt/t-Akt、GLUT-1以及关键糖酵解酶HK2、PFKM、LDHA、抗凋亡蛋白Bcl-2的表达均下调，促凋亡蛋白Bax和caspase-3上调（<0.01）。
rMETase可通过抑制PI3K/Akt/GLUT-1通路活性抑制有氧糖酵解，诱导凋亡并抑制SGC-7901细胞增殖，提示其作为胃癌治疗药物的潜力。
