The Role Of Mitochondria Fission And Fusion Abnormality In Arsenic Induced Malignant Transformation Of Human Mammary Epithelial Cells

Objective:Arsenic is a metalloid element widely distributed in nature,inorganic arsenic and its compounds are recognized as Class I carcinogens.Humans can be exposed to arsenic in a number of ways.The latest global epidemiological statistics on tumors have shown that breast cancer possesses the highest rates of incidence and mortality among the female population.The mechanisms of breast cancer are complex,and environmental factors are thought to enhances the risk of breast cancer occurrence.The association between low levels of environmental arsenic exposure and breast cancer has been reported in populations.Mitochondria are highly dynamic and plastic organelles that divide and fuse continuously within the cell.In recent years,abnormalities in mitochondrial division and fusion have been suggested to be involved in the development of breast cancer.However,whether this process plays a role in arsenic-induced breast cancer remains unclear.In this study,the kinetic process of mitochondrial division and fusion was investigated in the malignant transformed human normal mammary epithelial cells（MCF-10A）induced by long-term low-dose inorganic arsenic exposure,with the aim of providing a theoretical basis for exploring the association and potential mechanisms of arsenic exposure and breast cancer.Methods:1.Cell culture:MCF-10A cells were treated with 0.1μM sodium arsenite（Na As O₂）for 24 weeks,and the control group was the passage-matched cells without arsenic treatment.The cultural medium was changed every 2 days.Cell samples-treated with arsenic for different duration were collected.2.Detection of indicators related to malignant transformation:cells were evaluated for malignant transformation using scratch healing assay and soft agar clone formation assay.3.Observation of mitochondrial morphology:cells were treated with mitochondrial green fluorescent probe（Mito Tracker Green）and then confocal fluorescence microscopy was used to photograph the mitochondrial morphology.4.Determination of protein expression levels:cell samples treated with arsenic for different duration were collected and western blot was used to detect protein expression.5.Cytoskeleton observation:cells were treated with ghost pen cyclic peptide,and the expression and distribution of actin microfilaments（F-actin）were observed under an ortho-fluorescence microscope.6.Calcium ion(Ca²⁺)assay:Ca²⁺concentration was measured by flow cytometry.7.Calcineurin（CaN）activity assay:CaN activity was detected with calcineurin activity assay kit.8.Inhibitor intervention:mitochondrial division inhibitor Mdivi-1 was used to treat cells and related indicators were evaluated.9.Statistical analysis:Graph Pad Prism 8 was used to analyze data and form figures.Results:1.Long-term low-dose inorganic arsenic exposure resulted in MCF-10A cells acquired malignant phenotypes:cells treated with 0.1μM arsenic for 24 weeks showed enhanced clone formation ability and wound healing ability as that compared to control cells（P<0.05）.2.Long-term low-dose inorganic arsenic exposure resulted in elevated protein expression of extracellular regulated protein kinases（p-ERK1/2）:compared with the control cells,p-ERK1/2 protein expression in cells treated with arsenic for 16,20 and24 weeks was significantly increased（P<0.05）.3.Malignantly transformed MCF-10A cells induced by long-term low-dose arsenic exposure showed enhanced mitochondrial division and reduced mitochondrial fusion:compared with the control cells,the protein expression levels of dynamin related protein 1（DRP1）and mitochondrial fission protein 1（FIS1）did not change after 24 weeks of arsenic exposure.In contrast,the protein expression levels of phosphorylated DRP1 serine 616 site（p-DRP1 Ser616）was increased（P<0.05）,mitochondrial outer membrane fusion protein 1（Mitofusin 1,MFN1）and mitochondrial outer membrane fusion protein 2（Mitofusin 2,MFN2）were decreased（P<0.05）.Meanwhile,the mitochondria were fragmented and its network structure connections were reduced（P<0.05）.4.Long-term low-dose inorganic arsenic exposure resulted in enhanced expression of mitochondrial division-related proteins and reduced expression of fusion-related proteins during the malignant transformation of MCF-10A cells:the protein expressions of DRP1,p-DRP1 Ser616 and FIS1 were not significantly altered in the control cells.In the arsenic-treated cells,the expression levels of p-DRP1Ser616 did not change at 8 weeks of arsenic exposure,but it increased at 16,20 and24 weeks of arsenic exposure（P<0.05）.Meanwhile,the protein expression levels of MFN1 and MFN2 were significantly decreased in cells treated with arsenic for 24weeks（P<0.05）.5.Long-term low-dose inorganic arsenic exposure induced activation of the Ca²⁺-CaN signaling axis:compared to control cells,cytoplasmic Ca²⁺and CaN activity were increased in malignantly transformed MCF-10A cells（P<0.05）.Cytosolic actin depolymerisation factor（Cofilin）is a downstream effector protein of CaN.The protein level of Cofilin in cells treated with arsenic for 24 weeks was significantly higher as that compared to control group（P<0.05）,along with enhanced fluorescence intensity of F-actin,which is a downstream molecule regulated by Cofilin.6.Mdivi-1 inhibited enhanced mitochondrial division in malignantly transformed MCF-10A cells induced by long-term low-dose inorganic arsenic exposure:Mdivi-1is a specific inhibitor of DRP1.Control cells and 24-weeks arsenic-treated cells were divided into Mdivi-1 pretreatment or non-pretreatment group.Compared with the arsenic-treated cells without Mdivi-1 pretreatment,cells pretreated with Mdivi-1showed higher protein levels of DRP1 and p-DRP1 Ser616（P<0.05）,and increased mitochondrial connectivity as well as network branching（P<0.05）.7.Mdivi-1 inhibited the capacity of colony formation and migration in malignantly transformed MCF-10A cells induced by long-term low-dose inorganic arsenic exposure:significantly lower colony formation capacity and significantly lower scratch healing capacity were observed in the Mdivi-1-pretreated transformed cells as that compared to transformed cells without Mdivi-1 pretreatment（P<0.05）.8.Mdivi-1 inhibited increased cytoplasmic Ca²⁺in malignantly transformed MCF-10A cells induced by long-term low-dose inorganic arsenic exposure:the cytoplasmic Ca²⁺concentration was significantly lower in the Mdivi-1-pretreated transformed cells as that compared to transformed cells without Mdivi-1 pretreatment（P<0.05）.Conclusion:1.The protein expression of p-ERK1/2 was enhanced during malignant transformation of human mammary epithelial cells induced by long-term low-dose inorganic arsenic exposure,and the protein expression of p-DRP1,a downstream target of p-ERK1/2,was also gradually enhanced,and enhanced mitochondrial division as well as reduced mitochondrial fusion were observed for malignantly transformed cells.2.The cytoplasmic Ca²⁺-CaN-Cofilin signaling axis was activated in malignantly transformed cells induced by long-term low-dose inorganic arsenic exposure,and the expression of F-actin,a downstream molecule of Cofilin,was enhanced.3.Increased mitochondrial division may be involved in the malignant transformation of MCF-10A cells induced by long-term low-dose inorganic arsenic exposure by regulating cytoplasmic Ca²⁺concentration.