Study On The Inhibitory Effect Of Kudinoside D In The Leaves Of Ilex Latifolia On Breast Cancer Cells And Its Mechanism

Objective:To examine the effect of the triterpenoid saponin compound Kudinoside D(KD-D)in the leaves of Ilex latifolia on the proliferative activity of breast cancer cells and the intervention of KD-D in the programmed cell death pathway,to explore the possible mechanisms of action and to provide new strategies for the clinical treatment of breast cancer,as well as to provide a scientific basis and experimental foundation for the research of new targeted drugs.Methods:In this experiment,HCC-1806 and MDA-MB-468 breast cancer cells were used as the study subjects.The proliferation inhibitory effects of KD-D on different breast cancer cell lines were detected by CCK8 and EDU methods.The effects of KD-D on the morphology of breast cancer cells were observed by inverted microscope.The effects of KD-D on cell clone formation ability were detected by plate cloning assay.The effects of KD-D on cell migration ability were detected by scratch assay.The effects of KD-D on cell apoptosis were detected by Annexin V-FITC/PI assay.The effects of KD-D on cell mitochondrial membrane potential were detected by JC-1 staining.Immunofluorescence was used to detect the effect of KD-D on the protein expression of Cleaved Caspase-3,EGFR,HER3,HER4,LC3,P62,SREBP1 and SCAP in cells.Wsetern Blot was used to detect the effect of KD-D on the protein expression of Cleaved Caspase-3,PanAKT,Phosp-AKT,SREBP1,SCAP and LC3 II in cells.Biochemical kit was used to detect the effect of KD-D on high-density lipoprotein,low-density lipoprotein,total cholesterol,triglyceride content in cells.The autophagy double-labeled m RFP-EGFP-LC3 tandem fluorescent protein adenovirus was used to infect breast cancer cells to observe the effects of KD-D on autophagosome,autolysosome and autophagy flux in the cells.Immunofluorescence double-labeling assay was used to detect the effect of KD-D on the colocalization of ERBB family proteins in breast cancer cells.Result:1.CCK8 results showed that different concentrations of KD-D treated HCC-1806 and MDA-MB-468 cells for 24 h and 48 h,Cell activity was significantly inhibited(P<0.001)with increasing administration concentration and longer treatment time in a concentration-and time-dependent manner,indicating that KD-D has an inhibitory effect on proliferation activity of breast cancer cells.EDU results further showed that KD-D treatment significantly decreased the OD value(P<0.05),indicating that intracellular DNA synthesis was inhibited,suggesting that KD-D could significantly inhibit breast cancer cell proliferation.2.The results of cell cloning assay showed that KD-D inhibited the clone formation of HCC-1806 and MDA-MB-468 cells(P<0.001).3.The results of cell scratching assay showed that KD-D inhibited the migration of HCC-1806 and MDA-MB-468 cells(P<0.05).4.Annexin V-FITC/PI assay showed that KD-D induced apoptosis in HCC-1806 and MDA-MB-468 cells,and the proportion of early and late apoptosis was significantly increased(P<0.05).JC-1 staining showed that KD-D decreased the mitochondrial membrane potential in HCC-1806 and MDA-MB-468 cells(P<0.05).KD-D upregulated Cleaved Caspase-3 protein expression in HCC-1806,MDA-MB-468 cells(P<0.0001).KD-D upregulated Cleaved Caspase-3 expression in HCC-1806 cells was associated with activation of AKT signaling.5.KD-D upregulated LC3 II protein expression(P<0.0001)and downregulated P62 protein expression(P<0.0001)in HCC-1806 cells and increased the number of intracellular autophagosome and autolysosome(P<0.001),indicating that KD-D induced autophagy and activated autophagy flow in HCC-1806 cells.KD-D had no significant effect on LC3 and P62 protein expression in MDA-MB-468 cells(P>0.05).6.KD-D downregulated EGFR(P<0.05),HER3(P<0.001),HER4(P<0.0001)protein expression and attenuated EGFR-HER3 colocalization in MDA-MB-468 cells.KD-D upregulated HER3 protein expression in HCC-1806 cells(P<0.0001).7.KD-D upregulated Pan-AKT and Phosp-AKT protein expression in HCC-1806 cells(P<0.05).KD-D downregulated Pan-AKT protein expression in MDA-MB-468 cells(P<0.01),and had no significant effect on Phosp-AKT protein expression(P>0.05).8.KD-D upregulated SREBP1,SCAP protein expression in HCC-1806 cells associated with activation of AKT signaling.KD-D upregulated SREBP1,SCAP protein expression in MDA-MB-468 cells independent of PI3K/AKT/m TOR signaling pathway.KD-D downregulated triglyceride content in MDA-MB-468 cells(p<0.001),upregulated lowdensity lipoprotein(P<0.01)and total cholesterol content(P<0.01),and had no significant effect on high-density lipoprotein content(P>0.05).KD-D had no significant effect on triglyceride,low-density lipoprotein,high-density lipoprotein and total cholesterol content in HCC-1806 cells(P>0.05).Conclusion:1.KD-D inhibits the proliferation,cloning and migration of HCC-1806 and MDA-MB-468 cells.2.KD-D induces apoptosis in HCC-1806 and MDA-MB-468 cells,while KD-D upregulates Cleaved Caspase-3 protein expression in HCC-1806 cells is associated with activation of AKT signaling.3.KD-D induces autophagy in HCC-1806 cells.4.KD-D interferes with HCC-1806 and MDA-MB-468 cell death by affecting ERBBAKT-SREBP1-lipid metabolism pathway...