Effect Of Lycorine On Proliferation,autophagy And Apoptosis Of K562 Cells Resistant To Imatinib And Its Mechanism

Purpose: In this research,we investigated the effects of lycorine on the proliferation,apoptosis,and autophagy of K562 cells and imatine-resistant K562 cells,and preliminarily elaborated the possible molecular mechanism of lycorine in reversing imatine-resistant chronic leukemia from the level of autophagy,providing new ideas for clinical reversal of the drug resistance of targeted drugs for chronic myelogenous leukemia.Methods: The resistant K562/IM cell line was established by gradient induction.The sensitivity of K562 cells and K562/IM cells to imatinib and the morphological changes of K562/IM cells were detected by CCK-8 assay.The differential genes,biological processes and signaling pathways related to K562 cells and K562/IM cells were analyzed by bioinformatics.K562 cells were cultured in vitro,and the proliferation activity of K562 and K562/IM cells treated with lycorine or lycorine combined with imatinib was detected by CCK-8 assay;the autophagy of K562 cells treated with lycorine or rapamycin was detected by dyestyra mine（MDC）staining;the protein expression changes of Beclin-1,Atg5,LC3,and Caspase-3 in K562 cells treated with lycorine combined with autophagy agonists or inhibitors were detected by Western blot;the differential genes and biological processes and signaling pathways involved in K562/IM cells treated with lycorine and lycorine were analyzed using bioinformatics.The proliferation activity of K562/IM cells treated with lycorine or lycorine combined with imatinib and autophagy agonist/inhibitor was detected by CCK-8 assay,and the apoptosis of K562 cells and K562/IM cells treated with lycorine was detected by flow cytometry and apoptosis assay;MDC staining was used to detect autophagy in Lycorine treated K562 and K562/IM cells;Western blot was used to discover changes in P62,Beclin-1,Atg5,LC3,Bax,Bcl-2,Caspase-3,and P-gp protein expression in K562 cells and K562/IM cells.Results: The resistant cell line K562/IM was successfully constructed.The outcomes of CCK-8 demonstrated that the resistance index of K562/IM cells was 55.86 times higher than that of K562 cells after 72 hours of treatment（P<0.05）.Microscopic observation of cell morphology indicated that K562 cells displayed cell shrinkage,increased cytoplasmic granules and decreased cell volume,K562/IM（3μM）cells had smooth surface,and no obvious change in cell morphology after 72 hours of treatment.Bioinformatics analysis showed that compared with K562 group,K562/IM autophagy-related gene SESN3 was up-regulated,and differential genes were enriched in autophagy-related PI3K/Akt/MAPK/RAS and other pathways.The results of CCK-8 demonstrated that after 24 hours and 48 hours of lycorine treatment,K562 cell proliferation was inhibited in a dose-dependent pattern（P<0.05）;after 24 h,48h,and 72 h of lycorine combined with imatinib treatment,K562 cell and K562/IM cell proliferation was inhibited（P<0.05）;MDC staining results demonstrated that the fluorescence intensity of K562 cells treated with the autophagy inducer rapamycin was enhanced after 24 h,and the fluorescence intensity of K562 cells treated with lycorine was attenuated after 24 h.Western blot results revealed that after 24 h of lycorine treatment,Beclin-1,Atg5,and LC3 II protein expression was declined（P<0.05）,and Caspase-3 protein expression was raised（P<0.05）;bioinformatics analysis indicated that compared with K562/IM,lycorine treatment of K562/IM histophagy-related gene SQSTM1（P62）was up-regulated,and differential genes were enriched in autophagy-related PI3K/Akt/MAPK pathways.The results of CCK-8 revealed that after 24 h and 48 h of lycorine treatment,K562/IM cell proliferation was inhibited in a dose-dependent manner（P<0.05）;after24h and 48 h of lycorine combined with imatinib and autophagy agonists/inhibitors treatment,K562/IM cell proliferation was obviously inhibited,and the cell survival rate was the lowest in the combination group containing hydroxychloroquine（P<0.05）;the results of flow cytometry demonstrated that the cell cycle distribution of K562 and K562/IM was markedly arrested in G0/G1 phase after 24 h of lycorine treatment（P<0.05）;the outcomes of flow cytometry demonstrated that the apoptosis rate of K562 and K562/IM cells was significantly enhanced after 24 h of lycorine treatment（P<0.05）;the outcomes of MDC staining displayed that the fluorescence intensity of K562 and K562/IM cells was attenuated after 72 h of lycorine treatment,and the fluorescence intensity of K562/IM cells was higher than that of K562 cells;Caspase-3,P62,Bax/Bcl-2,and Bax protein expression was increased（P<0.05）,P-gp protein expression was declined in K562/IM cells（P<0.05）,and P-gp protein was not detected in K562 cells.Conclusions:1.The resistant cell line K562/IM was successfully constructed.Combined with differentially expressed genes and KEGG pathway enrichment analysis,it is suggested that the mechanism of resistant cell line K562 may be related to autophagy.2.Lycorine effectively inhibited the proliferation of K562 cells and K562/IM cells,arrested the cell cycle in GO/G1 phase,and induced apoptosis in vitro,and the mechanism may be related to the reduction of Bcl-2 expression,stimulation of Caspase-3 pathway,and thus exaltation of Bax/Bcl-2 expression.Lycorine inhibited autophagy thereby reversing drug resistance in K562/IM cells,and the mechanism may be related to down-regulation of autophagy pathway proteins Beclin-1,Atg5,LC3-II and up-regulation of p62 expression and then down-regulation of resistance-associated protein P-gp expression.