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

[Objective]Research team members of Prof. Shen Qun previously demonstrated that Xue-Fu-Kang compound preparation, which was a complex mainly containing three traditional Chinese herb--indigo, curcumae and puerariae radix could effectively inhibit the proliferation of chronic myeloid leukemia (CML) cell line--K-562 cells and induce the cells to apoptosis associated with the degradation of leukemiagenesis BCR/ABL fusion gene expression. Further, the application of active components of indigo, curcumae and to K-562 cells also showed that K-562 cells were induced to apoptosis via different signal pathways. On the other hand, MTT assay data also manifested that with the concentration of 50-200μg/ml Puerariae radix flavones(PRF), which is the active components of puerariae radix, could inhibit the proliferation of 4 sub-types cell lines (HL-60, Kasumi-1, NB4 and U937 respectively) of acute myeloid leukemia (AML). The cell inhibitory rate of NB4 cells was much higher than others. Then, whether the NB4 cells can be induced to apoptosis is still unknown and worth exploring.PartⅠThe molecular mechanism of PRF induces NB4 cells to apoptosis in vitro[Methods](1) Treated with the PRF concentration from 12.5-200μg/ml for 24,48 and 72 hours, the NB4 cells proliferation inhibitory rate was also detected by MTT assay;(2) Treated with 50μg/ml PRF for 48 hours, the NB4 cells were observed by Wright's and Hoechst 33258 staining;(3) Treated with 50-200μg/ml PRF for 48 hours, the NB4 cell cycle was detected by flow cytometry(FCM);(4) Treated with 12.5-200μg/ml PRF for 24 and 48 hours, the NB4 cells early-stage apoptotic rates were detected by FITC-Annexin V/PI double staining with FCM;(5) Treated with 50μg/ml PRF for 48 hours, the NB4 cells PML/RARa fusion gene and anti-apoptotic genes were detected by real time RT-PCR assay,1μM ATO treatment group acted as positive control;(6) Treated with 12.5-200μg/ml PRF for 48 hours, the NB4 cells apoptotic relative proteins were detected by Western blot assay,1μM ATO treatment group also acted as positive control;[Results]The IC50(50% inhibiting concentration) of NB4 cells for 24,48 and 72 hours were 108.75, 65.86 and 37.55μg/ml respectively. The NB4 cells displayed distinct apoptotic character by Hoechst 33258 and Wright's staining with 50μg/ml PRF treated for 48 hours, Cell cycle detection showed that NB4 cells were arrested in cell S phase. FITC-Annexin V/PI double staining indicated that 12.5-200μg/ml PRF could induce NB4 cells to apoptosis, which presented time-dose dependent manner. The results of real-time PCR documented that 50μg/ml PRF could degradate the expression level of PML/RARa fusion gene and down-regulate anti-apoptotic Bcl-2 and survivin gene. While combined with 1μM arsenic trioxide (ATO), these genes expression degradated more significantly. With Western Blot assay, the pro-Caspase3 protein expression decreased and the active p17 subunit increased when the NB4 cells were suffered from 12.5-200μg/ml PRF for 48 hours. Simultaneously, FasL and Caspase8 were up-regulated.PartⅡThe impact of PRF on NB4 cells Xenografts nude mice in vivo[Methods](1) The Xenografts nude mice models were established by subcutaneous inoculation of 4×106/ 0.2 ml NB4 cells into the right flank.(2) When leukemic transplantation tumor became measurable, the nude mice were intraperitoneally received PRF treatment (10mg/kg/d and 20mg/kg/d,2 weeks), ATO(1.5mg/kg/d,5 days a week,2 weeks) as positive control and comprehensive treatment (PRF 20mg/kg/d+ATO 1.5mg/kg/d,5 days a week,2 weeks).(3) The tumor body, weight and lifespan of Xenografts nude mice were measured; The paraffin sections of Xenografts nude mice liver, spleen and tumor body were stained by hematoxylin-eosin(HE) and observed with microscope.[Results]The results showed that although no significant diminution of tumor body and no tendency of body weight loss were seen, PRF could prolong survival of Xenografts nude mice, which presented synergetic effect combined with ATO.[Conclusion]PRF can induce the NB4 cells to apoptosis effectively. All these effects are triggered by the activation of JNK, which can increase the expression of FasL, recruit and activate Caspase8, finally activate effector Caspase3 following the delivery of induce-apoptotic factor and down-regulation of anti-apoptotic factors expression. Simultaneously, RPF also can degradate the expression of PML/RARa fusion gene at some extent. Therefore PRF appears to be a potential candidate for the treatment of APL.