Study On The Apoptosis Of NB4-R1Cells Induced By Puerariae Radix Flavones And Arsenic Trioxide In Vitro

【Background and Objective】Acute promyelocytic leukemia (APL)is a subtype of acute myeloid leukemia(AML). The cells from almost all patients have a balance reciprocal translocation between chromosomes15and17, which generates a fusion transcript joining the PML (promyelocyte) and RAR-a(retinoic acid receptor-α). APL cells have the unique ablility to undergo differentiation with exposure to ATRA and both differentiation and apoptosis with exposure to ATO,so APL represents a powerful example of a malignant disease that is highly curable with molecularly targeted therapy against its specific genetic abnormality. But the drug resistant to ATRA is a hard problem. Our colleagues showed that a degree of concentration of Puerariae radix flavones (PRF) can inhibit the proliferation and induce the apoptosis of NB4. The effect mechanism might be associated with up-regulation of FasL,Caspase8, Caspase3and activation of JNK, PARP, down-regulation of p38MAPK.Besides, we found low dose PRF(10,30,50ug/ml)±1μM ATO slso inhbit proliferation of NB4.There is a close relationship between the activation of JNK and the function on promoting apoptosis. We aim to explore the influence of the low doses PRF(10,30,50ug/ml)±1μM ATO combination on the proliferation and apoptosis of NB4-R1cell line in vitro.[Methods]NB4-R1cells were exposed to0,10,30,50μg/ml PRF±1μM ATO in24,48,72hours respectively. The cell proliferation inhibitory rates were detected by methyl thiazolyl tetrazolium (MTT) assay; morphologic changes were screened by optical microscope and fluorescence microscope; apoptosis with FITC-AnnexinV staining, cells cycle progression were measured by Flow cytometry (FCM); the NB4-R1cells apoptotic relative proteins were detected by Western blot assav.【Results】1. MTT data indicate that low doses PRF(10、30、50μg/ml)±1μM ATO groups inhibited NB4-R1cells proliferation. The combined group represented stronger inhibition than that of single herb group and the effect were a dose-dependent and time-dependent. Interestingly,1μMATO group showed some mild differentiaton performance,but this differentiation effect weakened with time extending.The quantitative analysis showed the inhibitory rates had statistical difference in the interior-group and in inter-group (p<0.01).2.2. By Wright’s staining, the NB4-R1cells show morphological change of apoptosis to varying degrees after exposed to different concentration of PRF±ATO, such as obscured structural、vacuoles、chromatin condensation、ruptured membranes、karyorrhexis achromatolysis and apoptotic bodies,30μg/ml,50μg/mlPRF±1μM ATO groups shows distinct apoptostic characters. PRF (10～50ug/ml)can disturb the cell cycle procedure and induce NB4-R1cell apoptosis.3.3. By the FITC-Annexin V/PI double staining method,we found the early apoptosis rates was increased in a dose-dependent manner.The (0,10,30,50μg/ml) PRF alone group displays a weak inhibititory effect on NB4-R1, early apoptotic rates werel.4%,1.8%,6.7%,8.3%, respectively.The early apoptosis rate of combined groups rose slightly. PRF(10-50ug/ml)±1μM ATO can disturb the cell cycle procedure.4.4. Cell cycle detection showed that NB-R1cells were arrested in cell S phase.The increase of Sub-G1ratio were significantly in combined groups. Besides, The50mg/mlPRF+1uMATO group showed marked hypodiploid cells (26.7%).5. With Western Blot assay, the pro-JNK protein expression active when the NB4-R1cells were suffered from10、30、50μg/ml PRF for48hours, Simultaneously, ERK1/2and P38were down-regulated.【Conclusion】 Low doses PRF (10,30,50μg/ml)±1μM ATO could induce NB4-R1cell apoptosis. The effects were a dose-dependent and time-dependent.The suppression effect of conbined groups were stronger than that of PRF alone groups. Its mechanisms might be probably associsated with inducing cell cycle block and cell apoptosis. The mechanism seems to have relationship with the activation of JNK and suppression of ERK1/2and P38.