Rac2, In The Role And Mechanism Of Diallyl Disulfide-induced Apoptosis In Human Leukemia

BackgroundThe main active component of the cancer-fighting allyl sulfides in garlic is apparently diallyl disulfide (DADS), which can induce apoptosis in many tumor cells. Previous studies in our laboratory confirmed that DADS could induce differentiation and apoptosis in human gastric cancer cells and human leukemia cells. Mechanisms of inducing apoptosis involve G2/M phase cell cycle arrest, histone acetylation, inhibition of extracellular signal-regulated kinases (ERKs) and activation of p38 signaling pathway. However, the exact mechanisms of DADS induction of tumor cell apoptosis need further investigation.Establishment of the model of apoptosis initiation Phase in human leukemia HL-60 cells induced by DADSThis research aim to investigate the apoptosis initiation phase in human leukemia HL-60 cells induced by DADS, establish the apoptosis initiation model.After incubation of HL-60 cells with 3.6mg·L-1 DADS, the effects of DADS on HL-60 cell growth inhibition were estimated by growth curve, inverted microscope, light microscope, flow cytometry method was used to determine the induction of apoptosis and the expression of actived caspase-3, DNA agarose electrophoresis was used to determine the induction of apoptosis, and protein expression of caspase-3, Bcl-2 was tested by western-blotting.Growth curve show the growth of HL-60 cells could not be inhibited at day l,but remarkably reduced the growth rate at day2 to day6 (24.1%,36.5%,44.2%,52%,53.6%, P<0.05), After HL-60 cells were exposed to DADS, the typical apoptotic morphological changes were observed under the light microscope and inverted microscope. Flow cytometry analysis showed that the apoptotic rate of DADS treated cells at day 1 and day 2 was 3.1%, 4.3%, with no difference from control cells(3.0%), while from day 3 to day 5, the apoptotic rate was 8.5%,15.2%,27.4%, respectively, have great difference from control cells, the DADS-withdrawal group from day 2 to day 5 the apoptotic rate was 7.9%, 12.4%,16.5%,18.8% respectively, significantly increased than the control group and the 1 day with DADS withdrawl(P<0.05).the expression of actived caspase-3 from day 2 to day 5 was 6.3%,10.0%,10.4%,14.9%,17.3%, respectively, was higher than control group and DADS treated 1 day(P<0.05), DNA agarose electrophoresis was showed the DADS treated group from day 4 and the DADS-withdrawal group from day 2 had DNA ladder, and western-blotting showed caspase-3 was upregulated and Bcl-2 was downregulated from day 2.These results suggested Diallyl disulfide could significantly induce apoptosis of human Leukemia HL-60 cells; The initiation phase of apoptosis triggered by 3.6mg/L DADS at day 2.The Differential Proteomic Expression Analysis of apoptosis initiation induced by diallyl disulfide in HL-60 cellsThis study was designed to explore the differential proteomic expression in apoptosis initiation phase in human leukemia HL-60 cells induced by diallyl disulfide and its related molecular mechanisms.A series of methods, including immobilized PH gradient-two dimensional polyacrylamide gel electrophoresis, Coomassie brilliant blue staining, PDQuest 2-DE software analysis, peptide mass fingerpringting based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and public domain NCBInr, EMBL and SWISS-PROT databases were used to separate and identify the differential proteomic expressions in apoptosis initiation phase in human leukemia HL-60 cells induced by diallyl disulfide.The results showed that the good 2-DE pattern including high resolution and reproducibility was obtained. After Coomassie brilliant blue staining, the 2-DE image analysis by PDQuest 2-DE software detected average (467±24) spots in HL-60 cell, and (385±18) spots in DADS treated HL-60 cell. And the average matching rate was 82% and 74% respectively. The differential proteomic expression analysis found that there were 387 spots matched and 193 spots unmatched between HL-60 and DADS treated HL-60 maps. The spots on treated group, whose quantity of expressed proteins was above two times compared to control, were 29.22 of them realized up-regulation while 7 of them realized down-regulation. Nine of the differential spots were cut off from Coomassie brilliant blue staining gel at random, measured with MAIDI-TOF-MS and searched in related database. The score of seven protein were above 64, The expression of Ras-related C3 botulinum toxin substrate 2; ACTB Protein; core-binding factor, beta subunit isoform 1; Small proline-rich protein 3; Esterase D/formylglutathione hydrolase and Pkci-Substrate Analog increased in DADS-treated cells. Elongation factor was down-regulation observed in the expression. These proteins function were associated with RNA transcription, protein degradation, transcription regulation, cytoskeleton, metabolism and oxidation-reduction.These data suggested DADS may up-regulate the expression of Ras-related C3 botulinum toxin substrate 2; ACTB protein; Core-binding factor, beta subunit isoform 1; Small proline-rich protein 3; Esterase D/formylglutathione hydrolase protein and Pkci-Substrate Analog protein in apoptosis initiation phase in human leukemia HL-60 cells and down-regulated of protein elongation factor in apoptosis initiation phase in human leukemia HL-60 cells induced by diallyl disulfide.Diallyl disulfide induces apoptosis in human leukemia HL-60 cells through activation of JNK mediated by reactive oxygenDiallyl disulfide (DADS) is a chemopreventive agent that can induce apoptosis in many tumor cells. Reactive oxygen species (ROS) are important mediators in apoptosis induced by various stimuli, including chemopreventive agents. The phosphotransferase c-JUN N-terminal kinase (JNK) has been shown to regulate apoptosis.In this study, we found that DADS-induced apoptosis in human leukemia HL-60 cells is mediated by ROS-activated JNK. The DADS-treated HL-60 cells showed a dose-and time-dependent decrease in cell viability and proliferation. Agarose gel electrophoresis of cells treated with 10.0 or 20.0 mg/L DADS for 24h showed a characteristic ladder pattern in their DNA. Levels of DADS-induced ROS, as measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescence, also showed dose-and time-dependent increases in HL-60 cells. Activity of JNK was induced by DADS in a dose-dependent manner; HL-60 cells exposed to10.0 mg/L DADS for 8 h showed maximum levels of phosphorylated JNK, which decreased when exposed for additional 4 hours. In contrast, Sp600125, a specific inhibitor of JNK, blocked apoptosis of HL-60 cells exposed to DADS. N-acetylcysteine (NAC), a known antioxidant, also decreased ROS generation, effectively blocked apoptosis, and decreased DADS-induced phosphorylated JNK levels.These results suggest that JNK is involved in DADS-induced ROS-mediated apoptosis in HL-60 cells. The role of Rac2 in diallyl disulfide induced human leukemia cells apoptosis and its related signaling pathways1. Upregulation of Rac2 during DADS-induced apoptosis in HL-60 cellsTreatment of HL-60 cells with 5,10 or 15 mg/L DADS for 24 h increased apoptosis compared with control (10.4,30.9,35.5 vs xx% apoptotic cells, respectively). Real-time PCR confirmed that there was significant upregulation of the mRNA of components of the NADPH oxidase complex in HL-60 cells after treatment with 10 mg/L DADS for 24 h, including CYBA (p22 subunit of cytochrome b 558), CYBB (gp91phox), NCF1 (p47phox), NCF2 (p67phox), NCF4 (p40phox) and RAC2 (Rac2), which is important for the activation of NADPH oxidase. Western blot results are shown in Fig. 1c,d. Compared with the control group, there was a significant upregulation of Rac2 protein in HL-60 cells treated with 10 and 15 mg/L DADS for 24 h.These results indicate that upregulation of Rac2 and activation of NADPH oxidase occur during DADS-induced apoptosis of HL-60 cells.2. Effect of Rac2 interference on DADS-induced apoptosis in HL-60 cellsTreatment of HL-60 cells with siRNAs resulted in inhibition of Rac2 expression. Results of western blot analysis and semiquantitative real-time PCR clearly demonstrate the effect of RNA interference; in these cells, Rac2 expression was inhibited by approximately 70%. In control cells, in which Mcl-1 siRNA was used instead of Rac2 siRNA, Mcl-1 was inhibited, but there was no effect on Rac2 expression. In DADS+ siRNA-treated group, Rac2 expression was also inhibited. As shown in Fig.2d, treatment of cells with 5,10 and 15 mg/L DADS for 24 h markedly reduced the percentage of apoptotic cells after inhibition of Rac2 with siRNA. The nuclear DNA of late-stage apoptotic cells degraded to regular nucleosome-sized fragments in multiples of 180 bp, as shown by their characteristic ladder pattern on agarose gel electrophoresis. Figure 2e shows that after cells had been treated with 10 and 15 mg/L DADS for 24 h, their DNA displayed characteristic ladder patterns on agarose gel electrophoresis, whereas siRNA-treated groups showed normal bands.These results indicate that pretreatment of HL-60 cells with Rac2 siRNA blocks DADS-induced apoptosis.3. Effect of Rac2 interference on DADS-induced ROS production in HL-60 cells Because DCFH-DA can be rapidly oxidized to highly fluorescent dichlorofluorescein (DCF) in the presence of ROS, the intensity of DCF fluorescence is proportional to the amount of intracellular ROS generated. Although DADS both activates NADPH oxidase and induces ROS production, whether NADPH is the source of the ROS generated remains to be determined. DCF fluorescence intensity was increased in HL-60 cells treated with 10 mg/L DADS for 8 h compared with control group, suggesting that intracellular levels of ROS were also increased. The marked stimulation by PMA of ROS production by DADS-treated HL-60 cells strongly implicates NADPH oxidase as the source of the elevated ROS, because PMA is known to activate latent NADPH oxidase.19 Meantime, PMA enhanced DADS-induced HL-60 cell apoptosis. It is known that DPI is a flavoprotein inhibitor of NADPH oxidase and, in the present study, DPI ameliorated DADS-induced HL-60 cell apoptosis. Furthermore, after the addition of DPI, there was no DADS-induced ROS production evident, even after PMA stimulation. Although DPI is regarded as an inhibitor of NADPH oxidase, it is not specific.24 Hence, in the presesnt study we inhibited Rac2 expression in HL-60 cells using siRNA. After suppression of Rac2, ROS production, as measured by fluorescence intensity, after DADS treatment and PMA stimulation was markedly reduced.These results indicate that NADPH oxidase is the main source of DADS-induced ROS production.4. Effect of Rac2 interference on the JNK signalling pathway in DADS-treated HL-60 cellsWestern blot analysis revelated that, in cells treated with DADS for 12 h, JNK, activator protein (AP)-1, caspase 3 and p38 levels were upregulated compared with untreated cells, whereas c-myc levels decreased. Interestingly, as shown in Fig.4b, JNK1 levels decreased when Rac2 expression was inhibited with siRNA in HL-60 cells (P<0.05), whereas there was no difference in JNK2 levels between Rac2-competent and Rac2 siRNA-inhibited DADS-treated cells. The expression of phosphorylated (p-) JNK1 and cleaved caspase 3 in HL-60 cells decreased when Rac2 expression was inhibited with siRNA. Activator protein-1 and caspase 3 levels were decreased in Rac2 siRNA-inhibited cells exposed to 10 mg/L DADS for 12 h compared with Rac2-competent, DADS-treated cells, whereas c-myc levels were increased and there was no change in p38 and p-p38 (P>0.05). These data suggest that Rac2 selectively activates the JNK pathway, but not the p38 pathway, in DADS-induced apoptosis in HL-60 cells.ConclusionIn conclusion, first, this study used the methods of inverted microscope, light microscope, flow cytometry, DNA agarose electrophoresis, western blot and so on to Establish the model of apoptosis initiation Phase in human leukemia HL-60 cells induced by DADS. Then, Total protein of HL-60 cells with or without 2-day treatment of 3.6 mg/L DADS was extracted, separated by two-dimensional polyacrylamide gel electrophoresis, and analyzed by PDQuest 2-DE software. Differentially expressed proteins were separated and identified by peptide mass fingerprinting analysis and bioinformatics. As compared with those in untreated HL-60 cells, 29 proteins were differentially expressed in DADS-treated HL-60 cells:22 were up-regulated and seven were down-regulated. Among nine proteins which were randomly selected for peptide mass fingerprinting analysis and bioinformatics, seven were meaningful. Rac2 was up-regulated in DADS-treated HL-60 cells. Then, RNA interference was used to test the effect of Rac2. 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.