Study On The Mechanism Of Multi-Drug Resistance Of Agaricus Blazei Extract FA-2-b-β To Reverse Acute T Lymphoblastic Leukemia

OBJECTIVE:To investigate the mechanism of reverse drug resistance of Agaricus blazei extract FA-2-b-β（acidic RNA-protein complex）on T cell acute lymphoblastic leukemia（T-ALL）.METHODS:FA-2-b-β（0,0.6,1.2,1.8,2.4,3,3.6 mg/m L）intervened on CCRF-CEM（sensitive strains）and CEM/C1（multi-resistance strains）of T-ALL.The cell proliferation inhibition rate was detected by CCK-8 assay.Flow cytometry was used to detect apoptosis,mitochondrial membrane potential,cell cycle,and intracellular rhodamine accumulation;q RT-PCR was used to detect ABCB1 and ABCG2（drug resistance-related genes）,CTNNB1and C-myc（Wnt/β-catenin signaling pathway-related genes）,BAX and BCL-2（apoptosis-related genes）;Western blot was used to detect the corresponding encoded proteins MDR1,BCRP,β-catenin,c-Myc,BAX,BCL-2;Immunofluorescence observation of membrane surface protein MDR1 and Cytochrome C in cytoplasmic.RESULTS:Different concentrations of FA-2-b-βsignificantly inhibited the proliferation of CCRF-CEM and CEM/C1 cells with time-concentration-dependent inhibition,and the r correlation coefficient values were-0.992 and-0.976 at 48h,respectively.Flow cytometry detection of apoptosis revealed that FA-2-b-β（1.2,1.8,and 2.4 mg/m L）promoted apoptosis on CCRF-CEM and CEM/C1 cells,with early and late apoptosis rates of 9.19/12.2%,20.0/35.2%,and 29.8/50.6%on CCRF-CEM and 42.2/17.7%,47.0/17.4%,and 51.1/19.6%.Different concentrations of FA-2-b-β（0,1.2,1.8,2.4 mg/m L）acting on CCRF-CEM cells,and blocked on S phase,which was 32.81%,39.3%,45.02%,51.18%,and CEM/C1 cells have been blocked on G₀/G₁ phase,which was 29.33%,36.68%,43.23%,and 52.06%,respectively.The expression of ABCB1,ABCG2,CTNNB1,C-myc,and BCL-2 was gradually decreased and BAX expression was gradually increased by using RT-q PCR for FA-2-b-βacting on CEM/C1 cells.Western blot revealed that MDR1,BCRP,β-catenin,c-Myc,and BCL-2 expression gradually decreased and BAX gradually increased.Flow cytometry revealed the mitochondrial membrane potential depolarization（red/green fluorescence ratio）was decreased by FA-2-b-β（0,1.2,1.8,2.4 mg/m L）on CEM/C1 cells,the ratio was 0.81,0.65,0.53,0.44,respectively.Immunofluorescence suggested that Cytochrome C in the cytoplasm of CEM/C1 cells gradually increased.In addition,Wnt pathway inhibitor ICG001（1μmol/L）significantly decreased the drug resistance genes ABCB1 and ABCG2;and revealed a significant decrease in rhodamine efflux.Immunofluorescence shows that the membrane surface protein MDR1 fluorescence was significantly diminished.CONCLUSION:Agaricus Blazei Extract FA-2-b-βinhibits cell proliferation,promotes apoptosis,regulates the cell cycle,reduces mitochondrial energy supply,and down-regulates MDR1 expression to reverse the resistance of CEM/C1,which all suggest it is through regulating the Wnt/β-catenin signaling pathway in T-ALL.