Comparison Of Glucose Metabolism In Liver Cancer Cells And Liver Cancer Stem Cells

The fatality rate of tumors is second only to cardiovascular and cerebrovascular diseases,and it is a malignant disease that seriously threatens human life and health.Tumor cells have unique energy metabolism characteristics,namely high glucose uptake,aerobic glycolysis,and high lactate production.Thus,tumors are also considered as a "metabolic disease".Studies confirmed that tumor tissues have heterogeneity.The presence of a small number of cells with stemness characteristic in tumor tissue is known as tumor stem cells(TSCs)or cancer stem cells(CSCs).Compared with ordinary tumor cells,CSCs have the potential for self-renewal and differentiation,and are resistant to chemoradiation.This is the root cause of tumor recurrence,metastasis and poor prognosis.CSCs targeted intervention is a new strategy for tumor treatment.The regulation of energy metabolism is closely related to stem cells differentiation.Studies have found that adult stem cells maintain their stemness by glycolysis,and progeny cells after differentiation rely on oxidative phosphorylation for glucose metabolism.In turn,regulating the energy metabolism of stem cells can also induce their differentiation.It can be seen that the stemness of stem cells and energy metabolism affect each other.However,the differences in energy metabolism between CSCs in tumor tissues and ordinary tumor cells and their relationship with cell phenotypes are currently lacking a systematic understanding.Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in humans,ranking third in the global mortality rate of malignant tumors and second in the mortality rate of malignant tumors in China.Liver cancer has the characteristics of poor curative effect,high recurrence and metastasis rate,extremely low survival rate,which seriously endangers patients’ lives and places a heavy burden on families and society.For clinical scientists,its prevention and treatment has always been a huge challenge.Therefore,we use human liver cancer cells HCCLM3 and enriched liver cancer stem cells(LCSCs)as research objects,systematically compares the differences in glucose metabolism,and explores the relationship between energy metabolism and LCSCs stemness.The main research contents and results are as follows:(1)Culture of liver cancer cells and enrichment and identification of liver cancer stem cellsHCCLM3 were routinely cultured in DMEM/H medium containing 10% fetal bovine serum.LCSCs were enriched from HCCLM3 by DMEM/F12 serum-free stem cell culture medium by in vitro tumor sphere culture and identified.The results showed that compared with HCCLM3,the enriched cells have good spheroidizing ability,colony forming ability,migration ability and drug resistance;the expression of CSCs surface markers CD133 and CD44 was significantly higher than that of HCCLM3,and the expression of stemness genes Sox2,Oct4,Nanog,c-Myc also increased significantly.The results showed that the enriched cells have the CSCs characteristics.(2)Comparison of glucose metabolism between liver cancer cells and liver cancer stem cellsThe glycolytic metabolism of HCCLM3 and LCSCs was compared by analysising the expression of glycolytic related enzymes and glucose uptake capacity.The results showed that the expression of glucose transporter 1(GLUT1),hexokinase 2(HK2),phosphofructokinase 1(PFK1),and pyruvate kinase(PKM)in LCSCs was significantly higher than that of HCCLM3,but the expression of lactate dehydrogenase A(LDHA)was significantly down-regulated;the glucose uptake capacity of LCSCs was also significantly higher than that of HCCLM3.The oxidative phosphorylation of HCCLM3 and LCSCs was further compared.The results showed that: pyruvate dehydrogenase complex(PDHC),mitochondrial biogenesis related gene peroxisome proliferator-activated receptor γ coactivator 1α(PGC1α),mitochondrial transcription factor A(TFAM)and nuclear respiratory factor 1(NRF1)were significantly up-regulated,and the levels of reactive oxygen species(ROS)were also significantly higher than those of HCCLM3.The above results showed that LCSCs have more vigorous glucose metabolism,and pyruvate produced by glycolysis enters mitochondria for oxidative phosphorylation.(3)Relationship between glucose metabolism of liver cancer stem cells and stemnessAiming at the difference between the glucose metabolism of LCSCs and HCCLM3,the relationship between glucose metabolism of LCSCs and stemness was continued to be investigated.After inhibiting the glycolysis of LCSCs by 2-deoxy-D-glucose(2-DG)treatment,the results showed that stemness genes expression of LCSCs and the CSCs surface markers CD133 and CD44 were up-regulated;PDHC and mitochondrial biogenesis related genes were also up-regulated;mitochondrial and ROS levels increased.After treatment of LCSCs with mitochondrial division inhibitor Mdivi-1,mitochondrial DNA(mtDNA)copy number and mitochondrial level in LCSCs were significantly inhibited;ROS levels were significantly down-regulated;stemness genes expression of LCSCs and CSCs surface markers CD133 and CD44 both were significantly reduced.The above results indicate that mitochondrial oxidative phosphorylation plays a decisive role in maintaining the stemness of LCSCs,and Mdivi-1 can reduce its stemness by inhibiting the level of oxidative phosphorylation of LCSCs.In summary,HCCLM3 and LCSCs have different phenotypes and metabolic characteristics.Mitochondrial oxidative phosphorylation plays a decisive role in the stemness maintaining of LCSCs,and its stemness characteristics can be down-regulated by inhibiting oxidative phosphorylation.The results of this study provide an experimental basis to better understanding the relationship between energy metabolism and the stemness of LCSCs,and provide new ideas and theoretical guidance for the treatment of liver cancer by clinically inducing liver cancer stem cell differentiation from a metabolic perspective.