| 박노관¹, 김유경¹, 정일하¹, 권진숙¹, 한아름¹, 심태진¹, 배장환¹,황경국¹, 김동운¹,김승택², 조명찬¹ |
objectives; Although recent studies suggest protective effects of erythropoietin(EPO) protein against doxorubicin(DOX)-induced cardiotoxicity, the mechanism was not known. We assessed the hypothesis that EPO gene therapy using lentivirus(Lenti-EPO) reduces DOX-induced oxidative stress and DOX-induced apoptosis by upregulation of survival signal. Methods; Recombinant lentiviral vectors containing rat EPO gene were made by cotransfection of 293 T cell with pRRL-cPPT-CMV-EPO-PRE-SIN, pMDL, pVSVG, and pREV plasmid. Neonatal rat CMCs(3×105 cells) were infected by lenti-EPO virus or lenti-GFP(1.5×103 IU) as control. Transfected CMCs by lenti-EPO virus(CMCs/EPO) or lenti-GFP(CMCs/GFP) were evaluated by confocal laser microscope, and measured EPO levels. Under the concentration of 1μM of DOX, CMCs/EPO were incubated for 24 hours. We measured the amount of oxidative stress by oxyblot, anti-oxidant peroxiredoxin(Prx) system, the extent of apoptosis by FACS and the anti-apoptotic survival signals by Western blot. Results: More than 90% of CMCs had GFP expression. The concentration of EPO from the supernatant of CMCs/EPO was 81.1 pg/ml. In CMCs/EPO after DOX exposure, nonspecific oxidation by oxyblot was decreased.(Optical density(OD) ratio of CMCs/EPO compared to CMCs/GFP:0.8) And, Prx was increased (OD ratio: 1.3) with its downstream NF-kB suppression (OD ratio:0.64). The cell death and apoptosis of CMCs/EPO were decreased by FACS analysis(31.1 vs 27.6%). The expressions of p-Akt, p-GSK-3ß, p-Erk and Bcl-2/Bax-αwere enhanced at CMCs/EPO by Western blot(OD ratios: p-Akt;1.4, p-GSK-3ß;1.3, p-Erk;1.2, Bcl-2/Bax-α;1.3). Caspase-3, the final executor of apoptosisl, was also decreased. Summaries and Conclusion: Although the precise mechansim is not elucidated in this study, doxorubicin-induced cardiotoxicity may be associated with oxidative stress. Lenti-viral EPO gene transfer reduces DOX-induced oxidative stress by upregulation of Prx, and attenuated CMCs’apoptosis via enhanced expressions of p-Akt, p-GSK-3ß pathway. These findings suggest lenti-EPO gene therapy has a considering potential as an effective treatment at DOX-induced cardiotoxicity.
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