title: Role of Ras-related C3 botulinum toxin substrate 2 (Rac2), NADPH oxidase and reactive oxygen species in diallyl disulphide-induced apoptosis of human leukaemia HL-60 cells.
abstract: 1. 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.
authors.affiliation: Cancer Research Institute, University of South China, Hengyang, Hunan Province, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Yi
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Ji
authors.forename: Hui
authors.initials: H
authors.lastname: Tan
authors.forename: Min
authors.initials: M
authors.lastname: Lin
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
authors.forename: Ling
authors.initials: L
authors.lastname: Wen
authors.forename: Yan-Hua
authors.initials: YH
authors.lastname: Ma
authors.forename: Qi
authors.initials: Q
authors.lastname: Su
mesh_terms: D000498:Allyl Compounds / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3 / Q000096:biosynthesis; D045744:Cell Line, Tumor; D053938:DNA Fragmentation; D004220:Disulfides / Q000494:pharmacology*; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D019255:NADPH Oxidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018808:Transcription Factor AP-1 / Q000096:biosynthesis; D015854:Up-Regulation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000096:biosynthesis; D020744:rac GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D000498:Allyl Compounds; D053148:Caspase 3; D004220:Disulfides; D048031:JNK Mitogen-Activated Protein Kinases; D019255:NADPH Oxidases; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D018808:Transcription Factor AP-1; D048051:p38 Mitogen-Activated Protein Kinases; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein; C489427:MYC protein, human; C028009:diallyl disulfide