Study On The Apoptosis Of NB4-R1 Cell Induced By PRF And ATO In Vitro

[Background and Objective]Acute promyelocytic leukemia (APL) is a subtype of acute myelocytic leukemia (AML). In the last three decades, the advent of molecular targeted therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has achieved an important progress in the treatment of APL, which make it from the high-risk leukemia to the one can be cured completely. But retinoic acid resistance is one of the reasons for the failure of induction treatment. Puerariae radix flavones (PRF) is one of the effective component of Chinese medicine Puerariae radix. Our preliminary study found that low concentration (10～50μg/ml) PRF could inhibit the proliferation and induce the apoptosis of NB4 cells, which is one of the acute promyelocytic leukemia cell line. And the mechanism might be associated with the interaction of JNK, p38 and ERK signal pathways. Besides, we found that low concentration PRF combined 1μM ATO could inhibit the proliferation of NB4 cells effectively, and the JNK protein is up-regulated. Based on the results above, the retinoic acid resistant cell line NB4-R1 cells is regarded as the research object in this study, we intend to explore the influence of the combination of PRF and ATO on the proliferation and apoptosis of NB4-R1 cells, and verify the possible role of JNK signal pathway in the NB4-R1 apoptosis.[Methods]1. NB4-R1 cells were exposed to 0,10,30,50μg/ml PRF±1μM ATO and 0.5μM ATO for 24,48,72 hours respectively, the cells proliferation inhibition rates were detected by MTT assay.2. NB4-R1 cells were exposed to 0,10,30,50μg/ml PRF±1μM ATO and 0.5μM ATO for 48 hours, the changes of early-stage cell apoptosis were detected by FITC-AnnexinV/PI double-staining.3. NB4-R1 cells were exposed to 0,10,30,50μg/ml PRF±1μM ATO for 48 hours, the relative proteins of JNK signal pathway were detected by Western Blot.[Results]1. MTT data indicated that 10,30,50μg/ml PRF±1μM ATO groups inhibit the proliferation of NB4-R1 cell, and the effects were in a time and concentration dependence manner. The IC50 (50% inhibiting concentration) for 24,48 and 72 hours are 238.09, 56.75,35.95μg/ml, respectively. The proliferation inhibition rates of PRF combine 1μM ATO groups were higher than the single PRF groups. On this basis, we reduce the concentration of ATO to 0.5μM and combine it with 0,10,30,50μg/ml PRF, compared with the single PRF groups, the proliferation rate did not change significantly.2. FCM (Flow Cytometry) indicated that 10,30,50μg/ml PRF could induce NB4-R1 cell apoptosis after incubating 48 hours, the early apoptosis rates are 1.5%,2.4%,4.9%, respectively. 1μM ATO combine 10,30,50μg/ml PRF,the early apoptosis rates are 6.0%, 12.6%,12.9%,19.3% respectively; and the early apoptosis rates of 0.5μM ATO combine 0,10,30,50μg/ml PRF are 2.5%,5.0%,5.0%,6.3%.3. Western Blot showed that the expression of JNK、p-JNK、cleaved caspase 3 were up-regulated while p38、ERK1/2、pro-caspase3、pro-caspase 9、p53 were down-regulated.[Conclusion]Lower concentration (10～50μg/ml) of PRF single or combine 1μM ATO could significantly inhibit the proliferation of NB4-R1 cell and induce apoptosis. The effects were in a dose-dependent and time-dependent manner. And the combination groups were stronger than that of PRF alone groups. The mechanism might be associated with the activation of JNK signal pathway as well as the down regulation of p38 and ERK.