Apoptotic effect of oridonin on NB4 cells and its mechanism.
The anti-proliferation effects of oridonin on acute promyelocytic leukemia (APL) cells and its mechanisms were studied in vitro. NB4 cells as well as fresh leukemia cells obtained from APL patients in culture medium were treated with different concentrations of oridonin. Cell growth inhibition, apoptosis and related pathways were assessed by MTT assay as well as flow cytometry (FCM) and western blot analysis. The data revealed that oridonin (over 16 micromol/L) could inhibit the growth of NB4 cells by induction of apoptosis. Marked changes of cell apoptosis were observed very clearly by using electron microscopy and DNA fragmentation analysis after the cells exposed to oridonin for 48 h; Western blotting showed cleavage of the caspase-3 zymogen protein (32-kDa) with the appearance of its 20-kDa subunit as well as a cleaved 89-kDa fragment of 116-kDa PARP when apoptosis occurred. The expression of Bcl-2 was down-regulated remarkably accompanied by the disruption of the mitochondrial membrane potential (delta(psi)m). The anti-proliferative and apoptosis-inducing effects by oridonin in fresh APL cells were also found remarkably using Trypan Blue dye exclusion method and Wright's staining. We concluded that oridoning has significant anti-proliferative and apoptosis-inducing effects on NB4 cells by activation of caspase-3 and cleavage of PARP as well as by down regulation of Bcl-2 and disruption of the delta(psi)m. Furthermore, oridonin demonstrated apparent cell growth inhibition effects on fresh APL cells in vitro. The results indicated that oridonin may serve as a potential anti-leukemia reagent.
Liu
Jiajun
J
Department of Hematology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, People's Republic of China. liujj@gzsums.edu.cn
Huang
Renwei
R
Lin
Dongjun
D
Wu
Xiangyuan
X
Peng
Jun
J
Lin
Qu
Q
Pan
Xianglin
X
Zhang
Maohong
M
Hou
Ming
M
Chen
Feng
F
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004224:Diterpenes / Q000494:pharmacology*; D045786:Diterpenes, Kaurane; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000139:chemically induced / Q000188:drug therapy* / Q000378:metabolism*; D015394:Molecular Structure; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000378:metabolism
D000970:Antineoplastic Agents; D004224:Diterpenes; D045786:Diterpenes, Kaurane; D019253:Proto-Oncogene Proteins c-bcl-2; C011959:oridonin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases