1.Effects of calcium and calmodulin dependent kinase against hypoxic neuronal injury.
Hui ZHOU ; Xiao-Mei SUN ; Xiao-Li LUO ; Meng MAO
Chinese Journal of Contemporary Pediatrics 2007;9(4):324-326
OBJECTIVETo study the effects of calcium and calmodulin dependent kinase against hypoxic neuronal injury and its possible mechanisms.
METHODSEmbryonic cortical neurons of 17-day pregnant embryo Sprague-Dawley rats were cultured in vitro and the cultured neurons were randomly allocated into different groups that exposed to hypoxia or hypoxia +calcium channel antagonist. Nimodipine and MK-801 were used to block the L-voltage sensitive calcium channel and NMDA receptor respectively before hypoxia. The methyl thiazolyl tetrazolium (MTT) method was used to determine the cell viability. Fluo-4AM, an intracellular calcium indictor, was used to detect the changes of intracellular calcium after hypoxia. The expressions of CaMKII and CaMKIV were detected by Western blot.
RESULTSThe cell viability of the nimodipine or MK-801-treated groups was significantly higher than that of the untreated hypoxia group. The intracellular calcium level of the nimodipine-treated group decreased rapidly after hypoxia. Compared to nimodipine treatment, MK-801 treatment could inhibit hypoxia-induced calcium influx for a longer time. Nimodipine treatment decreased the CaMKII expression while MK-801 treatment decreased the CaMKIV expression.
CONCLUSIONSNimodipine and MK-801 protect neurons from hypoxic injury possibly by the inhibition of CaMKII and CaMKIV expressions respectively.
Animals ; Calcium ; analysis ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinase Type 4 ; Calcium-Calmodulin-Dependent Protein Kinases ; antagonists & inhibitors ; physiology ; Cell Hypoxia ; Dizocilpine Maleate ; pharmacology ; Female ; Neurons ; pathology ; Neuroprotective Agents ; pharmacology ; Nimodipine ; pharmacology ; Rats ; Rats, Sprague-Dawley
2.Study the role of PD98059 in ovarian carcinoma cell line HO-8910.
Lin-lin XU ; Jin-hong MEI ; Jia-xiang CHEN ; Shan XU ; Hai-yan QIN ; Shan-shan WANG
Chinese Journal of Pathology 2008;37(9):625-626
Calcium-Calmodulin-Dependent Protein Kinases
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antagonists & inhibitors
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Carcinoma
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pathology
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Cell Cycle
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drug effects
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Cell Line
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Cell Line, Tumor
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Enzyme Inhibitors
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pharmacology
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Female
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Flavonoids
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pharmacology
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Gene Expression Regulation
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drug effects
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Humans
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Ovarian Neoplasms
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pathology
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Proto-Oncogene Proteins c-fos
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genetics
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metabolism
3.Potential mechanism underlying down-regulation of albumin expression induced by lipopolysaccharide in rat hepatocytes.
Xin-ying WANG ; Ning LI ; Wei-qin LI ; Jie-shou LI
Chinese Journal of Surgery 2004;42(6):362-365
OBJECTIVETo investigate the potential molecular mechanism underlying down-regulation of albumin expression induced by lipopolysaccharide (LPS) in rat hepatocytes.
METHODSThe albumin mRNA expression and albumin protein levels in the supernatant were observed at 0, 2, 8, 12 and 24 hours after 1 micro g/ml LPS treatment. The albumin mRNA expression was determined by RT-PCR, and albumin protein levels were assayed with EIA in the supernatant of hepatocytes pretreated with specific inhibitors of extracellular signal-regulated kinase (PD98059) and p38 kinase (SB203580).
RESULTSThe albumin mRNA expression was reduced to 70% of baseline value, meanwhile, the albumin protein concentration in the supernatant was reduced by 50% compared with the controls after 24 hours of LPS treatment. Pretreatment with PD98059 and SB203580 did significantly inhibit the reduction of albumin by LPS at the given dose.
CONCLUSIONThe results indicate that LPS can induce the reduction of albumin synthesis by down-regulation of albumin mRNA expression at transcription level, and the process may be related to the signal transducation of extracellular signal-regulated kinase and p38 kinase.
Albumins ; genetics ; metabolism ; Animals ; Calcium-Calmodulin-Dependent Protein Kinases ; antagonists & inhibitors ; Dose-Response Relationship, Drug ; Down-Regulation ; Enzyme Inhibitors ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Flavonoids ; pharmacology ; Hepatocytes ; drug effects ; metabolism ; Imidazoles ; pharmacology ; Lipopolysaccharides ; toxicity ; Male ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; Pyridines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; p38 Mitogen-Activated Protein Kinases
4.Synergistic effect of rapamycin (RPM) and PD98059 on cell cycle and mTOR signal transduction in human colorectal cancer cells.
Yan-Jie ZHANG ; Jing-Yuan FANG ; Dan-Feng SUN ; Shu-Liang ZHAO ; Guan-Feng SHEN ; Qing ZHENG ; Hong-Yin ZHU
Chinese Journal of Oncology 2007;29(12):889-893
OBJECTIVETo investigate the synergistic effect of rapamycin (RPM) and PD98059 on human colorectal cancer cells and its potential mechanisms.
METHODSThree human colorectal cancer cell lines SW480, HCT116 and HT29 were treated with RPM 10 nmol/L, PD98059 (10 micromol/L, 20 micromol/L, 40 micromol/L, 50 micromol/L), or RPM plus PD98059, respectively, and the sensitivity was analyzed by MTT assay. The cell cycle progression was evaluated by flow cytometry. Western blotting analysis was performed to examine the total and phosphorylated levels of mammalian target of rapamycin (mTOR) and its downstream translational signaling intermediates, 70 kDa ribosomal protein S6 kinase (p70s6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1).
RESULTSBoth RPM and PD98059 could inhibit viability of the three cell lines. The anti-proliferative effect of PD98059 exhibited a time/dose dependent manner and was strengthen by RPM. All the treatment with RPM, PD98059, and RPM + PD98059 induced arrest of cell cycle, although the arrest was confined at different cell cycle phases. In addition to their effect on proliferation and cell cycle, both inhibitors also reduced phosphorylation levels of mTOR, p70s6k, and 4E-BP1, as well as total 4E-BP1 levels in SW480 and HCT116 cells. That effect was reinforced when cells were treated with RPM plus PD98059 simultaneously, whereas total protein levels of mTOR and p70s6k remained unchanged.
CONCLUSIONRPM and PD98059 inhibit proliferation of colorectal cancer cells synergistically, and induce cell cycle arrest. The modulation of mammalian target of rapamycin signaling pathway is involved in its potential mechanisms.
Adaptor Proteins, Signal Transducing ; metabolism ; Antibiotics, Antineoplastic ; pharmacology ; Calcium-Calmodulin-Dependent Protein Kinases ; antagonists & inhibitors ; Cell Cycle ; drug effects ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; Drug Synergism ; Flavonoids ; pharmacology ; HCT116 Cells ; HT29 Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; antagonists & inhibitors ; metabolism ; Phosphoproteins ; metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases
5.Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system.
Yoshitaka FUKAYA ; Masayuki KURODA ; Yasuyuki AOYAGI ; Sakiyo ASADA ; Yoshitaka KUBOTA ; Yoshitaka OKAMOTO ; Toshinori NAKAYAMA ; Yasushi SAITO ; Kaneshige SATOH ; Hideaki BUJO
Experimental & Molecular Medicine 2012;44(5):330-339
Auto-transplantation of adipose tissue is commonly used for the treatment of tissue defects in plastic surgery. The survival of the transplanted adipose tissue is not always constant, and one of reasons is the accelerated apoptosis of the implanted preadipocytes. We have recently established highly homogeneous preadipocytes, named ccdPAs. The aim of the current study was to evaluate the regulation of the potency of platelet-rich plasma (PRP) on the apoptosis of ccdPAs in vitro. PRP stimulated the proliferation of the preadipocytes in a dose-dependent manner, and the stimulatory activity of 2% PRP was significantly higher than that of 2% FBS or 2% platelet-poor plasma (PPP). The presence of 2% PRP significantly inhibited serum starvation- or TNF-alpha/cycloheximide-induced apoptosis in comparison to 2% FBS or 2% PPP. DAPK1 and Bcl-2-interacting mediator of cell death (BIM) mRNAs were reduced in the preadipocytes cultured with 2% PRP in comparison to those cultured in 2% FBS. The gene expression levels were significantly higher in cells cultured without serum in comparison to cells cultured with 2% FBS, and the levels in the cells with 2% PRP were reduced to 5-10% of those in the cells without serum. These results indicated that ccdPAs exhibit anti-apoptotic activities, in addition to increased proliferation, when cultured in 2% PRP in comparison to the same concentration of FBS, and that this was accompanied with reduced levels of DAPK1 and BIM mRNA expression in in vitro culture. PRP may improve the outcome of transplantation of adipose tissue by enhancing the anti-apoptotic activities of the implanted preadipocytes.
Adipocytes/*cytology
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Adipose Tissue/cytology/metabolism
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Apoptosis/*physiology
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Apoptosis Regulatory Proteins/antagonists & inhibitors/metabolism
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Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors/metabolism
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Cell Culture Techniques/*methods
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*Cell Differentiation
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Cell Proliferation
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Cells, Cultured
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Gene Expression Regulation
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Humans
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Membrane Proteins/antagonists & inhibitors/metabolism
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*Platelet-Rich Plasma/metabolism/physiology
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Proto-Oncogene Proteins/antagonists & inhibitors/metabolism
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Tissue Transplantation
6.Reversion transcriptional expression of DAPK in bladder cancer T24 cells 5-aza-2'-deoxycytidine.
Ning-ru XU ; Chun-xiao LIU ; Shao-bo ZHENG ; Hu-lin LI ; Ya-wen XU ; Kai XU
Journal of Southern Medical University 2009;29(9):1882-1886
OBJECTIVETo investigate the methylation status of the promoter of resion death associated protein kinase (DAPK) gene in bladder cancer cell (T24), and study the effect of 5-aza-2'-deoxycytidine (5-aza-dc) on DAPK gene reactive expression in T24 and its inhibitory effect on T24.
METHODSThe bladder cancer cell T24 was treated with different doses of 5-aza-dc. The inhibitory effect and apoptosis rate were detected by MTT and flow cytometry, and the changes of DAPK mRNA and protein expression and the methylation status of DAPK promoter were assessed by RT-PCR, Western blotting, and methylation specific PCR, respectively.
RESULTSThe growth of bladder cancer cell was inhibited significantly and the maximal apoptosis rate detected by flow cytometry was (24.12-/+1.4)%. DAPK mRNA was not expressed in bladder cancer cell T24 in normal conditions. DAPK mRNA and protein re-expressed after 5-aza-dc (12.5 micromol/L) treatment in cell line T24 for 24 h, and DAPK promoter became unmethylated.
CONCLUSIONSThe promoter methylation can be an important factor for silencing the expression of DAPK in bladder cancer cell. 5-aza-dc can inhibit the growth and induce apoptosis of bladder cancer cells through reversing unmethylation status of DAPK promoter.
Apoptosis Regulatory Proteins ; genetics ; metabolism ; Azacitidine ; analogs & derivatives ; pharmacology ; Calcium-Calmodulin-Dependent Protein Kinases ; genetics ; metabolism ; Cell Line, Tumor ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; Death-Associated Protein Kinases ; Humans ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; genetics ; metabolism ; Transcriptional Activation ; drug effects ; Urinary Bladder Neoplasms ; metabolism ; pathology