The Study On Apoptosis Of The All-trans-retinoic Acid(ATRA)-sensitive (NB4) Cells Induced By Puerariae Radix Flavones In Vitro

[background]Puerariae radix is one of the main components of Xue-Fu-Kang, a the Chinese native medicine group produced by Jiangsu Province Hospital of TCM which has been the effective treatment in chronic myeloid leukemia and acute myeloid leukemia (AML). In the past five years, Prof. Shen Qun and her colleagues reported that four AML cells (Kasumi-1, HL-60, NB4and U937) proliferations were inhibited by Puerariae radix flavones (PRF), an active componet of Puerariae radix, but puerairin and Daidzin-the other two active components of Puerariae radix. The results showed that the NB4cells were markedly inhibited and arrested in S phase with PRF in vitro. PRF, acted synergy with ATO, could improve the life quality of Xenografts nude mice to some degrees in vivo.[Objective]To explore the effect of PRF±ATO on the proliferation and apoptosis in all-trans-retinoic acid (ATRA)-sensitive (NB4) cells, and the mechanism of PRF on retinoic receptor and the JNK signal pathway in the apoptosis of NB4cells.[Methods]The proliferation of NB4and NB4-R1exposed with PRF±ATO for24,48,72hours was determined by MTT. Then, the NB4and NB4-R1cells were exposed with PRF group and PRF+ATO group for48hours. Cell apoptosis was determined by Wright's staining and confocal laser technique; cell cycle and apoptosis were analyzed by flow cytometry (FCM). JNK, TNFα, ERK and p38MAPK were examined by western blotting with interrupting or without interrupting JNK signaling pathway by pharmacological inhibitor (SP600125) for48hours in NB4cells incubated with PRF.[Results]PRF (10,30,50μg/ml±ATO1μM) inhibited the proliferation of NB4and NB4-R1cells in time-and dose-dependent manners. In NB4cell line, PRF IC50in24,48,72h were39.82,27.45,19.27μg/ml, respectively; In the combined groups, IC50were29.30,21.08,7.56μg/ml, respectively. In NB4-R1cell line, PRF IC50were71.66,34.29,31.44μg/ml, respectively; In the combined groups, IC50were38.50,24.28,16.62μg/ml, respectively. The cells displayed distinct apoptotic characters by Wright's staining and confocal laser technique. Meanwhile, FITC-AnnexinⅤ/PI double staining indicated that PRF±ATO could induce NB4and NB4-R1cells to apoptosis, which presented dose dependent manner. Cell cycle process could be changed, with increase cell in S phase and sub-diploid peak. The results in combined groups were more significantly. Interestingly, NB4-R1cells treated with ATO1μM group for24h and48h were proliferated, for72h inhibited. PRF can induce apoptosis of NB4cells accompanied by increased JNK1, JNK2/3, p38MAPK, ERK and TNFα. JNK inhibiting suppressed the activation of JNK1, JNK2/3and p38MAPK, diminished ERK1/2and TNFa expression in PRF50μg/mI induced apoptosis, increased in10μg/ml and30μg/ml PRF groups.[Conclusion]These datas provide novel information on the PRF induced NB4cells and NB4-R1cells apoptosis.0μg/ml to50μg/ml PRF and also synergetic with1μM ATO can induce the apoptosis of NB4and NB4-R1cells. PRF may evoke activation of MAPKs signaling pathway, by enhancing TNFa activation. JNK, ERK and p38MAPK, the three members of MAPK signaling pathway, but not retinoic receptor, may play an important role and influence each other in NB4cells apoptosis induced by PRF.