| Objective To investigate the molecular mechanisms of the apoptosis resistance inducing by cytoplasmic M-CSF in human breast cancer MCF-7 cells.Methods MCF-7 cells,which stable express cytoplasmic M-CSF,were constructed in our laboratory.MCF-7 cells,MCF-7-C cells and MCF-7-M cells were maintained in RPMI 1640 supplemented with 10% NBCS.The effect of cytoplasmic M-CSF on the expression of Nrf2 and HO-1 was observed by Western blot in human breast cancer MCF-7 cells.The treatment of LY294002(a specific PI3 K inhibitor)and SH6(a specific Akt inhibitor)was used to analyze whether PI3K/Akt signalings mediate effect of cytoplasmic M-CSF on expression of Nrf2 and HO-1.The subcellular lacalization of Nrf2 was detected by immunofluorescence and Western blotting.Hoechst33342 staining and flow cytometry were utilized to evaluate the effect of cytoplasmic M-CSF on the apoptosis in MCF-7 cells treated with ADM.Results The expression of HO-1,total Nrf2 and nuclear Nrf2 was markedly higher,and cytoplasmic Nrf2 was significantly less in MCF-7-M cells than in MCF-7 cells or MCF-7-C cells(p<0.05).There was little in the expression of HO-1 and Nrf2 between MCF-7 cells and MCF-C cells(p<0.05).Red fluorescence,which labelled Nrf2,aggregated at the nucleus in MCF-M cells and scatteringly localized at whole MCF-7cells and MCF-7-C cells.The expression of HO-1,total Nrf2 and nuclear Nrf2 was dramatically less,and cytoplasmic Nrf2 was significantly higher in MCF-7-M cells treated with LY294002 and SH6,concomitance with red fluorescence scatteringly localizing at whole MCF-7-M cells,and with nuclear fluorescence attenuation.The poptosis rate of MCF-7-M cells is,respectively,7.14%,18.77%,19.96% and24.27%,after the treatment without ADM or with ADM,HO-1-si NC+ADM,and HO-1-si RNA+ADM.The apoptosis induced by HO-1-si RNA is significantly higher in MCF-7-M cells after the incubation with ADM than in MCF-7-M cells after the incubation with ADM alone or HO-1-si NC+ADM(p<0.01).The apoptosis rate of MCF-7-M cells is,respectively,18.77%,19.17%,27.33% and 27.97%,after the treatment with ADM,or ADM+DMSO,ADM+LY294002 and ADM+SH-6.The apoptosis induced by LY294002 or SH-6 dramatically more increased in MCF-7-M cells with ADM incubation than in MCF-7-M cells with ADM alone or ADM+DMSO incubation(p<0.01).Conclusions Cytopasmic M-CSF induced the expression of HO-1 and Nrf2,and the nuclear translocation of Nrf2 in MCF-7 cells.PI3K/Akt signalings was involved in the expression of HO-1 and Nrf2,and the nuclear translocation of Nrf2 induced by Cytopasmic M-CSF.The apoptosis resistance indued by Cytopasmic M-CSF was mediated by PI3K/Akt / Nrf2/ HO-1signalings in MCF-7 cells with ADM treatment. |
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