• Lan Yi, Xiao-Xia Ji, Hui Tan, Min Lin, Yi Tang, Ling Wen, Yan-Hua Ma, and Qi Su.
• Cancer Research Institute, University of South China, Hengyang, Hunan Province, China.
• Clin. Exp. Pharmacol. Physiol. 2010 Dec 1; 37 (12): 1147-53.

Abstract1. Diallyl disulphide (DADS) has potential as a chemopreventive and therapeutic agent. Previous studies have reported that Ras-related C3 botulinum toxin substrate 2 (Rac2), a regulatory subunit of the NADPH oxidase complex, is upregulated in DADS-induced apoptosis in human leukaemia HL-60 cells. The aim of the present study was to investigate the role of Rac2, NADPH oxidase and reactive oxygen species (ROS) in DADS-induced apoptosis. 2. Expression of the Rac2 gene along with that of five other genes of NADPH oxidase subunits were in HL-60 cells measured by Sybergreen quantitative real-time polymerase chain reaction. RNA interference was used to test the effect of Rac2. Protein expression was evaluated using western blot analysis and ROS levels were measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescence. DNA fragmentation and flow cytometry analysis were used to detect apoptotic cells. 3. Levels of Rac2 gene and protein were significantly upregulated and NADPH oxidase was activated in DADS-induced apoptosis. Pretreatment of HL-60 cells with small interfering (si) RNAs to inhibit Rac2 blocked DADS-induced apoptosis. Diallyl disulphide-induced intracellular ROS production was increased in phorbol myristate acetate-stimulated cells, but decreased in Rac2 siRNA-treated cells. In Rac2 siRNA-treated cells, activator protein-1 and caspase 3 levels decreased, c-myc protein levels were increased and p38 protein levels were unchanged compared with Rac2-competent, DADS-treated cells. 4. These results demonstrate that NADPH oxidase is the main source of DADS-induced ROS. In addition, Rac2 selectively activates the c-Jun N-terminal kinase pathway, but not the p38 pathway, in DADS-induced apoptosis. So, Rac2, NADPH oxidase and ROS have a critical role in DADS-induced apoptosis in human leukaemia HL-60 cells.© 2010 The Authors. Clinical and Experimental Pharmacology and Physiology © 2010 Blackwell Publishing Asia Pty Ltd.

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