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Background and Objectives: Cholesteatomas are chronically progressive keratinizing epithelial lesions that locally destroy tissue and erode bone. Surgery is the primary treatment for cholesteatomas; however, tumor recurrence and complications have prompted the search for more effective medical therapies. Halofuginone (HF) inhibits multiple pathways implicated in cholesteatoma pathogenesis. Therefore, the dose- and time-dependent cytotoxic effects of HF on the spontaneously transformed human adult skin keratinocyte (HaCaT) cell line were investigated, and the IC50 values of HF were determined as a preliminary step in evaluating the potential of using HF as a pharmacological treatment for cholesteatoma.Materials and Methods: An in vitro experiment was conducted using HaCaT cells, which is an immortalized human keratinocyte cell line. Cells were treated with HF at 0.1 μM to 100 μM for 24 and 48 hours. Cell viability was measured via an MTT assay, and the IC50 values were calculated using nonlinear regression analysis.Results: HF treatment substantially dose- and time-dependently reduced HaCaT cell viability. The IC50 values at 24 and 48 hours were 2.74 μM and 0.24 μM, respectively, reflecting a more than ten-fold increase in cytotoxic potency over time. The differences between the treatment groups were significant at both time points (p=0.017 and p=0.001 at 24 and 48 hours, respectively).Conclusions: HF exerts potent cytotoxic effects on keratinocytes, with efficacy increasing with exposure duration. These findings support the further study of HF as a pharmacological agent for modulating epithelial proliferation in cholesteatomas and related disorders.

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