Proteomics Analysis And Mitochondrial Metabolic Study In Hepatocellular Carcinoma Cells Surviving After Incubation With The Highest Dose Of Sorafenib

Hepatocellular carcinoma(HCC)is a common malignant tumor that seriously threatens human health worldwide,and its incidence and mortality are increasing.For patients with early HCC,tumor resection or liver transplantation remains the preferred treatment choice,although most patients diagnosed with advanced or metastatic disease do not qualify for curative treatments.Therefore,molecular targeted therapy is an important treatment option for HCC.Sorafenib is the first targeted drug that has been approved by the United States Food and Drug Administration(FDA)for use in HCC treatment since 2007,prolonging median patient survival by 2-3 months.And today,sorafenib is still recommended as the standard first-line systemic therapy for HCC patients with advanced disease according to the US National Comprehensive Cancer Network(NCCN)Clinical Practice Guidelines.However,the objective tumor response rates were only 2%to 3%in sorafenib-treated patients.It is supposed that a certain fraction of HCC cells that are initially insensitive to sorafenib treatment,may exist in patients.Therefore,the study of the molecular mechanisms of HCC cells surviving the first incubation with the highest dose of sorafenib is expected to be beneficial for more effective individualized therapy without the side-effects of sorafenib that are caused by increase in the dose alone.In this study,we aimed to explore the molecular mechanisms of HepG2cells surviving the first treatment with the highest dose of sorafenib.For this purpose,we generated low-insensitive(LI),middle-insensitive(MI)and high-insensitive(HI)hep G2 cells surviving long-term exposure(72 h)to low(IC₂₀),medium(IC₅₀),and high(IC₈₀)concentrations of sorafenib,respectively.Next,we used the Tandem Mass Tag(TMT)-based quantitative proteomics to describe the global intracellular protein changes in these surviving cells in order to search for the key molecules or signaling pathways that are associated with cells against sorafenib.Guided by proteomics results,the metabolic analysis of the mitochondrial function and glycolysis in 3 types of surviving cells were employed to further analyze the changes in the mitochondrial energy metabolism of HepG2 and huh7 cells treated with the highest dose of sorafenib to reveal the molecular mechanisms by which the hepatoma cells survive the initial treatment with the highest dose of sorafenib.Part?Proteomics study of the HepG2 cells that survive the initial treatment with the highest dose of sorafenibObjective:To establish a quantitative proteomics method for HepG2cells as well as to evaluate the molecular mechanisms of HepG2 cells surviving the first treatment with the highest dose of sorafenib.Methods:Proliferation assays of sorafenib on hep G2 cells was conducted and the IC₂₀,IC₅₀,and IC₈₀ values of sorafenib(the concentration that inhibited the cell growth by 20%,50%,and 80%,respectively)were calculated by using the SPSS software.The HepG2 cells were cultured with IC₂₀,IC₅₀,and IC₈₀ of sorafenib for 72 h.The HepG2 cells that survived the long-term incubation with 3 different concentrations of sorafenib were collected and are referred to as LI_72h,MI_72h and HI_72h HepG2 cells,respectively.TMT peptide-labeling coupled with the n LC-HRMS technique was employed to identify and quantify the proteomic changes and the change regularities in the 3 types of surviving cells.And then combined with bioinformatic methods,we investigated the survival mechanisms of the HepG2 cells that survived the first treatment with the highest dose of sorafenib as well as the key molecules and signaling pathways that were affected by sorafenib.Results:1.Effects of sorafenib on HepG2 cell proliferationSorafenib inhibited the HepG2 cell proliferation in a concentration-or time-dependent manner.After 72 h of incubation,the IC₂₀,IC₅₀,and IC₈₀values of sorafenib for HepG2 cells were 2.25,4.72,and 9.94?M,respectively.2.Influence of sorafenib on proteomics of HepG2 cellsBy using a TMT-based quantitative approach,a total of 4,100 proteins were identified and quantified in 3 types of surviving cells and control cells.Cluster analysis according to the patterns of the identified proteins was performed to identify the host factors targeted by sorafenib in the LI_72h,MI_72h,and HI_72h cells as well as the control cells.It was found that 520 proteins were significantly and concentration-dependently upregulated by sorafenib(P=3E^-61 and P=4E^-32).The bioinformatic analysis of 520 proteins was performed.Gene ontology(GO)enrichment analysis revealed that there were102 proteins involved in the most significantly enrichment term of“mitochondrion”(P=7.77E^-21);Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis indicated that the“metabolic pathways”were the most significantly enriched ones with the p-value of 1.24E^-7 and that it included 80 proteins that mainly participated in glycometabolism.The results of transcription factor(TF)prediction revealed that NRF1(P=1.79E^-19)and YY1(P=2.75E^-92),the canonical masterregulators of mitochondrial biogenesis,may take charge of the mitochondria protein expression response to sorafenib.Furthermore,protein-protein interaction analysis of 80 proteins in the metabolic pathways revealed that 33 proteins were associated with mitochondrial energy metabolism pathway.Especially,12 of them were more closely related to the mitochondrial respiratory chain and were involved in oxidative phosphorylation(OXPHOS).Brief summary:Our analysis revealed that the expression of 520 proteins was significantly and concentration-dependently upregulated by sorafenib in HepG2 cells.Among these,33 proteins with the highest reliability were mainly involved in the mitochondrial metabolic pathway,and 12 of these proteins were closely related to the mitochondrial OXPHOS.TFs NRF1and YY1 were found to regulate the expression of most sorafenib-related mitochondrial respiratory chain proteins.These changes may reflect the mechanism of HepG2 cells that survive the initial treatment with the highest dose of sorafenib.Part ? Mitochondrial metabolic study of HepG2 cells incubated with sorafenibObjective:Based on the proteomics outcomes,the metabolic changes in the mitochondrial respiratory function and glycolysis in HepG2 cells that survived the long-term treatment with sorafenib(IC₂₀,IC₅₀,and IC₈₀)were analyzed to reveal the changes in the energy metabolism of HepG2 cells that survived the exposure to the highest concentration of sorafenib and whether these changes were related to the drug dose.Methods:To understand the changes in the cellular mitochondrial functions and glycolysis in LI,MI,and HI HepG2 cells,the oxygen consumption rate(OCR)and the extracellular acidification rate(ECAR)were analyzed in 3 types of surviving cells at 48 h and 72 h.Meanwhile,to find the targets of sorafenib on the mitochondrial respiratory chain,the cultured HepG2 cells were employed to observe the effects of short-term treatment with sorafenib on the mitochondrial electron transport system(ETS)and the OXPHOS capacity by using high-resolution respirometry in the presence or absence of metabolic inhibitors.Results:1.OCR measurements in HepG2 cells surviving long-term treatment with sorafenibSorafenib(IC₂₀,IC₅₀,and IC₈₀)significantly inhibited the mitochondrial respiratory function,including the basal respiration,ATP production,maximal respiration,and spare respiratory capacity in a concentration-dependent manner in LI,MI,and HI HepG2 cells when compared with those in the control cells,with the maximal in hibition by 93.5%,100%,97.6%,and 99.8%,respectively,in HI_72h HepG2 cells(P<0.01).Moreover,the inhibition of the basal respiration by sorafenib was found to be time-dependent in LI,MI,and HI HepG2 cells at 72 h when compared to 48 h(P<0.01).2.ECAR measurements in HepG2 cells surviving the long-term treatment with sorafenibSorafenib concentration-dependently inhibited glycolysis and the glycolytic capacity of LI,MI,and HI HepG2 cells when compared with those in the control cells with the maximal inhibition by 67.6%and 77.4%,respectively(P<0.01).However,no significant difference was noted in the glycolysis and glycolytic capacity between the HI_48h and HI_72h HepG2 cells(P>0.05).3.Cellular energy phenotype analysis of HepG2 cells surviving the long-term treatment with sorafenibThe basal OCR/ECAR ratios of HI_72h HepG2 cells significantly decreased when compared with those of the control cells(0.35 vs 1.44,P<0.01),suggesting that the surviving HepG2 cells underwent a metabolic phenotype switch from the mitochondrial OXPHOS to glycolysis in order to maintain their energy requirements in the presence of the highest-dose sorafenib treatment.4.Effects of short-term treatment with sorafenib on the mitochondrial respiratory systemSorafenib at three different concentrations(corresponding to IC₂₀,IC₅₀,and IC₈₀)inhibited the complex I(CI)OXPHOS capacity and CI ETS capacity in a concentration-dependent manner with the maximal inhibition by 59.7%and 79.2%,respectively(P<0.01).Sorafenib inhibited CI&CII OXPHOS capacity with the maximal inhibition by 20.1%(P<0.05).Sorafenib significantly enhanced the leak respiration and CI leak,and the maximal enhancement was 214.9%and 399.8%(P<0.01).5.Effects of sorafenib in combination with a glycolysis inhibitor on HepG2 cell proliferationA treatment with 0.1 m M 2-DG(2-deoxy-D-glucose)alone did not affect HepG2 cell proliferation(P>0.05),but its combination with IC₂₀ sorafenib or IC₈₀ sorafenib resulted in a significant inhibition of the cell proliferation by20.1%or 35.1%when compared with the single treatment with IC₂₀ sorafenib or IC₈₀ sorafenib(P<0.01).6 Effects of sorafenib in combination with a CII inhibitor on HepG2 cell proliferationA treatment with 0.5 m M or 5 m M malonic acid alone could significantly influence the HepG2 cell proliferation by 9.4%or 17.8%(P<0.05 and P<0.01).In addition,5 m M malonic acid combination with IC₅₀ sorafenib or IC₈₀sorafenib resulted in a significant inhibition of the cell proliferation by 50.5%or 85.9%when compared with that in the control;this combination resulted in a significant inhibition of the cell proliferation by 12.8%or 38.3%when compared with that by the single treatment with IC₅₀ sorafenib or IC₈₀sorafenib(P<0.05).However,the 0.5 m M or 1 m M malonic acid combination with sorafenib did not show any synergistic effect when compared with that by the single treatment with sorafenib(P>0.05).Brief summary:At clinically relevant concentrations sorafenib significantly inhibited mitochondrial respiratory function and glycolysis of HepG2 cells,preferentially and selectively inhibited mitochondrial CI of HepG2 cells,and caused an uncoupling of oxidative phosphorylation.The majority of HepG2cells had strong potential of mitochondrial OXHPOS,which would be fully utilized to maintain cell survival when exposed to low concentration(IC₂₀)of sorafenib.A few HepG2 cells had an ability to change their energy metabolism phenotype from mitochondrial OXPHOS to glycolysis to maintain energy supply,and this mechanism might be related to the escape and recurrence of liver cancer during sorafenib chemotherapy.Part ? Mitochondrial metabolic study in Huh7 cells incubated with sorafenibObjective:The Huh7 cells were cultured in sorafenib(IC₂₀,IC₅₀,and IC₈₀)to analyze the metabolic changes in the mitochondrial respiratory function and glycolysis in the surviving cells under long-term treatment with sorafenib as well as to compare the effects of different concentrations of sorafenib on the HepG2 cells and Huh7 cells.Methods:Proliferation assays of sorafenib on Huh7 cells was determined,followed by the determination of the IC₂₀,IC₅₀,and IC₈₀ values of sorafenib that could inhibit cell growth by 20%,50%,and 80%,respectively,which were calculated by the SPSS software analysis.After long-term treatment with sorafenib(IC₂₀,IC₅₀,and IC₈₀),the OCR and ECAR were analyzed in LI,MI,and HI Huh7 cells to comprehend the changes in the cellular mitochondrial function and glycolysis in these surviving cells at 48 h and 72 h.The CCK-8assay was performed to investigate the cell viability of a combination of 2-DG and long-term treatment with sorafenib in Huh7 cells.Results:1.Effects of sorafenib on Huh7 cell proliferationSorafenib inhibited the Huh7 cell proliferation in a concentration-or time-dependent manner.After 72 h of incubation,the IC₂₀,IC₅₀,and IC₈₀values of sorafenib for Huh7 cells were found to be 1.50,3.38,and 6.50?M,respectively.2.OCR measurements in Huh7 cells surviving long-term treatment with sorafenibSorafenib(IC₂₀,IC₅₀and IC₈₀)significantly inhibited mitochondrial respiratory function,including the basal respiration,ATP production,maximal respiration,and spare respiratory capacity in a concentration-dependent manner in LI,MI,and HI Huh7 cells when compared with those in the control cells with the maximal inhibition by 77.4%,94.6%,91.7%,and 100%,respectively(P<0.01).Moreover,the inhibition of basal respiration and maximal respiration by sorafenib was found to be time-dependent in the HI Huh7 cells at 72 h when compared to that at 48 h(P<0.01).3.ECAR measurements in Huh7 cells surviving long-term treatment with sorafenibSorafenib concentration-dependently inhibited glycolysis and the glycolytic capacity in LI,MI,and HI Huh7 cells when compared with that in the control cells,with the maximal inhibition by 38.6%and 38.8%,respectively,in the HI Huh7 cells at 72 h(P<0.01).However,no significant difference was noted in glycolysis and glycolytic capacity between the HI_48hand HI_72h Huh7 cells(P>0.05).4.Effects of different concentrations of sorafenib on HepG2 and Huh7cellsThe basal OCR/ECARratios of the control Huh7 cells(1.58±0.07)was significantly higher than that of the control HepG2 cells(1.44±0.03).In contrast to LI_72h or MI_72h HepG2 cells,the basal OCR/ECAR ratios of Huh7cells that survived the corresponding treatment with sorafenib that significantly decreased.Especially,when HCC cells were incubated with a high concentration of sorafenib(IC₈₀)for 72 h,the basal OCR/ECAR of HI Huh7 cells was 0.58±0.05,which was significantly higher than that of HI HepG2 cells(0.35±0.15),indicating significant differences in the metabolic response to sorafenib between the two cell lines.5.Effects of sorafenib in combination with a glycolysis inhibitor 2-DG on Huh7 cell proliferationA treatment with 0.1 m M 2-DG alone did not affect Huh7 cell proliferation(P>0.05),but its combination with IC₂₀ sorafenib or IC₈₀sorafenib resulted in a significant inhibition of the cell proliferation by 14.3%or 22.4%when compared with the single treatment with IC₂₀ sorafenib or IC₈₀sorafenib(P<0.01).Brief summary:The energy metabolism phenotype of Huh7 cells was found to be more dependent on the mitochondrial OXPHOS in comparison to that of HepG2cells.The inhibitory effects of sorafenib on the mitochondrial respiratory function and glycolysis in Huh7 cells were weaker than that in HepG2 cells,especially on glycolysis.The combination of sorafenib and a glycolysis inhibitor 2-DG displayed a synergistic anti-cancer effect on the human HCC cell lines(HepG2 and Huh7cells)and significantly reduced hepatoma cells survival from sorafenib treatment.Conclusions:The expression of 520 proteins was significantly and concentration-dependently upregulated in HepG2 cells treated with long-term incubation of sorafenib at clinically relevant concentrations.Among these,33proteins with the highest reliability were mainly involved in the mitochondrial metabolic pathway,and 12 proteins of these pathway were closely related to mitochondrial OXPHOS.TFs NRF1and YY1 regulated the expression of most sorafenib-related mitochondrial respiratory chain proteins.Sorafenib significantly inhibited mitochondrial respiratory function and glycolysis of HepG2 cells,preferentially and selectively inhibited the mitochondrial CI of HepG2 cells,and caused an uncoupling of oxidative phosphorylation.The majority of HepG2 cells had strong potential of mitochondrial OXHPOS,which would be fully utilized to maintain cell survival when exposed to a low concentration of sorafenib.A few HepG2 cells had an ability to change their energy metabolism phenotype from mitochondrial OXPHOS to glycolysis to maintain energy supply.Energy metabolism phenotype of Huh7 cells was more dependent on mitochondrial OXPHOS in comparison to HepG2 cells.The inhibitory effects of sorafenib on the mitochondrial respiratory function and glycolysis in Huh7 cells were weaker than that in HepG2 cells,especially on glycolysis.The combination of sorafenib and a glycolysis inhibitor,2-DG,displayed a synergistic anti-cancer effect on human HCC cell lines(both HepG2 and Huh7cells)and significantly reduced hepatoma cells escape from sorafenib treatment.