Effect Of Hypoxia On Glycolysis Metabolism And Angiogenesis Of Liver Tumor After TACE Surgery

Objective:To explore the hypoxia status in tumor tissues after transarterial chemoembolization(TACE)treatment for liver tumor.To clarify the relationship between hypoxia and changes of glycolysis metabolism and tumor angiogenesis in liver tumors after TACE treatment.Methods:In this study,the human hepatoma cell line Hep G2 cells were used for in vitro experiments.Hep G2 cells were treated with hypoxia(1% O2)at different times(0 h,4 h,8 h,16 h,24 h)through a three-gas incubator.The proliferation of hepatoma cells was detected under hypoxia at different times by CCK-8 assay.The m RNA expression levels of hypoxia-related genes(ARNT,HIF1 A,EPAS1,HIF3 A,EGLN2,EGLN1 and EGLN3)and key molecules in glycolytic metabolism(SLC2A1,SLC2A3,SLC2A4,HK2,PFKL,PKM,PDK1,LDHA,SLC16A1 and SLC16A4)was detected by RT-q PCR after hypoxia at different times.The protein expression levels of Glut4,HK2,PFKL,PKM2,LDHA,PDK1,MCT1,MCT4,HIF-1α and PHD3 was detected by WB.Lactic acid production and glucose consumption in cell culture medium supernatant were determined by lactic acid and glucose assay kits,respectively.The expression level of VEGF protein in cell culture medium supernatant was detected by ELISA.One-way ANOVA was used for statistical analysis and Dunnett’s test was used for multiple comparisons.Based on the above in vitro experimental studies,Hep G2 cells were treated with hypoxia(1% O2)combined with pirarubicin for 24 hours for in vitro to mimic in vivo TACE treatment for liver tumor.Firstly,the effect of pirarubicin on the proliferation of Hep G2 cells was detected by CCK-8,and pirarubicin was selected for follow-up experiments on the IC50 concentration of Hep G2 cells.The cell experiments were divided into control group,pirarubicin group,hypoxia group,hypoxia combined with pirarubicin group.Protein expression levels of Glut1,HK2,PFKL,PKM2,LDHA,PDK1 and MCT1,HIF-1α and PHD3 was detected by WB.The expression level of VEGF protein in cell culture medium supernatant was detected by ELISA.One-way ANOVA was used for the group comparisons,and LSD test was used for the post-hoc comparisons.In vivo experiments,this study used New Zealand white rabbits to establish liver tumor model and then liver tumors were treated with TACE procedure.First,a VX2 liver allograft tumor model was established under ultrasound guidance.The rabbits VX2 liver tumor models after tumorigenesis were randomly divided into control group(n=10)and experimental group(n=10).Transfemoral approach hepatic arteriogram was performed within 2-3 weeks after implanting,the control group was perfused with normal saline,and the experimental group was injected with the mixed emulsion of lipiodol and pirorubicin via hepatic artery to the embolization endpoint.On the day before and 14 days after procedure: monitored rabbit’s weight.Rabbit blood samples were collected through the intermediate auricular artery and used to measure ALT and AST levels.And MRI imaging under anesthesia was detected to measure the size of rabbit tumors.All rabbits were sacrificed 14 days after procedure and collected tissue samples including the tumor and surrounding liver tissues.H&E staining was used to observe tissue cell morphology.Oil red O staining was used to observe lipiodol deposition in tumors.Immunofluorescence staining was used to observe the hypoxia state and protein levels of Glut4,HK2,PFKL,PKM2,LDHA,PDK1,MCT1,HIF-1,PHD3 and CD34.Western blot(WB)observes protein expression levels of Glut4,HK2,PFKL,PKM2,LDHA,and MCT1 in tumor tissues.The statistical analysis was performed using an independent sample t-test.Results:In vitro experimental studies,the increase of hypoxia times,the results of CCK-8indicated that the proliferative activity of Hep G2 cells increased.RT-q PCR test results show that the hypoxia-related genes ARNT,EPAS1 and HIF3 A showed an increasing trend,expression levels of EGLN1 and EGLN3 increased significantly,and HIF1 A and EGLN2 did not increase or decrease significantly.The m RNA expression levels of key molecules SLC2A1,SLC2A3,SLC2A4,HK2,PFKL,PKM,PDK1,LDHA,SLC16A1 and SLC16A4 in glycolytic metabolism gradually increased.The expression of Glut1,Glut4,HK2,PFKL,PKM2,LDHA,PDK1,MCT1 and MCT4 was detected by WB,and the results showed that the expression of the above molecules showed a gradual increase trend.The expression levels of HIF-1α and PHD3 proteins showed an increasing trend.The results of lactic acid determination showed that the lactic acid content in the supernatant gradually increased.Glucose assay kit results suggested that glucose consumption increased.The expression of VEGF in cell culture medium supernatant also gradually increased.The CCK-8 results showed that the IC50 of pirarubicin for 24 hours of treatment of Hep G2 cells was 0.63 umol/L,and 0.5 umol/L and 1.0 umol/L of pirarubicin were selected around the pirarubicin IC50 for subsequent experiments.It was divided into control group(cells without any treatment),pirarubicin 0.5 umol/L group,pirarubicin1.0 umol/L group,hypoxia 24 hours group,hypoxia 24 hours combined with pirarubicin 0.5 umol/L group,and hypoxia 24 hours combined with pirarubicin 1.0umol/L group.There was no significant difference between pirarubicin treatment and control group(all P > 0.05).The expressions of Glut1,HK2,PFKL,PKM2,PDK1,LDHA and MCT1 increased with hypoxia alone and hypoxia combined with pirarubicin,and there were statistically significant differences in Glut1,HK2,PFKL,PDK1,LDHA and MCT1(all P < 0.05),while no statistically significant differences in PKM2(P > 0.05).Glut1,HK2,PFKL,PKM2,PDK1,LDHA and MCT1 protein expressions were increased in hypoxia alone and hypoxia combined with pirarubicin group compared with pirarubicin alone group(1.0umol/L).Glut1,HK2,PFKL,PKM2 and PDK1 were significantly different(all P < 0.05),while LDHA and MCT1 were not significantly different(all P > 0.05).There was no significant difference between hypoxia alone and hypoxia combined with pirarubicin group(all P > 0.05).The results of WB detection of PHD3 and HIF-1α indicated that compared with the control group,there was no statistical difference in the expression of HIF-1α and PHD3 of pirarubicin alone(P > 0.05).The expression of PHD3 in 1.0 umol/L group was significantly different(P < 0.05).HIF-1α and PHD3 expressions were increased in hypoxia alone and hypoxia combined with pirarubicin(all P < 0.05).Compared with pirarubicin alone group 1.0 umol/L,the expressions of PHD3 and HIF-1α in hypoxia combined with pirarubicin group were significantly increased(all P < 0.05).Compared with hypoxia alone,there were no significant differences in the expressions of PHD3 and HIF-1α of hypoxia combined with pirarubicin(all P > 0.05).VEGF content in the supernatant of cell culture medium was detected by ELISA: compared with the control group,the expression of hypoxia alone and hypoxia combined with pirarubicin groups increased,and the difference was statistically significant(all P <0.05).Compared with pirarubicin alone group 1.0 umol /L,VEGF expression in hypoxia combined with pirarubicin group was significantly increased(all P < 0.05).There was no significant difference in VEGF expression between hypoxia group and hypoxia combined with pirarubicin group(P > 0.05).Twenty VX2 liver tumor model rabbits were successfully established under ultrasound guidance,and the modeling success rate was 100%.Modeling followed by hepatic arteriography was with a 100% success rate.Compared with the control group,the experimental group had decreased body weight,increased blood ALT and AST,and decreased tumor volumes.H&E staining observed a large number of tumor cell necrosis in the experimental group,but only a little tumor necrosis in the control group.Oil red O staining showed deposition of iodide oil in tumor vessels,and oil red O staining in tumor cells in experimental group was significantly more than that in control group.Hypoxic probe was used to detect hypoxia intra-and peri-tumor tissues and liver tissues.Compared with positive control group,hypoxia intra-and peri-tumor tissues increased.Immunohistochemical staining of HIF-1α and PHD3 indicated that the tumor tissue in the experimental group was significantly higher than that in the control group(all P < 0.05).WB detected Glut4,HK2,PFKL,PKM2,LDHA,PDK1 and MCT1 in glycolytic metabolism.The results indicated that the experimental group was significantly higher than the control group(all P < 0.05).Immunohistochemical staining showed that Glut4,HK2,PKM2,LDHA and MCT1 in the experimental group were higher than those in the control group(all P < 0.05).CD34 staining in the experimental group was significantly higher than that in the control group(all P < 0.05).Conclusions:1.Hypoxia at different time promoted the protein expressions of Glut1,HK2,PFKL,PKM2,LDHA,PDK1,and MCT1 in the glycolytic metabolism of hepatocellular carcinoma cells,enhanced the glucose uptake and extracellular lactic acid production of hepatocellular carcinoma cells,and promoted the expression of VEGF protein in the supernatant of cell medium.2.Hypoxia caused increased glycolytic metabolism and VEGF protein expression of hepatoma cells,which increased angiogenesis ability to a certain extent,which may be related to the induction of PHD3/HIF-1α/VEGF regulatory axis by hypoxia.3.TACE treatment of VX2 liver allograft tumor tissues hypoxia;Hypoxia may be associated with increased protein levels of Glut1,Glut4,HK2,PFKL,PKM2,LDHA,PDK1,MCT1 and MCT4 and increased microvascular density intra-and peritumor tissues after TACE procudure.