ABSTRACT
Objective:Vacuolar ATPase (V-ATPase) was found within the membranes and internal organelles of a vast array of eukaryotic cells,and was related to various kinds of highly metastatic tumors.LASS2/TMSG1 gene was a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3M in 1999 by our laboratory.It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of V-ATPase (ATP6V0C).In this study,To use RNA interference to suppress the expression of ATP6V0C and try to further investigate the molecular mechanism of ATP6V0C in tumor metastasis and its relationship with LASS2/TMSG1 gene.Methods and Results:The expression level of ATP6V0C mRNA and protein in high metastatic potential prostate cancer cell lines (PC-3M-1E8 and PC-3M) was significantly higher than that in low metastatic potential prostate cancer cell lines (PC-3M-2B4 and PC-3),the expression level in PC-3M-1E8 being the highest.Follow-up tests selected PC-3M-1E8 cells for gene silencing.The expression and secretion of MMP-2 and the expression of MMP-9 in ATP6V0C siRNA transfected PC-3M-1E8 cells displayed no obvious change,but the activity of secreted MMP-9 was abated noticeably compared with the controls (P ＜ 0.05).Extracellular hydrogen ion concentration and V-ATPase activity in interference group were both reduced significantly compared with the controls (P ＜ 0.05).The migration and invasion capacity of ATP6V0C siRNA interfered cells in vitro were diminished significantly compared with the controls (P ＜ 0.05).Furthermore,a dramatic reduction of LASS2/TMSG1 mRNA and protein level after transfection of siRNA in PC-3M-1 E8 cells was discovered (P ＜ 0.05).Confocal immunofluorescence showed a vast co-localization of ATP6V0C protein and LASS2/TMSG1 protein in plasma and membrane.The co-localization signals of control group were much stronger than those of interference group.Conclusion:Specific siRNA silencing of ATP6V0C gene inhibits the invasion of human prostate cancer cells in vitro by mechanism of inhibiting V-ATPase activity and then reducing the extracellular hydrogen ion concentration,inhibiting MMP-9 activation and affecting ECM degradation and reconstruction.Meanwhile,ATP6V0C and LASS2/TMSG1 have interaction and it is likely that ATP6V0C functions as a feedback regulator of LASS2/TMSG1.
ABSTRACT
AIM:To investigate the inhibitory effect of ethanol extract of propolis (EEP) on oxidized low-density lipoprotein (ox-LDL)-induced vascular endothelial cell apoptosis and the underlying molecular mechanisms.METHODS:Human umbilical vein endothelial cells (HUVECs) were pretreated with EEP (7.5,15 and 30 mg/L) or 4-phenylbutyric acid (PBA,4 mmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin (TM,4 mg/L) for 24 h.The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC/PI double staining,respectively.The activities of lactic dehydrogenase (LDH) in the medium and caspase-3 in the HUVECs were measured.The protein and mRNA levels of C/EBP homologous protein (CHOP),a proapoptotic molecule under endoplasmic reticulum stress (ERS),and its downstream Bcl-2 were examined by Western blot and real-time PCR,respectively.RESULTS:Like PBA (an ERS inhibitor),EEP protected HUVECs from ox-LDL-induced injury in a dose-dependent manner,as assessed by the increased cell viability and the decreased LDH release,apoptotic rate and caspase-3 activation.The decrease in cell viability and the increases in LDH release,apoptotic rate and caspase-3 activation induced by TM,an ERS inducer,were also attenuated by EEP.Moreover,EEP suppressed ox-LDL-induced CHOP upregulation and Bcl-2 downregulation,and this effect was similar to that of PBA.Similarly,EEP significantly suppressed TM-induced CHOP upregulation both at the protein and mRNA levels.CONCLUSION:EEP may protect HUVECs from ox-LDL-induced apoptosis,and the mechanism is at least partially involved in suppressing CHOP-mediated ERS-associated apoptotic pathway.
ABSTRACT
AIM:To investigate the inhibitory effect of ethanol extract of propolis (EEP) on oxidized low-density lipoprotein (ox-LDL)-induced vascular endothelial cell apoptosis and the underlying molecular mechanisms.METHODS:Human umbilical vein endothelial cells (HUVECs) were pretreated with EEP (7.5,15 and 30 mg/L) or 4-phenylbutyric acid (PBA,4 mmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin (TM,4 mg/L) for 24 h.The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC/PI double staining,respectively.The activities of lactic dehydrogenase (LDH) in the medium and caspase-3 in the HUVECs were measured.The protein and mRNA levels of C/EBP homologous protein (CHOP),a proapoptotic molecule under endoplasmic reticulum stress (ERS),and its downstream Bcl-2 were examined by Western blot and real-time PCR,respectively.RESULTS:Like PBA (an ERS inhibitor),EEP protected HUVECs from ox-LDL-induced injury in a dose-dependent manner,as assessed by the increased cell viability and the decreased LDH release,apoptotic rate and caspase-3 activation.The decrease in cell viability and the increases in LDH release,apoptotic rate and caspase-3 activation induced by TM,an ERS inducer,were also attenuated by EEP.Moreover,EEP suppressed ox-LDL-induced CHOP upregulation and Bcl-2 downregulation,and this effect was similar to that of PBA.Similarly,EEP significantly suppressed TM-induced CHOP upregulation both at the protein and mRNA levels.CONCLUSION:EEP may protect HUVECs from ox-LDL-induced apoptosis,and the mechanism is at least partially involved in suppressing CHOP-mediated ERS-associated apoptotic pathway.
ABSTRACT
Objective To analyze the effect of Yishenkang capsule on urinary protein in patients with chronic glomerulonephritis uraemia. Methods 100 patients with chronic glomerulonephritis uraemia from March 2013 to April 2016 were divided into two groups with lottery method and 50 cases for each group. Two groups were treated with dialysis and routine treatment, the treatment group was added Yishenkang capsule to analyze its effect. Results After treatment, 24 h urinary protein of two groups decreased than before treatment, both Alb and Hb increased than before treatment, and the treatment group was better than that of the control group with statistical significance (P<0.05). SCr, BUN and UA level of two groups decreased than before treatment, the treatment group was lower than that of the control group with statistical significance (P<0.05). The level of inflammation in the control group was significantly higher than that before treatment, the treatment group decreased and lower than that of the control group with statistical significance (P<0.05). There were no significant differences in adverse reactions between two groups. Conclusion Yishenkang capsule could decrease the excretion of urine protein in patients with chronic nephritis uraemia, improve the renal function and has few adverse reactions.
ABSTRACT
In order to prepare the monoclonal antibodies (MAbs) by the recombinant phosphoprotein (p24) of Borne dis ease virus (BDV) as immunogen,the spleen cells of immunized balb/c mice with the recombinant BDV p24 were fused with the myeloma cells SP2/0.The hybridoma cell lines secreting MAbs against p24 were obtained after selected by indirect ELISA and subcloned for 3 times.The MAbs were prepared by intraperitoneal injection in mice,purified by affinity chromatography,identified by western blot and immunofluorescence assay (IFA) for specificity.The purified MAbs against BDV p24 from ascites belonged to IgG1 showed a purity of 98％ and 93％,and a titer of 1 ∶ 81 000,and specific reaction with the BDV p24 restructured and expressed in Oligodendroglia cells (OL).The MAbs against BDV p24,with high specificity and sensitivity,were prepared successfully,which laid a basis for the study of diagnostic reagents and pathogenic mechanism of BDV.
ABSTRACT
Objective To analyze the effect of Yishenkang capsule on urinary protein in patients with chronic glomerulonephritis uraemia. Methods 100 patients with chronic glomerulonephritis uraemia from March 2013 to April 2016 were divided into two groups with lottery method and 50 cases for each group. Two groups were treated with dialysis and routine treatment, the treatment group was added Yishenkang capsule to analyze its effect. Results After treatment, 24 h urinary protein of two groups decreased than before treatment, both Alb and Hb increased than before treatment, and the treatment group was better than that of the control group with statistical significance (P<0.05). SCr, BUN and UA level of two groups decreased than before treatment, the treatment group was lower than that of the control group with statistical significance (P<0.05). The level of inflammation in the control group was significantly higher than that before treatment, the treatment group decreased and lower than that of the control group with statistical significance (P<0.05). There were no significant differences in adverse reactions between two groups. Conclusion Yishenkang capsule could decrease the excretion of urine protein in patients with chronic nephritis uraemia, improve the renal function and has few adverse reactions.
ABSTRACT
In order to prepare the monoclonal antibodies (MAbs) by the recombinant phosphoprotein (p24) of Borne dis ease virus (BDV) as immunogen,the spleen cells of immunized balb/c mice with the recombinant BDV p24 were fused with the myeloma cells SP2/0.The hybridoma cell lines secreting MAbs against p24 were obtained after selected by indirect ELISA and subcloned for 3 times.The MAbs were prepared by intraperitoneal injection in mice,purified by affinity chromatography,identified by western blot and immunofluorescence assay (IFA) for specificity.The purified MAbs against BDV p24 from ascites belonged to IgG1 showed a purity of 98％ and 93％,and a titer of 1 ∶ 81 000,and specific reaction with the BDV p24 restructured and expressed in Oligodendroglia cells (OL).The MAbs against BDV p24,with high specificity and sensitivity,were prepared successfully,which laid a basis for the study of diagnostic reagents and pathogenic mechanism of BDV.