Polychlorinated Biphenyl Quinones Promotes Metastasis In Breast Cancer By Regulating Matrix Metalloproteinase And Epithelial-mesenchymal Transition

Polychlorinated biphenyls(PCBs),a persistent industrial pollutant,are a group of structurally including 209 congeners related chlorinated aromatic hydrocarbons.Although the production of PCBs was banned in Western Europe and North America in the 1970s and in China in 1983,they have still widely existed in our environment and continued to enter the human body,which are typical representative of persistent pollutants.Polychlorinated biphenyl quinones(PCBQ),a metabolite of polychlorinated biphenyls,can cause more toxic effects than the parent compound.Breast cancer is one of the most common causes of cancer related death for women in the world.The incidence of breast cancer in China is lower than that of western countries,but it has the fastest growing in the world.One of the main causes of death in patients with breast cancer is tumor metastasis.The rate of localized breast cancer can reach 98%in five years.Conversely,women diagnosed with distant metastasis have a survival rate of only27%.This is related to many numerous factors,including pollutants in the environment.The purpose of this study is to analyze the impact of PCB29-pQ on breast cancer metastasis,and to elucidate the possible molecular mechanisms.Part?:In this part we explored the role of PCBs in breast cancer metastasis.We selected MDA-MB-231 and 4T1 cells as the research object in vitro experiments,and then constructed 4T1 xenograft model in vivo experiments.First,the two cells were treated with low concentration(0-4?M)of PCB29-pQ for 24 h,respectively using normal conditions and 3D conditions.Then CCK8 was used to identify the cell viability.The results showed that low concentration of PCB29-pQ had no significant cytotoxicity on MDA-MB-231 and 4T1 cells.Secondly,to determine the effect of PCB29-pQ on the migration capability of MDA-MB-231 and 4T1 cells,Wound healing assay and Transwell assay was used to examine.The cells stimulated with different concentrations PCB29-pQ for 24 h.Results showed that PCB29-pQ markedly induced the migration of MDA-MB-231 and 4T1 cells after treatment for 24 h and had the most obvious effect in4?M PCB29-pQ.Then,we used the matrigel-coated 24-well plate to evaluate the effect of PCB29-pQ on MDA-MB-231 and 4T1 cells invasion.We found that compared with the control group,the PCB29-pQ group increased the invasiveness of cell lines significantly and reached its maximum with 4?M PCB29-pQ after 24 h.These results indicate that PCB29-pQ can promote migration and invasion of MDA-MB-231 and 4T1cells.We explored several other polychlorinated biphenyls and their metabolites.The effects of PCB3,PCB3-Q,PCB15 and PCB15-Q on the migration and invasion ability of MDA-MB-231 cells were compared.The migration and invasion ability of PCB3-Q and PCB15-Q groups increased,but the effect was still lower than PCB29-pQ group.Finally,we established a mouse xenograft model with 4T1-luc breast cancer cells and BALB/C nude mice,which gavaged different doses of PCB29-pQ.Then,the mice were monitored using bioluminescence imaging.We found that the mice treated with PCB29-pQ had stronger fluorescence signal than the control group in the lung.After 15days,the fluorescent signal had been detected in mouse liver.HE staining showed that the lung and liver of PCB29-pQ group had more obvious cancer lesions.According to the above results we can conclude that low concentrations of PCB29-pQ can promote the breast cancer metastasis in vitro and in vivo.Part?:In this part we researched the molecular mechanism of breast cancer metastasis that through MMP-2/-9 by PCB29-pQ.AKT/ERK/p38/NF-?B signaling pathways played an important role in this process.Immunofluorescence and Western Blot experiments showed that both MMP-2 and MMP-9 in MDA-MB-231 and 4T1cells were significantly up-regulated after 24 h treatment.Through gelatin zymography assay,we found that the activities of MMP-2 and MMP-9 in both cells were significantly raised compared with the control group.By immunohistochemistry,results showed that MMP-2/-9 significantly increased in breast cancer graft model.MT1-MMP was obviously going up when the cell treated PCB29-pQ.The results of Western Blot and immunofluorescence showed that PCB29-pQ can induce the expression of related proteins in NF-?B signaling pathway.PCB29-pQ can reduce cytoplasmic p65 while increased nuclear p65 and also increased NF-?B transcriptional activity.When suppressed the expression of NF-?B,the MMP-2/-9 significantly decreased.Subsequently,the phosphorylation status of ERK,p38 and AKT was analyzed by Western Blot.PCB29-pQ was found to promote the phosphorylation of ERK,p38 and AKT.Moreover,after adding the inhibitors of ERK,p38 and AKT,the expression of MMP-2 and MMP-9 were decrease.The wound healing and Transwell invasion assay demonstrated that the migration and invasion characteristics of MDA-MB-231 cells induced by PCB29-pQ significantly reduced in the presence of NF-?B,ERK,p38 and AKT inhibitors.These results indicated that AKT/ERK/p38/NF-?B pathways were involved in the expression of MMP-2 and MMP-9.In the end,we used NAC to block ROS accumulation in MDA-MB-231 and 4T1 cells.Indeed,the cascade of protein cascades caused by PCB29-pQ has been reduced following pre-treatment with NAC.It wasindicatedthatROSwasimportantlywiththeactivationofthe AKT/ERK/p38/NF-?B signaling pathways,and it is possible to regulate these signaling pathways in the upstream of the molecule.Part?:To further explore the mechanism of polychlorinated biphenyraquinones induced invasion and migration of MDA-MB-231 cells with the presence of PCB29-pQ.We analyzed the phenomenon of epithelial mesenchymal transition that regulates tumor metastasis.First,the markers of epithelial mesenchymal were detected by Western Blot and immunofluorescence.It was found that in the presence of PCB29-pQ,the quantity of E-cadherin expression decreased under the concentration gradient,while N-cadherin and Vimentin increased significantly.Then the Wnt/?-catenin pathway was detected.The cell lysates in nuclear and cytosolic fractions were detected by Western blot and Immunofluorescence demonstrated that?-catenin accumulation into nucleus.After adding the inhibitor of Wnt/?-catenin,it reversed the trend of E-cadherin,N-cadherin and Vimentin under the action of PCB29-pQ.These experiments showed that Wnt/?-catenin may be involved in the regulation of PCB29-pQ to promote the EMT of MDA-MB-231.In addition,we also detected the CD44~+/CD24~-ratio of MDA-MB-231by flow cytometry,and found that the number of CD44~+/CD24~-cells was increase under the treatment of PCB29-pQ.These results suggest that the epithelial cells of breast cancer cells can be induced by PCB29-pQ.The three-part experiments above indicate that PCB29-pQ induced breast cancer metastasis,mainly through the AKT/ERK/p38/NF-?B signaling pathways to mediated MMP-2 and MMP-9 release,and through the Wnt/?-catenin pathway to regulate the Epithelial-mesenchymal transition of breast cancer cells.Through this research,we provides more theoretical basis for the study of PCB29-pQ induced breast cancer characteristics,and presents new evidence for the relation between PCBs and breast cancer.