Inhibition of apoptotic signals in overgrowth of human gingival fibroblasts by cyclosporin A treatment.
Cyclosporin A (CsA), an immunosuppressive drug, has overgrowth effects on human gingival fibroblasts (HGF) in vitro. However, the molecular mechanism responsible for the CsA-induced gingival overgrowth remains still unclear. The present study is aimed to investigate the correlation with the apoptotic signal pathway in CsA-induced overgrowth of HGF. CsA-treated HGF were assessed for cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, for reactive oxygen species (ROS) detection by flow cytometry, for proliferation ability using the 5-bromo-20-deoxyuridine (BrdU), for caspase activities biochemically, for expression of apoptotic signal molecules such as cytochrome c, Fas and Fas-L and Bcl-2 family by Western blotting and VDAC by RT-PCR. CsA increased the cell viability, but not the number of BrdU-positive HGF, indicating that CsA fails to induce the proliferation of HGF. CsA also decreased the intracellular reactive oxygen species level in HGF. This was accompanied by that the antiapoptotic protein Bcl-2 was upregulated whereas the proapoptotic protein Bax was downregulated. Moreover, CsA downregulated VDAC, a mitochondrial transition pore, and decreased the level of cytochrome c released from the mitochondria into the cytosol and activation of caspase-3 and -9 associated with mitochondria-mediated apoptosis. On the other hand, Fas-L level and caspase-8 activation, the major mediator of the death receptor-mediated apoptosis, were diminished in the CsA-treated HGF. CsA inhibits the apoptotic signal molecules such as cytochrome c, caspases and Fas-L with the regulation of Bcl-2 family whereas it has no effect on cell division, which can contribute to overgrowth of HGF. These findings suggest that the decreased apoptosis plays a more important role than the increased cell proliferation in the CsA-induced overgrowth of HGF.
Jung
Ji-Yeon
JY
Dental Science Research Institute, School of Dentistry, Chonnam National University, Gwangju 500-757, South Korea.
Jeong
Yeon-Jin
YJ
Jeong
Tea-Sul
TS
Chung
Hyun-Ju
HJ
Kim
Won-Jae
WJ
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000008:administration & dosage / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D053222:Fas Ligand Protein / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005881:Gingiva / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000008:administration & dosage / Q000494:pharmacology*; D008928:Mitochondria / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C505661:FASLG protein, human; D053222:Fas Ligand Protein; D007166:Immunosuppressive Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D016572:Cyclosporine; D045304:Cytochromes c; D020169:Caspases