Mechanism Of Targeted Therapy With Berberine For Childhood Chemoresistant Acute Lymphoblastic Leukemia

Part I. Molecular Mechanism of Apoptosis Induced by Berberine in Childhood p53-Null Leukemia CellsObjective p53defects have been associated with chemoresistance in human cancer. Berberine has been shown to exert cell cycle arrest and pro-apoptotic activity in numerous cancer cell lines. The objective of this study is to investigate the molecular mechanism of apoptosis induced by berberine in childhood p53-null leukemia cells.Methods EU-4cells, a p53-null leukemia cell line, were chosen to carry out the experiments.(1) EU-4cells were incubated with different doses of berberine for72hours, and the final doses of berberine were0,1,10,50and100μmol/L, respectively. Then the cell viability of EU-4cells was detected by cell counting kit-8, the apoptosis rates were detected by flow cytometry, the morphology of apoptotic cells was analyzed by fluorescence microscopy, the expression levels of Bcl-2family protein (Bax and Bcl-xl) and X-linked inhibitor of apoptosis protein (XIAP) were tested by western blot, and caspase-3activity was measured using the caspase-3activity assay kit.(2) EU-4cells were incubated with100μmol/L berberine for0,24,48and72hours, respectively. The expression of PARP, caspase-3and XIAP were tested by western blot.(3) EU-4cells were pre-incubated with50μmol/L Z-VAD-FMK for30min, and the effect of berberine on apoptosis rate was detected by flow cytometry.(4) EU-4cells were incubated with different doses of doxorubicin for48hours, and the final doses of doxorubicin were0,0.1,0.5and1.0μmol/L, respectively. The expression of murine double minute2(MDM2) and XIAP were tested by western blot.(5) The XIAP expression was down-regulated by XIAP small interfering RNA (siRNA) or berberine, and the effects of XIAP down-regulation or combined with doxorubicin on apoptosis rates of EU-4cells were detected by flow cytometry.Results (1) Berberine induces potent dose-dependent apoptosis in EU-4cells (P<0.05), and induces the expression of apoptosis related protein.(2) Berberine induces caspase-dependent apoptosis, and increases caspase-3activity significantly (P<0.05).(3) Berberine down-regulates XIAP expression in a dose-and time-dependent manner.(4) Doxorubicin does not have pro-apoptotic effect on EU-4cells, and does not down-regulate the expression of MDM2and XIAP.(5) Inhibition of XIAP expression is responsible for the apoptotic effect on EU-4cells, and knockdown of XIAP increases the sensitivity of EU-4cells to doxorubicin induced apoptosis (P<0.05).Conclusions Berberine induces EU-4cells apoptosis in a p53-independent manner. Inhibition of XIAP leading to caspase-3activation is responsible for the apoptotic effect on EU-4cells. Part II. Mechanism of X-linked Inhibitor of Apoptosis Protein Expression Regulated by Berberine in Leukemia cellsObjective Berberine induces apoptosis in leukemia cells by regulation X-linked inhibitor of apoptosis protein (XIAP). The objective of this study is to investigate the mechanism of X-linked inhibitor of apoptosis protein expression regulated by berberine in leukemia cells.Methods (1) EU-4cells were incubated with100μmol/L berberine for0,24,48and72hours, respectively. Polymerase chain reaction (PCR) was performed to investigate the XIAP mRNA levels. After treatment with actinomycin D in the presence or absence of berberine, the degradation rate of XIAP mRNA was examined by pulse-chase assay.(2) EU-4cells were incubated with different doses of berberine for72hours, and the final doses of berberine were0,1,10,50and100μmol/L, respectively. Western blot was performed to investigate the expression of murine double minute2(MDM2) and phospho-MDM2and the cellular redistribution of MDM2. EU-4cells were incubated with100μmol/L berberine for0,24,48and72hours, respectively. MDM2mRNA was tested by reverse transcription-PCR.(3) The MDM2expression was down-regulated by MDM2small interfering RNA (siRNA). The effect of down-regulation MDM2on XIAP expression was tested by western blot.(4) After treatment with cycloheximide, the degradation rate of XIAP protein was examined by pulse-chase assay. Co-immunoprecipitation was performed to test XIAP ubiquitination after berberine treatment.Results (1) Berberine treatment does not regulate XIAP mRNA expression levels and stability.(2) Berberine down-regulates MDM2expression in a dose-and time-dependent manner. Berberine may regulate MDM2expression at the transcriptional level. Berberine decreases cytoplasmic MDM2expression.(3) Down-regulation MDM2by siRNA decreases XIAP expression.(4) Berberine promotes the ubiquitination of XIAP, and thus reduces the stability of XIAP protein and promotes its ubiquitin-mediated proteasomal degradation. Conclusions Berberine down-regulates XIAP expression at the post-transcriptional level. Specifically, berberine may inhibits XIAP translation by down-regulation MDM2protein, and promotes XIAP protein ubiquitin-mediated proteasomal degradation.