Effects And Mechanism Of Polychlorinated Biphenyls On Cervical Cancer Cell(HeLa) Survival

Polychlorinated diphenyls(PCBs),also known as chlorinated biphenyls,are compounds formed by the substitution of a chlorine atom for a hydrogen atom on a biphenyl.PCBs are widely used in the industry as hydraulic oils,solvents,plastics,paints and heat carriers.The compound has been identified as a contaminant for almost all components of the global ecosystem,including fish,wildlife and human adipose tissue,breast milk and serum,and its hazards have caused global concern.The most representative of PCBs are 3,3'4,4',5-pentachlorobiphenyl(PCB126),2,3',4,4',5-pentachlorobiphenyl(PCB118)or2,2',4,4',5,5'-hexachlorobiphenyl(PCB153).Both PCB126 and PCB118 are pentachlorobiphenyl,which are highly toxic and belong to coplanar,dioxins-like polychlorinated biphenyls.PCB153 is a non-dioxin(non-coplanar)congener.Cervical cancer is currently the most common gynecological malignant tumor.In recent years,the incidence rate has been rising.In our country,an estimated 98,900 cases of cervical cancer occur each year,accounting for about 20% of the total number of new cases worldwide,and nearly 30,500 women die of cervical cancer.Studies have shown that the exposure of PCBs is related to the pathogenesis of cervical cancer.Therefore,this study investigated the effects and mechanisms of PCBs exposure on cervical cancer cell survival and aerobic glycolysis.It will provide a theoretical basis for evaluating the correlation between PCB126,PCB118 or PCB153 and the risk of cervical cancer development.The research includes the following parts:Part one: Effects of exposure of PCB126,PCB118 or PCB153 on survival and aerobic glycolysis of cervical cancer cells.He La cells were treated with PCB126,PCB118 or PCB153 at concentrations of 0.01,0.1,1,10 and 100 n M for 24 or 48 h,and the survival of He La cells was detected by MTT assay.The results showed that treatment of PCB126,PCB118 or PCB153 at 0.01,0.1,1,10 and 100 n M for 24 h had no significant effect onthe survival rate of He La cells.After 48 h of PCB126,PCB118 or PCB153 treatment,PCB126 with a concentration of 0.1 and 1 n M,PCB118 with 1and 10 n M and PCB153 with 1 n M significantly improved the survival rate of He La cells.After exposure of cervical cancer cells with 1 n M PCB126,PCB118 or PCB153 for 48 h,glucose and lactic acid kits were used to detect the amount of glucose consumed in the culture medium and the amount of lactic acid produced were measured,and as a result,it was revealed that glucose in the medium was largely consumed and the amount of lactic acid produced was significantly increased.In addition,real-time fluorescent quantitative PCR was used to detect expression of key factors of aerobic glycolysis in cervical cancer cells.Such as: glucose transporter 1 GLUT1,lactate dehydrogenase LDHA and pyruvate dehydrogenase kinase PDK.The results showed that m RNA expression levels of GLUT1,LDHA and PDK were significantly increased.The above results indicated that exposure of PCB126,PCB118 or PCB153 can promote cervical cancer cells survival and aerobic glycolysis.Part two: Effects of PCB126,PCB118 or PCB153 on pyruvate kinase PKM2.Cervical cancer cells were treated with 1 n M PCB126,PCB118 or PCB153 for 48 h,respectively.The effects of PCB126,PCB118 or PCB153 on pyruvate kinase PKM2 in He La cells were detected by real-time fluorescent quantitative PCR and western blotting.The results indicated that PCB126,PCB118 or PCB153 exposure increased the m RNA and protein levels of PKM2 in He La cells.In addition,detection of pyruvate kinase activity revealed that PCB126,PCB118 or PCB153 exposure inhibited the enzymatic activity of PKM2.Part three: PCB126,PCB118 or PCB153 regulate PKM2-mediated aerobic glycolysis through oxidative stress to promote He La cells survival.He La cells were treated with 1 n M PCB126,PCB118 or PCB153 for 48 h,respectively,and ROS levels in He La cells were detected.The results showed that exposure of PCB126,PCB118 or PCB153 up-regulated thelevels of ROS in He La cells,and the m RNA expression levels of NADPH oxidase subunits NOX2,p40 phox and p47 phox which promote ROS were also significantly increased.In addition,when NAC was added,the contents of glucose and lactic acid in the medium were measured.The results showed that the glucose consumption and the increase of lactic acid production induced by PCBs were reversed,and the m RNA level of GLUT1,LDHA and PDK showed a downward trend.In addition,after treatment of He La cells with PCBs and antioxidant NAC for 48 h,the up-regulated PKM2 protein levels induced by PCB126,PCB118 or PCB153 were reversed.MTT assay also showed that PCB126,PCB118 or PCB153-induced He La cells survival was down-regulated after NAC addition.The above results indicated that PCB126,PCB118 or PCB153 promote the survival of cervical cancer cells by PKM2-mediated aerobic glycolysis process,which is mediated by oxidative stress.In summary,PCB126,PCB118 or PCB153 can promote the survival of cervical cancer cells by PKM2-mediated aerobic glycolysis,which is mediated by oxidative stress.These findings may be an important mechanism for PCBs promoting cervical cancer development.