The Study On Apoptosis of the All-Trans-Retinoic Acid (ATRA)-Sensitive (NB4) Cells Induced by Puerariae Radix Flavones in Vitro
Authors: Qun Shen , Ou Ji 
Published: October 2019

Abstract
Abstract 4922

Background
Puerariae radixis one of the main components of Xue-Fu-Kang, a 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, NB4 and U937) proliferations were inhibited by Puerariae radix flavones (PRF), an active component of Puerariae radix, but puerairin and Daidzin, the other two active components. The results showed that PRF can markedly inhibit the NB4 cells proliferation with arresting in S phase 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 NB4 cells.

Methods
The cell vitality of NB4-R1 cells exposed with 0, 10, 30, 50μg/ml PRF±1μM ATRA for 24, 48, 72 hours and the proliferation of NB4 and NB4-R1 exposed with PRF±ATO for 24, 48, 72 hours were determined by MTT. Then, the NB4 and NB4-R1 cells were exposed with PRF group and PRF+ATO group for 48 hours. Cell apoptosis was determined by Wright's staining and confocal laser technique; cell cycle and apoptosis were analyzed by flow cytometry (FCM). JNK, TNFÁ, ERK and p38MAPK were examined by western blotting with interrupting or without interrupting JNK signaling pathway by pharmacological inhibitor (SP600125) for 48 hours in NB4 cells incubated with PRF.

Results
PRF (10, 30, 50μg/ml ± ATRA 1μM) inhibited the proliferation of NB4-R1 cells in time-dependent manners. There was no statistically difference in PRF IC50 between PRF group and PRF+ATRA group in 24, 48 and 72 hours (54. 25¡¢26. 68¡¢28. 55μg/ml, vs 59. 55¡¢28. 43¡¢26. 59μg/ml). PRF (10, 30, 50μg/ml ± ATO 1μM) inhibited the proliferation of NB4 and NB4-R1 cells in time- and dose-dependent manners. In NB4 cell line, PRF IC50 in 24¡¢48¡¢72h were 39. 82¡¢27. 45¡¢19. 27μg/ml, respectively; In the combined groups, IC50 were 29. 30¡¢21. 08¡¢7. 56μg/ml, respectively. In NB4-R1 cell line, PRF IC50 were 71. 66¡¢34. 29¡¢31. 44μg/ml, respectively; In the combined groups, IC50 were 38. 50¡¢24. 28¡¢16. 62μg/ml, respectively. The cells displayed distinct apoptotic characters by Wright's staining and confocal laser technique. Meanwhile, FITC-Annexin¢õ/PI double staining. The results indicated that PRF±ATO could induce NB4 and NB4-R1 cells to apoptosis, which presented dose dependent manner. Cell cycle process could be changed, with increasing cell in S phase and sub-diploid peak. The results in combined groups were more significantly. Interestingly, NB4-R1 cells treated with ATO 1μM group for 24h and 48h were proliferated, for 72h inhibited. PRF can induce apoptosis of NB4 cells accompanied by increased JNK1, JNK2/3, p38MAPK, ERK and TNFÁ. JNK inhibiting suppressed the activation of JNK1, JNK2/3 and p38MAPK, diminished ERK1/2 and TNFÁ expression in PRF 50μg/ml group, increased in 10μg/ml and 30μg/ml PRF groups.

Conclusion
These datas provide novel information on the PRF induced NB4 cells and NB4-R1 cells apoptosis. 0μg/ml to 50μg/ml PRF and also synergetic with 1μM ATO can induce the apoptosis of NB4 and NB4-R1 cells. PRF may evoke activation of MAPKs signaling pathway, by enhancing TNFÁ 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 NB4 cells apoptosis induced by PRF.

Disclosures:
No relevant conflicts of interest to declare.