18‑α‑glycyrrhetinic acid induces apoptosis in gingival fibroblasts exposed to phenytoin

Abstract

Phenytoin (PHT)-induced gingival overgrowth is caused by the increased proliferation and reduced apoptosis of gingival fibroblasts in inflammatory gingiva. Licorice has long been used as a component of therapeutic preparations. It inhibits cell proliferation, induces cell apoptosis and has anti-inflammatory effects. 18-α-glycyrrhetinic acid (18α-GA), the active compound in licorice, promotes apoptosis in various types of cells. The present study determined whether 18α-GA affects apoptosis in gingival fibroblasts exposed to PHT. The present study aimed to establish a basis for the therapeutic application of 18α-GA to treat the gingival overgrowth induced by PHT. Human gingival fibroblasts from healthy donors were cultured to semi-confluence and then stimulated in serum-free DMEM containing PHT with or without 18α-GA for subsequent experiments. Apoptotic cells were detected by ELISA. Analysis of the distribution of cell cycle phases and the apoptotic cell population was performed by flow cytometry. The expression levels of mRNAs and proteins of apoptotic regulators were measured using reverse transcription-quantitative PCR and western blotting, respectively. Caspase (CASP) activities were assessed by an ELISA. Treatment with 18α-GA markedly increased the number of apoptotic cells, reduced BCL2 mRNA expression, increased CASP2 and receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1) domain containing adaptor with death domain, Fas (TNFRSF6)-associated via death domain, RIPK1, tumor necrosis factor receptor superfamily; member 1A, TNF receptor-associated factor 2, CASP2, CASP3 and CASP9 mRNA expression, and also upregulated the protein expression levels and activities of caspase-2, caspase-3 and caspase-9. These results demonstrated that 18α-GA induced apoptosis through the activation of the Fas and TNF pathways in the death receptor signaling pathway in gingival fibroblasts treated with PHT. 18α-GA exhibited therapeutic potential for the treatment of PHT-induced gingival overgrowth.

Keywords: 18α-GA; PHT; apoptosis; death receptor pathway; gingival fibroblast; gingival overgrowth.

Figure 1

A representative cell image from four independent experiments is shown. Cells were cultured in an atmosphere of 5% CO₂/95% air at 37˚C in D-MEM (high glucose) with L-glutamine and Phenol Red supplemented with 10% FBS, 50 units/ml penicillin and 50 µg/ml streptomycin to reach semi-confluence. Cells were routinely passaged using 0.05 w/v% trypsin-0.53 mmol/l EDTAx4Na Solution with Phenol Red. Scale bar, 100 µm.

Figure 2

Relative apoptotic cell number in gingival fibroblasts treated with PHT in the presence or absence of 18α-GA. After semiconfluent cells were treated with 0.25 µM PHT with or without (control) 10 µM 18α-GA in serum-free D-MEM for 24, 48 and 72 h, the quantification of apoptotic cells was performed by detecting the absorbance at 550 nm using APOPercentage Dye. After normalization to 0 h, the fold change compared with the control was determined. Data are presented as the mean ± SEM. ^*P<0.05 compared with the control using Welch's t-test (n=4). 18α-GA, 18-α-glycyrrhetinic acid; PHT, phenytoin.

Figure 3

Analysis of the apoptotic cell population (sub-G₁) and distribution of cell cycle phases of gingival fibroblasts cultured in the presence or absence of 18α-GA. Semiconfluent cells were incubated in serum-free D-MEM containing phenytoin (0.25 µM) with or without (control) 18α-GA (10 µM) for 48 h and then subjected to flow cytometric analysis. A representative dot plot from four independent experiments is shown. The detailed values of sub-G₁ and cell cycle parameters are shown in the table at the bottom. The data are presented as the mean ± SEM. ^*P<0.05 compared with the control using Welch's t-test (n=4). 18α-GA, 18-α-glycyrrhetinic acid; FL2-A, fluorescence pulse signal 2-area; FL2-W, fluorescence pulse signal 2-width.

Figure 4

mRNA expression levels of apoptotic regulators in gingival fibroblasts treated with PHT in the presence or absence of 18α-GA. Semiconfluent cells were incubated in serum-free D-MEM containing PHT (0.25 µM) with or without (control) 18α-GA (10 µM) for 12 h, after which reverse transcription-quantitative PCR analysis was performed. Relative quantification was performed using the 2^-∆∆Cq method. After normalization to GAPDH, RNA ratios in treated vs. control cultures were determined. Data are presented as the mean ± SEM. ^*P<0.05 compared with the control using Welch's t-test (n=4). (A) Anti-apoptotic genes. (B) Pro-apoptotic genes. 18α-GA, 18-α-glycyrrhetinic acid; BIRC3, baculoviral IAP repeat containing 3; CASP, caspase; CFLAR, CASP8 and FADD-like apoptosis regulator; CRADD, CASP2 and RIPK1 domain containing adaptor with death domain; FADD, Fas (TNFRSF6)-associated via death domain; PHT, phenytoin; RIPK1, receptor (TNFRSF)-interacting serine-threonine kinase 1; TNFRSF1A, tumor necrosis factor receptor superfamily; member 1A; TRAF2, TNF receptor-associated factor 2.

Figure 5

Protein expression levels of caspases in gingival fibroblasts treated with PHT in the presence or absence of 18α-GA. Semiconfluent cells were incubated in serum-free D-MEM containing PHT (0.25 µM) with or without (control) 18α-GA (10 µM) for 24 h and then assessed using western blotting, after which the fold change compared with the control was determined. The band images shown are representative of results from four independent experiments. Data are presented as the mean ± SEM. ^*P<0.05 compared with the control using Welch's t-test (n=4). 18α-GA, 18-α-glycyrrhetinic acid; PHT, phenytoin.

Figure 6

Caspase activity in gingival fibroblasts treated with PHT in the presence or absence of 18α-GA. After semiconfluent cells were treated with 0.25 µM PHT with or without (control) 10 µM 18α-GA in serum-free D-MEM for 24 h, caspase activities were assessed by detecting the absorbance at 405 nm, after which the fold change compared with the control was determined. Data are presented as the mean ± SEM. ^*P<0.05 compared with the control using Welch's t-test (n=4). 18α-GA, 18-α-glycyrrhetinic acid; PHT, phenytoin.

Figure 7

Schematic representation of apoptosis accelerated by 18α-GA in gingival fibroblasts treated with phenytoin. 18α-GA induced the upregulation of FADD and caspase-3, leading to an increase in the apoptotic Fas pathway. 18α-GA also induced the upregulation of RIPK1, CRADD, caspase-2, caspase-9 and caspase-3 in the TNF pathway, which resulted in apoptosis acceleration. Furthermore, 18α-GA decreased BCL2, which increased caspase-9. Purple (components of death-inducing signaling complex), red (antiapoptotic factors) and yellow (caspases) ellipses denote the molecules analyzed in the present study. The blue or red large arrows denote upregulation or downregulation, respectively, following 18α-GA treatment. Hyphens denote the molecules that are unaffected by 18α-GA treatment. 18α-GA, 18-α-glycyrrhetinic acid; BIRC3, baculoviral IAP repeat containing 3; CFLAR, CASP8 and FADD-like apoptosis regulator; CRADD, CASP2 and RIPK1 domain containing adaptor with death domain; FADD, Fas (TNFRSF6)-associated via death domain; RIPK1, receptor (TNFRSF)-interacting serine-threonine kinase 1; TNFRSF1A, tumor necrosis factor receptor superfamily; member 1A; TRADD, TNFRSF1A associated via death domain; TRAF2, TNF receptor-associated factor 2.