Effects and Mechanism Study of Kudinoside D on Proliferation,Apoptosis and Autophagy of Human HCC-1806 Breast Cancer Cells
Objective To investigate the effect and mechanism of kudinoside D(KD-D)on proliferation,apoptosis and autophagy of human HCC-1806 breast cancer cells.Methods Human HCC-1806 breast cancer cells were treated with different concentrations of KD-D,and the cell viability was detected by CCK-8 method.EdU method was used to detect cell proliferation ability;the ability of cell clone formation was detected by crystal violet staining.Apoptosis was detected by flow cytometry(Annexin V-FITC/PI).Mitochondrial membrane potential was detected by JC-1 staining.The protein expressions of Cleaved Caspase-3,LC3 and P62 were detected by immunofluorescence.The protein expressions of Cleaved Caspase-3,Pan-AKT,Phosp-AKT and LC3Ⅱ were detected by Western Blot.Autophagy double-labeled mRFP-EGFP-LC3 adenovirus infection assay was used to detect cell autophagy flow.Results Compared with the control group,HCC-1806 cells were treated with 12.5,25,50,100,200,400 μmol·L-1 KD-D for 24 and 48 hours.With the increase of drug concentration and treatment time,the cell activity was significantly decreased(P＜0.001),the intracellular absorbance value was significantly decreased(P＜0.001),and the cell proliferation was inhibited.The cell clone formation counts in 60 and 80 μmol·L-1 KD-D groups were significantly decreased(P＜0.001).The proportion of early and late apoptosis in 50,100,150 μmol·L-1 KD-D groups were significantly increased(P＜0.05,P＜0.001).The proportion of red fluorescence in 40,60 and 80 μmol·L-1 KD-D groups were significantly decreased(P＜0.001),the proportion of green fluorescence was significantly increased(P＜0.01,P＜0.001),and the mitochondrial membrane potential of HCC-1806 cells decreased.The protein expression of Cleaved Caspase-3 in 60,80,100 μmol·L-1 KD-D group were significantly up-regulated(P＜0.001),and the protein expressions of Pan-AKT and Phosp-AKT in 60 μmol·L-1 KD-D group were significantly up-regulated(P＜0.001).In the 60 μmol·L-1 KD-D group,the number of LC3 protein fluorescent dots was significantly increased(P＜0.001),the average fluorescence intensity of P62 protein was significantly decreased(P＜0.001),the expression of LC3Ⅱ protein was significantly up-regulated(P＜0.001),the number of autophagosomes and autolysosomes were significantly increased(P＜0.01,P＜0.001),and the autophagic flow was activated.Compared with 60 μmol·L-1 KD-D group,the expression of Cleaved Caspase-3 and Pan-AKT protein in KD-D+AKT inhibitor(Afuresertib)group was significantly down-regulated(P＜0.01,P＜0.001),and the expression of Phosp-AKT protein was significantly up-regulated(P＜0.001).Conclusion KD-D can inhibit the proliferation of human breast cancer HCC-1806 cells and induce apoptosis and autophagy.The apoptosis of HCC-1806 cells induced by KD-D may be related to the activation of AKT signal and the expression of Cleaved Caspase-3.
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