Mitochondria And Estrogen Play A Role In The Biological Behaviour Of Breast Cancer Cells In Vitro

ObjectiveEpidemiologic study has showed that estrogen plays a great role in the progress of breast cancer.Estrogen receptor is one gene which modifies cell growth and provides evidence for endocrine therapy.Estrogen is consisdered to elicit different growth responses through binding to ER.Mitochondria are important targets of estrogen action and mitochondria have been implicated in the control of cell proliferation.MtDNA(mitochondrial DNA)is the only extranuclea genetic substance and has been one of the research hot spots in recent years.It is commonly considered that ER is necessary for estrogen action and provides as index for both endocrine therapy and prognosis.However,some of the breast cancer patients who are verified as ER negative have benefited from endocrine therapy.For these reasons,it is of great importance to investigate the correlations among mitochondia,mtDNA,estrogen and ER and explore the possible mechanisms.Methods(1)Expression and location of ER was detected using immunofluorescence double staining method and laser scanning confocal microscopy(LSCM)in MCF-7 human breast cancer cells after treated with estrogen.(2)The rho⁰ derivative of human cancer cell line MDA-MB-231 which is ER negative were generated by treating the cultured cells with ethidium bromide and veritifed using auxotroph and PCR methods. The morphology was confirmed by transmission electron microscope(TEM),cell growth was determined by MTT,cell movement was analyzed by cell scratch assay, mitochondrial membrane potential was measured by flow cytometry(FCM),the sensitivities to chemotherapy were carried out by Collagen Gel Droplet Embeded Culture-Drug Sensitivity Test(CD-DST),P-glycoprotein(P-gp)and breast cancer resistance protein(BCRP)expression were evaluate by immunohistochemistry,in MDA-MB-231and rho⁰MDA-MB-231 cells,respectively.(3)After treated with estrogen,cell proliferation was evaluated by CD-DST assay,cell cycle distribution was determined by FCM,the morphology was confirmed by TEM,in MDA-MB-23 land rho⁰MDA-MB-231 cells,respectively.(4)After treated with estrogen,the differently expressed mtDNA genes were examined with the mtDNA oligonucleotide microarray in MCF-7 and MDA-MB-231 cells,respectively.Results1.Besides located in the nucleoli of MCF-7 cells after treated with estrogen,more ER signals were found in the cytoplasm.2.The deletion of mtDNA was confirmed by non-selective culture and PCR.Compared with MDA-MB-231 cells,the derived rho⁰MDA-MB-231 cells showed different characteristics,including a larger number of swollen mitochondria,stronger proliferation and movement ability,higher clone formation ability,decreased mitochondrial membrane potential,relatively resistance to chemical drugs and higher expressions of MDR related proteins.3.Estrogen stimulated the proliferation of MDA-MB-231 cells,while inhibited the rho⁰ MDA-MB-231 cell growth in a dose-dependent manner.G₂/M phase arrest, swollen and myelinationed mitochondria were manifested in rho⁰MDA-MB-231 cells,whereas S phase arrest,expanded rough endoplasmic reticulum and more ribosomal particles were manifested in MDA-MB-231 cells after estrogen exposure.4.After exposure to estrogen,the ND4,ND4L and COXâ…¡encoded by mtDNA gene were increasing expressed in MCF-7 cells,while no statistically differential gene expression was showed in MDA-MB-231 cells.Conclusions1.rho⁰MDA-MB-231 cell line lacking mtDNA was successfully established.2.MtDNA might play a role in the formation of malignant and mulitdrug resistant phenotypes of human breast cancer MDA-MB-231 cells.3.Besides the typical nuclear ER pathway,estrogen may take action through other signal pathway which involves mitochondria or mitochondrial DNA.