ABSTRACT
The present study focused on the biologic effects of tempol on anti-inflammatory and nitric oxide generation in contusion spinal cord injury (SCI). The animal model of SCI was induced by dropping a 10-g rod (2.0 mm in diameter) at a height of 25 mm. Tempol was injected intraperitoneally a dose of 100 mg/kg at 15 min before SCI. Controls was injected with saline. The contused spinal segments were removed according to time courses, and the expression level of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was analyzed along with the size of irreversibly damaged region. After SCI, the relative amounts of COX-2 and iNOS mRNA were peaked at 8 h after post-injury, and then decreased up to 7 days post-injury, and normal level at 14 days. Expression of COX-2 protein was peaked at 8 h post-injury. With the tempol pre-treatment, the immunoreactivity of COX-2 and nitrotyrosine in paraffin-embedded tissue slices was profoundly decreased. The irreversibly damaged area of the spinal cord was peaked at 3 days after SCI. With tempol pre-treatment, the irreversibly damaged area shows a statistically significant decrease at 3 days after SCI. These evidences indicate that tempol pre-treatment reduces irreversibly damaged area on the contusion SCI in rat. The mechanisms of biologic reactions of tempol might be related to the decreased expressions of COX-2 and iNOS in spinal cord cells, neurons and glia. It is expected that the tempol effect on the SCI is not only antioxidant activity but also anti-inflammatory reaction.
Subject(s)
Antioxidants/pharmacology , Cyclic N-Oxides/pharmacology , Cyclooxygenase 2/biosynthesis , Neuroprotective Agents/pharmacology , Nitric Oxide Synthase Type II/biosynthesis , Reactive Oxygen Species/metabolism , Spinal Cord Injuries/enzymology , Animals , Anti-Inflammatory Agents/pharmacology , Blotting, Western , Disease Models, Animal , Male , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Spin Labels , Spinal Cord Injuries/pathologyABSTRACT
Natural isoflavones and flavones are important dietary factors for prostate cancer prevention. We investigated the molecular mechanism of these compounds (genistein, biochanin-A and apigenin) in PC-3 (hormone-independent/p53 mutant type) and LNCaP (hormone-dependent/p53 wild type) prostate cancer cells. A cell growth rate and apoptotic activities were analyzed in different concentrations and exposure time to evaluate the antitumor activities of genistein, biochanin-A and apigenin. The real time PCR and Western blot analysis were performed to investigate whether the molecular mechanism of these compounds are involving the p21 and PLK-1 pathway. Apoptosis of prostate cancer cells was associated with p21 up-regulation and PLK-1 suppression. Exposure of genistein, biochanin-A and apigenin on LNCaP and PC-3 prostate cancer cells resulted in same pattern of cell cycle arrest and apoptosis. The inhibition effect for cell proliferation was slightly greater in LNCaP than PC-3 cells. In conclusion, flavonoids treatment induces up-regulation of p21 expression, and p21 inhibits transcription of PLK-1, which promotes apoptosis of cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Cell Cycle Proteins/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Flavonoids/pharmacology , Prostatic Neoplasms/pathology , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Apigenin/pharmacology , Cell Cycle/drug effects , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Gene Expression Regulation, Neoplastic/drug effects , Genistein/pharmacology , Humans , Male , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/metabolism , Transcription, Genetic/drug effects , Polo-Like Kinase 1ABSTRACT
The aim of the present study is to investigate the effect of genistein on human neuroblastoma SK-N-MC cells. MTT proliferation assay, LDH cytotoxicity assay, flow cytometric analysis, real-time quantitative RT-PCR and western blotting were used to investigate the effect of genistein on cell survival, cellular toxicity, cell cycle progression, and mRNA and protein alterations of selected DNA damage-, cell cycle- and apoptosis-related genes in SK-N-MC cells. Genistein suppressed cell proliferation, increased LDH release and modulated cell cycle distribution through accumulation of cells at G2/M- and S-phase and sub-G0 (cell death) with a concurrent decrease of cells at G0/G1 phase. Genistein increased the MDC1 (Mediator of DNA damage Checkpoint protein 1), p53, p21(waf1/cip1), Cdc2 and Bax mRNA levels in a dose-dependent manner. However, PLK1 (Polo-Like Kinase 1) and Cyclin B1 mRNAs were down-regulated after genistein treatment. Furthermore, Genistein did not alter Chk2 (Checkpoint Kinase 2), Bcl-2 and Cdc25C mRNA levels. On western blotting analyses; genistein increased the protein level of MDC1, p53, p21(waf1/cip1), and Bax in a dose-dependent manner. Genistein also increased the phosphorylation of Chk2 and Cdc25C at Thr-68 and Ser-216, respectively. In addition, consistently with PLK1 down-regulation, the phosphorylation of Cdc25C at Ser-198 was markedly decreased after genistein treatment. Additionally, Chk2, Cdc25C, Cyclin B1, p-Cyclin B1 (Ser-147), and Cdc2 as well as Bcl-2 proteins were down-regulated after genistein treatment. Altogether, these results suggest for the first time the involvement of MDC1 up-regulation after genistein treatment in DNA damage-induced Chk2 activation- and PLK1 down-regulation-mediated apoptosis and cell cycle checkpoint pathways.
Subject(s)
Apoptosis/drug effects , Cell Cycle Proteins/metabolism , Cell Cycle/drug effects , DNA Damage/drug effects , Genistein/pharmacology , Neuroblastoma/chemically induced , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Adaptor Proteins, Signal Transducing , Base Sequence , CDC2 Protein Kinase/metabolism , Checkpoint Kinase 2 , Cyclin B/metabolism , DNA Damage/physiology , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Down-Regulation , G2 Phase/drug effects , Humans , S Phase/drug effects , Up-Regulation , bcl-2-Associated X Protein/metabolism , cdc25 Phosphatases/metabolism , Polo-Like Kinase 1ABSTRACT
Acute spinal cord injury (SCI) is two-step process that first involves the primary mechanical injury and then the secondary injury is induced by various biochemical reactions. Apoptosis is one of secondary SCI mechanisms and it is thought to play an important role for the delayed neuronal injury. The enhanced formation of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of apoptosis in SCI. The level of .iNOS mRNA peaked at 6 hr after SCI and it declined until 72 hr after SCI in a rat model. Double-immunofluorescence staining revealed that iNOS positive cells were stained for ED-1, synaptophysin, GFAP, and oligodendrocyte marker. The terminal deoxynucleotidyl-transferase-mediated dUDP-biotin nick end-labeling (TUNEL) positive cell count was higher for the 72 hr post-SCI group than for the 24 hr post-SCI group. This cell count was also higher going in the caudal direction than in the rostral direction from the epicenter, and especially for the 72 hr group. Treatment with a selective iNOS inhibitor resulted in the reduction of TUNEL-positive cells at the lesion site. These findings suggest that nitric oxide generated by the iNOS of macrophages, neurons, oligodentrocytes, and astrocytes plays an important role for the acute secondary SCI that results from apoptotic cell death.
