The Role And Mechanism Of Autophagy In The Treatment Of Laryngeal Carcinoma Cells By Asparaginase

Objective:Head and neck cancer is a common threat to human health of malignant tumors,and laryngeal cancer is one of the most important head and neck cancer,and the incidence of laryngeal cancer is still increasing year by year.Despite the continuous improvement of the treatment of laryngeal cancer,its five-year survival rate has not improved significantly.Therefore,looking for new therapies for the treatment of laryngeal cancer is imminent.Tumor cells can not synthesize asparagine and must rely on exogenous asparagine to survive.Asparaginase can inhibit the synthesis of protein in tumor cells by degrading asparagine,leading to the death of tumor cells,which has been used in leukemia,lymphoma and melanoma treatment.However,asparaginase treatment for laryngeal cancer is rarely reported.Cell autophagy,a very conserved mechanism in eukaryotes,dependent on the lysosomal protein degradation pathway.When amino acids,sugar and other nutrients lack,pH or osmotic changes,autophagy can clear the cells within the wrong folding or aggregation of proteins,damaged or aging organelles to maintain the cell internal steady state.Recent studies have shown that autophagy plays a dual role in tumor therapy,promoting tumor cell apoptosis or protecting tumor cells.The study of autophagy was not published when asparaginase acted on laryngeal cancer cells.Methods:1.The effect of asparaginase on laryngeal cancer cells was evaluated by MTT assay,and the effects of the combination of autophagy inhibitor CQ and the combined inhibitor of apoptosis on cell toxicity were investigated by using asparaginase on Tu212 and Tu686 cells for 48 hours.2.Flow cytometry was used to detect cell apoptosis.Asparaginase combined with or without autophagy inhibitor CQ on the apoptosis of Tu212 and Tu686 cells;the effect of antioxidant NAC on apoptosis of laryngeal carcinoma induced by asparaginase.3.Transmission of electron microscopy(TEM)was used to observe the production of autophagosomes in Tu212 and Tu686 cells by asparaginase.4.The expression of asparagine synthetase(ASNS)in Tu212 and Tu686 cells was detected by Western blot.The expression of autophagic protein LC3-II in laryngeal squamous cell carcinoma was detected after the combination of asparaginase and CQ.The changes of expression of Caspase 3 and PARP were detected.5.Confocal microscopy was used to detect the production of autophagy and autophagic flux.Results:Laryngeal squamous cell carcinoma(LSCC)Tu212 and Tu686 aspartate synthase expression was absent;asparaginase by depletion of asparagine on laryngeal cancer cells Tu212 and Tu686 cells can be significantly killed.Asparaginase can induce significant apoptosis in laryngeal carcinoma cells and the cleaved of Caspase 3,PARP and Caspase inhibitor z-VAD-fmk significantly attenuated asparaginase-induced apoptosis of Tu212 and Tu686 cells,suggesting that asparaginase-induced apoptosis is dependent on the Caspase pathway.Asparaginase can induce the appearance of autophagosomes in laryngeal squamous cell carcinoma,induce LC-II expression,and produce autophagic flux.Asparaginase-induced inhibition of Akt / mTOR and activation of signaling pathway Erk1 / 2,suggesting that Akt / mTOR signaling pathway and Erk1 / 2 signaling pathway are involved in asparaginase-induced laryngeal cell autophagy.It is noteworthy that the combination of autophagic inhibitor CQ in laryngeal cancer cells significantly enhanced the asparaginase on laryngeal cancer cells Tu212 and Tu686 growth inhibition and apoptosis.This suggests that autophagy plays a protective role in the process when asparaginase was exposed on laryngeal cancer cells.Antioxidant NAC can significantly inhibit asparagine-induced cell autophagy,and asparagine-induced cytotoxicity is also significantly reduced,indicating that ROS plays an important role in asparaginase-induced autophagy and cytotoxicity.In a word: Asparaginase has significant cytotoxicity to laryngeal cancer cells Tu212 and Tu686.Asparaginase can induce autophagy in laryngeal cancer cells.Inhibiting cell autophagy significantly enhanced the killing effect of asparaginase on laryngeal squamous cell.Therefore,for the treatment of laryngeal cancer to provide new ideas.