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1.
Zhonghua Bing Li Xue Za Zhi ; 39(2): 100-5, 2010 Feb.
Article in Chinese | MEDLINE | ID: mdl-20388375

ABSTRACT

OBJECTIVES: To investigate molecular mechanisms of PAR-1 regulation on intracellular Ca²(+) mobilization in lung giant cell carcinoma cells in vitro and its involvement in tumor metastasis. METHODS: Free intracellular Ca²(+) ([Ca²(+)]i) was measured in lung giant cell carcinoma PLA801C and PLA801D cells by confocal microscopy. Sense and anti-sense PAR-1 expression vectors were transfected into PLA801C (C+)and PLA801D(D-) cells, respectively. The effects of PAR-1 expression were investigated by thrombin and TRAP-induced mobilization of [Ca²(+)]i in the C+ and D-cells. RESULTS: There were significant differences of the mean values of [Ca²(+)]i between PLA801D (59.55) and PLA801C cells (35.46, P < 0.01). The mean [Ca²(+)]i of C+ cells (45.77) was significantly higher than that of its control CV cells (35.46, P < 0.05), and the mean [Ca²(+)]i of D-cells (48.42) was significantly lower than that of its control DV cells (59.55, P < 0.05). The peaks of [Ca²(+)]i of C+ and CV cells were 48.19 ± 9.84 and 45.64 ± 9.87 (P < 0.05) respectively at 80 s and 100 s after thrombin treatment, but were 111.31 ± 25.00 and 52.93 ± 11.21 (P < 0.05) respectively at 60 s after TRAP treatment. The peaks of [Ca²(+)]i of D- and DV cells were 40.71 ± 5.89 and 61.07 ± 21.36 (P < 0.05) respectively at 60 s after thrombin treatment, but were 84.98 ± 11.23 and 102.58 ± 21.48 (P < 0.05) respectively at 40 s after TRAP treatment. CONCLUSIONS: The high metastatic potential of PLA801D and PLA801C may be related to [Ca²(+)]i of the tumor cells. PAR-1 may play an important role in the metastasis of lung giant cell carcinoma cells by up-regulating the intracellular Ca²(+).


Subject(s)
Calcium/metabolism , Carcinoma, Giant Cell/metabolism , Lung Neoplasms/metabolism , Receptor, PAR-1/metabolism , Calcium Signaling/drug effects , Carcinoma, Giant Cell/pathology , Cell Line, Tumor , DNA, Antisense/genetics , Humans , Lung Neoplasms/pathology , RNA, Messenger/metabolism , Receptor, PAR-1/genetics , Receptor, PAR-1/physiology , Receptors, Thrombin/metabolism , Thrombin/pharmacology , Transfection , Up-Regulation
2.
Zhonghua Bing Li Xue Za Zhi ; 36(5): 313-7, 2007 May.
Article in Chinese | MEDLINE | ID: mdl-17706138

ABSTRACT

OBJECTIVE: To study the functional aspects of protease-activated receptor 1 (PAR-1) gene involved in tumor metastasis. METHODS: Two human lung giant cell carcinoma cell lines PLA801C (low metastasis potential) and PLA801D (high metastasis potential) were chosen as in-vitro human cancer model systems. Sense and anti-sense expression constructs of PAR-1 gene (pC/PAR1s and pC/PAR1as) were transfected into PLA-801C and PLA-801D cells by lipofection. PAR-1 expression was determined by RT-PCR and western blot analysis. MTT growth, flow cytometry analysis, fibronectin adhesion, and matrigel invasion assays were used to study the effect of PAR-1 expression on the proliferation, adhesion, and invasion of the transfected cells. RESULTS: Appropriate up-regulation or down-regulation of protein expression of PAR-1 was observed in both transfected cell lines (PLA801C and PLA801D) to express PAR-1s or PAR-1as, respectively. Expression of the sense PAR-1 markedly increased cellular proliferation, adhesion and invasion of PLA-801C cells. In contrast, anti-sense PAR-1 significantly inhibited cell growth, adhesion and invasion capabilities, along with cell arrest at G0/G1 phase of the PLA-801D cells. CONCLUSIONS: Successful up- and down- regulation of expression of PAR-1 can be achieved by in-vitro transfection of sense and antisense PAR-1 constructs. PAR-1 may enhance metastasis of lung cancer through its regulation of cellular proliferation, adhesion and invasion. Down-regulation of expression of PAR-1 may provide a new therapeutic strategy against lung carcinoma.


Subject(s)
Carcinoma, Giant Cell/pathology , Cell Adhesion , Cell Proliferation , Lung Neoplasms/pathology , Receptor, PAR-1/genetics , Carcinoma, Giant Cell/metabolism , Cell Cycle , Cell Line, Tumor , DNA, Antisense , Down-Regulation , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/metabolism , Neoplasm Invasiveness , RNA, Messenger/metabolism , Receptor, PAR-1/metabolism , Transfection
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 15(3): 671-4, 2007 Jun.
Article in Chinese | MEDLINE | ID: mdl-17605891

ABSTRACT

Thrombin is a multifunctional serine protease that plays a key role in a variety of physiological and pathological conditions. In addition to the role in hemostasis and coagulation, thrombin has other numerous biological activities affecting inflammation, immune responses, tissue repair and wound healing. Apart from its physiological role thrombin activates the oncogenic potential of both normal and malignant cells and leads a metastatic phenotype. It is a potent mitogen for many tumor cells. It potentiates the proliferative response of tumor cells to some growth factors, increases the adhesive properties to the platelets and invasion processes of tumor cells to the extracellular matrix, enhances the metastatic capacity of tumor cells, activates angiogenesis and remodels the microenvironment of the tumor. The cellular biological effects of thrombin are mediated at least in part by a new subfamily of G-protein-coupled receptors designated proteinase-activited receptors (PARs). Thrombin has a bilateral effect on tumor cells:enhanced growth at low concentration, impaired growth/apoptosis at higher concentration. In this papers, the biological function of thrombin, thrombin and tumors, and thrombin receptors etc were reviewed.


Subject(s)
Neoplasm Metastasis , Neoplasms/pathology , Thrombin/physiology , Animals , Humans , Neoplasm Invasiveness , Neoplasms/enzymology , Receptors, Thrombin/physiology
4.
Ai Zheng ; 25(7): 798-804, 2006 Jul.
Article in Chinese | MEDLINE | ID: mdl-16831267

ABSTRACT

BACKGROUND & OBJECTIVE: Antioncogene p16 is one of the most important genes used in tumor gene therapy. Apoptosis induced by adenovirus mediated overexpression of p16 in cancer cells has been confirmed in various p16 gene inactive cancers. Studies have indicated that p16 gene is frequently inactive in human primary hepatocarcinoma. Therefore this study was to investigate the effect of exogenous p16 gene driven by alpha fetoprotein (AFP) core promoter on human hepatocellular carcinoma cells and further explore the potentials of p16 in hepatocellular carcinoma gene therapy. METHODS: The recombinant replication-defective adenovirus Ad-AFP-p16 containing p16 gene downstream the AFP core promoter-AF0.3 was constructed and infected hepatocellular carcinoma cells. The expression of p16 was detected by Western blot. The effects of exogenous p16 gene on cell growth and apoptosis were measured by MTT, flow cytometry and DNA ladder in vitro. Subcutaneous injection of mouse hepatocarcinoma cell line H22 infected with Ad-AFP-p16 was applied to observe the effect of Ad-AFP-p16 on tumorigenesis in vivo. RESULTS: Over-expression of exogenous p16 gene was confirmed in hepatocarcinoma cells infected with Ad-AFP-p16. Cell growth was inhibited by (94.42+/-11.70)% and (94.99+/-6.74)% in Be1-7402 and HepG2 cells on the 6th day after virus infection; 39.57% and 39.75% apoptotic cells were also induced respectively in these two cell lines on the 2nd day. Moreover, the infection of Ad-AFP-p16 significantly inhibited the tumorigenesis of mouse hepatocarcinoma cell line H22 in vivo. The tumor volumes of control, Ad-GFP, Ad-AFP-p16 and Ad-CMV-p16 groups were (1.54+/-0.65)cm(3), (1.71+/-1.01)cm(3), (0.25+/-0.39)cm(3) and (0.25+/-0.45)cm(3), respectively. CONCLUSION: The expression of p16 gene driven by AFP promoter can induce apoptosis in hepatocarcinoma cells.


Subject(s)
Adenoviridae/genetics , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Genes, p16 , Liver Neoplasms/pathology , alpha-Fetoproteins/genetics , Animals , Apoptosis , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/therapy , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p16/genetics , Female , Gene Transfer Techniques , Genetic Therapy/methods , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/therapy , Mice , Neoplasm Transplantation , Plasmids , Promoter Regions, Genetic , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics
5.
Zhonghua Bing Li Xue Za Zhi ; 35(1): 24-8, 2006 Jan.
Article in Chinese | MEDLINE | ID: mdl-16608645

ABSTRACT

OBJECTIVE: To explore the correlation between expression of PAR-1 and metastasis of human lung carcinoma. METHODS: Expression levels of PAR-1 were examined in surgically resected lung carcinoma specimens and corresponding lymph nodes by RT-PCR and immunohistochemistry, combined with morphometric methodology and clinicopathologic profiles. RESULTS: Strong PAR-1 staining was detected in the periphery of carcinoma nests, adenocarcinomatous emboli, foci of atypical adenomatous hyperplasia adjacent to the adenocarcinoma and atypical proliferation of duct epithelium of bronchial mucous glands. The expression rates of PAR-1 were 73.8% (59/80) and 63.9% (23/36) by immunohistochemistry and RT-PCR respectively. The percentage of PAR-1 protein expression cells was significantly higher in tumors with metastasis (85.7%, 48/56) than those without (45.8%, 11/24). Morphometric study demonstrated that there were significant differences of PAR-1 protein expression levels between tumors with metastatic and those without, primary and metastatic carcinomas, primary carcinomas and benign lung tissues adjacent to the carcinoma. No significant correlation was found between PAR-1 expression level and tumor size, histological types and tumor grades. The positive rate of PAR-1 mRNA expression in the metastatic group was significantly higher than that of the non-metastatic group (78.3%, 18/23 v.s. 38.5%, 5/13). CONCLUSION: PAR-1 expression may play an important role in determining the malignant phenotypes of lung cancers and significantly contribute to their initiation, progression and metastasis.


Subject(s)
Adenocarcinoma/metabolism , Carcinoma, Squamous Cell/metabolism , Lung Neoplasms/metabolism , Receptor, PAR-1/biosynthesis , Adenocarcinoma/pathology , Adult , Aged , Carcinoma, Squamous Cell/pathology , Female , Humans , Lung Neoplasms/pathology , Lymphatic Metastasis , Male , Middle Aged , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptor, PAR-1/genetics
6.
Zhonghua Zhong Liu Za Zhi ; 26(10): 590-3, 2004 Oct.
Article in Chinese | MEDLINE | ID: mdl-15634517

ABSTRACT

OBJECTIVE: To screen genes differentially expressed in two human giant-cell lung cancer lines of same origin but with different metastasis potentials. METHODS: Suppression subtractive hybridization (SSH) was done twice on two giant-cell lung cancer lines, PLA-801C and PLA-801D (hereafter abbreviated as C and D), of same origin but with low (C) and high (D) metastatic potentials. In the first round, SSH C was used as tester and D as driver, while in the second round, the tester and driver were interchanged. The sequences acquired from both rounds of SSH were spotted on glass slides respectively and screened by hybridizing with two-color fluorescence probes. Clones that had different expression levels on chips were also confirmed by RNA dot blot or Northern blot. RESULTS: There were 16 sequences with high expression in C as compared to those in D, and 79 sequences with high expression in D compared to those in C. After sequencing, most of them were found to be highly homologous to those encoding the following proteins: (1) cytokines and their receptors; (2) kinases and related proteins; (3) other proteins including enzymes, heat shock proteins, receptors, proteins of cell skeleton and mitochondria, products of oncogenes, etc; (4) some proteins deduced from gene sequences with yet unknown functions. CONCLUSION: The alterations in expression of some known genes, including HSP70, AXL receptor tyrosine kinase and 14-3-3zeta, might have impact on metastasis of giant-cell lung cancer. Whether some differentially expressed genes newly revealed are metastasis-related needs further study.


Subject(s)
Carcinoma, Giant Cell/genetics , Carcinoma, Giant Cell/secondary , Gene Expression Profiling , Lung Neoplasms/genetics , Lung Neoplasms/pathology , 14-3-3 Proteins/metabolism , Carcinoma, Giant Cell/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , HSP70 Heat-Shock Proteins/metabolism , Humans , Lung Neoplasms/metabolism , Neoplasm Metastasis , Nucleic Acid Hybridization , Oncogene Proteins/metabolism , Proto-Oncogene Proteins , Receptor Protein-Tyrosine Kinases/metabolism , Axl Receptor Tyrosine Kinase
7.
Zhonghua Zhong Liu Za Zhi ; 25(6): 531-4, 2003 Nov.
Article in Chinese | MEDLINE | ID: mdl-14690554

ABSTRACT

OBJECTIVE: To study the metastasis-associated molecules differentially expressed in highly and poorly metastatic sublines and the mechanism of metastasis in lung giant cell carcinoma. METHODS: Highly and poorly metastatic sublines (PLA801D and PLA801C)were used as metastasis model. Cell motility and invasion assay in vitro were first compared between the two sublines. Then, gelatin zymography analysis was used to determine the MMP-2 and MMP-9 activity. The protein expression level of secreted MMP-2, MMP-9, TIMP-1, TIMP-2 and intracellular expression level of p53, p16, PCNA, CD44(V6) isomeride, E-cadherin, CK18, nm23-H1 as well as the mRNA expression level of MMP-2, MMP-9, TIMP-1, TIMP-2, VEGF were compared through Western blot. Semi-quantitative RT-PCR analysis was used to determine the intracellular mRNA expression of MMP-2, MMP-9, TIMP-1, TIMP-2 and VEGF. RESULTS: The in vitro cell invasion potential of highly metastatic subline PLA801D was significantly higher than that of poorly metastatic subline PLA801C by about 4 folds, while the cell motility potential was similar. The secreted MMP-2 activity was notably higher in PLA801D, which was initiated by the higher expression of MMP-2 at protein and mRNA level. In addition, the expression level of p53, PCNA, CK18 protein and VEGF mRNA were significantly higher, while the expression level of p16, E-cadherin and nm23-H1 protein were significantly lower in PLA801D. Some molecules such as MMP-9, TIMP-1, TIMP-2, CD44(V6) isomeride, which had been reported to be associated with tumor metastasis, were not observed to change significantly between the two sublines. CONCLUSION: There are significant differences in metastatic potential and phenotypes between highly and poorly metastatic sublines of lung giant cell carcinoma. Some differentially expressed molecules might be playing roles in promoting or inhibiting metastasis of lung giant cell carcinoma, which may be useful to elucidate the mechanism of metastasis.


Subject(s)
Carcinoma, Giant Cell/pathology , Lung Neoplasms/pathology , Neoplasm Metastasis , Carcinoma, Giant Cell/metabolism , Cell Line, Tumor , Humans , Interleukin-8/genetics , Lung Neoplasms/metabolism , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Neoplasm Invasiveness , RNA, Messenger/analysis , Tissue Inhibitor of Metalloproteinase-1/analysis , Vascular Endothelial Growth Factor A/genetics
8.
Zhonghua Zhong Liu Za Zhi ; 25(5): 448-52, 2003 Sep.
Article in Chinese | MEDLINE | ID: mdl-14575567

ABSTRACT

OBJECTIVE: To construct replication selective adenovirus AdhepE1 targeting human melanoma and observe its specific killing of human melanoma cells in vitro. METHODS: Adenovirus E1 region, the murine tyrosinase promoter and enhancer DNA sequences were acquired respectively by PCR cloning. The shuttle plasmid of replication-selective adenovirus targeting human melanoma was constructed by DNA recombination. Replication-selective adenovirus AdhepE1 was generated by homologous recombination. The human melanoma cell line SK-Mel-1 and hepatocellular carcinoma cell line HepG2 were attacked separately by lower dose of AdhepE1. Change of cell morphology was observed and the surviving cells were calculated. The expression of E1A was assayed by RT-PCR to verify the specific-replication of AdhepE1. RESULTS: Replication selective adenovirus AdhepE1 targeting human melanoma was acquired by PCR. Human melanoma cell line SK-Mel-1 was sensitive to oncolytic killing of AdhepE1 whereas HepG2 was little responsive. The results of RT-PCR suggested that AdhepE1 replicated specifically in human melanoma cells. CONCLUSION: AdhepE1 can selectively kill human melanoma cells.


Subject(s)
Adenoviridae/genetics , Genetic Therapy , Melanoma/therapy , Virus Replication , Animals , Cell Line, Tumor , Humans , Liver Neoplasms/therapy , Melanoma/virology , Mice , Reverse Transcriptase Polymerase Chain Reaction
9.
Zhonghua Zhong Liu Za Zhi ; 25(4): 348-52, 2003 Jul.
Article in Chinese | MEDLINE | ID: mdl-12921563

ABSTRACT

OBJECTIVE: To study the function of IL-18 in promoting metastasis of lung cancer. METHODS: The differential expression of IL-18 protein or mRNA level between highly and poorly metastatic sublines of human lung giant cell carcinoma metastatic model was detected by Western blot, semi-quantitative RT-PCR and northern blot analysis. The poorly metastatic PLA801C subline or highly metastatic PLA801D subline was transfected with constructed IL-18 sense or IL-18 antisense expressed plasmid by lipofectamine stable transfection technique. The metastasis-related effect mediated by IL-18, the metastatic phenotype differences, cell motility and cell invasion potential in vitro determined by MICS system and the expression level of metastasis-associated biomarkers detected by Western blot analysis, were compared between IL-18 stably transfectants and mock control, i.e. between PLA801C/IL-18(S) and PLA801C/pcDNA3.1, or between PLA801D/IL-18(As) and PLA801D/pcDNA3. RESULTS: IL-18 was only present in highly metastatic PLA801D subline at either protein or mRNA level, which implied that IL-18 might play a role in promoting metastasis of lung cancer. After IL-18 sense expressed plasmid was transfected into poorly metastatic PLA801C subline, IL-18 fused protein with myc tag detected by Western blot analysis using either IL-18 or myc tag monoclonal antibody. In addition, cell motility ability in vitro was significantly increased about 3 times and E-cadherin protein was significantly down-regulated at about 50% in PLA801C/IL-18(S) transfectants compared with mock control. While IL-18 expressed plasmid was transfected into highly metastatic PLA801D subline, IL-18 protein and mRNA were simultaneously decreased by 30%. In addition, cell invasion ability in vitro was significantly decreased at about 75% and E-cadherin protein was significantly up-regulated in PLA801D/IL-18(As) transfectants compared with mock control. CONCLUSION: IL-18 might play a role in enhancing tumor metastasis of lung cancer by down-regulating E-cadherin protein expression.


Subject(s)
Carcinoma, Giant Cell/secondary , Interleukin-18/biosynthesis , Lung Neoplasms/pathology , Neoplasm Metastasis , Cadherins/metabolism , Carcinoma, Giant Cell/metabolism , Cell Line, Tumor , Cell Movement , DNA, Antisense/genetics , Gene Expression Regulation, Neoplastic , Humans , Interleukin-18/genetics , Lung Neoplasms/metabolism , Neoplasm Invasiveness , Neoplasm Metastasis/genetics , Plasmids , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Transfection
10.
Exp Hematol ; 30(3): 237-44, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11882361

ABSTRACT

OBJECTIVE: Following activation of T cells, phosphorylation of tyrosine residues occurs through a complex signaling process involving protein tyrosine kinases, phosphatases, and a variety of adapter molecules including Grb2. We have attempted to identify new signaling molecules that are important for the activation response. METHODS: Using a protein interaction screening protocol based on phage display, T-cell signaling components that associate with the adapter molecule, Grb2, the lymphoid-specific tyrosine phosphatase Lyp was identified. Using transcriptional reporter assays, the role of Lyp in T-cell activation was studied by overexpression of wild-type or catalytically inactive mutants of Lyp. RESULTS: A GST fusion containing the C-terminal SH3 domain of Grb2 bound to the nucleotide exchange factor Sos or Grb2-associated binder 2 (Gab2). In contrast, the N-terminal SH3-containing fusion bound to the protein tyrosine phosphatase Lyp. Grb2 was co-immunoprecipitated with Lyp in 293T cells overexpressing both proteins. Using Northern blot analysis, Lyp was found to be expressed predominantly in hematopoietic tissue, including spleen, lymph node, thymus, peripheral blood leukocytes, bone marrow, and fetal liver. Two human T-cell lines, Jurkat and HuT78, expressed both Lyp mRNA and protein. Overexpression of wild-type Lyp or a catalytically inactive, substrate-trapping mutant (D195A) in Jurkat cells inhibited transcriptional activity initiated by anti-CD3 and anti-CD28 antibodies. In contrast, two other catalytically inactive mutants (R233M or C227S) had no effect. CONCLUSION: These data demonstrate a novel interaction between the phosphatase Lyp and the adaptor Grb2 and are consistent with a negative regulatory role for Lyp in T-cell signaling.


Subject(s)
Adaptor Proteins, Signal Transducing , Homeostasis , Lymphocyte Activation , Protein Tyrosine Phosphatases/physiology , Proteins/metabolism , T-Lymphocytes/immunology , Amino Acid Sequence , Blotting, Northern , Cell Line , GRB2 Adaptor Protein , Gene Expression , Glutathione Transferase/genetics , Humans , Immunosorbent Techniques , Jurkat Cells , Molecular Sequence Data , Mutagenesis, Site-Directed , Organ Specificity , Peptide Fragments/genetics , Peptide Library , Polymerase Chain Reaction , Protein Tyrosine Phosphatase, Non-Receptor Type 22 , Protein Tyrosine Phosphatases/analysis , Protein Tyrosine Phosphatases/genetics , Proteins/genetics , RNA, Messenger/analysis , Recombinant Fusion Proteins/metabolism , Signal Transduction , Transfection
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