Chrysin Induces Autophagy By Promoting ROS-mediated Akt/mTOR Signaling Pathway Inactivation In Endometrial Cancer

Endometrial cancer tends to be an aggressive malignancy,and limited therapeutic options and poor prognosis for patients with advanced-stage force researchers to search for effective treatment alternatives.Chrysin is a natural flavonoid,which has been proved to have significant anti-cancer effect in other cancers.However,the role of chrysin in endometrial cancer and its related mechanism remains largely unclear.Objective: To explore the anti-cancer mechanism of chrysin in endometrial cancer cells.Methods: Human endometrial cancer cells HEC-1A and Ishikawa were cultured for the following experiments:1.Cell viability assay,clone formation assay and flow cytometry analysis were used to detect the effect of chrysin on the proliferation and apoptosis of the two cell lines.2.The autophagosomes and autophagosolysosomes of two cell lines treated with chrysin were observed by transmission electron microscopy.3.The fluorescence intensity of LCB of the two cell lines treated with chrysin was observed by immunofluorescence.4.Western blotting analysis was used to detect the expression of autophagy related proteins under the action of chrysin alone or CQ.5.The cell viability assay and flow cytometry were used to detect the proliferation ability and apoptosis of the two cell lines under the action of chrysin combined with CQ.6.ROS detection kit and western blotting analysis were used to detect the expression of ROS and autophagy related proteins under the action of chrysin alone or in combination with NAC.7.Western blotting analysis was used to detect the expression of chrysin in autophagyrelated AKT/mTOR pathway proteins.Results:1.The results of cell viability assay,clone formation assays and flow cytometry analysis showed that chrysin inhibited proliferation and induced apoptosis in EC cells.2.The results of transmission electron microscopy showed that the number of autophagosomes and autophagosolysosomes in EC cells increased.3.The results of immunofluorescence experiments showed that LC3 intracellular localization and abundant LC3 spots were detected in the EC cells treated with chrysin,while the control group did not show significant fluorescence intensity.4.The protein expressions of LC3 B Ⅱ and Beclin 1 in EC cells were up-regulated,and the protein expression of P62 was down-regulated.The expression of LC3 B Ⅱ was further elevated after the combination of CQ.5.The results of cell viability assay and flow cytometry showed that the inhibition of proliferation and induction of apoptosis of EC cells were further enhanced after the addition of CQ.6.Chrysin promoted ROS accumulation in EC cells.The expression of LC3 B Ⅱ was down-regulated after addition of NAC.7.Chrysin inhibited the expression of proteins related to the Akt/mTOR pathway.After adding NAC,the expression of pathway-related proteins increased.Conclusion: Chrysin induced autophagy by promoting ROS-mediated Akt/mTOR signaling pathway inactivation in EC cells,and combination treatment with autophagy inhibitors further enhanced the anti-cancer effect of chrysin.