[Inducement effect of Meisoindigo on apoptosis of leukemia cell line HL-60 and its mechanism].
OBJECTIVE
Meisoindigo is a powerful drug used in treating chronic myeloid leukemia (CML), but little is known about the mechanisms. This study was to investigate the inducement effect of meisoindigo on apoptosis of myelocytic leukemia cell line HL-60, and explore the possible mechanisms.


METHODS
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].


CONCLUSIONS
Meisoindigo induces apoptosis of HL-60 cells which may relate to regulation of caspases pathway and bcl-2 family proteins.
Wang
Yi
Y
State Key Laboratory of Oncology in Southern China, Guangzhou, Guangdong 510060, P. R. China.
Zhu
Xiao-Feng
XF
Xiao
Zhi-Jian
ZJ
Wang
Hong-He
HH
Zhou
Jun-Min
JM
Mei
Yu-Ping
YP
Deng
Rong
R
Jiang
Wen-Qi
WQ
Liu
Zong-Chao
ZC
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D061945:Caspase Inhibitors; D007211:Indoles; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D045304:Cytochromes c; C047330:N-methylisoindigotin; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases