ABSTRACT
Objective: To investigate the effect of mycophenolate mofetil(MMF)on proliferation and apoptosis of cyst-lining epithelial cells in patients with autosomal dominant polycystic kidney disease (ADPKD), and to compare its effect with that of rapamycin (RAPA) in vitro. Methods: Primary cultured cyst-lining epithelial cells were treated with MMF and RAPA at different concentrations(0, 0.005, 0.05, 0.5, 5 μg/ml)for 48 h or 72 h. The inhibitory effects of them on the cells were evaluated by MTT assay; the cell cycle distribution and apoptotic ratio were determined by flow cytometry. The morphological changes of cyst-lining epithelial cells were observed under transmission electron microscope. Results: Both MMF and RAPA significantly inhibited the proliferation of cyst-lining epithelial cells in a dose- and time dependent manner. After 48 h treatment, the cells were blocked at S phase by MMF and at G0/G1 phase by RAPA. Both drugs induced cell apoptosis, with the maximal apoptotic rate being (5.53 ± 0.27)% for MMF and (4.36 ± 0.10)% for PAPA. Typical morphological changes of apoptotic cells were observed under electron microscope. Conclusion: MMF can effectively inhibit proliferation and induce apoptosis of cyst-lining epithelial cells, but its inhibitory effect is weaker than that of RAPA.
ABSTRACT
Objective To initially investigate the mechanism of COX-2 inhibitor inducing cell apoptosis through the observation of celecoxib (CXB),a specific COX-2 inhibitor,inducing apoptosis of cyst lining epithelial cells of human polycystic kidney.Methods (1)Primarily cultured cell was divided into control group and CXB group to evaluate the proliferative state by Brdu assay.(2)The cell apoptosis was observed by transmitted electronic microscope after being cultured in CXB 2?10~(-5) mol/L for 24,48 hours.(3)The cell apoptosis and apoptotic rate were detected by TUNEL assay.(4) The cell apoptotic rate were measured by AnnexinV,PI-labeled flow cytometry after being cultured in CXB 2?10~(-5) mol/L for 0,24,48 hours.(5)Protein expression of Bax,Bcl-2,caspase 3 was examined by Western blotting.Results (1)The Brdu assay revealed that CXB inhibited cell growth in a concentration-dependent manner,with the maximum growth inhibition ratio of 63.9% when treated by CXB 2?10~(-5) mol/L for 24 h.(2)Typical morphological changes of apoptotic cell were apoptotic body, nuclear concentration,chromatin aggregation,endochylema vacuolization and ravinement under eletrou microscope.(3)TUNEL assay showed that the apoptotic rate was (2.8?0.2)% in control group,and (28.5?1.6)%,(48.5?1.2)% in CXB group for 24,48 hours respectively,with significant differences to control group(P＜0.05).(4) AnnexinV,PI-labeled flow cytometry showed that,in 0,0.5,1,2?10~(-5) mol/L CXB group,the apoptotic rates were (3.15?0.05)%,(7.15?0.11)%,(7.76?0.08)%, (12.15?0.07)% for 24 hours respectively,and (13.53?0.21)%,(18.36?0.17)%,(24.87?0.25)%, (53.66?0.32)% for 48 hours respectively.Significant differences were found among corresponding groups(all P＜0.01 ).(5) Extracted total cell protein in every group and more protein of Bax,Bcl-2 expressed in CXB-treated group was detected by Western blotting than that in control group. Conclusions CXB can inhibit the proliferation of cyst liner epithelial cells in a time- and concentration- dependent manner,and induce cell apoptosis through increasing the ratio of Bax/Bcl-2.CXB is hopeful to become an effective drug to treat ADPKD.
ABSTRACT
Objective:To investigate the effect of mycophenolate mofetil(MMF)on proliferation and apoptosis of cyst-lining epithelial cells in patients with autosomal dominant polycystic kidney disease(ADPKD),and to compare its effect with that of rapamycin(RAPA)in vitro.Methods: Primary cultured cyst-lining epithelial cells were treated with MMF and RAPA at different concentrations(0,0.005,0.05,0.5,5 ?g/ml)for 48 h or 72 h.The inhibitory effects of them on the cells were evaluated by MTT assay;the cell cycle distribution and apoptotic ratio were determined by flow cytometry.The morphological changes of cyst-lining epithelial cells were observed under transmission electron microscope.Results: Both MMF and RAPA significantly inhibited the proliferation of cyst-lining epithelial cells in a dose-and time-dependent manner.After 48 h treatment,the cells were blocked at S phase by MMF and at G0/G1 phase by RAPA.Both drugs induced cell apoptosis,with the maximal apoptotic rate being(5.53?0.27)% for MMF and(4.36?0.10)% for PAPA.Typical morphological changes of apoptotic cells were observed under electron microscope.Conclusion: MMF can effectively inhibit proliferation and induce apoptosis of cyst-lining epithelial cells,but its inhibitory effect is weaker than that of RAPA.
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Objective To investigate the antiproliferative effect of HMG CoA reductase inhibitor on ADPKD cyst-lining epithelial cells and its possible mechanism. Methods Cell viability was assayed by MTT. Membrane-bounded p2lras was detected by immunoblotting. The expression of c-jun and c-fos was examined by immunocytochemistry. Results After ADPKD cyst-lining epithelial cells were exposed to HMG CoA reductase inhibitor, proliferation was markedly inhibited( P
ABSTRACT
Objective:To investigate whether celecoxib(CXB), a specific COX-2 inhibitor,can inhibit the proliferation of cyst lining epithelial cells through blocking mitogen-activated protein kinase(MAPK) signal transduction pathway.Methods: Primarily cultured cells were treated with different concentrations of CXB(0,2.5?10~(-6),5?10~(-6),1?10~(-5),2?10~(-5),3?10~(-5),4?10~(-5),5?10~(-5)mol/L) and the proliferative status was evaluated by BrdU assay.The levels of vascular endothelial growth factor(VEGF) were measured by enzyme-linked immunosorbent assay(ELISA);the production of proliferating cell nuclear antigen(PCNA) and phosphoMAPK were measured by real-time reverse transcription-PCR assay;and the expression of PCNA,MAPK and phospho-MAPK protein was detected by Western blotting.Results: BrdU assay revealed that CXB inhibited cell growth in a concentration-dependent manner;the maximum inhibition rate([63.9?1.2]%) was found when cells were treated with 2?10~(-5) mol/L CXB for 24 h.VEGF secretion by cyst lining epithelial cell was reduced by CXB in a concentration and time-dependent manner.The mRNA and protein levels of PCNA,phospho-MAPK in CXBtreated group were lower than those in control group(with no CXB treatment).Conclusion: CXB can obviously inhibit the proliferation of cyst lining epithelial cell and the secretion of VEGF,which might be through interfering with the phosphorylation of MAPK and partly blocking MAPK signal transduction pathway.
