Overexpression Of Mitofusin-2Gene Enhances Photodynamic Therapy Effect On Human Breast Cancer Cell Line T47D

Objective:This study aims to investigate the effects and possible mechanisms of mitofusin-2(Mfn-2) gene overexpression on PDT sensitivity in breast cancer cells, the predicted outcome of this study will provide novel ideas in clinical breast cancer phtotodymic therapy.Methods:The mRNA expression of Mfh-2gene in breast cancer cells was detected by Real-time PCR. And the protein level of Mfh-2gene in breast cancer cells was detected by Western blotting. Plasmid pEGFP-Mfn-2and control plasmid pEGFP-N2were transfected respectively into human breast cancer T47D cells by Lipofectamin-eTM2000in vitro. T47D cells were cultured and treated with different concentration of photosensitizer methylene blue, then lighted with630nm wave length laser meter. MTT assay was used to measure the inhibitory effect of the PDT and overexpression of Mfh-2gene on human breast cancer T47D cells. Cell apoptosis and mitochondrial membrane potential (△Ψ m) of T47D cells were assayed by flow cytometry. Fluore-scence microscope was used to survey the variation of mitochondrial membrane pote-ntial (△Ψm). Laser scanning confocal fluorescence microscope was applied to obs-erve the morphological ultrastructure of mitochondria.Results:1. Real-time PCR showed that Mfn-2mRNA was highly expressed in Hcc38cells compared with the normal breast cell line [(1±0.05) vs (8.82±0.26), P<0.01], and lower expressed in breast cancer cell lines including T47D, MDA-MB-231, MCF-7, MDA-MB-435[(1±0.05) vs (0.50±0.06),(0.15±0.01),(0.93±0.08),(0.14±0.01),(0.06±0.01),P<0.05].2. Western-blotting results showed Mfn-2mRNA was highly expressed in Hcc38cells compared with the normal breast cell line, and lower expressed in other breast cancer cell lines including T47D, MDA-MB-231, MCF-7, MDA-MB-435.3. After transfecting T47D cells with pEGFP-Mfn-2, Real-time PCR and Western-blotting revealed that the gene of Mfn2-transfected cells had higher expression of Mfn-2gene [(0.48±0.07) vs (0.50±0.06),(946±41.72), P<0.05] after48h.4. Phase contrast microscope showed that pEGFP group with adherence growth and higher breeding speed had integrated cell membrane and limpid borderline; the cell morphology of pEGFP-Mfn-2group and pEGFP+PDT group appeared sev-eral alterations:cell borderline became round, vacuole generated in cytoplasm; the pEGFP-Mfn-2+PDT group showed that cell membrane disrupted, cells were solved and dead.5. MTT assay showed that mitofusin-2gene transfection can enhance the photody-namic therapy sensitivity of methylene blue in human breast cancer T47D cells,[5μm/ml methylene blue, the pEGFP-Mfn-2+PDT group(46.50±1.72)%vs the pEGFP+PDT group (56.93±2.05)%, P<0.05J.6. Flow cytometry assay showed that the cell apoptosis rates in the pEGFP-Mfn-2+PDT group was higher compared with the pEGFP+PDT group[5μm/ml methyl-ene blue, the pEGFP-Mfn-2+PDT group (81.2±2.1)%vs the pEGFP+PDT group (68.8±2.6)%,P<0.05].7. Flow cytometry assay showed that the mitochondrial membrane potential was lower in the pEGFP-Mfn-2+PDT group compared with the pEGFP+PDT group[5μm/ml methylene blue, the pEGFP-Mfn-2+PDT group (81.2±2.1)%vs the pEGFP+PDT group (68.8±2.6)%, P<0.05].8. Laser scanning confocal fluorescence microscope showed that both pEGFP-Mfn-2group and pEGFP+PDT group could cause mitochondrial fusion, and the pEGFP-Mfn-2group could induce mitochondria disintegrating and lose the normal morphology.Conclusions:1. Compared with the normal breast cell line, Mfn-2gene was highly expressed in Hcc38cells, and lower expressed in other breast cancer cell lines.48h after pEGFP-Mfn-2transfection, the expression level of Mfn-2mRNA and protein were significantly upregulated in T47D cells.2. MTT assay showed that mitofusin-2gene transfection can enhance the the photody namic therapy sensitivity in human breast cancer T47D cells. Flow cytom etry assay showed that combination of mitofusin-2gene transfection with PDT induced cell apoptosis more apparently and significantly decreased the mitochondrial membrane potential. 3. Mfn-2gene can be exploited to a new potential target for photodynamic therapy, which owing to the normal morphology aleration of mitochondria and via mitochondrial apoptosis signaling pathway.