Effects Of Arsenic Trioxide On Apoptosis And The Mechanism Of Reversal Effect In K562/A02 Cell

Objective:To study the expression of multidurg resistance gene(mdr1),P-glycoprotein (P-gp) and vascular endothelial growth factor (VEGF) during apoptosis induced by Arsenic trioxide (As2O3) and explore the molecule mechnaism of reversal of apoptosis resistance by Arsenic trioxide in multidurg- resistant human leukemia K562/ A02 cell.Methods:The K562/A02 cell was cultured in RPMI1640 at different concentration of As2O3 (0μmol/L,0.5μmol/L,2.0μmol/L,5.0μmol/L) and harvested in 24 h, 48 h, 72 h. The cell morphology was assayed by inverted microscope,Wright's stain,laser scanning confocal microscope(LSCM). The cell apoptosis was examined by flow cytometer(FCM). The cell proliferating activity was assessed by MTT assay. The expression of mdr1 mRNA was examined with reverse ranscription-polymerase chain raction(RT-PCR). The P-gp was examined by immunohistochemistry assay . The VEGF levels were measured with enzyme-linked immunosorbent assay (ELISA) in cultural supernatants of K562/A02 cell。Results: As2O3 could inhibit the proliferation of multidrug-resistant K562/A02 cell and induced the cell to undergo apoptosis in a dose - and time-dependent.As2O3 also could decrease the number of K562/A02 cell and increase the number of K562/A02 cell debris. The cell morphology showed typical morphological changes of apoptosis in K562/A02 cells the apoptosis-induction of As2O3,such as chromatin condensation,nuclear fragmentation and so on. After treatment with 0.5μmol/L of As2O3 for 72h h,the inhibitory rate of the multidrug-resistant K562/A02 cell was (13.23±2.56)% (P < 0.05).After treatment with 5.0μmol/L of As2O3 for 72h h,the inhibitory rate of the multidrug-resistant K562/A02 cell was(61.78±2.27)% (P < 0.05). The Annexin V-FITC/PI double staining showed apoptosis rate of K562/A02 cells was obviously increased in a dose - and time-dependent. After treatment with 5.0μmol/L of As2O3 for 24h,48 h and 72 h,the apoptosis rate of the cells were(31.58 ±0.92)%,(36.82±1.13)% and(53.95±1.04)% , respectively. Constitutive expression of mdr1 mRNA,P-gp and VEGF were detected by RT-PCR,immunohistochemistry assay and ELISA respectively. After treatmeat with As2O3 for 48h,the expression of mdr1 mRNA and P-gp showed significent decrease (P < 0.05).Incubated with 0.5μmol/l As2O3 for 48h, K562/A02 cells exhibited decreased mdr1 mRNA and P-gp expression, grey scale ratio and grey scale numerus were 0.363±0.079 and108.71±3.54 ,respectively( P < 0.05)After been treated with 5.0μmol/L As2O3 for 72h , the expression of mdr1 mRNA and P-gp in K562/A02 cells became decrease futher( P < 0.05), grey scale ratio and grey scale numerus were 0.139±0.018 and132.04±3.88 ,respectively( P < 0.05).After been treated with 0.5μmol/l As2O3 for 24h , the expression of VEGF in K562/A02 cells became decrease to(405.02±62.44) pg/ml (P < 0.05) After been treated with the same time, when incubated with5.0μmol/l As2O3,the expression of VEGF was least .After been treated with the same level, when incubated As2O3 for 72 h,the expression of VEGF was least.Conclusion:⑴As2O3 can inhibit the proliferation of multidrug-resistant K562/A02 cell and induce the cell to undergo apoptosis in a concentration-dependent (0.5μmol/L -5.0μmol/L) and time-dependent(24h-72h).⑵As2O3 can inhibit the expression of mdr1mRNA,P-gp and VEGF in order to overcome the resistance in multidrug-resistant cell in a concentration-dependent (0.5μmol/L -5.0μmol/L) and time-dependent(24h-72h).