Effect Of Sulfonylmorpholine Hydrazone 6p On Esophageal Cancer Cell Apoptosis And Its Underlying Mechanisms

Background: Esophageal cancer is a common malignant tumor of digestive tract,with a high incidence in China,expecially in Henan province.Currently,5-Fluorouracil and doxorubicin are the main drugs used for esophageal cancer in clinic.However,these drugs have strong side effects and are easy to induce drug resistance in esophageal cancer cells.Therefore,it is necessary to find new and more selective small molecular compounds for the treatment of esophageal cancer.In recent years,some acylhydrazone compounds have been found to be promising candidates for the research of anti-tumor small molecules.Therefore,our research group synthesized 20 acylhydrazone derivatives and screened out compound 6p by MTT method which showed great cytotoxicity on esophageal cancer cells.So we investigated the effect and mechanism of compound 6p in this study.Methods: We detected the effects of these 20 acylhydrazone compounds on the survival rates of esophageal cancer(EC9706 and EC109),liver cancer(SMMC-7721),prostatic cancer(PC3),breast cancer(MCF7),gastric cancer(MGC803)by MTT.The effect of compound 6p on the clone formation of EC9706 and EC109 in esophageal cancer cells was detected by cloned formation and crystal violet staining.The effect of compound 6p on the cell cycle of EC9706 and EC109 cells in esophageal cancer cells was detected by the PI staining andflow cytometry.The morphological changes of the apoptosis of EC9706 and EC109 in esophageal cancer cells induced by compound 6p were detected by Hoechst 33342 staining and fluorescence microscopy.Annexin-V/FITC /PI staining and flow cytometry were used to detect the effect of compound 6pon the apoptosis of EC9706 and EC109 in esophageal cancer cells,and we also use the Caspase inhibitor Z-VAD-FMK to reverse the effect of compound 6p.The effect of compound 6p on ROS production in EC9706 and EC109 of esophageal cancer cells were detected by DCFH-DA probe staining and flow cytometry,meanwhilewe use the reactive oxygen scavenger NAC to reverse the action of the compound6 p.The effects of compound 6p on the mitochondrial membrane potential of EC9706 and EC109 in esophageal cancer cells were detected by JC1 staining and fluorescence microscopy.The effects of compound 6p on EC9706 and EC109 cycle and apoptosis related proteins in esophageal cancer cells were detected by Western Blot.The safety of compound 6p was evaluated by mice acute toxicity test and HE staining.Results: 1.The 20 acylhydrazones compounds showed significant inhibitory effects on a variety of tumor cells,and compound 6p had a strong inhibitory effect on EC9706 and EC109 in esophageal cancer cells in time and concentration dependent way.2.Compound 6p can significantly inhibit the clonal formation ability of EC9706 and EC109 cells in a concentration dependent manner.Compound 6p induced G0/G1 phase arrest in esophageal cancer cells EC9706 and EC109,and increased the expression of cyclin related protein P27.3.Compound 6p can reduce the mitochondrial membrane potential of EC9706 and EC109 cells,induce the increase of active oxygen level in EC9706 and EC109 cells,induce apoptosis of esophageal cancer cells,and reactive oxygen scavenger NAC can reverse the increase of reactive oxygen species and apoptosis induced by compound 6p.4.Compound 6p induced the increase of expression of apoptotic protein P53,Bax and t Bid,the decrease of the expression of anti apoptotic protein Bcl2,and the activation of Caspase8/9/3 and PARP,which did not affect the expression of antioxidant family protein Prxs.5.The high dose of compound 6p used in mice does not affect the survival of mice and there is no obvious damage to the viscera of mice.Conclusion: The acylhydrazone compound 6p can increase the expression of cyclin protein p27 in the EC9706 and EC109 of esophageal cancer cells,block esophageal cancer cells in G0/G1 phase,which inhibit cell proliferation.Compound 6p can reduce the mitochondrial membrane potential and induce the production of reactive oxygen species in the EC9706 and EC109 cells,but it does not affect the expression levels of the Prx family.Compound 6p can increase the cleavage of Caspase-9 and Caspase-8,and lead to activation of mitochondria and death receptor apoptosis pathway,resulting in apoptosis of EC9706 and EC109 cells.Compound 6p also has certain biosafety.Compound 6p could be used as a lead compound for further optimization in esophageal cancer.