ABSTRACT
BACKGROUND: Oxidized low density lipoprotein (oxLDL) is involved in the development of vascular diseases. Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glyero-3-phosphorylcholine) is a key component of oxLDL. METHODS: In the present study, we evaluate whether oxLDL can regulate migration of human bone-marrow derived stem cells (hBMSCs) and characterize the role of PAF in the oxLDL-induced migration. RESULTS AND CONCLUSIONS: oxLDL stimulated chemotaxis of hBMSCs in vitro. Treatment of the cells with BN52021, a specific antagonist of PAF receptor (PAF-R), completely blocked the cell migration induced by PAF, but not platelet-derived growth factor (PDGF-BB). Using PAF-R-specific small interfering RNA, it was demonstrated that silencing of endogenous PAF-R expression significantly attenuated cell migration induced by PAF, but not PDGF-BB, suggesting the specific involvement of PAF-R in the oxLDL-induced cell migration. In addition, PAF-induced migration of hBMSCs was abrogated by pretreating cells with mitogen-activated protein kinase (MAPK) inhibitors, including the MEK inhibitor U0126, the p38 MAPK inhibitor SB202190, and the JNK inhibitor SP600125. Moreover, adenoviral overexpression of a dominant negative mutant of p38 MAPK blocked PAF-stimulated migration. Taken together, these results suggest that PAF plays a pivotal role in the oxLDL-induced recruitment of hBMSCs through mechanisms involving PAF-R-dependent activation of MAPKs.
Subject(s)
Bone Marrow Cells/cytology , Lipoproteins, LDL/metabolism , Mesenchymal Stem Cells/cytology , Platelet Membrane Glycoproteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Anthracenes/pharmacology , Becaplermin , Butadienes/pharmacology , Cell Movement , Humans , Imidazoles/pharmacology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Nitriles/pharmacology , Platelet Activating Factor/pharmacology , Platelet Membrane Glycoproteins/antagonists & inhibitors , Platelet Membrane Glycoproteins/genetics , Platelet-Derived Growth Factor/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-sis , Pyridines/pharmacology , RNA Interference , RNA, Small Interfering/metabolism , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Diallyl disulfide (DADS) is the most prevalent oil-soluble sulfur compound in garlic and inhibits cell proliferation in many cancer cell lines. Here we examined DADS cytotoxicity in a redox-mediated process, involving reactive oxygen species (ROS) production. In the present study, p53-independent cell cycle arrest at G2/M phase was observed with DADS treatment, along with time-dependent increase of cyclin B1. In addition, apoptosis was also observed upon 24-h DADS treatment accompanied by activation of p53. In HCT-116 cells, DADS application induced a dose-dependent increase and time-dependent changes in ROS production. Scavenging of DADS-induced ROS by N-acetyl cysteine or reduced glutathione inhibited cell cycle arrest, apoptosis and p53 activation by DADS. These results suggest that ROS trigger the DADS-induced cell cycle arrest and apoptosis and that ROS are involved in stress-induced signaling upstream of p53 activation. Transfection of p53 small interfering RNA prevents the accumulation of cleaved poly(ADP-ribose) polymerase and sub-G1 cell population by 65% and 35%, respectively. Moreover, DADS-induced apoptosis was also prevented by treatment with oligomycin, which is known to prevent p53-dependent apoptosis by reducing ROS levels in mitochondria. These results suggest that mitochondrial ROS may serve as second messengers in DADS-induced apoptosis, which requires activation of p53.
Subject(s)
Allyl Compounds/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Colonic Neoplasms/pathology , Disulfides/pharmacology , Antioxidants/metabolism , CDC2 Protein Kinase/metabolism , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin B/metabolism , Cyclin B1 , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Flow Cytometry , HCT116 Cells , Humans , Oligomycins/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Time Factors , Tumor Suppressor Protein p53/metabolismABSTRACT
We investigated the differential role of diphenyleneiodonium (DPI), which is widely used as an inhibitor of NADPH oxidase, on the activation of cell cycle regulators in the cell cycle progression. DPI efficiently blocked the transition from G0/G1 to S phase by serum stimulation in quiescent HCT-116 (wild-type p53) and HL-60 (null p53) cells. Concomitant with G0/G1 arrest, HCT-116 cells treated with DPI resulted in strong and sustained upregulation of p53 and p21. p53- or p21-deficient HCT-116 cells using a small interfering RNA (siRNA) significantly increased the progression into S phase by stimulation of DPI, compared with DPI alone. However, the silencing of p53 resulted in more efficient transition into S phase than the silencing of p21 siRNA and significantly inhibited p21 upregulation by DPI stimulation. Interestingly, brief exposure to DPI did not change p53 expression, but showed transient upregulation of p21 and G0/G1 arrest. These results suggest that p53 upregulation sustains G0/G1 cell cycle arrest and p21 upregulation by DPI stimulation in HCT-116 cells. In HL-60 cells, DPI also induced p21 upregulation in a p53-independent manner and the increase of p21 expression seems to be regulated by DPI-mediated ERK activation. Cyclin D1 expression was not significantly affected by DPI treatment in HCT-116 cells. However, in HL-60 cells, DPI irreversibly impaired cyclin D1 upregulation by serum stimulation and a much greater fraction of cells arrested in G0/G1 was observed in HL-60 cells than in HCT-116 cells at 24 h after brief DPI treatment. These results suggest that cyclin D1 is an important regulatory factor in the inhibition of cell cycle progression by DPI in HL-60 cells.
Subject(s)
Cell Cycle/drug effects , Enzyme Inhibitors/pharmacology , NADPH Oxidases/antagonists & inhibitors , Onium Compounds/pharmacology , Tumor Suppressor Protein p53/metabolism , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin D1/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Dose-Response Relationship, Drug , Extracellular Signal-Regulated MAP Kinases/metabolism , HCT116 Cells , HL-60 Cells , Humans , NADPH Oxidases/metabolism , Neoplasms/enzymology , Neoplasms/pathology , RNA Interference , RNA, Small Interfering/metabolism , Signal Transduction/drug effects , Time Factors , Transfection , Tumor Suppressor Protein p53/genetics , Up-RegulationABSTRACT
To understand the role of adventitial fibroblasts (AF) in diabetic vascular diseases, the importance of high glucose (HG, 25mM) on matrix metalloproteinase-2 (MMP-2) production in AF was determined. HG enhanced mRNA, protein and gelatinolytic activity of MMP-2. The enhanced MMP-2 activity was significantly attenuated not only by a PI3K inhibitor but also by an Akt inhibitor. These HG-induced MMP-2 responses were markedly reduced in Akt1-deficient (1KO) cells. The diminished HG-induced MMP-2 responses were completely restored by re-expression of Akt1. Both the reporter activity and electrophoretic mobility shift assay for activator protein-1 and nuclear factor-kappa B (NF-kappaB) were enhanced by HG, but NF-kappaB were not increased in 1KO cells. Furthermore, HG-induced MMP-2 responses were markedly suppressed by NF-kappaB decoy oligodeoxynucleotides. Based on these results, it is suggested that HG augments MMP-2 production via PI3K/Akt1/NF-kappaB pathway.