The Mechanism Of Celastrol Enhancing The Sensitivity Of Dox In Leukemia Multi-drug Resistant K562/ADR Cell

Objective:In this study,the anti-leukemia effect of Celastrol on human leukemia MDR line K562/ADR cells and the mechanism of enhancing the sensitivity of K562/ADR cells to Dox were studied to provide experimental data for exploring new therapeutic drugs for leukemia.Methods:Human leukemia cell line K562 and its MDR line K562/ADR were cultured in vitro.Screening differentially expressed mRNAs by RNA sequencing of K562 and K562/ADR cells;K562 and K562/ADR cells were treated with different concentrations of Dox and Celastrol,respectively,and cell viability was detected by CCK-8 assay after 48 hours.The apoptosis of K562/ADR cells was detected using flow cytometry.The expression levels of death-related genes Bcl-2 and Bax were detected by qRT-PCR and Western Blot.The cell cycle of K562/ADR cells was detected using flow cytometry CCK-8 assay was performed to detect the viability of K562/ADR cell which was treated by Dox coupled Celastrol after 48h.K562/ADR cells were treated with Celastrol to detect the correlation between the expression of drug resistance-related genes and drug concentration.Results:1.RNA-Seq detected up-regulated and down-regulated mRNAs of 751and 1050 respectively in differentially expressed mRNAs;Treatment of K562and K562/ADR cells with different doses of Dox,the IC₅₀ values were 0.04and 11.328?M,which suggests that K562/ADR cells were resistant to Dox.Celastrol inhibited the growth of K562 and K562/ADR cells in a dose-dependent manner.With the increase of the Celastrol concentration,the viability of K562 and K562/ADR cells gradually decreased.The IC₅₀ values of Dox for K562 and K562/ADR cells were 1.91 and 2.72?M at 48h,respectively.2.There was a significantly increased population of apoptotic cells in K562/ADR treated with Celastrol for 48h,compared with the untreated cones.Additionally,Bcl-2 was significantly down-regulated and Bax was markedly up-regulated in K562/ADR cells after Celastrol treatment.3.Celastrol induced phase arrest after treatment with celastrol for 48h,compared with untreated cells.4.After 48 hours of combined treatment with different concentrations of Celastrol and Dox,the viability of K562/ADR cells in the combined treatment group was significantly inhibited,compared with the single treatment group.5.Celastrol down-regulated the expression of ABCB1 dose-dependently.With the increase of the Celastrol dose,the mRNA level of ABCB1 gradually decreased.Conclusion:1.Celastrol can exert anti-K562/ADR cell leukemia therapy by inhibiting cell viability,promoting cell apoptosis,inducing cell cycle arrest,and reversing drug resistance.2.The anti-leukemia effect of Celastrol may be closely related to inhibiting ABCB1,Bcl-2,up-regulating Bax expression,and inducing leukemia cell apoptosis.