Identify effects of phenytoin and losartan on TGF‐beta using human gingival fibroblast‐1 (HGF‐1) cells and establish HGF‐1 cell culture as a model to study drug‐induced gingival hyperplasia

Abstract

PurposeThe main objectives of this study were 1) to evaluate the effects of phenytoin (antiepileptic drug) and losartan (TGF‐beta inhibitor) on cytotoxicity, ATP, and TGF‐beta in human gingival fibroblasts (HGF‐1) cells, 2) to identify whether losartan inhibits gingival overgrowth caused by phenytoin and 3) to establish HGF‐1 cell culture as a model to study druginduced gingival hyperplasia.MethodsHGF‐1 cells obtained from ATCC were grown to 90% confluency in DMEM media supplemented with PenStrep, and bovine serum (FBS 10%). Cells were incubated in 5% CO2, 37°C and 95% relative humidity. For cytotoxicity, LDH leakage in the medium was measured using the CytoTox 96 Non‐radioactive Cytotoxicity Assay Kit, and for ATP, total % ATP was measured using CellTiter‐Glo Luminescent Cell Viability Assay Kit. For ATP and LDH measurements, cells were treated with various concentrations of phenytoin (0, 10, 25, 50, 100 and 200 μM) and losartan (0, 25, 50, 100, 250 and 500 μM) dissolved in DMSO, and incubated for 24h (30,000 cells/well in 96 well plates), 5% CO2, 37°C and 95% relative humidity. Final DMSO concentration was 0.1%. To identify effects on TGF‐beta, cells were treated with DMSO (0.1% final concentration), Phenytoin (100 μM), Losartan (100 μM), and Phenytoin (100 μM) + Losartan (100 μM). After 24h treatment of cells, TGF beta was measured as per supplier’s protocol using ELISA assay kit (Thermo Fisher, Grand Island, NY). All experiments were carried out as triplicate measurements and mean of triplicates were considered as one experiment (n). Minimum of three repetitions (n = 3) were carried out unless otherwise noted in results.ResultsPreliminary results show that none of the drug concentrations had a cytotoxic effect on HGF‐1 cells. No increase in ATP levels were observed for losartan. However, phenytoin showed greater increase in percent ATP levels when compared to vehicle control. Percent ATP levels for losartan and phenytoin were in the range of 101 ± 4 to 113.5 ± 8.2% and 135 ± 11.1 to 138.1 ± 16.5% respectively. Data from one TGF‐beta experiment showed no increase in TGF‐beta levels with phenytoin treatment, however losartan, and losartan plus phenytoin treatment showed 1.3 and 2‐fold increase in TGF beta levels over untreated cells respectively.ConclusionOur study suggests that phenytoin causes cell proliferation as noted from increased ATP levels. Preliminary results form TGF‐beta experiments were inconclusive and further experiments are needed to ascertain effects of losartan and phenytoin on TGF‐beta in HGF‐1 cells and identify whether HGF‐1 cells can be used as a model to study drug‐induced gingival hyperplasia.Support or Funding InformationThis project is supported by College of Pharmacy, Roseman University of Health Sciences, South Jordan, Utah 84095