Study On The Mechanism Of Liver Cancer Cells Apoptosis Differentially Induced By Shikonin Through PKM2/HIF1α Pathway

Backgroud:Liver cancer is one of the most common malignant tumors,and the choice of treatment options are limited for advanced stage of liver cancer.Systemic chemotherapy is often the first choice for patients’treatment of advanced liver cancer.However,due to the drug resistance mechanisms,the treatment efficacy is not satisfaction.Therefore,it is urgent to find new targets to improve the therapeutic efficacy and improve the prognosis of patients.Glucose metabolic reprogramming has gradually become one of the hallmarks of liver cancer.With the proposal of"Warburg effect",aerobic glycolysis has been widely concerned,especially in liver cancer,in which the enhancement of aerobic glycolysis directly leads to a variety of malignant phenotypes such as tumor cell proliferation,drug resistance and metastasis.Targeting energy metabolism,especially glucose metabolism,may become a promising therapeutic treatment in liver cancer.PKM2 is the key enzyme of glycolysis,and exists in various forms,such as monomers,dimers and tetramers.The tetramer of PKM2 is of high enzyme activity in cytoplasm to catalyze glycolysis,while dimer of PKM2,the enzyme activity of which is low but can be located to the nucleus,playing the role of activating transcription activity of various proteins including HIF1α,which participates in glucose metabolism regulation.The transformation between two forms of PKM2 is crucial for activating glycolysis,promoting macromolecular biogenesis and maintaining cell proliferation.More and more attention has been paid to the role of PKM2 in tumor cells,and inhibitors targeting PKM2 are also emerging.Shikonin has been proved to be an inhibitor of PKM2 in a variety of cancer cells and can specifically inhibit its catalytic activity and glycolysis.Although the"Warburg effect"has been widely accepted,with the progress of research,it has been gradually found that mitochondrial oxidative metabolism is of great importance for the survival of tumor cells.It has been reported that tumor cell metabolism often exhibits a glycolysis/OXPHOS mixed metabolic phenotype.This kind of mixed phenotype is more plastic,and it is easier to form an optimal metabolic balance state in response to external stimuli,which is helpful for tumor cell proliferation,metastasis and chemotherapy resistance.However,the conversion between glycolysis and mitochondrial oxidative metabolism is still a field worth exploring.It has been found in liver cancer that the circadian rhythm gene Neuronal PAS domain protein 2(NPAS2)can target both HIF1αand PGC1α,upregulate glycolysis while inhibiting PGC1α-mediated mitochondrial biogenesis and oxidative phosphorylation,and promote glucose metabolic reprogramming.Overexpression of HIF1αcan reverse the upregulation of PGC1αtranscription mediated by NPAS2knockdown,suggesting that there may be a regulatory relationship between HIF1αand PGC1αat the transcription level,and mediate the conversion between glycolysis and mitochondrial oxidative phosphorylation.In this study,shikonin was used as a tool to propose the mechanism of shikonin regulating metabolic pathway through PKM2 and HIF1αexpression in the nucleus,which may explain the mechanism of difference in sensitivity of liver cancer to shikonin.At the same time,these results provide more theoretical basis for targeting glycolytic enzymes and improving the clinical treatment of liver cancer.Objective:In this study,shikonin was used as a tool to detect two kinds of liver cancer cells with different sensitivity to shikonin.By observing the expression of mitochondrial related genes and analyzing the subcellular localization of PKM2/HIF1α,the mechanism of apoptosis induced by glucose metabolism recombination induced by PKM2/HIF1αpathway was explored,which provides a new idea for the treatment of liver cancer.Methods:Two kinds of liver cancer cells Hep G2 and HCCLM3 were selected in this study,in which Hep G2 was more sensitive to shikonin.Based on the comparison of glucose metabolism phenotypes of two kinds of cells,the relationship between the differential regulation of glucose metabolism and sensitivity to shikonin in two kinds of cells treated with shikonin was explored,and the following experiments were conducted:1.Hep G2,BEL-7402,SMMC-7721 and HCCLM3 cells were treated with shikonin at different concentrations.MTT assay was used to compare the activity of these cells.A suitable concentration of 15μM(the half maximal inhibitory concentration for Hep G2 cell)was selected for subsequent experiments.2.To compare the metabolism phenotype of Hep G2 and HCCLM3 cells,q PCR experiment was used to detect the m RNA expression levels of mitochondrial biogenesis,mitochondrial oxidative phosphorylation and glycolysis-related genes;Western Blot assay was applied to evaluate the expression levels of oxidative phosphorylation and glycolysis-related proteins;the ATP level and extracellular oxygen consumption rate were detected by a luminometer;ROS was used to evaluate the cell oxidation level by the flow cytometry;3.Two kinds of liver cancer cells Hep G2 and HCCLM3 were treated with shikonin 15μM for 6 h,12 h or 24 h.Flow cytometry was used to analyze the level of cell apoptosis and MMP,the expression of apoptosis-related proteins was detected by Western Blot assay.ROS level was determined with DCFH-DA by flow cytometry.4.Hep G2 and HCCLM3 cells were treated with shikonin 15μM for different time.NAD~+/NADH,ATP levels and extracellular oxygen consumption rate were detected by CLARIOstar fluorescence plate reader.Lactate content in the culture medium was detected to evaluate cell glycolysis level.Expression levels of genes and proteins related to mitochondrial biogenesis were detected by q PCR and Western Blot assay.5.Hep G2 and HCCLM3 cells were treated with shikonin 15μM for different time,the protein expression and subcellular localization of PKM2 and HIF1αwere detected by Western Blot and immunofluorescence staining,and the expression of HIF1αdownstream target gene PDK1 was detected by q PCR.6.After treated with shikonin 15μM and ROS inhibitor N-acetylcysteine(N-acetylcysteine)10m M,the level of apoptosis and mitochondrial membrane potential were detected by flow cytometry,and the level of ATP was detected by CLARIOstar fluorescence plate reader.Results:1.By comparison,ROS levels of Hep G2 cells was higher than HCCLM3.Gene expression levels involved in mitochondrial biogenesis,and mitochondrial OXPHOS levels were higher(including higher oxygen consumption and higher complex protein expression).However,PKM2,the critical enzyme of glycolysis and ATP content in HCCLM3 cells were higher,and antioxidant level was higher(NAD~+was more abundant).2.After shikonin treatment,apoptosis rate and expression of apoptosis-related proteins were higher in Hep G2 cells than that in HCCLM3 cells.3.ROS level was increased,and mitochondrial membrane potential and oxygen consumption rate were decreased in Hep G2 cells after shikonin treatment.However,shikonin induced the increase of ROS and a decrease in mitochondrial membrane potential in HCCLM3 cells.4.The expression of proteins related to the mitochondrial biogenesis pathway was upregulated at 6 h and 12 h but down-regulated at 24 h in Hep G2 cells after shikonin treatment.However,the expression of proteins related to the mitochondrial biogenesis pathway of HCCLM3 was down-regulated after shikonin treatment.5.The expression of PKM2/HIF1αprotein in nucleus,the expression of HIF1αdownstream target gene PDK1 m RNA level,PKM2 protein level and lactate production were increased in HCCLM3 cells after shikonin treatment.The nucleus expression of PKM2/HIF1αand lactate production were decreased in Hep G2 cells.Conclusions:1.There was a difference in the level of glucose metabolism between Hep G2and HCCLM3 cells.Oxidative phosphorylation level was higher in Hep G2 cells and glycolysis level was higher in HCCLM3 cells.2.Hep G2 cells were more sensitive to shikonin than HCCLM3 cells.3.High level of ROS was induced by shikonin in Hep G2 cells,which damaged the mitochondrial function and activated the mitochondrial biogenesis pathway.However,the oxidative damage was not alleviated but aggravated continuously.At the same time,the production of lactate,the level of glycolysis and the level of ATP were decreased.4.The nuclear expression of PKM2/HIF1αwas increased,activated the process of glycolysis,down-regulated mitochondrial biogenesis and reprogrammed glucose metabolism in HCCLM3 cells after shikonin treatment,which was an important reason for maintaining ROS levels and energy.In this study,shikonin was used as a tool to explore the effects of shikonin on glucose metabolism in two kinds of liver cancer cells with different glucose metabolism phenotypes.The glycolysis level was higher in HCCLM3 cells,in which the expression level of glycolysis-related genes was increased through PKM2 and HIF1αprotein expression in nucleus,and on the other hand,mitochondrial biogenesis was down-regulated to reprogram glucose metabolism after shikonin treatment.Fundamentally,the difference may be related to the basic expression levels of PKM2/HIF1αand PGC1αin liver cancer cells.The regulation of metabolism by nuclear expression of PKM2 and HIF1αin liver cancer cells may provide a basis for explaining the difference in sensitivity of liver cancer cells to shikonin.These results may provide more possibilities for improving the clinical therapeutic effect of liver cancer and enhancing the chemosensitivity of liver cancer by targeting glycolytic enzyme.The combination of PKM2/HIF1αand PGC1αas a marker may provide a theoretical basis for determining the metabolic type and malignant degree of liver cancer.