Experimental Study On Inhibition Of Notch-1 Pathway And Arsenic Trioxide（As₂O₃） In Reversing Drug Resistance Of HepG2/ADM Cells

Objective:Researching the biocharacteristics,drug resistance and angiogenic mimic（VM）formation ability of As₂O₃（IC50）dose combined with Notch-1 pathway blocker（γ-secretase inhibitor MW167）on human hepatoma HepG₂ cells and HepG₂/ADM resistant cells The influence of the effect on the Notch-1signaling pathway was further explored to further investigate the mechanism of As₂O₃ reversing the multidrug resistance of tumors,and provide experimental data for enriching the multidrug resistance of tumors.Method:The human hepatoma HepG₂ cells and HepG₂/ADM resistant cells were divided into four groups（blank group,As₂O₃ group,MW167 group,As₂O₃+MW167 combined group）,and MTT assay was used to detect cell proliferation inhibition rate under different protocols;Transwell The chamber method was used to determine the invasive ability of the two groups under different protocols.The scratch healing experiment was used to determine the cell migration ability of the two groups under different protocols.2.HepG₂ cells and HepG₂/ADM resistant cells were cultured in vitro to construct VM,and VM was identified by PAS staining.The VMs constructed from hepatocellular carcinoma HepG₂ cells and HepG₂/ADM resistant cells were used alone and in combination,and the number,length and area of VMs were recorded.HepG₂ cells and HepG₂/ADM resistant cells were cultured,and the gel was inoculated for 2 hours.Interventions were performed using different drug treatment groups.After 48 h of inoculation of the drug,an inverted microscope was observed to record the development of changes in the number,length and area of the VM.Western-blot was used to detect the expression of related proteins such as Vimentin,VE-cadherin,MMP-2,Mcl-1,MDR-1,Cyclin D₁,Notch-1,Hes-1 and NICD.4.Statistical software spss19.0 analysis data.Result:The proliferation inhibition rate of cells was detected by MTT assay,and the treatment group was higher than the control group（p<0.05）.The cell invasion ability was measured by Transwell chamber:combination group<As₂O₃<γ-secretase inhibitor MW167 group<Control group（p<0.05）;cell migration ability:combination group<As₂O₃<γ-secretase inhibitor MW167 group<control group（p<0.05）.2.Construction of human hepatocellular carcinoma HepG₂ and HepG₂/ADM cells VM under different therapeutic regimens,VM three-dimensional culture results showed that the number,length,and area of VMs of HepG₂ and HepG₂/ADM cells were observed under high power microscope after 48H treatment.The combination group<As₂O₃<γ-secretase inhibitor MW167 group<control group（p<0.05）.The results of western-blot showed that the expression levels of Notch-1,Hes-1,NICD and protein were decreased in the blank group,and the expressions of MDR-1 and Mcl-1 were decreased（p<0.05）.The expression of the death-related protein Cyclin D₁was decreased（p<0.05）.（VM）vascular mimicry and（EMT）epithelial-interstitial-associated protein VE-cadherin expression increased,MMP-2,Vimentin expression decreased（p<0.05）.The expression of each protein in the combined group was compared with the control group（p<0.01）.In conclusion:1.As₂O₃（IC50）combined with MW167 reverses the drug resistance of liver cancer HepG₂/ADM cells and is closely related to reducing the occurrence of EMT in vitro,reducing the ability of cell migration and invasion,and inhibiting the occurrence and development of three-dimensional constructing VM in vitro.2.As₂O₃（IC50）and Notch-1 pathway inhibitor-MW167 can reverse the drug resistance mechanism of liver cancer HepG₂/ADM cells.There may be a common target or a common pathway effect.