Experimental Study On Induction Of The Growth Inhibition And Apoptosis Of Human Acute Myeloid Leukemia Cells By 5, 7-Dimethorychrysin

Objective To observe the effect of 5,7-dimethorychysin(dMChR) in inhibition of human acute myeloid leukemia cell (HL-60) proliferation and in induction of HL-60 cell apoptosis, and approached if activation of PPARγand regulation of PTEN-Akt signal transduction pathway were involved in the effect of dMChR on HL-60 cells. Methods HL-60 cell line was cultured in vitro and the inhibitory effect of dMChR on proliferation of HL-60 cell lines was measured with 3-(4,5-dimethylthiazol -z-yl)-2,5-dipheny tertrazolium bromide (MTT) colorimetric assay. The inhibition of the growth by dMChR in HL-60 cells was used to test by the cell counting after trypan blue staining. The fluorescence microscope using AO/EB fluorescence staining was used to observe the morphologic changes of apoptosis induced by dMChR in HL-60 cell line. The apoptosis rate of HL-60 cell line induced by dMChR was analyzed using flow cytometry (FCM) with PI staining. The influence of dMChR on the PPARγ, PTEN and p-AKt protein expression of HL-60 cells was analyzed with western blot.Results MTT assay indicated dMChR (6.25μM, 12.5μM, 25.0μM, 50.0μM, 100.0μM) treated HL-60 cells for 48 hours, HL-60 cells were significantly inhibited in a dose-dependent manner. The potency of dMChR was similar to arabinosylcytosin (Ara-C), and was higher than that of lead compound. The cell counting data had shown that all the 12.5μM, 25.0μM and 50.0μM dMChR inhibited the growth of HL-60 cells in a concentration-dependent manner, and 25.0μM dMChR prolonged the population doubling time of HL-60 cells from 21.5h to 31.7h. After treatment with dMChR, typical morphologic changes of apoptosis like karyopycnosis and nuclear fragmentation could be observed by fluorescence microscope using AO/EB fluorescence staining. FCM with PI staining indicated the apoptosis rate of HL-60 cell lines treated with dMChR (12.5μM,25.0μM,50.00μM) for 48 hours were 6.18±2.4%, 16.3±3.5% and 50.1±5.6% respectively and treated with dMChR (25.00μM) were higher than that with ChR (4.23±1.1%). Western blot indicated that after 0, 4, 8, 24 hours of action of dMChR (25.00μM), PPARγand PTEN protein expression of HL-60 cells were increased and p-AKt protein was decreased in a time-dependent manner. The pre-incubation of GW9662, blocker of PPARγ, could efficiently antagonize inhibition of proliferation and induction of apoptosis on HL-60 by dMChR, and eliminated or weakened the regulative effect of dMChR on the p-AKt protein expression of HL-60 cells.Conclusion 5,7-dimethorychysin (dMChR) can inhibit the proliferation and induce the apoptosis of human acute myeloid leukemia cell line (HL-60) by activating PPARγand increasing expression of PTEN and inhibiting phosphorylation of AKt. This effect was strong than that of lead compound (ChR).