南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (01): 27-33.doi: 10.12122/j.issn.1673-4254.2020.01.05

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重组甲硫氨酸酶调控 PI3K/Akt/Glut-1 通路抑制有氧糖酵解促进胃癌细胞凋亡

周立强，李世豪，刘 力，周 祺，袁宜武，辛 林   

  • 出版日期:2020-02-18 发布日期:2020-01-20
  • 基金资助:

Recombinant methioninase regulates PI3K/Akt/Glut-1 pathway and inhibits aerobic glycolysis to promote apoptosis of gastric cancer cells

  

  • Online:2020-02-18 Published:2020-01-20

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摘要： 目的 探讨重组甲硫氨酸酶（rMETase）对胃癌细胞增殖抑制作用及潜在的分子机制。方法 rMETase（终浓度为0.00、1.25、 2.50 mmol/L）处理胃癌SGC-7901细胞72 h，采用CCK-8法检测SGC-7901细胞活力，倒置显微镜下观察SGC-7901细胞形态变化，平板细胞克隆形成实验检测SGC-7901细胞克隆形成率，流式细胞术分析胃癌细胞凋亡情况及细胞周期变化，比色法和荧光酶标仪检测rMETase对胃癌细胞葡萄糖吸收和乳酸、ATP释放的影响，Western blot分析rMETase对SGC-7901细胞PI3K/Akt通 路、GLUT-1、糖酵解相关蛋白及凋亡蛋白的影响。结果 rMETase能够抑制SGC-7901细胞的增殖和克隆形成、诱导胃癌细胞S期周期阻滞、促进细胞凋亡（P<0.05）；随着rMETase的浓度的增加，细胞吸收的葡萄糖降低，并伴随糖酵解产物乳酸和ATP的下降（P<0.001）；Western blot结果显示，rMETase作用SGC-7901细胞后，PI3K、p-Akt/t-Akt、GLUT-1，糖酵解关键酶HK2、PFKM、LDHA、抑凋亡Bcl-2的蛋白表达下调，促凋亡蛋白Bax、Caspase-3表达上调（P<0.01）。结论 rMETase可以通过抑制PI3K/Akt/GLUT-1通路的活性，抑制胃癌细胞有氧糖酵解，诱导胃癌细胞凋亡，抑制SGC-7901细胞增殖，rMETase可能是一种潜在的胃癌治疗药物。

Abstract: Objective To investigate the molecular mechanism of recombinant methioninase (rMETase) in promoting apoptosis of gastric cancer cells. Methods 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. Results rMETase significantly inhibited the proliferation and clonal formation, promoted cell apoptosis, and induced cell cycle arrest in S phase in SGC-7901 cells (P<0.05). With the increase of rMETase concentration, the cells showed obviously decreased glucose intake accompanied by decreased glycolysis and ATP concentration (P<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 (P<0.01). Conclusion 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.