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. Research Methods and Procedures: Mature 3T3-L1 adipocytes were incubated with ajoene at concentrations up to 200 mu M. Viability and apoptosis were quantified using an MTS-based cell viability assay and an enzyme-linked munosorbent 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. Discussion: 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.