Abstract
NF-kappaB promotes cell survival against external stress such as radiation. We examined whether NF-kappaB decoy transfection enhances the antiproliferative effects of radiation on vascular smooth muscle cells (VSMCs) in vitro. The irradiation induced activation or nuclear translocation of NF-kappaB p65 in VSMCs was confirmed by immunofluorescence. NF-kappaB decoy transfection resulted in inhibition of the radiation-induced NF-kappaB activation in VSMCs and the subsequent reduction of transcription and translocation of ICAM, iNOS, and TNF-alpha, downstream molecules under the control of NF-kappaB. By using MTT assay, NF-kappaB decoy augmented the antiproliferative effects of radiation, where the effect of low dose radiation (2 and 8-Gy) of the cells transfected with NF-kappaB decoy was equivalent to the high dose (16-Gy) irradiated non-transfected cells at 48 h after irradiation: 1.06+/-0.16, 1.11+/-0.22, 1.20+/-0.25, respectively. The decrease in proliferation and survival of the radiation treated cells by flow cytometry analysis showed that NF-kappaB inhibition did not show any additive effects on the cell cycle of the irradiated VSMCs, while apoptosis was significantly increased after NF-kappaB decoy transfection in the irradiated VSMCs (apoptosis fraction: 13.33+/-2.08% vs. 26.29+/-7.43%, for radiation only vs. radiation+NF-kappaB decoy transfection, P < 0.05). In addition, at 48 h, NF-kappaB decoy transfection dose dependently (10 microM vs. 20 microM) inhibited proliferation of 16Gy-irradiated VSMCs, and showed greater antiproliferative efficacy than 100 microM sulfasalazine, a specific NF-kappaB inhibitor. These results indicate that NF-kappaB inhibition reduces proliferation and survival of irradiated VSMCs, likely by increased apoptosis rather than additive cell cycle arrest and suggest the possibility of adjunctive gene therapy using NF-kappaB decoy to improve efficacy and to decrease the adverse effects of intracoronary radiation therapy.
Highlights
Restenosis is one of the most important problems in coronary angioplasty, and its major mechanisms are vascular remodeling and neointimal proliferation (Rajagopal and Rockson, 2003)
The inhibition of vascular smooth muscle cell (VSMC) proliferation, which constitutes the major portion of the neointima, is the main target of therapy in restenosis prevention
VSMCs are relatively resistant to radiation (Brenner et al, 1996) compared with other cells and intracoronary radiation has been shown to have some adverse side effects at high doses such as late thrombosis and coronary artery aneurysm (Condado et al, 1999; Farb et al, 2003)
Summary
Restenosis is one of the most important problems in coronary angioplasty, and its major mechanisms are vascular remodeling and neointimal proliferation (Rajagopal and Rockson, 2003). The inhibition of vascular smooth muscle cell (VSMC) proliferation, which constitutes the major portion of the neointima, is the main target of therapy in restenosis prevention. Intracoronary radiation therapy (or coronary brachytherapy), is one of the most promising anti-restenotic modalities due to its antiproliferative, proapoptotic effects along with cell cycle arrest (Teirstein et al, 1997). NFκB was initially considered as a pro-apoptotic factor because of its rapid activation in response to apoptotic signals and its involvement in the expression of apoptotic genes, such as TNF-α and c-myc, recent work have revealed an anti-apoptotic effect of NF-κB in response to a variety of apoptotic stimuli (Chen et al, 2001) including radiation. This in vitro study was performed to elucidate the additive effects of NF-κB inhibition on the proliferation and apoptosis of irradiated VSMCs, and its effect on the cell cycle
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