After treatment of
meisoindigo, the proliferation of HL-60 cells was detected by
trypan blue exclusion assay, and DNA fragmentation by
agarose electrophoresis; cell morphology was observed under fluorescent microscope. Cell apoptosis and the expression of Fas were detected by flow cytometry. The expression of
Caspase-3,
Caspase-8,
Caspase-9, PARP, Bcl-2, Bax and the concentration of
cytochrome c in cytosol were analyzed by Western blot.
RESULTS:
Meisoindigo inhibited proliferation and induced apoptosis in HL-60 cells. When treated with 20 micromol/L
meisoindigo for 12-48 h, the proliferation of HL-60 cells was significantly inhibited. When treated for 1 h, the apoptosis rate of HL-60 cells was (3.70+/-0.56)%; the apoptosis rate was significantly higher in HL-60 cells treated for 3, 6, and 12 h than in control cells [(19.80+/-1.13)%, (29.20+/-2.69)%, and (47.05+/-7.70)% vs. (2.65+/-0.78)%, P<0.05]. When treated with
meisoindigo for 3 h, typical changes of apoptosis, such as
chromatin condensation and
DNA ladder, were detected in HL-60 cells. The positive rate of Fas was significantly higher in cells treated with 20 micromol/L
meisoindigo for 1 h than in control cells [(21.30+/-1.27)% vs. (9.35+/-0.21)%, P<0.05].
Meisoindigo activated
Caspase-3,
Caspase-8,
Caspase-9 and PARP, down-regulated the expression of Bcl-2, up-regulated the expression of Bax and the concentration of
cytochrome c. Furthermore, pretreatment of
caspase-3 inhibitor
z-DEVD-fmk partially reversed the inhibitory effect of
meisoindigo on cell proliferation, and decreased cell apoptosis; when treated with
meisoindigo for 5 h, the apoptosis rate was significantly higher in pretreated cells than in cells without pretreatment [(29.8+/-5.4)% vs. (16.5+/-5.5)%, P<0.05]; when treated with
meisoindigo for 12 h, the alive cell number was significantly lower in pretreated cells than in cells without pretreatment [(1.80+/-0.14) x 10(5)/ml vs. (3.57+/-0.18) x 10(5)/ml, P<0.05].
CONCLUSION: