ABSTRACT
To investigate the cell proliferation inhibition and apoptosis induced by berberine a-hydroxy f-decanoylethyl sulfonate (HB) on MDA-MB-231 cells in vitro, and the inhibitory effect of HB on the expression of poly adenosine diphosphate RNA polymerase (PARP), MTT assay was used to detect the viability of MDA-MB-231 cells and cell cycle was examined by flow cytometry. The results showed that HB could significantly inhibit the proliferation of MDA-MB-231 cells, and mildly arrested cell cycle progression at S phase. The IC50S for 24, 48 and 72 h treatment were 4.65, 1.46 and 0.75 mg.L-1 (7.55, 2.37 and 1.22 micromol.L-1), respectively. Annexin V-FITC/PI double staining assay showed that HB increased apoptotic ratio of MDA-MB-231 cells. Western blotting analysis showed the expressions of procaspase-3, procaspase-9 and PARP were decreased after HB treatment, while their fragment increased. The results suggest that HB can inhibit the growth and induce apoptosis of MDA-MB-231 cells, which may be associated with inhibition of the expression of procaspase-3, procaspase-9 and PARP.
Subject(s)
Female , Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Berberine , Pharmacology , Caspase 3 , Metabolism , Caspase 9 , Metabolism , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Inhibitory Concentration 50 , Poly(ADP-ribose) Polymerases , Metabolism , Triple Negative Breast Neoplasms , Metabolism , PathologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of enoyl coenzyme A hydratase-1 (Ech1) on the proliferation and invasion ability of mouse hepatocarcinoma Hca-P cells in vitro.</p><p><b>METHODS</b>Recombinant pcDNA3.1(+)-Ech1 gene and pcDNA3.1(+) were transfected into Hca-P cells by cationic liposomes introduction. Clone of PEch1 cells that stably expressing Ech1 and clone of control Pvector cells were screened by G418. The Ech1 expression was identified subsequently by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The malignant behaviors of the cell lines were compared by proliferation, invasion and migration test.</p><p><b>RESULTS</b>The cell line Hca-P cells stably expressing Ech1 gene was constructed. The relative expression of Ech1 mRNA in the PEch1 group was 3.21 ± 0.43 and in the Pvector group was 1.44 ± 0.03, with a significant difference between the two groups (P = 0.029). The results of ELISA revealed that the expression of Ech1 protein was 0.140 ± 0.005 in the PEch1 group, 0.088 ± 0.003 in the Pvector group, and 0.078 ± 0.006 in the Hca-P group, showing a significant difference between the PEch1 group and the Pvector and Hca-P groups (P < 0.05). Transwell migration test showed that the number of penetrated cells in the PEch1 group was 143.00 ± 7.25 cells, significantly higher than that of the Pvector group (95.73 ± 3.88 cells) and un-treated Hca-1 group (106.67 ± 3.54 cells, both P < 0.05). The Transwell invasion assay showed that the number of penetrated cells was 77.20 ± 5.46 cells in the PEch1 group, significantly higher than 46.34 ± 4.35 cells in the Pvector group and 49.80 ± 5.21 cells in the un-treated Hca-1 group (both P < 0.05).</p><p><b>CONCLUSIONS</b>The results showed that overexpressed Ech1 in Hca-P cells may significantly increase the cell proliferation in a time-dependent manner. The up-regulation of Ech1 may increase to some extent the migration and invasion capacity of Hca-P cells. The efforts aiming at up-regulation of Ech1 expression may become a therapeutic target in the treatment of hepatocarcinoma.</p>
Subject(s)
Animals , Mice , Carbon-Carbon Double Bond Isomerases , Genetics , Metabolism , Cell Movement , Cell Proliferation , Liver Neoplasms, Experimental , Pathology , Neoplasm Invasiveness , Plasmids , RNA, Messenger , Metabolism , Recombinant Proteins , Genetics , Metabolism , Transfection , Tumor Cells, Cultured , Up-RegulationABSTRACT
To examine the differential expression pattern of Ech1 protein in mouse Hca-F and Hca-P hepatocarcinoma cell lines with high and low rates of lymphatic metastasis, respectively, and to investigate the relationships between Ech1 expression and adhesion of Hca-F cells. Fluorescence two-dimensional difference in-gel electrophoresis (2D DIGE) and mass spectrometry were used to detect Ech1 expression. Ech1 gene silencing was achieved by stable transfection of Hca-F cells with a plasmid vector harboring short hairpin RNA (shRNA) targeting Ech1, pGPU6/GFP/Neo-shRNA-Ech1. Ech1 mRNA and protein expressions were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting analysis, respectively. Adhesive properties of cells were assessed by hematoxylin-eosin staining and fluorimetric detection of extracellular matrix (ECM) proteins. Endogenous Ech1 protein level was remarkably higher in the highly metastatic Hca-F cell line than in the Hca-P cell line (2.7-fold by 2D DIGE; 1.5-fold by Western blotting). shRNA-induced silencing of Ech1 significantly reduced the adhesion ability of Hca-F cells, as evidenced by decreased absorbance values of fibronectin and collagen I (Hca-F cells vs. pGPU6/GFP/Neo-shRNA-Ech1 cells: 1.42+/-0.26 vs. 1.01+/-0.27 and 1.14+/-0.07 vs. 0.90+/-0.09, respectively; P less than 0.05). Down-regulation of Ech1 can inhibit the adhesive capacity of metastatic Hca-F cells.
Subject(s)
Animals , Mice , Carbon-Carbon Double Bond Isomerases , Genetics , Metabolism , Carcinoma, Hepatocellular , Genetics , Pathology , Cell Adhesion , Cell Line, Tumor , Down-Regulation , Electrophoresis, Gel, Two-Dimensional , Gene Expression Regulation, Neoplastic , Liver Neoplasms , Genetics , Pathology , Lymph Nodes , Pathology , Lymphatic Metastasis , Plasmids , RNA, Messenger , Genetics , Metabolism , RNA, Small Interfering , Genetics , Pharmacology , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To study the expression of enoyl CoA hydratase 1 (ECH1) and the effect when down-regulation of ECH1 gene expression in mouse hepatocarcinoma cell.</p><p><b>METHODS</b>Immunofluorescence was used for detecting the expression of ECH1, and stably transfected Hca-F cells with pGPU6/GFP/Neo-shRNA-ECH1 expression plasmids. Cell proliferation was assessed by Cell counting kit-8 (CCK8) assay. The Boyden-transwell assay (8 µm pore size) was performed to analyze the inhibitory effect of shRNA on Hca-F cell migration and invasion.</p><p><b>RESULTS</b>ECH1 expression was obtained in the cytoplasm and upregulated expression in Hca-F cells than that in Hca-P cells. The down-regulation of ECH1 could inhibit the cell proliferation of Hca-F cells, decrease the number of cell pass through Transwell (27.07 ± 17.49) compared with scramble-negative (72.38 ± 18.83) and Hca-F controls (59.06 ± 30.33), decrease the migration capacities of Hca-F cells, increase the ratio of Hca-F cells in S phase (86.1%) compared with scramble-negative (75.8%) and Hca-F controls (66.2%) and decrease the ratio of G(1) phase (9.4%) compared with scramble-negative (24.2%) and Hca-F controls (30.3%).</p><p><b>CONCLUSION</b>ECH1 serves as a potential critical factor attributes to tumor lymphatic metastasis.</p>
Subject(s)
Animals , Mice , Cell Cycle , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cytoplasm , Down-Regulation , Enoyl-CoA Hydratase , Genetics , Metabolism , Gene Expression Regulation, Neoplastic , Liver Neoplasms, Experimental , Pathology , Lymphatic Metastasis , Plasmids , RNA, Small Interfering , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To study the effects of silencing CLIC1 gene expression on the proliferation and invasion of Hca-F cells.</p><p><b>METHODS</b>The mouse CLIC1 cDNA sequence was retrieved from NCBI. Three shRNA sequences were designed and cloned into pGPU6/GFP/Neo plasmids. The plasmids were transfected into Hca-F cells with Lipofectamine 2000. Cell Counting-8 (CCK-8) kit and transwell chamber were used to study the effects of CLIC1 on the proliferation and invasion of Hca-F cells.</p><p><b>RESULTS</b>The pGPU6/GFP/Neo-shRNA-3 plasmid effectively repressed the expression of CLIC1 mRNA. Inhibition of CLIC1 gene expression led to decreased cell proliferation and reduced invasion.</p><p><b>CONCLUSION</b>CLIC1 is essential for the proliferation and invasion of Hca-F cells.</p>
Subject(s)
Animals , Mice , Carcinoma, Hepatocellular , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Chloride Channels , Genetics , Metabolism , Gene Expression Regulation, Neoplastic , Genetic Vectors , Liver Neoplasms , Metabolism , Pathology , Neoplasm Invasiveness , Plasmids , Genetics , RNA Interference , RNA, Messenger , Genetics , Metabolism , RNA, Small Interfering , Genetics , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To study the localization and expression of CLIC1 in mouse hepatocarcinoma ascites cell lines with different metastatic potentials.</p><p><b>METHODS</b>Mouse hepatocarcinoma ascites models (a high potential of lymphatic metastasis cell line-Hca-F, and a low potential of lymphatic metastasis cell line-Hca-P) were investigated using fluorescent two-dimensional difference-gel electrophoresis (2-D DIGE) and mass spectrometry for detecting the localization and expression of CLIC1. Immunofluorescence, immunocytochemistry and Western blot were used to assess CLIC1 protein status in the two cell lines.</p><p><b>RESULTS</b>CLIC1 expression was obtained in the cytoplasm and plasma membrane of cells in both cell lines. 2-D DIGE showed that CLIC1 was overexpressed in Hca-F cells, 1.6 folds higher than that of the Hca-P cells. Hca-F cells also had a higher integral membrane CLIC1 in the Hca-P cells.</p><p><b>CONCLUSIONS</b>Although CLIC1 expression is detected in both Hca-F and Hca-P cell lines, a higher protein expression level is present in Hca-F cells. CLIC1 may play an important role in tumor metastasis.</p>
Subject(s)
Animals , Mice , Ascites , Metabolism , Pathology , Blotting, Western , Cell Line, Tumor , Cell Membrane , Metabolism , Chloride Channels , Metabolism , Cytoplasm , Metabolism , Immunohistochemistry , Liver Neoplasms, Experimental , Metabolism , Pathology , Lymphatic Metastasis , Mice, Inbred Strains , Two-Dimensional Difference Gel ElectrophoresisABSTRACT
<p><b>OBJECTIVE</b>To study the expression of COX-2 and pregnancy associate plasma protein A (PAPP-A) in coronary arteries and their relationship with acute coronary syndrome.</p><p><b>METHODS</b>Twenty-one autopsy cases with acute coronary syndrome encountered during the period from 2002 to 2007 were enrolled into the study. Another 21 autopsy cases without evidence of acute coronary syndrome were used as the controls. The right and left coronary arteries of each group were dissected, embedded and processed as paraffin sections. Immunohistochemical study for CD68 and alpha-actin was performed to highlight the presence of macrophages and smooth muscle cells, respectively. The expression of COX-2 and PAPP-A was evaluated.</p><p><b>RESULTS</b>In the acute coronary syndrome group, COX-2 was localized mainly in the cytoplasm of endothelial cells, macrophages and smooth muscle cells. COX-2 expression in the cytoplasm of smooth muscle cells (28.60%) was significantly higher than that in the control group (4.76%, chi(2) = 14.13, P< 0.05). There was a positive correlation on COX-2 and PAPP-A expression in smooth muscle cells of the media layer of coronary arteries in acute coronary syndrome group (r = 0.88, P < 0.05). The expression of PAPP-A in smooth muscle cells of the media layer in coronary arteries not associated with plaque formation, was higher than that when there were atherosclerotic plaques (chi(2) = 10.36, P < 0.05).</p><p><b>CONCLUSION</b>In coronary arteries, COX-2 and PAPP-A play certain roles in the pathogenesis of acute coronary syndrome.</p>
Subject(s)
Adult , Aged , Female , Humans , Middle Aged , Pregnancy , Young Adult , Acute Coronary Syndrome , Metabolism , Pathology , Autopsy , Coronary Vessels , Metabolism , Pathology , Cyclooxygenase 2 , Metabolism , Myocytes, Smooth Muscle , Metabolism , Plaque, Atherosclerotic , Metabolism , Pregnancy-Associated Plasma Protein-A , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis.</p><p><b>METHODS</b>A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastasis Hca-F and its synogenetic cell line Hca-P with low metastatic potential was constructed by suppression subtractive hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed.</p><p><b>RESULTS</b>Ten differentially expressed cDNA fragments of Hca-F with high potential of lymphatic spreading were obtained, two of which were newly identified ones.</p><p><b>CONCLUSION</b>SSH is a useful technique to detect genes of differential expression and an effective method to clone novel genes.</p>
Subject(s)
Animals , Mice , Carcinoma, Hepatocellular , Genetics , Pathology , Cell Line, Tumor , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Library , Liver Neoplasms, Experimental , Genetics , Pathology , Lymphatic Metastasis , Genetics , Mice, Inbred Strains , Nucleic Acid Hybridization , Methods , RNA, Messenger , GeneticsABSTRACT
Aim The effects of antibiotics and anticoagulants on mouse peritonaeum were ob-served to explore the factor of the peritoneal dialysis related sclerosing peritoni-tis. Methods The experimental models of peritoneal dialysis were established in miceby infusing different kind of drugs to the peritoneal cavity and the changes of the peri-toneal membrane for each drug at different time were observed by the autopsy and lightmicroscope for several weeks. Results Amikacin, Cefradine, Zinacef, Ciprofloxacin,Heparin and Urokinase could induce sclerosing changes of peritoneal membrane such asloss of peritoneal mesothelum infiltration of inflammatory cells and of proliferation fibrecell.These changes were irreversible after the drugs were stoped.Conclusion Thedrugs commonly used in peritoneal dialysis may in different degree result in peritonealsclerosis.