Preliminary Study On The Mechanism Of Action Of Maduramycin On A549 Cells

Lung cancer remains at the forefront of global cancer incidence and mortality.The incidence of non-small cell lung cancer is very high,accounting for about 85% of lung cancer incidence.In fact,non-small cell lung cancer is a great threat to human life and health.Studies have shown that some polyether ionophore antibiotics have anti-tumor activity and inhibit cancer stem-like cells growth.Therefore,this study selected maduramycin as the test drug,in order to explore the anticancer activity of maduramycin and its mechanism.In this study,A549 cells were used as the main research object to analyze the anticancer activity and explore its mechanism.Firstly,MTT assay,colony formation assay,microscope observation were used to explore the effect of maduramycin on cell proliferation;Wound Healing and Transwell cell migration assay were used to explore the effect of maduramycin on cell migration.Secondly,The mechanism of apoptosis induced by maduramycin was preliminarily investigated.The apoptosis morphology of cells were observed by Acrdine orange staining and hematoxylin-eosin staining.Annexin V-FITC/PI was used to analyze the apoptosis rate after maduramycin treatment.The effect of some caspase enzymes on apoptosis was investigated by caspase activity detection and additional caspase inhibitor detection of cell viability.The reactive oxygen assay kits were used to detect ROS levels.The changes of apoptosis-related factors at the protein level were analyzed by Western blot.Finally,the effect of maduramycin on autophagy was preliminarily analyzed by Western blot.The experimental results showed that compared with Wi-48 cells,maduramycin has a stronger cytotoxic effect on A549 cells,obviously inhibits the proliferation and migration of A549 cells,and is time-dose-dependent.After maduramycin treatment,it could be observed that A549 cells have apoptosis characteristics in the cell morphology observation experiment.The late apoptosis of the cells and the apoptosis rate increased with the increase of maduramycin concentration detected by Annexin V-FITC/PI.The activities of caspase-3/4/8/9 enzymes significantly increased except for caspase-6,and the inhibitory ability of maduramycin against cell proliferation was decreased after the addition of caspase-3/4/8/9 enzymes inhibitor.The level of ROS was increased.Maduramycin resulted in a dose-dependent activation of cleavage of PARP.Furthermore,the expression of apoptosis-related protein Bax increased,and the expression of anti-apoptotic protein Bcl-2 decreased.Besides,autophagy related proteins,ULK,Beclin1,ATG5 and ATG7 protein expression lowered in different degrees,but the expression of LC3-?/LC3? were activated.Conclusions:1.Maduramycin could inhibit the proliferation and migration of A549 cells;2.Maduramycin could induce apoptosis in A549 cells,and the process might be related to mitochondrial pathways;3.Maduramycin blocks autophagy flux in A549 cells.