Molecular mechanisms of apoptosis induced by Scorpio water extract in human hepatoma HepG2 cells.
OBJECTIVE
To clarify the mechanism underlying the anti-mutagenic and anti-cancer activities of Scorpio water extract (SWE).


METHODS
Human hepatoma HepG2 cells were incubated with various concentrations of SWE. After 24-h incubation, cytotoxicity and apoptosis evaluations were determined by MTT and DNA fragmentation assay, respectively. After treatment with SWE, mitochondrial membrane potential (MMP) was determined by measuring the retention of the dye 3,3'-dihexyloxacarbocyanine (DiOC(6)(3)) and the protein expression including cytochrome C and poly-(ADP-ribose) polymerase (PARP) were measured by Western blotting. Caspase-3 and -9 enzyme activities were measured using specific fluorescence dyes such as Ac-DEVD-AFC and Ac-LEHD-AFC.


RESULTS
We found that treatment with SWE induced apoptosis as confirmed by discontinuous DNA fragmentation in cultured human hepatoma HepG2 cells. Our investigation also showed that SWE-induced apoptosis of HepG2 cells were associated with intracellular events including disruption of MMP, increased translocation of cytochrome C from mitochondria to cytosol, activation of caspase-3, and PARP. Pre-treatment of N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), a caspase-3 specific inhibitor, or cyclosporin A (CsA), an inhibitor of MMP disruption, completely abolished SWE-induced DNA fragmentation.


CONCLUSIONS
These results suggest that SWE possibly causes mitochondrial damage, leading to cytochrome C release into cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic cell death in HepG2 cells. These results further suggest that Scorpio may be a valuable agent of therapeutic intervention of human hepatomas.
Kwon
Kang-Beom
KB
Department of Physiology, School of Oriental Medicine, Wonkwang University, Iksan, 570-749 Republic of Korea.
Kim
Eun-Kyung
EK
Lim
Jung-Gook
JG
Jeong
Eun-Sil
ES
Shin
Byung-Cheul
BC
Jeon
Young-Se
YS
Kim
Kang-San
KS
Seo
Eun-A
EA
Ryu
Do-Gon
DG
D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D016572:Cyclosporine / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008113:Liver Neoplasms; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D009842:Oligopeptides; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D016572:Cyclosporine; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases
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