Molecular mechanisms of apoptosis induced by ajoene in 3T3-L1 adipocytes.
OBJECTIVE
Determine the biochemical pathways involved in induction of apoptosis by ajoene, an organosulfur compound from garlic.


METHODS
Mature 3T3-L1 adipocytes were incubated with ajoene at concentrations up to 200 microM. Viability and apoptosis were quantified using an MTS-based cell viability assay and an enzyme-linked immunosorbent assay for single-stranded DNA (ssDNA), respectively. Intracellular reactive oxygen species (ROS) production was measured based on production of the fluorescent dye, dichlorofluorescein. Activation of the mitogen-activated protein kinases extracellular signal-regulating kinase 1/2 (ERK) and c-Jun-N-terminal kinase (JNK) was shown by Western blot. Western blot was also used to show activation of caspase-3, translocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus, and cleavage of 116-kDa poly(ADP-ribose) polymerase (PARP)-1.


RESULTS
Ajoene induced apoptosis of 3T3-L1 adipocytes in a dose- and time-dependent manner. Ajoene treatment resulted in activation of JNK and ERK, translocation of AIF from mitochondria to nucleus, and cleavage of 116-kDa PARP-1 in a caspase-independent manner. Ajoene treatment also induced an increase in intracellular ROS level. Furthermore, the antioxidant N-acetyl-L-cysteine effectively blocked ajoene-mediated ROS generation, activation of JNK and ERK, translocation of AIF, and degradation of PARP-1.


CONCLUSIONS
These results indicate that ajoene-induced apoptosis in 3T3-L1 adipocytes is initiated by the generation of hydrogen peroxide, which leads to activation of mitogen-activated protein kinases, degradation of PARP-1, translocation of AIF, and fragmentation of DNA. Ajoene can, thus, influence the regulation of fat cell number through the induction of apoptosis and may be a new therapeutic agent for the treatment of obesity.
Yang
Jeong-Yeh
JY
Department of Animal and Dairy Science, University of Georgia, Athens, GA 30602-2771, USA.
Della-Fera
Mary Anne
MA
Nelson-Dooley
Cass
C
Baile
Clifton A
CA
D017667:Adipocytes / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004220:Disulfides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013454:Sulfoxides
D051033:Apoptosis Inducing Factor; D004220:Disulfides; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D013454:Sulfoxides; C048980:ajoene; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D020929:Mitogen-Activated Protein Kinase Kinases; D020169:Caspases