Anti-proliferative effect of Juglone from Juglans mandshurica Maxim on human leukemia cell HL-60 by inducing apoptosis through the mitochondria-dependent pathway.
Induction of apoptosis in tumor cells has become the major focus of anti-tumor therapeutics development. Juglone, a major chemical constituent of Juglans mandshurica Maxim, possesses several bioactivities including anti-tumor. Here, for the first time, we studied the molecular mechanism of Juglone-induced apoptosis in human leukemia HL-60 cells. In the present study, HL-60 cells were incubated with Juglone at various concentrations. Occurrence of apoptosis was detected by Hoechst 33342 staining and flow cytometry. Expression of Bcl-2 and Bax mRNA was determined by quantitative polymerase chain reaction (qPCR). The results showed that Juglone inhibits the growth of human leukemia HL-60 cells in dose- and time-dependent manner. Topical morphological changes of apoptotic body formation after Juglone treatment were observed by Hoechst 33342 staining. The percentages of Annexin V-FITC-positive/PI negative cells were 7.81%, 35.46%, 49.11% and 66.02% with the concentrations of Juglone (0, 0.5, 1.0 and 1.5 microg/ml). Juglone could induce the mitochondrial membrane potential (DeltaPsim) loss, which preceded release of cytochrome c (Cyt c), Smac and apoptosis inducing factor (AIF) to cell cytoplasm. A marked increased of Bax mRNA and protein appeared with Juglone treatment, while an evidently decreased of Bcl-2 mRNA and protein appeared at the same time. These events paralleled with activation of caspase-9, -3 and PARP cleavage. And the apoptosis induced by Juglone was blocked by z-LEHD-fmk, a caspase-9 inhibitor. Those results of our studies demonstrated that Juglone-induced mitochondrial dysfunction in HL-60 cells trigger events responsible for mitochondrial-dependent apoptosis pathways and the elevated ratio of Bax/Bcl-2 was also probably involved in this effect.
Xu
Hua-Li
HL
Department of Pharmacology, School of Pharmaceutical Sciences, Jilin University, Changchun 130021, Jilin Province, PR China.
Yu
Xiao-Feng
XF
Qu
Shao-Chun
SC
Zhang
Rui
R
Qu
Xiang-Ru
XR
Chen
Yan-Ping
YP
Ma
Xing-Yuan
XY
Sui
Da-Yuan
DY
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D031324:Juglans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D024101:Mitochondrial Proteins / Q000378:metabolism; D009285:Naphthoquinones / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
D000972:Antineoplastic Agents, Phytogenic; D051033:Apoptosis Inducing Factor; D051017:Apoptosis Regulatory Proteins; D061945:Caspase Inhibitors; C412403:DIABLO protein, human; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D009285:Naphthoquinones; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; C005134:juglone