Study On The Mechanism Of Acridine Derivative A23 Against Multidrug Resistance In K562/ADM

Background: Chemotherapy is one of the most important methods for cancer therapy,while multidrug resistance is the primary cause of the chemotherapy failure.The genomic instability of chronic myelogenous leukemia or ant other reasons lead to the resistance to chemotherapeutic drugs.Acridine derivatives are capable of interacting with double-stranded DNA by their inherent structural features,therefore they are used in wide therapeutic domains.There was more and more research on apply of acridine derivatives in antitumor for the past few years.Objective: In this study,the effect of acridine derivative A23 on K562/ADM,which is lymphoblast with character of multidrug resistance from bone marrow of chronic myelogenous leukemia patient will be evaluated,and the mechanisms of overcoming mutildrug resistance will be explored.Methods: MTT assay was used to certify the multidrug resistance of K562/ADM,and to detecte the growth inhibition of A23 in K562/ADM and the effect of 3-MA,NAC or GSH on the growth inhibition of K562/ADM after treated with A23.Inverted microscope was used to observe morphological change of K562/ADM after treated with A23.HCS was used to observe morphological change of K562/ADM nucleus stained by hoechst 33342 after treated with A23.FITC Annexin V/PI,Rh 123,MDC and Lyso-Tracker Red staining were used to study the change of apoptosis ratio,mitochondrial membrane potential and autophagy ratio in K562/ADM after treated with A23,measured by FCM.FITC Annexin V/PI,MDC and Lyso-Tracker Red staining were used to study the effect of 3-MA,NAC or GSH on the apoptosis ratio and autophagy ratio in K562/ADM after treated with A23,measured by FCM.Rh 123/Hoechst 33342,MDC,Lyso-Tracker Red/ Hoechst 33342,DCFH-DA/ Hoechst 33342,DHE/ Hoechst 33342 staining were used to study the chang of the function of P-gp,autophagy ratio and the level of ROS in K562/ADM after treated with A23,measured by HCS.DCFH-DA/ Hoechst 33342 and DHE/ Hoechst 33342 staining were used to study the effect of 3-MA,NAC or GSH on the level of ROS in K562/ADM after treated with A23,measured by HCS.Western blotting was used to detected the change of autophagy related proteins(ATG5,ATG7,Beclin-1,LC3-II)and apoptosis related protein(Caspase-3,Caspase-9,Bax,Bcl-2,XIAP),cytochrome c in K562/ADM after treated with A23 in the presence or absence of 3-MA,NAC or GSH.Results: 1.We demonstrateed that A23 had anti-proliferative effects in time and dose dependent manners on K562/ADM,which was multidrug resistant,without impact of P-gp function.2.Apoptotic body,nuclear condensation and breakage,mitochondrial membrane potential decreasing were obversed in K562/ADM after incubated with A23.A23 induced apoptosis on K562/ADM with the change of apoptotic protein expression levels in time and dose dependent manners.3.A23 induced autophagy on K562/ADM with the change of autophagy protein expression levels in time and dose dependent manners.After inhibition of autophagy,apoptotic ratio and pro-apoptotic protein expression levels had lower trend and anti-apoptotic protein expression levels had higher trends,which meaned the autophagy induced by A23 promoted apoptosis in K562/ADM.4.A23 could increase the amount of reactive oxygen species(ROS)in dose dependent manner.After scavenging of ROS with antioxidant,apoptotic ratio and pro-apoptotic protein expression levels had lower trend and anti-apoptotic protein expression levels had higher trends,which meaned the ROS induced by A23 promoted apoptosis in K562/ADM.5.After scavenging of ROS with antioxidant,which was induced by A23 in K562/ADM,autophagy ratio and autophagy protein expression levels decreased,which meaned ROS promoted autophagy.6.After inhibition of autophagy,the level of ROS showed a decreased tendency in K562/ADM after incubated with A23,which meaned autophagy promoted producing ROS.Conclusions: Acridine compound A23 showed significantly cytotoxic effects on K562/ADM which was mutildrug resistant.The death of K562/ADM was via apoptosis,which was initiate by ROS and induced by aotophagy,which was the result of A23 overcoming multidrug resistance.