Relationship Between PTEN-Akt Pathway And 8-nitrochyrsin In Inhibiting The Proliferation Of HL-60 Cells

Objective To observe the effect of 8-nitrochyrsin in inhibition of HL-60 cell proliferation and in induction of HL-60 cell apoptosis, analyzed correlation between PTEN-Akt signal transduction pathway and 8-nitrochrysin in inhibition of HL-60 cell proliferation.Methods HL-60 cell line was cultured in vitro and the inhibitory effect of ChR and NOChR on proliferation of HL-60 cell lines was measured with 3-(4,5-dimethylthiazol–2-yl)-2,5-dipheny tertrazolium blue (MTT) colorimetric assay. The inhibition of the growth by ChR and NOChR in HL-60 cells was used to test by the cell counting after trypan blue staining. The apoptosis rate of HL-60 cell line induced by NOChR was analyzed using flow cytometry (FCM) with PI staining. The fluorescence microscope using AO/EB fluorescence staining was used to observe the morphologic changes of apoptosis induced by ChR and NOChR in HL-60 cell line. Characteristic The influence of NOChR on the PTEN and p-AKt protein expression of HL-60 cells was analyzed with western blot. Results MTT assay indicated that NOChR markedly inhibited proliferation of HL-60 cells in a concentration-dependent manner, and when IC50 was 1.34μM, the potency of NOChR was 5.3 times than that of lead compound, chrysin (ChR, IC50 was 7.1μM), and was similar to all-trans retinoic acid (ATRA IC50 =1.33μM). The cell counting data had shown that NOChR significantly inhibited the growth of HL-60 cells in a time-dependent manner. Flow cytometry after PI staining indicated that apoptosis in HL-60 cells was induced by NOChR in a time- and concentration-dependent manner. The apoptosis rate of HL-60 cell lines treated with NOChR (1.0μM) for 48 hours was 23.16%, while the apoptosis rate treated with ChR(1.0μM) and ATRA were merely 5.8% and 9.75%respectively. After treatment with NOChR for 48h, typical morphologic changes of apoptosis could be observed by fluorescence microscope using AO/EB fluorescence staining, and apoptotic index of HL-60 cells was increased in concentration-dependent manner. Western blot indicated that NOChR can efficiently up-regulated PTEN and down-regulated p-AKt protein expression of HL-60 cells in a concentration- and time-dependent manner, and the pre-incubation of GW9662, blocker of PPARγ, can antagonize the up-regulating effect of 8-nitrochrysin on PTEN protein expression of HL-60 cells.Conclusion1) 8-nitrochyrsin can significantly inhibit the proliferation and the growth of human acute myeloid leukemia cell line(HL-60) in a concentration-dependent manner.2) 8-nitrochyrsin efficiently induce apoptosis of HL-60 cells.3) This effect of induction of the growth inhibition on HL-60 cells may be associated with activation of PPARγ, up-regulation of PTEN and inhibition of p-AKt by 8-nitrochyrsin.