Appropriate Concentration Of Copper Inhibits Apoptosis Of Hepatoma Cells By Activating Mitophagy

Objective:Copper is a heavy metal commonly used in industry.Our country’s copper consumption has always been ranking the first in the world.Environmental copper pollution is becoming more and more serious.Recent studies have shown that the level of copper in tumor tissues and serum in patients with hepatocellular carcinoma（HCC）has increased,and copper may be closely related to the development of HCC,but the details of the mechanism are not yet available.Autophagy helps to cope with the pressure in tumor cells and tumor microenvironment.Moderate mitophagy can clear the damaged mitochondria,maintain the survival and growth of tumor cells,and promote the progression of HCC.With the help of serum starvation model and nude mice subcutaneous xenograft tumor model,this study explored the regulation of copper on mitophagy and further explored the molecular mechanism of copper promoting the development of HCC,to provide etiological basis for preventing the deterioration of HCC patients,slowing the progression of HCC and effectively reducing the burden of HCC.Methods:1.To explore the effect of appropriate concentration of Cu²⁺on proliferation and apoptosis of HepG2 cells.In order to clarify the effect of Cu²⁺on the proliferation and apoptosis of hepatoma cells,Cell Counting Kit-8（CCK8）reagent was used to detect the cell viability of HepG2cells under different concentrations of Cu²⁺exposure at different time points.The mRNA expression of cell proliferation-related indicators was detected by q RT-PCR after 96h treatment with different concentrations of Cu²⁺,including Ki67,proliferating cell nuclear antigen（PCNA）and cellular myelocytomatosis oncogene（C-MYC）.The protein expression levels of Ki67 and Cleaved-caspase3 were detected by Western Blot.The apoptosis level of HepG2 cells was determined by Annexin V-FITC/PI double staining after 96h treatment with different concentrations of Cu²⁺.2.Serum starvation method was used to investigate the mechanism of optimal concentration of Cu²⁺exposure inhibiting HepG2 cell apoptosis.1)Serum starvation method was used to simulate the tumor microenvironment of nutrient deficiency,and CCK8 reagent was used to detect the viability of HepG2 cells exposed to serum starvation combined with different concentrations of Cu²⁺for different times.The following experiments were conducted after serum starvation combined with exposure of different concentrations of Cu²⁺for 18h.The mRNA expression level of Ki67was detected by q RT-PCR.Mitochondrial transmembrane potential was detected by JC-1flow cytometry.The protein expression levels of Bcl-2,BAX,Apaf1,Cleaved-caspase9and Cleaved-caspase3 were detected by Western Blot.Mitochondrial superoxide indicator（Mito SOX）was used to measure mitochondrial reactive oxygen species（mt ROS）levels.Malondialdehyde（MDA）detection reagent was used to determine the content of MDA in hepatoma cells.2)Aiming at the mitophagy pathway,Kaplan-Meier Plotter database was used to analyze the relationship between the expression level of FUN14 domain protein 1（FUNDC1）and the survival of liver cancer patients.After serum starvation combined with different concentrations of Cu²⁺exposure for 18h,Western Blot was used to detect the protein expression levels of ULK1,p-S17-FUNDC1 and LC3-Ⅱ,and the expression levels of TIMM23,p-S17-FUNDC1 and LC3-Ⅱafter pretreatment with autophagy inhibitor Baf-A1.Transmission electron microscopy was used to detect the number of autolysosomes,and fluorescence staining was used to detect the colocalization of mitochondria and lysosome to verify the autophagic flux.3)HepG2 cells were transfected with sh-FUNDC1 plasmid to construct FUNDC1knockdown model.Under serum starvation condition,the expression levels of mitophagy pathway and mitochondrial apoptosis pathway related proteins in sh-NC group,sh-FUNDC1 group,sh-FUNDC1+Cu²⁺group and sh-FUNDC1+TEMPO group were detected by Western Blot.TUNEL fluorescence staining was used to detect the apoptosis level of the four groups and the Baf-A1 pretreatment group after transfection with sh-NC plasmid（sh-NC+Baf-A1）,and Mito SOX fluorescence staining was used to detect the mt ROS level.3.Hep3B human hepatocellular carcinoma cell subcutaneous transplantation tumor model in nude mice was used for verification.Human Hep3B hepatoma cells were used to construct nude mice subcutaneous xenograft tumor model.After tumor formation,they were divided into Control group（Control）and free drinking water exposed to 35μM of Cu²⁺group(Cu²⁺（35μM）)according to tumor volume and body weight.The tumor volume was recorded during the experiment.The experiment was terminated 21 days later,and the tumors and organs were stripped and weighed.The maximum tumor volume was no more than 2000 mm³.Body weight and tumor volume were recorded every 3 days during this period.Expression levels of Ki67 and Cleaved-caspase3 were determined by immunohistochemistry.TUNEL fluorescence staining was used to detect apoptosis of tumor cells.ROS fluorescence staining was used to detect ROS levels.Mitochondrial autophagosomes in tumor tissue were observed by transmission electron microscopy.The expression levels of mitophagy and mitochondrial apoptosis pathways were detected by Western Blot.Results:1.Appropriate concentration of Cu²⁺exposure could inhibit the apoptosis of HepG2 cells.35μM of Cu²⁺treatment for 96h showed increased viability of HepG2 cells,but the mRNA expression levels of proliferation-related indicators Ki67,PCNA and C-MYC were down-regulated,and the protein expression level of Ki67 was also decreased,indicating that the increased viability of cells was not caused by promoting proliferation.Instead,apoptosis-related indicators were detected.At 35μM,the apoptosis rate of HepG2 cells was decreased by flow cytometry,and Cleaved-caspase3 protein expression was down-regulated,suggesting that the increased cell viability may be related to apoptosis inhibition.2.The appropriate concentration of Cu²⁺could upregulate the mitophagy pathway to clear excessive mt ROS,thus inhibiting the apoptosis of HepG2 cells.1)Serum starvation combined with 100μM of Cu²⁺exposure for 18 h increased cell viability of HepG2 cells.At this time point,there was no significant difference in the mRNA expression level of Ki67 among different treatment groups.Serum starvation increased the loss of mitochondrial transmembrane potential,upregulated the expression levels of pro-apoptotic proteins of Apaf1,Cleaved-caspase9 and Cleaved-caspase3,and increased the contents of mt ROS and MDA in HepG2 cells.Cu²⁺at 100μM significantly reduced the loss of mitochondrial transmembrane potential,decreased the contents of mt ROS and MDA in cells,and downregulated the protein expression of BAX,Apaf1,Cleaved-caspase9 and Cleaved-caspase3.2)Kaplan-Meier Plotter database analysis showed that HCC patients with higher FUNDC1 expression level had shorter OS and DSS.Serum starvation down-regulated ULK1 protein expression,but ULK1–p-S17-FUNDC1–LC3-Ⅱmitophagy pathway was still expressed to a certain extent.Cu²⁺could up-regulate the expression of ULK1 in a dose-dependent manner,and activate the pathway at 75 and 100μM,and the expression level of p-S17-FUNDC1 increased.After pretreatment with Baf-A1,the protein expressions of TIMM23,p-S17-FUNDC1 and LC3-Ⅱincreased compared with those without Baf-A1,and the expressions of p-S17-FUNDC1 and LC3-Ⅱwere increased more significantly in the serum starvation combined with 100μM Cu²⁺treatment group.It was proved that the autophagic flow was unimpeded and Cu²⁺could upregulate the ULK1–p-S17-FUNDC1–LC3-Ⅱmitophagy pathway.Under electron microscope,serum starvation combined with 100μM of Cu²⁺treatment resulted in the increase in the number of autolysosomes.Mitochondrial and lysosome fluorescence staining colocalization was also significantly increased,further demonstrating the integrity of autophagy flow.3)Compared with the sh-NC group,protein expression level of p-S17-FUNDC1 was decreased,and expression levels of pro-apoptotic proteins BAX and Cleaved-caspase3were higher in the sh-FUNDC1 group.Compared with sh-FUNDC1 group,sh-FUNDC1+Cu²⁺group significantly up-regulated the expression of ULK1–p-S17-FUNDC1–LC3-Ⅱpathway,while sh-FUNDC1+TEMPO group had no significant effect on the mitophagy pathway.Both Cu²⁺and TEMPO decreased the expressions of pro-apoptotic proteins of BAX and Cleaved-caspase3.The effect of mitophagy up-regulation caused by Cu²⁺played the same role as mitochondria-specific antioxidant TEMPO.TUNEL fluorescence results showed that the apoptosis level was significantly increased after the inhibition of autophagy by Baf-A1.The apoptosis level of sh-FUNDC1 group was increased but not as high as that of sh-NC+Baf-A1 group,while the apoptosis level was significantly decreased by Cu²⁺and TEMPO.The change trend of mt ROS was consistent with that of apoptosis.3.35μM Cu²⁺could upregulate the mitophagy pathway and reduce the apoptosis of tumor tissue cells.Compared with Contral group,the tumor volume and weight of Cu²⁺（35μM）group were increased,and the tumor weight/body weight showed an increasing trend.The body weight and organ index of Cu²⁺（35μM）group were not significantly different,suggesting that the dose of 35μM was a tolerable non-toxic dose for nude mice.There was no significant difference in the expression of Ki67 between the two groups,but the expression level of Cleaved-caspase3 was decreased in Cu²⁺（35μM）group,and the results of TUNEL staining were consistent with that of Cu²⁺（35μM）group.The content of ROS was decreased but not statistically significant.Under electron microscope,mitochondrial autophagosomes were observed in both groups,and lysosomes in Cu²⁺（35μM）group appeared near the autophagosomes.The expression of the ULK1–p-S17-FUNDC1–LC3-Ⅱpathway was increased and the expression of pro-apoptotic proteins of BAX and Cleaved-caspase3 was downregulated in Cu²⁺（35μM）group.Conclusion:1.Appropriate concentration of copper could inhibit the apoptosis of hepatoma cells in the condition of nutrient deficiency,but not promote the proliferation of hepatoma cells,showing the phenomenon of increased cell vitality.2.Serum starvation could cause mitochondrial damage,increase the content of the intracellular mt ROS,and induce apoptosis of hepatoma cells through mitochondrial apoptosis pathway.3.Appropriate concentration of copper could upregulate mitophagy mediated by ULK1–p-S17-FUNDC1–LC3-Ⅱpathway,clear damaged mitochondria and reduce intracellular mt ROS level,so as to resist the endogenous apoptosis of hepatoma cells induced by serum starvation.