Phenytoin-induced gingival overgrowth caused by death receptor pathway malfunction.
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 semiconfluence 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 was 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.
Department of Biochemistry and Molecular Biology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
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http://orcid.org/0000-0002-5122-3736
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Takeuchi
Department of Pharmacology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
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Matsumoto
Department of Preventive and Public Oral Health, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
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Arikawa
Department of Preventive and Public Oral Health, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
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Taguchi
Department of Preventive and Public Oral Health, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
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Nakayama
Department of Preventive and Public Oral Health, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
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Nasu
Department of Biochemistry and Molecular Biology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
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Hiratsuka
D000927:Anticonvulsants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D000075747:Baculoviral IAP Repeat-Containing 3 Protein / Q000378:metabolism; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics / Q000378:metabolism; D053420:CRADD Signaling Adaptor Protein / Q000235:genetics; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D053200:Fas-Associated Death Domain Protein / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts; D015870:Gene Expression; D005881:Gingiva / Q000166:cytology; D019214:Gingival Overgrowth / Q000139:chemically induced; D006801:Humans; D010672:Phenytoin / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics; D047992:TNF Receptor-Associated Factor 2 / Q000235:genetics / Q000378:metabolism
D000927:Anticonvulsants; C000595707:BCL2 protein, human; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D053420:CRADD Signaling Adaptor Protein; C104252:CRADD protein, human; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; C101338:PSMD2 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D047888:Receptors, Tumor Necrosis Factor, Type I; D047992:TNF Receptor-Associated Factor 2; C506235:TNFRSF1A protein, human; D010672:Phenytoin; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; C506249:RIPK1 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D020169:Caspases
apoptosis; death receptor; gingival fibroblast; gingival overgrowth; phenytoin