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

[Background and Objective] Acute promyelocytic leukemia (APL), a subtype of acute myeloic leukemia(AML), have a balance reciprocal translocation between chromosomes 15 anc 17,which generates a fusion transcript joining the PML(promyelocyte) and RARα(retinoic acic receptor a). The bleeding caused by coagulation dysfunction and disseminated intravasculai coagulation (DIC) was the fatal complication in the early phase. Leukemic promyelocytes have the unique ability to undergo differentiation with exposure to all-trans retinoid acid (ATRA) and both differentiation and apoptosis to arsenic trioxide (ATO). In the past 20 years, a cure rate of approximately 90% of patients who survive induction with ATRA and ATO, achieve complete remission (CR) can be expected. Our colleagues showed that Puerariae radix flavones (PRF)(25-200μg/ml), the active component of Puerariae radix, might inhibit the proliferation and induce the apoptosis of APL cell lines NB4. The effect mechanism might be associated with up-regulation of FasL, Caspase 8, Caspase 3 and activation of JNK, down-regulation of MAPK p38. Above all, we aim to explore the influence of the lower doses PRF and 1μM ATO combination on the proliferation and apoptosis of NB4 cell line in vitro.[Methods] NB4 cells were treated with 0,10,30,50μg/ml PRF±μM ATO in 24,48,72 hours 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 and mitochondrial transmembrane potentials with JC-1 dye were measured by Flow cytometry (FCM); the NB4 cells apoptotic relative proteins were detected by Western blot assay.[Results] NB4 cells proliferation was inhibited in a dose-and time-dependent manners exposed in lower doses PRF (10,30,50μg/ml) alone or combined with 1μM ATO. The inhibitory rates had statistical difference in the interior-group and in inter-group (p<0.01). By Wright's and DAPI staining, the NB4 cells display distinct apoptostic characters such as pyknosis, karyorrhexis and apoptotic bodies. Cell counts were less and apoptotic bodies were more in 30μg/ml±1μM ATO, 50μg/ml±1μM ATO groups. The early apoptosis was increased in a dose-dependent manner in PRF alone groups and combined groups, only statistical significance between combined groups (p<0.05). The proportion of mitochondrial transmembrane potential damaged cells were not increased significantly between the groups (p>0.05). With Western Blot assay, the pro-JNK protein expression active when the NB4 cells were suffered from 10,30,50μg/ml PRF+ 1μM ATO for 48 hours. [Conclusion] (10,30,50μg/ml) PRF alone or combined with 1μM ATO could induce NB4 cell apoptosis. Moreover, coordination of 1μM ATO and PRF considerably enhanced apoptosis of NB4 cell. The mechanism seems to have relationship with the activation of JNK, without mitochondrial apoptosis way.