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由于高复发率、浸润和转移,胃癌总体5年生存率仍然很低[1]。线粒体作为氧气传感器以及三磷酸腺苷(adenosine triphosphate,ATP)的产生者的重要性已成为癌症研究的重点[2-3]。有氧糖酵解涉及己糖激酶(hexokinase,HK)和磷酸果糖激酶(phosphofructokinase,PFK)[4],其在肿瘤中过表达,可被许多癌蛋白调节,从而促进肿瘤增殖、迁移和化学抗药性[5-6]。癌细胞的代谢和有氧糖酵解被认为是癌细胞的特异性靶标,为癌症治疗提供了新的视角[7]。癌细胞具有较高的糖酵解速率,因此阻断该能量产生途径似乎是有选择地杀死癌细胞的合理方法[8]。糖酵解产生的能量不如氧化磷酸化产生的能量有效,故癌细胞需要更多的葡萄糖来支持更高的糖酵解速率[9]。WZB117是一种合成的小分子葡萄糖运转蛋白抑制剂,其下调葡萄糖运转蛋白以及细胞内ATP和糖酵解酶的水平,从而发挥抗癌活性[10-11]。本研究即探讨糖酵解抑制剂WZB117诱导人胃癌细胞系MGC-803的凋亡的机制,以为临床诊治提供依据。
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24 h时和48 h时,WZB117组较对照组细胞增殖均降低,差异有统计学意义(P < 0.01)(见表 1)。
分组 n 24 h吸光度值 48 h吸光度值 对照组 20 168.16±28.37 235.74±45.11 WZB117组 20 129.33±12.28 145.29±19.87 t — 5.62 8.21 P — < 0.01 < 0.01 表 1 2组细胞增殖检测比较(x±s)
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WZB117组较对照组细胞凋亡率升高,差异有统计学意义(P < 0.01),WZB117组较对照组细胞活力降低,差异有统计学意义(P < 0.01)。TUNEL分析显示,WZB117诱导的细胞的凋亡增加(见图 1、表 2)。
分组 n 细胞凋亡率/% 细胞活性/% 对照组 20 20.72±3.52 78.36±9.05 WZB117组 20 41.18±6.33 58.19±10.03 t — 12.63 6.68 P — < 0.01 < 0.01 表 2 细胞凋亡与活力测定(x±s)
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评估糖酵解细胞中ATP产生,12 h和24 h时WZB117组较对照组ATP含量均降低,差异有统计学意义(P < 0.01)(见表 3)。
分组 n 12 h 24 h 对照组 20 316.47±64.58 354.37±55.18 WZB117组 20 164.22±25.69 91.35±26.47 t — 9.80 19.21 P — < 0.01 < 0.01 表 3 ATP含量检测(x±s;nmol/mg蛋白)
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WZB117组较对照组Bcl-2表达降低,差异有统计学意义(P < 0.01),WZB117组较对照组Bax、caspase-3和Cyt-c表达升高,差异有统计学意义(P < 0.01)(见图 2、表 4)。
分组 n Bcl-2 Bax caspase-3 Cyt-c 对照组 20 1.95±0.36 1.06±0.12 1.14±0.18 1.08±0.14 WZB117组 20 1.17±0.15 1.84±0.31 1.79±0.24 1.83±0.25 t — 8.94 10.19 9.69 11.71 P — < 0.01 < 0.01 < 0.01 < 0.01 表 4 Bcl-2、Bax、caspase-3和Cyt-c蛋白的表达量(x±s;积分光密度值)
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WZB117组较对照组HK和PFK表达均降低,差异有统计学意义(P < 0.01)(见图 3、表 5)。
分组 n HK PFK 对照组 20 1.71±0.25 1.85±0.31 WZB117组 20 0.94±0.12 1.01±0.15 t — 12.42 10.91 P — < 0.01 < 0.01 表 5 糖酵解酶HK和PFK蛋白表达量(x±s;积分光密度值)
糖酵解抑制剂通过抑制糖酵解和促进线粒体调节的途径诱导胃癌细胞凋亡
Glycolysis inhibitor induces apoptosis of gastric cancer cells by inhibiting glycolysis and promoting mitochondria-regulated pathway
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摘要:
目的探究糖酵解抑制剂WZB117通过抑制糖酵解和促进线粒体调节的凋亡途径诱导人胃癌细胞系MGC-803的凋亡的机制。 方法处于对数生长期的胃癌MGC-803细胞用于实验。根据实验要求,将培养的细胞分为2组:对照组(正常培养的胃癌细胞),WZB117组(用20 μg/mL的葡萄糖运转蛋白抑制剂处理的胃癌细胞)。通过MTS测定试剂盒检测细胞的增殖能力;通过CCK-8测定细胞活力,TUNEL分析细胞细胞凋亡;通过测定ATP含量检测线粒体功能;通过免疫印迹分析Bcl-2、Bax、caspase-3和Cyt-c蛋白和糖酵解相关酶己糖激酶(HK)和磷酸果糖激酶(PFK)蛋白的表达。 结果24 h时和48 h时WZB117组较对照组细胞增殖降低(P < 0.01)。WZB117组较对照组细胞凋亡率升高(P < 0.01),WZB117组较对照组细胞活力降低(P < 0.01)。12 h时和24 h时WZB117组较对照组ATP含量均降低(P < 0.01)。WZB117组较对照组Bcl-2蛋白表达降低(P < 0.01),WZB117组较对照组Bax、caspase-3和Cyt-c的蛋白表达升高(P < 0.01)。WZB117组较对照组HK和PFK表达降低(P < 0.01)。 结论WZB117通过抑制糖酵解途径和减少线粒体的ATP产能诱导胃癌细胞系MGC-803的凋亡。 Abstract:ObjectiveTo investigate the mechanism of glycolysis inhibitor WZB117-induced apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptotic pathway. MethodsGastric cancer cell line MGC-803 was used in this study, and exponential growth cells were used in the experiment.According to the experimental requirements, the cultured cells were divided into control group(normally cultured cells) and WZB117 group(cells treated with 20 μg/mL glucose transporter inhibitor).The proliferation of cells was detected by MTS kit.Cell viability was measured by CCK-8 and apoptosis was analyzed by TUNEL.Mitochondrial function was detected by ATP content.The expression of Bcl-2, Bax, caspase-3, Cyt-c and glycolysis-related enzymes cells in hexokinase(HK) and cells in phosphofructokinase(PFK) were analyzed by Western blotting. ResultsThe proliferation of WZB117 group was lower than that in control group at 24 h and 48 hours(P < 0.01).The apoptosis rate in WZB117 group was higher than that in control group(P < 0.01), and the cell viability in WZB117 group was lower than that in control group(P < 0.01).The ATP content in WZB117 group was lower than that in control group at 12 h and 24 h(P < 0.01).The expression of Bcl-2 protein in WZB117 group was lower than that in control group(P < 0.01), and the expression of Bax, caspase-3 and Cyt-c protein in WZB117 group was higher than that in control group(P < 0.01).The expression of HK and PFK in WZB117 group was lower than that in control group(P < 0.01), indicating that WZB117 could inhibit the expression of enzymes related to glycolysis. ConclusionsWZB117 induces apoptosis of gastric cancer cell line MGC-803 by inhibiting glycolysis pathways and reducing mitochondrial ATP production. -
Key words:
- gastric neoplasms /
- WZB117 /
- glycolysis /
- energy metabolism /
- mitochondria
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表 1 2组细胞增殖检测比较(x±s)
分组 n 24 h吸光度值 48 h吸光度值 对照组 20 168.16±28.37 235.74±45.11 WZB117组 20 129.33±12.28 145.29±19.87 t — 5.62 8.21 P — < 0.01 < 0.01 表 2 细胞凋亡与活力测定(x±s)
分组 n 细胞凋亡率/% 细胞活性/% 对照组 20 20.72±3.52 78.36±9.05 WZB117组 20 41.18±6.33 58.19±10.03 t — 12.63 6.68 P — < 0.01 < 0.01 表 3 ATP含量检测(x±s;nmol/mg蛋白)
分组 n 12 h 24 h 对照组 20 316.47±64.58 354.37±55.18 WZB117组 20 164.22±25.69 91.35±26.47 t — 9.80 19.21 P — < 0.01 < 0.01 表 4 Bcl-2、Bax、caspase-3和Cyt-c蛋白的表达量(x±s;积分光密度值)
分组 n Bcl-2 Bax caspase-3 Cyt-c 对照组 20 1.95±0.36 1.06±0.12 1.14±0.18 1.08±0.14 WZB117组 20 1.17±0.15 1.84±0.31 1.79±0.24 1.83±0.25 t — 8.94 10.19 9.69 11.71 P — < 0.01 < 0.01 < 0.01 < 0.01 表 5 糖酵解酶HK和PFK蛋白表达量(x±s;积分光密度值)
分组 n HK PFK 对照组 20 1.71±0.25 1.85±0.31 WZB117组 20 0.94±0.12 1.01±0.15 t — 12.42 10.91 P — < 0.01 < 0.01 -
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