The Effects And Mechanism Of PCB118 On Liver Cancer Cell Proliferation And Adhesion

Polychlorinated biphenyls(PCBs)is a kind of persistent organic pollutants.PCB118,one of the PCBs congeners,is the most typical poly biphenyl.PCB118 is a direct indicator of PCBs used in environmental assessment.At present,PCB118 has been detected in the rivers,plants and fish,even in the human breast milk.Liver cancer is one of malignant tumors which severly impair human health.The incidence of liver cancer is increasing every year.Researches have found that PCBs can lead to the occurrence of liver cancer in mammals,but its mechanism is not still clear.The aim of this study was to explore the effects of PCB 118 exposure on the proliferation and adhesion of human hepatocellular carcinoma cells and involved molecular mechanisms.The research contents mainly include the following parts:Part one:After SMMC-7721 cells were treated with different concentrations of PCB118(10-12～10-6M)for 4 days,MTT assay and Trypan blue staining cell counts method were used to detect cell viability and proliferation.The results showed that PCB118(10-10～10-8M)could significantly promote the proliferation of hepatocellular carcinoma cells,and the numbers of cell clones were also increased after PCB118 treatment for 6 days.We found that medium glucose consumption and lactate production were significantly increased after PCB118(10-10,10-9 and 10-8M)treatment for 4 days.Meanwhile,the expression of key factors in Warburg effect,such as hexokinase HK2,pyruvate dehydrogenase kinase PDK,glucose transporter GLUT1 and lactate dehydrogenase LDHA,were up regulated.These results suggested that PCB 118 exposure promotes the Warburg effect of hepatocellular carcinoma cells.Furtherly,we found that the expression of pyruvate kinase M2(PKM2),the key enzyme of Warburg effect,was up-regulated,and its nuclear translocation was occurred.After using RNA interference to knock down PKM2,we found that PCB118 induced-Warburg effect and cell proliferation were inhibited.These results revealed that PCB118 promotes hepatocellular carcinoma cell proliferation by Warburg effect mediated by PKM2.Part two:After treatments with 10-10,10-9 and 10-8 M PCB118 for 4 days,the oxidative stress levels of hepatoma cells SMMC-7721 were detected.The results indicated that PCB118 exposure led to elevated ROS levels along with decreased SOD activity and GSH contents.We also found that NADPH oxidase subunits Noxl,Nox2,p22phox,p40phox,p47phox and p67phox expression levels were up regulated.To further demonstrate the role of oxidative stress in Warburg effect and cell proliferation induced by PCB118,we used antioxidants to antagonize oxidative stress induced by PCB118 and then detected the influence of PCB118 on the Warburg effect and cell proliferation.The results showed that the addition of antioxidants significantly inhibited the up regulation of PKM2 expression induced by PCB 118.Consistent with this,Warburg effect and cell proliferation induced by PCB118 were significantly inhibited.These results revealed that PCB118 regulates the Warburg effect by oxidative stress,thus contributing to the proliferation of hepatoma cell SMMC-7721.Part three:The mRNA expression of the aryl hydrocarbon receptor AhR was up regulated after PCB118(10-10,10-9 and 10-8M)treatment.To further demonstrate the role of AhR in the Warburg effect and cell proliferation induced by PCB 118,the RNA interference technique was used to knock down the AhR.After AhR knockdown,the mRNA and protein expressions of PKM2 induced by PCB 118 were significantly decreased.Consistent with this,PCB118 induced-Warburg effect and cell proliferation was obviously inhibited.These results suggested that PCB118 could adjust the Warburg effect through the aryl hydrocarbon receptor pathway,thus contributing to the proliferation of hepatoma cells SMMC-7721.Part four:In order to explore the effect and mechanism of PCB 118 on human hepatoma cell adhesion.BEL-7402 cells were exposed to PCB118(10-11～10-7M)for 4 and 6 days respectively.Then the cell-matrix adhesion assay and cell aggregation experiments were used to study the effects of PCB118 on cell-matrix adhesion and cell-cell adhesion in BEL-7402 cells.The results showed that the cell-matrix adhesion ability of human hepatocellular carcinoma cell line BEL-7402 was significantly increased and cell-cell adhesion ability was significantly reduced after treatment with 10’10,10-9 and 10-8M PCB118 for 6 days.Meanwhile,the CD29 and N-cadherin expression levels were up regulated along with the down regulation of E-cadherin.These results suggested that PCB118 exposure may affect cell adhesion,which may be an important mechanism for the promotion of tumor invasion and metastasis.In summary,PCB118 can regulate PKM2 mediated-Warburg effect through oxidative stress and aryl hydrocarbon receptor pathway,which then promotes the proliferation of hepatocellular carcinoma cells.In addition,PCB118 can change the cell adhesion ability of hepatocellular carcinoma.These effects may be important mechanisms to promote the development of liver cancer.