The Difference Of DCA Sensitivity In Hepatocellular Carcinoma Cells

Objective:A variety of tumor cells,including liver cancer cells show "aerobic glycolysis" characteristics,that is,under aerobic conditions also rely on glycolysis to produce ATP,while mitochondrial oxidative phosphorylation(OXPHOS)function reduced,this Metabolic remodeling of tumor cells is called "Warburg e ffect".Recent studies have shown that drug resistance in tumor cells is associated with glucose metabolism remodeling,and different tumor cells have different metabolic phynotype,some studies suggest that metabolic changes in tumor cells may be associated with drug resistance.Pyruvate dehydrogenase kinase(PDK1),a key enzyme for glucose metabolism,is highly expressed in most tumor cells and has a poor prognosis with PDK1 overexpression.PDK1 phosphorylates pyruvate dehydrogenase(PDH)to inhibit its activity(because the inhibition of PDH is associated with "Warburg effect"),thus limiting the entry of pyruvate into mitochondrial,which is known as an important part of tumor cell metabolism reprogramming.More and more studies have shown that PDK1 plays an important role in tumor cell glucose metabolism.Dichloroacetate(DCA)is a small molecule,which enhances PDH activity by inhibiting PDK resulting in a metabolic flux from glycolysis to OXPHOS and thereby activate the apoptosis of mitochondrial pathway.Howe ver,not all tumor cells are sensitive to the effect of DCA,in our invetigation,we selected two hepatocellular carcinoma cells,we found that compared with HepG2 cells,BEL-7402 cells have significantly higher expression of PDK1 but not sensitive to DCA.,suggesting that the dependence of hepatocarcinoma cells on PDK1 is related to the resistance of tumor drugs,but the specific mechanism is not clearIn this study,we analyzed the differences of metabolic phynotype between HepG2 and BEL-7402 cells.At the same time by interfering with tumor cells glucose metabolism,induced the mitochondrial stress and increased cell drug sensitivity,to provide idea for individual clinical treatment.Methods:(1)MTT assay was used to detect the survival rate of HepG2 and BEL-7402 cells.Apoptosis was detected by flow cytometry.Apoptosis-related protein was detected by Western Blot.(2)Colorimetric method was used to determine the levels of glucose and lactic a in the culture medium of two hepatocellular carcinoma cells respectively.q PCR was used to detect the expression of glycolase related enzymes.The activity of LDH was detected by LDH activity kit.Flow cytometry was used to detect the contents of ROS and citrate in mitochondria was detected by Colorimetric method.Western Blot was used to detect the expression of glycolytic protein.(3)Colorimetric method was used to detect the changes of glucose uptake and lactic acid production in HepG2 and BEL-7402 cells.q PCR and Western Blot were used to detect the gene and protein expression of glycolysis.The activity of LDH was detected by LDH activity kit.Western Blot was used to detect the expression of hypoxia inducible factor HIF-1α.(4)Flow cytometry was used to detect the changes of ROS and membrane potential in mitochondria.The content of citrate in mitochondria was detected by Colorimetric method.Morphological changes of mitochondria were observed by laser confocal microscopy after Mito Tracker staining.(5)The sh-PDK1 plasmid was constructed according to the principle of RNAi and verified by Western Blot and PCR.sh-PDK1 inhibited the expression of PDK1,and the changes of ROS in mitochondria were observed by inverted fluorescence microscopy.The glucose uptake and lactic acid production were detected by spectrophotometry.The expression of glycolytic related proteins were detected by Western Blot.Results:(1)DCA could inhibit the survival of HepG2 cells in a dose-dependent manner and promote its apoptosis.BEL-7402 cells were significantly inhibited in DCA 80 m M,and the apoptosis was not obvious.(2)The levels of lactate and LDH in BLC-7402 cells were higher than those in HepG2 cells and BEL-7402 cells.The levels of lactate and LDH in BLC-7402 cells were also higher than those in HepG2 cells.Meanwhile,mitochondria ROS and citrate levels were lower than those of HepG2 cells.(3)DCA could inhibit the expression of protein and genes such as glucose uptake and lactate production,LDH activity and related factors such as HK2,LDHA,HIF-1α,GLUT1 and GLUT4 in HepG2 and BEL-7402 cells,and the inhibitory effect on HepG2 was more obvious.(4)DCA could induce the formation of ROS and citrate in the mitochondria of HepG2 cells,leading to the decrease of membrane potential and the change of BEL-7402 cells.At the same time,DCA could inhibit the mitochondrial fusion of HepG2 cells and fragment the mitochondria.The mitochondria of BEL-7402 cells were fragmented,and 80 m M DCA could make the mitochondria began to reticulate.(5)Construction of sh PDK1 recombinant plasmid and transfection of HepG2 and BEL-7402 cells,inhibition of PDK1 expression,can inhibit HepG2 glycolysis related genes and proteins expression,reduce extracellular liquid glucose and lactic acid production,and increase mitochondrial ROS production;BEL-7402 cells,inhibition of PDK1 expression can also reduce the expression of most of the glycolytic factors.Conclusions:(1)The sensitivity of two hepatocellular carcinoma cells to DCA may be related to the difference of metabolic phynotype induced by PDK1 dependent.(2)The glucose metabolic pattern of BEL-7402 cells was not significantly affected by DCA and they were less sensitive to DCA.(3)BEL-7402 cells are not sensitive to DCA and may be associated with a lower degree of dependence on PDK1 pathway.