Effects And Mechanism Of Proliferation By Low Dose Diethylhexyl Phthalate In MCF-7 Cells

ObjectiveIn recent years,chemical substances attracted considerable attentions due to the advantages and disadvantages effects of low dose,studies have shown that low dose may be a stimulation;but there are opposite views,that negative effects should not be ignored,especially the low dose endocrine disruptors.Most researches of the environmental endocrine disruptors(EEDs)are about the toxic produced in high dose,the dose related to environment(low dose)is insufficient so far,the biological effects of phthalate acid esters play a role as endocrine disrupting activity.Plasticizer DEHP has a variety of toxicity,whether low dose DEHP has protective effect,or will increase the susceptibility of disease is still unclear.This study select DEHP and its metabolites MEHP as the representatives of EEDs,by cell proliferation pathway and estrogen receptor in aspects of mechanism research,which would value the biological effects in cells with low dose,then provide more comprehensive scientific clues to further studies about the relation between endocrine disruptors and other diseases.MethodsIn this study,the expression of estrogen receptor in human breast cancer cell line MCF-7 as the research object,to reveal the proliferation by different concentrations of DEHP and its metabolite MEHP,and the effects of estrogen receptor antagonist TAM.17(3-estradiol as positive control.1.Detect the effects of DEHP,MEHP on the growth of MCF-7 cells through CCK-8 assay,the migration ability of MCF-7 cells were determined by cell scratch assay.2.LDH activity assay exam the change of cell function,mainly to observe whether there is damage to cell membrane.3.The apoptosis of MCF-7 cells treated with DEHP,MEHP after 24 and 48hours was determined by flow cytometry.4.PCR analysis of estrogen receptors and PGE2 signal pathway factors Akt,PKA,PI3K,and CREB in MCF-7 cells treated with MEHP.demonstrate mechanism about the low dose environmental endocrine disruptors phthalic acid ester.Results1.DEHP and MEHP could induce the proliferation in MCF-7 cells,the effect of 24h exposure to E2 and 0.1?mol/L MEHP on cell proliferation was significant increased(P<0.05),100?mol/L DEHP on cell proliferation was significant decreased(P<0.05).The effects of 48h exposure to E2,0.1?mol/L DEHP and 0.1?mol/L MEHP on cell proliferation was significant increased in the scratch test(P<0.05).2.The LDH activity of 24h and 48h exposure to 0.1 ?mol/L MEHP was decreased.3.The apoptosis rate of DEHP 100?mol/L dose group was increased(P<0.05);E2 and 0.1?mol/L MEHP decreased the apoptosis rate significantly after 24h exposure(P<0.05),100?mol/L MEHP increased the apoptosis rate significantly after 48h(P<0.05).4.The mRNA expression of ER alpha and ER beta in MCF-7 cells exposure to 1?mol/L MEHP were significant decreased(P<0.05).5.After blocking the estrogen receptor,the changes of proliferation,migration,LDH activity and total apoptosis rate of the cells with different doses DEHP and MEHP were disappeared.6.After blocking the estrogen receptor,the expression of PGE2 signaling pathway factors,such as Akt,PKA,PI3K and CREB had no obvious change.' The expression levels of 0.1 ?mol/L dose group at different times were still significant lower than the control(P<0.05).ConclusionsDEHP and MEHP could induce the proliferation of MCF-7 cells in the low dose,as the cell apoptosis rate decreased,Reduce damage effect to the cell membrane,the MCF-7 cells displayed proliferation by MEHP,activate the PGE2 signaling pathway through estrogen receptors mainly,regulating Akt/PI3K pathway,play the estrogen role.