Phenytoin-induced gingival overgrowth caused by death receptor pathway malfunction

Abstract

Objective

In this study, we investigated the role of phenytoin (PHT) in death receptor-induced apoptosis of gingival fibroblasts to clarify the mechanism of PHT-induced gingival overgrowth.

Methods

Human gingival fibroblasts were cultured to semi-confluence and treated with PHT (0.025, 0.1, 0.25, and 1.0 μM) for 48 h, and then the apoptotic cell numbers were relatively determined by absorptiometry. After 24 h of 0.25 μM PHT treatment, caspase activity was measured by absorptiometry, apoptotic and cell cycle phase distribution was analyzed by flow cytometry, expression levels of apoptotic genes were quantified by real-time qPCR, and expression of apoptotic proteins was detected by western blot analysis. After 48 h of 0.25 μM PHT treatment, appearance of apoptotic cells were detected by TUNEL assay.

Results

PHT treatment decreased the proportion of apoptotic cells in gingival fibroblasts compared to a serum-free control culture in response to the protein changes as follows: PHT upregulated c-FLIP and, in turn, downregulated FADD, caspase-8, and caspase-3; PHT upregulated c-IAP2 and downregulated TRAF2; PHT downregulated caspase-9 and caspase-3 via decreased RIPK1 activity and increased Bcl-2 activity.

Conclusions

PHT-induced gingival overgrowth may result from the above-mentioned mechanisms involving apoptosis inhibition in gingival fibroblasts.

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