RESUMO
Objective To investigate the effects and possible mechanisms of Mfn-2 gene overexpression on photodynamic therapy (PDT) sensitivity of T47D cells in human breast cancer.Methods pEGFP and pEGFP-Mfn-2 were transfected into human breast cancer T47D cells,and then the mRNA and protein expression of Mfn-2 gene in T47D cells were detected by real-time PCR (RT-PCR) and Western blotting in pEGFP group and EGFP-Mfn-2 group,respectively.After pEGFP and pEGFP-Mfn-2 were transfected into human breast cancer T47D cells for 48 hours,methyl thiazolil tetracolium (MTT) assay was used to measure the PDT sensitivity of T47D cells in human breast cancer in pEGFP group and pEGFP-Mfn-2 group.pEGFP + PDT group and pEGFP-Mfn-2 + PDT group were obtained by PDT irradiating pEGFP and pEGFP-Mfn-2 which were transfected into human breast cancer T47D cells.Cell apoptosis and mitochondrial membrane potential of T47D cells were assayed by flow cytometry in pEGFP + PDT group and pEGFP-Mfn-2 + PDT group.Laser scanning confocal fluorescence microscope was applied to observe the morphological ultrastructure of mitochondria in pEGFP group,pEGFP-Mfn-2 group,pEGFP + PDT group,and pEGFP-Mfn-2 + PDT group.Results RT-PCR showed that after transfecting T47D cells with pEGFP,the expression of Mfn-2 mRNA was 1.01 ±0.12.After transfecting T47D cells with pEGFP-Mfn-2,the expression of Mfn-2 mRNA was 1 067.00 ±41.72.There was statistical significance (t =67.541,P ＜ 0.001).Western blotting revealed that compared with the pEGFP group,the pEGFP-Mfn-2 transfection group had higher expression of Mfn-2 gene in T47D cells.MTT assay showed that pEGFP-Mfn-2 transfection significantly enhanced the PDT sensitivity of T47D cells in hunan breast cancer compared with the pEGFP + PDT group.When the concentration of methylene blue was 5.00 μmol/ml,the survival rate of the pEGFP +PDT group and pEGFP-Mfn-2 + PDT group were (59.96％ ± 1.21％) vs.(46.50％ ± 1.72％),with significant difference (t =34.403,P ＜ 0.001).Flow cytometry assay showed that the cell apoptosis rates in pEGFP-Mfn-2 + PDT group was markedly higher compared with the pEGFP + PDT group [(81.21 ± 2.13)％ vs.(68.82 ±2.64)％,P=0.024],with statistical significance.Also,the mitochondrial membrane potential was obviously lower in pEGFP-Mfn-2 +PDT group compared with the pEGFP +PDT group [(1.37 ±0.12)％ vs.(23.33 ± 1.86)％,P＜0.001],with statistical significance.Laser scanning confocal fluorescence microscope showed that cells in pEGFP group showed network structure,both pEGFP-Mfn-2 group and pEGFP + PDT group could cause mitochondrial fusion,and the pEGFP-Mfn-2 + PDT group could induce mitochondrial disintegration and lose its normal morphology completely.Conclusion Mfn-2 may enhance the PDT sensitivity of T47D cells in human breast cancer,which is possibly related with the normal morphology alteration of mitochondria and the inducement of mitochondrial apoptosis.