Impacts of Evodiamine on Rituximab Resistance in Diffuse Large B-Cell Lymphoma Cells by Regulating the Shh/Gli1 Signaling Pathway
Objective:To investigate the impacts of evodiamine on rituximab(RIT)resistance in diffuse large B-cell lymphoma(DLBCL)cells by regulating the Shh/Gli family zinc finger protein 1(Gli1)signal pathway.Methods:OCI-LY10 cells were cultured in vitro and their RIT resistant cell line OCI-LY10/RIT was constructed using gradient drug addition method.All cells were treated with 0,1,5,10,20 and 30 μmoL/L of evodiamine,CCK-8 method was applied to determine the activity of OCI-LY10 and OCI-LY10/RIT cells in each group and to screen the optimal concentration of evodiamine.OCI-LY10/RIT cells were ran-domly grouped into control group,RIT group,RIT+evodiamine group,RIT+empty group,and RIT+evodia-mine+Shh overexpression group,after grouping and processing,real-time fluorescence quantitative PCR and immunoblotting experiments were applied to detect the expression of Shh/Gli1 pathway related mRNA and pro-tein of cells in each group;CCK-8 method and Edu staining were applied to detect cell proliferation in each group;flow cytometry was applied to detect cell apoptosis in each group;Western blot was applied to detect the expression of apoptotic proteins(Cleaved Caspase-3,Bax)and drug resistance proteins[multiple drug resistance related protein 5(MRP5),P-glycoprotein(P-gp)]in each group.OCI-LY10/RIT cells were cultured in vitro and randomly grouped into a control group,a evodiamine group,an empty group,and a evo-diamine+Shh overexpression group,after grouping and processing,the CCK-8 method was applied to detect the survival rate of OCI-LY10/RIT cells in each group under 0,16,32,64,128,256 and 384 μg/mL RIT treatment,and calculate their drug resistance index.Results:Compared with the control group,the apoptosis rate,the expression of Cleaved Caspase-3 and Bax proteins in the RIT+evodiamine group increased(P<0.05),the expression of Shh,Gli1 mRNAs and proteins,survival rate,Edu positive rate,and the expression of MRP5 and P-gp proteins decreased(P<0.05);there was no obvious change in all cell indicators in the RIT group and the RIT+empty group(P>0.05).Compared with the RIT group,the apoptosis rate,the expression of Cleaved Caspase-3 and Bax proteins in the RIT group and the RIT+empty group increased(P<0.05),the expression of Shh,Gli1 mRNAs and proteins,survival rate,Edu positive rate,and the expression of MRP5 and P-gp proteins decreased(P<0.05);there was no obvious change in all indicators of cells in the RIT+ empty group(P>0.05).Compared with the RIT+evodiamine group,the apoptosis rate,the expression of Cleaved Caspase-3 and Bax proteins in the RIT+evodiamine+Shh overexpression group decreased(P<0.05),the expression of Shh,Gli1 mRNAs and proteins,survival rate,Edu positive rate,and the expression of MRP5 and P-gp proteins increased(P<0.05).Compared with the control group,the cell resistance index in the evodiamine group decreased(P<0.05),the drug resistance index of cells in the empty group showed no obvious change(P>0.05);compared with the Evodiamine group,the evodiamine+Shh overexpression group showed an increase in cell resistance index(P<0.05).Conclusion:Evodiamine can down-regulate the ex-pression of Shh/Gli1 pathway related proteins,thereby reducing RIT resistance in DLBCL cells,inducing ap-optosis and inhibiting proliferation of RIT resistant DLBCL cells under RIT treatment.
Diffuse large B-cell lymphomaEvodiamineShh/Gli1RituximabResistance
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