The Study On The Human Acute Myeloid Leukemia Kasumi-1 Cells Apoptosis Induced By Puerariae Radix Flavones In Vitro

Xue-Fu-Kang compound preparation, which applied in treatment of chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) with high white cell count is sourced from the NO.1 hospital affiated to Nanjing Unvesity of Chinese Medicine. Many experiments results showed that not only Xue-Fu-Kang compound preparation, but also its three main components--indigo, curcumae and puerariae radix could effectively inhibit the proliferation of CML cell line-K-562 cells and induce the cells to apoptosis associated with the degradation of leukemiagenesis BCR/ABL fusion gene expression. Based on the well resultant of protophase study above, puerariae radix flavones (PRF) was chosed for further study in the potential anti-AML capacity. In the study, MTT data manifest that PRF (50～200μg/ml) could inhibit the proliferation of 4 sub-types of AML cell lines—HL-60, Kasumi-1, NB4 and U937 respectively. Meanwhile,50μg/ml and 200μg/ml concentration of PRF could also impact their cell cycle process. It presents that the proportion of G1/G0 phase cells decreases, S phase cells increases and sub-diploid peak appeares. It demonstrats that PRF can selectively inhibit the proliferation of 4 AML cell lines and block cell cycle process, especially in NB4 and Kasumi-1 cells. In the following study, we found that PRF cloud activate JNK, recruit and activate Caspase8, finally activate effector Caspase3 following the delivery of induce-apoptotic factor, degradate the expression of PML/RARαfusion gene at some extent, which might induced NB4 cells to apoptosis.The Kasumi-1 cells which have AML1-ETO leukemigenesis fusion gene were selected to explore the mechanism of PRF. MTT data indicate that PRF could inhibit the proliferation of Kasumi-1 cells in concentration range from 10μg/ml to 500μg/ml. With the increasing of concentration and action time, the vital of Kasumi-1 cells break down absloutly. But significant difference could be seen statistically only in concentration manner. Computed with SPSS software,10%,50% and 90% inhibiting concentration are 29.25μg/ml,158.07μg/ml and 618.60pμg/ml. So the concentration of 50μg/ml,200μg/ml and 500μg/ml were chosed for follow study. Treated with different concentration of PRF, the Kasumi-1 cells display distinct apoptotic character by Hoechst 33258 staining. FITC-AnnexinV/PI double staining indicates that PRF can induce Kasumi-1 cells to apoptosis, which presents dose-dependent manner. With Western Blotting assay, the Caspase3 and Caspase9 protein are activated. Simultaneously anti-apoptotic Bcl-2 protein is down-regulated and apoptotic Bim protein is up-regulated. The results of semi-quantity PCR suggest that PRF can't degradate the expression level of leukemiagenesis AML1-ETO fusion gene.In a word, the apoptotic induction effects of PRF on Kasumi-1 cells are co-related with the down-regulation of anti-apoptotic factors and the activation of caspase cascade reaction. It has no matter with the expression of AML1-ETO leukemigenesis fusion gene.