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.

Involvement of calreticulin in cell proliferation, invasion and differentiation in diallyl disulfide-treated HL-60 cells

“…The actions of DADS include the regulation of cell cycle arrest, induction of apoptosis and cell differentiation, and inhibition of cell invasion (5–7). Previous studies at the Cancer Research Institute, University of South China (Hengyang, China) confirmed that DADS can inhibit the proliferation of human leukemia cells in vivo in a dose-dependent manner (8,9). DADS exhibits a dual effect: A medium dose (>1.25 mg/l) can induce apoptosis in human leukemia cells (8,9), and a small dose (<1.25 mg/l) can induce human leukemia cell differentiation (2).…”

“…Previous studies at the Cancer Research Institute, University of South China (Hengyang, China) confirmed that DADS can inhibit the proliferation of human leukemia cells in vivo in a dose-dependent manner (8,9). DADS exhibits a dual effect: A medium dose (>1.25 mg/l) can induce apoptosis in human leukemia cells (8,9), and a small dose (<1.25 mg/l) can induce human leukemia cell differentiation (2). The mechanisms of inducing differentiation involve: G 2 /M-phase cell cycle arrest; histone acetylation; the regulation of regulatory gene expression, including signal transducer and activator of transcription 3, v-myc avian myelocytomatosis viral oncogene homolog, Fos proto-oncogene and Jun proto-oncogene regulation; and the upregulation of cyclin-dependent kinase inhibitor 1 expression (10–12).…”

Anticancer Mechanism of Sulfur-Containing Compounds

“…In the active state, it binds to different effector proteins including p67 phox and NOS2, regulates apoptosis, and increases the production of ROS by NADPH oxidase [58][59][60]. Accordingly, it was observed that NADPH oxidase and ROS play a pivotal role in DADS-induced apoptosis [38,39,57]. Indeed, cotreatment of lung carcinoma A549 cells with DADS (200 μM) and N-acetyl cysteine (NAC), which is a precursor of GSH, completely abrogated DADS-induced apoptosis [61].…”

“…For some Allium compounds, an antileukemia activity was demonstrated. For example, DADS (68 μM) and ajoene (20 μM) induce apoptosis in human acute myeloid leukemia cells (HL-60) involving JNK [37][38][39], ERK, and p38 signaling pathways [37,40]. Accordingly, pretreatment of colon cancer cells (COLO 205) with a JNK inhibitor leads to a decrease in the percentage of apoptotic cells induced by DADS [41].…”

Diallyl disulfide down‐regulates calreticulin and promotes C/EBPα expression in differentiation of human leukaemia cells

“…Although many patients benefit from these treatments, these methods are rarely curative for the very few residual disseminated tumour cells . The induction of differentiation is a desired consequence of chemopreventive and therapeutic agents, as it often results in the elimination of premalignant or malignant cells . Indeed, numerous studies have focused on selectively killing tumour cells through the induction of differentiation .…”