Anti-growth Activity And Mechanism Of Isoalantolactone On Head And Neck Squamous Cell Carcinoma Cell Line UM-SCC-10A

ObjectiveTo study the effect of Isoalantolactone on head and neck squamous cell carcinoma(HNSCC) cell line UM-SCC-10 A and its mechnisms.MethodsAfter the UM-SCC-10 A cells and normal mouse splenocytes were treated with different concentrations of isoalantolactone(0,6.25,12.5,25,50,100 μM), we used MTT assay to evaluate its anti-proliferative effect on tumor cells and cytotoxic effect on normal cells; We further confirmed the effect of isoalantolactone on UM-SCC-10 A cells by analyzing the percentage of living and dead cells using trypan blue staining and a cell viability analyzer; We also used Hoechst33258 staining to observe cell nucleus morphology; used Annexin V/PI staining kit and flow cytometry to determine whether isoalantolactone-induced cell death be caused by apoptosis or necrosis; In order to explain whether isoalantolactone-induced apoptosis be associated with cell cycle arrest, we examined the change of DNA content in each cell cycle phase on drug-treated UM-SCC-10 A cells using flow cytometry; To investigate whether isoalantolactone-induced cell apoptosis be associated with mitochondrial dysfunction, we analyzed the change of mitochondrial membrane potential(MMP) on drug-treated UM-SCC-10 A cells by Rhodamine(Rho 123) staining using flow cytometry; To further explore the mechanisms, we performed western blot to observe the protein expression of p53 、 p21 、 Cyclin D 、Cytochrome c(Cyto c)、Bax、Bcl-2 and Caspase-3(Casp-3) on UM-SCC-10 A cells after they were treated with different isoalantolactones; In addition, we used caspase inhibitor Z-VAD-FMK pretreatment to observe whether caspase-dependent apoptosis pathway be involved.ResultsThe MTT assay showed that isoalantolactone inhibited UM-SCC-10 A cell growth, while had little effect on normal mouse splenocytes; Immunostaining identified that isoalantolactone induced UM-SCC-10 A cell apoptosis but not necrosis; To analyze the molecular mechanism, flow cytometry and western blot assay indicated that drug-incubated led to cell cycle arrest, up-regulation of p53 and p21, down-regulation of Cyclin D; Furthermore, measurement of MMP and western blot assay revealed that isoalantolactone significantly increased proteins expression of Bax, Casp-3 and Cyto c, while a concomitant decrease in MMP and protein expression of Bcl-2, which suggested that the mitochondria pathway might be involved in isoalantolactone-induced cell apoptosis on UM-SCC-10 A cells; No significant early or late apoptosis were observed in UM-SCC-10 A cells pretreated with the caspase inhibitor Z-VAD-FMK, but G1-phase arrest was still observed.ConclusionsTaken together, our findings suggested that isoalantolactone inhibited UM-SCC-10 A cells growth with the possible mechnism of inducing caspase-dependent apoptosis via a mitochondrial pathway and being associated with cell cycle arrest in the G1 phase. Therefore, isoalantolactone may become a potential drug for treating HNSCC.