Effect of hinokitiol in ameliorating oral cancer: in vitro and in silico evidences

Odontology. 2024 Nov 14. doi: 10.1007/s10266-024-01020-1. Online ahead of print.

Abstract

The study aimed to evaluate the anticancer potential of hinokitiol in treating oral cancer by using in vitro models and examining its interaction with the Pim-1 protein through in silico methods. Hinokitiol was applied to KB-1 oral squamous carcinoma cells, where the half-maximal inhibitory concentrations (IC50) was determined. Morphologic changes in treated cells were observed using phase contrast microscopy, while acridine orange/ethidium bromide (AO/EB) staining was used to assess nuclear changes and apoptosis. Flow cytometry was employed to analyze the cell-cycle progression. Given the high expression of Pim-1 in oral squamous carcinoma cells, molecular docking and simulation were performed to evaluate hinokitiol's binding affinity and stability with the Pim-1 protein. To compare its effects, hinokitiol was also tested on non-cancerous pre-adipocytes (3T3-L1), providing insights into its selective cytotoxicity between healthy and cancerous cells. Hinokitiol treatment resulted in cytotoxic effects on KB-1 oral squamous carcinoma cells, with an IC50 of 30 µg/mL after 24 and 48 hs of exposure. Morphologic studies showed reduced cell population and density. In contrast, hinokitiol exhibited lower toxicity and caused fewer morphological changes in non-cancerous 3T3-L1 pre-adipocytes. Apoptosis was confirmed through acridine orange/ethidium bromide staining, while flow cytometry revealed cell-cycle arrest in the Synthesis phase (S) and Gap 2 phase/ Mitosis Phase (G2/M) phases. Molecular docking showed strong binding of hinokitiol to Pim-1, and simulations confirmed the interaction's stability. These findings suggest hinokitiol selectively targets cancer cells and effectively inhibit Pim-1, supporting its potential as an oral cancer treatment.

Keywords: Hinokitiol; Molecular dynamics; Oral cancer; Pim-1.