Induction of Non-apoptosis by α,β-Unsaturated Ketones in OSCC Cell Lines

Objective: There are at least three types of cell death, apoptosis, autophagy and necrosis. However, most of previous studies have focused on the apoptosis induced by various chemicals, but not on that of other types of cell death. We have recently shown that the type of cell death induced by hundreds of natural and synthetic compounds depends on the chemical structure of inducers and the sensitivity of target cells. We report here that most of α,β-unsaturated ketones selectively induce non-apoptotic cell death in human oral squamous cell carcinoma (OSCC) cell lines. Methods: OSCC cell lines (HSC-2, HSC-3, HSC-4) and normal oral cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) were cultured in DMEM supplemented with 10% FBS. The dose-response curve was used to determine the 50% cytotoxic concentration (CC₅₀). Tumor-specificity index was calculated by the ratio of mean CC₅₀ for normal cells to that for OSCC cell lines. Apoptosis markers used were internucleosomal DNA fragmentation (measured by agarose gel electrophoresis) and caspase-3,-8, -9 activation (measured by cleavage of substrates). Autophagy markers used were formation of autophagosome and secondary lysosome (detected by acridine orange staining, LC3-GFP accumulation and transmission electron microscopy). Results: 4,4-Dimethyl-2-cyclopenten-1-one, α-methylene-γ-butyrolactone, 5,6-dihydro-2H-pyran-2-one, 3,3,3-trifluoro-2-hydroxy-1-phenyl-1-propanone, codeinone, morphinone, vitamin K₁ (2-methyl-3-phytyl-1,4-naphthoquinone), vitamin K₂ (the phytyl side chain of vitamin K₁ replaced by 2 isoprenyl units), vitamin K₃ (2-methylnaphtoquinone), all of which have α,β-unsaturated ketone structure, did not induce DNA fragmentation, nor caspase activation in OSCC cell lines, but with occasional induction of secondary lysosome. These compounds showed relatively lower tumor-specificity, as compared with conventional chemotherapeutic agents. Conclusion: α,β-Unsaturated ketones generally induce non-apoptosis in OSCC cell lines, and there was no clear-cut correlation between the non-apoptosis-inducing activity of test compounds and the tumor-specificity.