缺氧条件下肝癌细胞和正常肝细胞能量代谢通路的差异

The difference of energy metabolism pathways between normal hepatocytes and hepatoma cells under hypoxia

目的比较缺氧条件下肝癌细胞与正常肝细胞能量代谢通路的差异,探索肿瘤细胞的耐缺氧机制。方法采用环境缺氧(0.2%O2缺氧培养箱)模拟肿瘤的体内环境,比较肝癌细胞株HepG2与原代分离的小鼠肝脏细胞在不同缺氧时间下的能量代谢情况。流式细胞术检测各组活性氧(reactive oxygen species,ROS)水平,MTT法检测各组细胞缺氧后细胞活力及生存率,real-time PCR方法检测己糖激酶2(hexokinase 2)、异柠檬酸脱氢酶(isocitrate dehydrogenase)等糖代谢关键酶及p53、TIGAR/Tigar、SCO2/Sco2基因的mRNA水平,NADH比色法检测乳酸脱氢酶(lactate dehydrogenase)活性,氧电极法检测各组细胞耗氧量,高效液相色谱法检测细胞内ATP含量。结果缺氧后,肿瘤细胞HepG2与正常小鼠肝脏细胞相比生存率更高,活性氧增幅更小。两种细胞在缺氧下耗氧量都下降,但肝癌细胞的ATP产量代偿性增加,而正常肝细胞的ATP显著下降;有氧氧化相关酶基因在缺氧小鼠肝脏细胞中代偿性上调,而在缺氧HepG2细胞中下降;糖酵解相关酶基因在缺氧HepG2细胞中上调更迅速,幅度更大;进一步研究表明,缺氧时,小鼠肝脏细胞p53、Tigar、Sco2基因表达上调,而HepG2细胞p53、TIGAR、SCO2基因表达下调。结论在缺氧应激状态下,正常细胞主要依赖有氧氧化补偿能量,肿瘤细胞则主要依赖糖酵解。ROS或可通过调节肿瘤细胞p53/TIGAR、p53/SCO2通路及糖酵解关键酶活性促进Warburg效应与肿瘤耐缺氧能力。

Objective To compare the difference between energy metabolism pathways of hypoxic normal hepatocytes cells and tumor cells,and try to understand the underlying mechanism of tumor hypoxia resistance. Methods We adopted environmental hypoxia（Hypoxia incubator with0.2%O2）to mimic the tumor environment in vitro,and compared the difference of energy metabolism response to hypoxic stress between HepG2 cells and mice hepatocytes.Flow cytometry was used to detect the level of different kinds of ROS.MTT was used to compare the viability of cells before and after hypoxia.The mRNA level of p53,Tigar,SCO2,and key enzymes in the metabolism including hexokinase 2 and isocitrate dehydrogenase,were detected by real-time PCR.NADH colorimetric assay was used to detect lactate dehydrogenase activity.Oxygen electrode was used to detect the oxygen consumption rate of cells.HPLC was used to detect the ATP levels in cells. Results Under moderate hypoxia,HepG2 cells had a higher survival rate and underwent a more steady reactive oxygen species（ROS）fluctuation.The oxygen consumption rate declined in both types of cells,ATP production in hepatoma cells increased with cellular proliferation being maintained,but decreased significantly in normal liver cells.The results also showed that key enzymes involved in aerobic oxidation were compensatively increased in normal liver cells but decreased in hepatoma cells under hypoxia.The key enzymes involved in glycolysis were both upregulated in response to hypoxia,but the hepatoma cells increased more significantly.Further studies showed that in response to hypoxia,the mRNA expression of p53,Tigarand Sco2 were all upregulated in normal liver cells but p53,TIGARand SCO2 downregulated in hepatoma cells. Conclusions Under hypoxia stress,normal cells mainly depend on aerobic oxidation to compensate the energy,but hepatoma cells mainly rely on glycolysis.ROS may enhance glycolysis through p53/TIGAR and p53/SCO2 pathways,so as to promote the Warburg effect and hypoxia tolerance.