Background and objective: We reported previously that cyclosporine A induces a high level of expression of p21 in rat gingival keratinocytes and in OECM1 cells. In this study, the apoptosis of gingival keratinocytes after treatment with cyclosporine A was evaluated using the same models.
Material and methods: Forty Sprague-Dawley rats with right edentulous ridges were assigned into cyclosporine A (30 mg/kg) and control groups. Four weeks later, gingivae were screened for expression of apoptotic genes using microarray analyses and DNA fragmentation. The expression of bcl2-associated X protein (Bax), apoptosis-inducing factor (AIF) and Caspase 3 mRNAs, and the expression of Bax, AIF, Caspase 9 and Fas proteins, were analyzed using the reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. Apoptosis in OECM1 cells (keratinocytes of a gingival carcinoma cell line), after treatment with cyclosporine A, was evaluated by 4',6-diamidino-2-phenylindole (DAPI) staining and flow cytometry, whereas the expression of Bax, AIF, Caspase 3 and 8, Bcl-2 and Fas proteins were examined using western blotting.
Results: According to microarray analyses, the expression of certain apoptotic genes was altered in the gingiva of rats who received cyclosporine A, and increased number of DNA fragments were detected. Expression of mRNA or protein for Bax, AIF and Caspase 3 and 9 in the gingivae of rats increased after treatment with cyclosporine A. An increased number of apoptotic bodies and of OECM1 cells in the sub-G1 phase was observed after treatment with cyclosporine A. Increased expression of AIF, Bax and Caspase 3 protein, but not of bcl-2, Caspase 8 or Fas protein, was observed in cells after treatment with cyclosporine A.
Conclusion: Based on the above findings, we suggest that cyclosporine A might enhance the apoptosis of gingival keratinocytes, mainly via the mitochondrial pathway.