Cinobufacini, an aqueous extract from Bufo bufo gargarizans Cantor, induces apoptosis through a mitochondria-mediated pathway in human hepatocellular carcinoma cells.
OBJECTIVE
Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG(2) and Bel-7402.


METHODS
The effects of cinobufacini on cell proliferation of HepG(2) and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Deltapsim) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis.


RESULTS
Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Deltapsim) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP).


CONCLUSIONS
The present study indicated that cinobufacini can induce apoptosis of HepG(2) and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.
Qi
Fanghua
F
Department of Traditional Chinese Medicine, Provincial Hospital affiliated with Shandong University, Jinan 250021, China.
Li
Anyuan
A
Zhao
Lin
L
Xu
Huanli
H
Inagaki
Yoshinori
Y
Wang
Dongliang
D
Cui
Xiaoyan
X
Gao
Bo
B
Kokudo
Norihiro
N
Nakata
Munehiro
M
Tang
Wei
W
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D002023:Bufo bufo / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002681:China; D003574:Cytochrome c Group / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015398:Signal Transduction; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D003574:Cytochrome c Group; D051028:bcl-2-Associated X Protein; C076615:cytochrome c''; D000246:Adenosine Diphosphate Ribose; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; D053453:Caspase 9