四种肝(癌)细胞系糖代谢的流量解析
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摘要
目的分析人正常肝细胞(HL-7702)、不同转移潜能的肝细胞癌细胞系(SMMC-7721、HCCLM3)以及肝癌门静脉癌栓细胞系(CSQT2)的糖代谢流量及相关基因表达。方法利用稳定同位素~(13)C标记的葡萄糖与谷氨酰胺作为营养源,结合酶活实验,对四种细胞进行糖代谢流量解析。结果 MTT与Transwell实验证实了HL-7702、SMMC-7721、HCCLM3、CSQT2细胞的增殖与迁移能力依次增加。酶活实验与标记实验结果均显示,糖酵解与三羧酸循环的代谢活性在四种细胞中依次增加,具体表现为葡萄糖消耗与乳酸产生的增加,代谢流量比率在多种糖代谢产物中的上升,尤其是在CSQT2细胞中达到最高。结论随着肝癌细胞的恶性程度及转移能力的增加,糖酵解与三羧酸循环的代谢活性增加。代谢流量分析可能用以判断肝癌的发生发展与转移。
Objective To analyze glucose metabolic flux and related gene expression of human normal liver cells(HL-7702), hepatoma cell lines with different metastasis degree(SMMC-7721, HCCLM3) and hepatic portal vein tumor thrombus cell line CSQT2. Methods Stable isotope ~(13)C labeled glucose and glutamine were used as the nutrient source. Label assay and enzymes activity assay were employed to analyze glucose metabolic activity and flux of the four cell lines. Results MTT and Transwell assay confirmed the proliferation and migration abilities were increased successively in HL-7702, SMMC-7721, HCCLM3 and CSQT2 cells. Label assay and enzyme activity assay showed that metabolic activity in glycolysis and tricarboxylic acid cycle(TCA cycle) was successively increased in the four cells, mainly reflected by the increase of glucose consumption and lactate production, as well as metabolic flux and ratio of glucose metabolism in hepatoma cell lines compared with normal liver cells, and reached the highest level in CSQT2 cell line. Conclusion The metabolic pathway of glycolysis and TCA cycle is activated as malignant and metastasis degrees are increased in liver cancer cell lines. Metabolic flux analysis may help to diagnose the development and metastasis of liver cancer.
Objective To analyze glucose metabolic flux and related gene expression of human normal liver cells(HL-7702), hepatoma cell lines with different metastasis degree(SMMC-7721, HCCLM3) and hepatic portal vein tumor thrombus cell line CSQT2. Methods Stable isotope ~(13)C labeled glucose and glutamine were used as the nutrient source. Label assay and enzymes activity assay were employed to analyze glucose metabolic activity and flux of the four cell lines. Results MTT and Transwell assay confirmed the proliferation and migration abilities were increased successively in HL-7702, SMMC-7721, HCCLM3 and CSQT2 cells. Label assay and enzyme activity assay showed that metabolic activity in glycolysis and tricarboxylic acid cycle(TCA cycle) was successively increased in the four cells, mainly reflected by the increase of glucose consumption and lactate production, as well as metabolic flux and ratio of glucose metabolism in hepatoma cell lines compared with normal liver cells, and reached the highest level in CSQT2 cell line. Conclusion The metabolic pathway of glycolysis and TCA cycle is activated as malignant and metastasis degrees are increased in liver cancer cell lines. Metabolic flux analysis may help to diagnose the development and metastasis of liver cancer.
引文
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[2]Kaibori M,Ishizaki M,Matsui K,et al.Predictors of microvascular invasion before hepatectomy for hepatocellular carcinoma[J].J Surg Oncol,2010,102(5):462-8.
[3]Benjamin DI,Cravatt BF,Nomura DK.Global profiling strategies for mapping dysregulated metabolic pathways in cancer[J].Cell Metab,2012,16(5):565-77.
[4]Wang T,Hu HS,Feng YX,et al.Characterisation of a novel cell line(CSQT-2)with high metastatic activity derived from portal vein tumour thrombus of hepatocellular carcinoma[J].Br J Cancer,2010,102(11):1618-26.
[5]Ma L,Tao Y,Duran A,et al.Control of nutrient stress-induced metabolic reprogramming by PKCzeta in tumorigenesis[J].Cell,2013,152(3):599-611.
[6]Huang Q,Tan Y,Yin P,et al.Metabolic characterization of hepatocellular carcinoma using non-targeted tissue metabolomics[J].Cancer Res,2013,73(16):4992-5002.
[7]Llovet JM,Bruix J.Novel advancements in the management of hepatocellular carcinoma in 2008[J].J Hepatol,2008,48 Suppl 1:S20-S37.
[8]邱继刚,樊嘉,刘银坤,等.肝癌门静脉癌栓相关小分子的比较蛋白质组学分析[J].中华实验外科杂志,2009,26(2):181-3.[Qiu JG,Fan J,Liu YK,et al.Comparative proteome analysis of PVTT realated small molecules[J].Zhonghua Shi Yan Wai Ke Za Zhi,2009,26(2):181-3]
[9]Haschemi A,Kosma P,Gille L,et al.The sedoheptulose kinase CARKL directs macrophage polarization through control of glucose metabolism[J].Cell Metab,2012,15(6):813-26.
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