Involvement of mitochondrial pathway in NCTD-induced cytotoxicity in human hepG2 cells.
BACKGROUND
Norcantharidin, the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of anti-cancer effects. However, the detailed mechanisms underlying this process are generally unclear. The aim of this study was to investigate the mechanism of NCTD-induced apoptosis in HepG2 cells.


METHODS
The cytotoxicity was measured by MTT assay for cellular viability and by flow cytometry. The mitochondrial membrane potential and reactive oxygen species production was evaluated by flow cytometry analysis. The role of caspase activities were assayed using caspase apoptosis detection kit . Western blot analysis was used to evaluate the level of Cyto-C, Bcl-2, Bax, Bid, caspase 3, -9, -8 and PARP expression


RESULTS
After treatment with NCTD, a decrease in the viability of HepG2 cells and increase in apoptosis were observed. NCTD-induced apoptosis was accompanied by an increase in ROS production, loss of mitochondrial membrane potential and release of cytochrome c(cyto-c) from the mitochondria to the cytosol and down-regulation of anti-apoptotic protein Bcl-2 levels with concurrent up-regulation in pro-apoptotic protein Bax levels. However, another pro-apoptotic molecule, Bid, showed no change in such same treatment. NCTD-increased activity of caspase 9,caspase 3 and the subsequent cleavage caspase substrate PARP were also observed. The expression levels of pro-caspase-8 were not changed after NCTD treatment.


CONCLUSIONS
These results indicate that NCTD induced cytotoxicity in HepG2 cells by apoptosis, which is mediated through ROS generation and mitochondrial pathway.
Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
Cheng
C
Chang
You-Qing
YQ
Zhu
Juan-Juan
JJ
Mei
Shi-Quan
SQ
Liu
Jun
J
Luo
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D005434:Flow Cytometry; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000972:Antineoplastic Agents, Phytogenic; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004365:Drugs, Chinese Herbal; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; C069741:norcantharidin; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases