OBJECTIVETo investigate the relationship between the expression of COX-2 and liver cancer and construct a recombinant adenovirus encoding human COX-2 antisense RNA, and then to investigate its effects on liver cancer cell proliferation.

METHODSThe expression of COX-2 in 34 cases of hepatocellular carcinoma and in SMMC-7402 and SMMC-7721 cell lines was studied by using immunohistochemical techniques. The shuttle plasmid encoding anti-sense COX-2 was constructed by using cloning COX-2 cDNA fragment in the reverse direction into the pHCMVSPIA. Then the plasmid pJM17 and the shuttle plasmid were co-transferred into 293 cells with lipofectamine for homologous recombination to acquire recombinant adenovirus (Ad-AShcox-2), which was confirmed by PCR. Human hepatocellular carcinoma cell lines SMMC-7402 and SMMC-7721 were transduced in vitro. The cell apoptosis and cell cycle were analyzed by flow cytometry. The cell proliferation was determined by colony-forming efficiency.

RESULTSWe observed COX-2 expression in 82.4% of the hepatocellular carcinomas and SMMC-7402 cell line, but no COX-2 expression in the SMMC-7721 cell line. In addition, the recombinant adenovirus encoding anti-sense COX-2 fragment Ad-AShcox-2 was obtained with a titer of 1.06 x 10(12) PFU/ml. Ad-AShcox-2 reduced the expression of COX-2 and enhanced the percentage of cells into G1/G0 phase in the SMMC-7402 cell line. The difference of apoptotic index between the Ad-AShcox-2 group and the control group was statistically significant in SMMC-7402 but not in SMMC-7721. Similarly, colony-forming rates of SMMC-7402 and SMMC-7721 cell lines after Ad-AShcox-2 being transferred were (2.7+/-0.94)% and (33.6+/-4.24)%, respectively.

CONCLUSIONBy reducing the expression of COX-2 in hepatocellular carcinoma cells with the expression of COX-2, the cells could be inhibited.

Adenoviridae ; genetics ; Apoptosis ; physiology ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cyclooxygenase 2 ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; pathology ; Membrane Proteins ; biosynthesis ; genetics ; RNA, Antisense ; biosynthesis ; genetics

OBJECTIVETo study the expression of cyclooxygenase-2 (COX-2) gene in breast cancer and its clinicopathologic characteristics.

METHODSWith beta-actin gene as reference, the COX-2 mRNA was monitored in 30 specimens of breast cancer tissue and adjacent normal breast tissue by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe COX-2 mRNA expression was significantly upregulated in most breast cancer tissues with range of 0.05 - 0.91 (median 0.56), which was rare in normal breast tissue with range of 0 - 0.09 (median 0). The difference of COX-2 mRNA expression between cancer and normal breast tissue was significant (rank sum test, P < 0.05). COX-2 overexpression in breast cancer was related to its lymph node metastasis (P < 0.05) but not to age, tumor size, pathologic grade or pathologic type (P > 0.05).

CONCLUSIONThe level of COX-2 mRNA expression is obviously higher in the breast cancer tissue than that in normal breast tissue. COX-2 overexpression may play a crucial role in the carcinogenesis, development of cancer and lymph node metastasis in breast cancer patients.

Adult ; Biomarkers, Tumor ; biosynthesis ; genetics ; Breast Neoplasms ; enzymology ; metabolism ; Cyclooxygenase 2 ; Female ; Gene Expression ; Humans ; Isoenzymes ; biosynthesis ; genetics ; physiology ; Membrane Proteins ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; genetics ; physiology ; RNA, Messenger ; biosynthesis ; RNA, Neoplasm ; analysis ; Reverse Transcriptase Polymerase Chain Reaction

Osteosarcoma is the most common primary bone tumor, but the pathogenesis is not well understood. While cyclooxygeanse-2 (COX-2) is known to be closely associated with tumor growth and metastasis in several kinds of human tumors, the function of COX-2 in osteosarcoma is unclear. Therefore, to investigate the function of COX-2 in osteosarcoma, we established stable cell lines overexpressing COX-2 in U2OS human osteosarcoma cells. COX-2 overexpression as well as prostaglandin E(2) treatment promoted proliferation of U2OS cells. In addition, COX-2 overexpression enhanced mobility and invasiveness of U2OS cells, which was accompanied by increases of matrix metalloproteinase-2 and -9 (MMP-2 and -9) activities. Selective COX-2 inhibitors, NS-398 and celecoxib, inhibited cell proliferation and abrogated the enhanced mobility, invasiveness and MMP activities induced by COX-2 overexpression. These results suggest that COX-2 is directly associated with cell proliferation, migration and invasion in human osteosarcoma cells, and the therapeutic value of COX-2 inhibitors should be evaluated continuously.
Bone Neoplasms/*enzymology/pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclooxygenase 2/biosynthesis/*physiology ; Cyclooxygenase 2 Inhibitors/pharmacology ; Dinoprostone/pharmacology ; Enzyme Activation ; Humans ; Matrix Metalloproteinase 2/metabolism ; Matrix Metalloproteinase 9/metabolism ; Neoplasm Invasiveness ; Nitrobenzenes/pharmacology ; Osteosarcoma/*enzymology/pathology ; Pyrazoles/pharmacology ; Sulfonamides/pharmacology

BACKGROUNDMechanical stress plays an important role in the maintenance of bone homeostasis. Current hypotheses suggest that interstitial fluid flow is an important component of the system by which tissue level strains are amplified in bone. This study aimed to test the hypothesis that the short-term and appropriate fluid shear stress (FSS) is expected to promote the terminal differentiation of pre-osteoblasts and detect the expression profile of microRNAs in the FSS-induced osteogenic differentiation in MC3T3-E1 cells.

METHODSMC3T3-E1 cells were subjected to 1 hour of FSS at 12 dyn/cm(2) using a parallel plate flow system. After FSS treatment, cytoskeleton immunohistochemical staining and microRNAs (miRNAs) were detected immediately. Osteogenic gene expression and immunohistochemical staining for collagen type I were tested at the 24th hour after treatment, alkaline phosphatase (ALP) activity assay was performed at 24th, 48th, and 72 th hours after FSS treatment, and Alizarin Red Staining was checked at day 12.

RESULTSOne hour of FSS at 12 dyn/cm(2) induced actin stress fiber formation and rearrangement, up-regulated osteogenic gene expression, increased ALP activity, promoted synthesis and secretion of type I collagen, enhanced nodule formation, and promoted terminal differentiation in MC3T3-E1 cells. During osteogenic differentiation, expression levels of miR-20a, -21, -19b, -34a, -34c, -140, and -200b in FSS-induced cells were significantly down-regulated.

CONCLUSIONThe short-term and appropriate FSS is sufficient to promote terminal differentiation of pre-osteoblasts and a group of miRNAs may be involved in FSS-induced pre-osteoblast differentiation.

Actins ; chemistry ; Alkaline Phosphatase ; metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Core Binding Factor Alpha 1 Subunit ; genetics ; Cyclooxygenase 2 ; genetics ; Gene Expression Profiling ; Mice ; MicroRNAs ; physiology ; Osteoblasts ; cytology ; Osteogenesis ; Stress, Mechanical ; Stress, Physiological

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. In this study, we investigated the effects of 15d-PGJ2, a high-affinity ligand of PPAR-gamma, on dedifferentiation and on inflammatory responses such as COX-2 expression and PGE2 production in rabbit articular chondrocytes with a focus on ERK-1/-2, p38 kinase, and PPAR-gamma activation. We report here that 15d-PGJ2 induced dedifferentiation and/or COX-2 expression and subsequent PGE2 production. 15d-PGJ2 treatment stimulated activation of ERK-1/-2, p38 kinase, and PPAR-gamma. Inhibition of ERK-1/-2 with PD98059 recovered 15d-PGJ2-induced dedifferentiation and enhanced PPAR-gamma activation, whereas inhibition of p38 kinase with SB203580 potentiated dedifferentiation and partially blocked PPAR-gamma activation. Inhibition of ERK-1/-2 and p38 kinase abolished 15d-PGJ2-induced COX-2 expression and subsequent PGE2 production. Our findings collectively suggest that ERK-1/-2 and p38 kinase oppositely regulate 15d-PGJ2-induced dedifferentiation through a PPAR-gamma-dependent mechanism, whereas COX-2 expression and PGE2 production is regulated by ERK-1/-2 through a PPAR-gamma-independent mechanism but not p38 kinase in articular chondrocytes. Additionally, these data suggest that targeted modulation of the PPAR-gamma and mitogen-activated protein kinase pathway may offer a novel approach for therapeutic inhibition of joint tissue degradation.
Animals ; Cartilage, Articular/*cytology ; Cell Differentiation/drug effects ; Chondrocytes/cytology/*drug effects/metabolism ; Cyclooxygenase 2/*analysis ; Dinoprostone/biosynthesis ; Mitogen-Activated Protein Kinase 1/*physiology ; Mitogen-Activated Protein Kinase 3/*physiology ; PPAR gamma/*physiology ; Prostaglandin D2/*analogs & derivatives/pharmacology ; Rabbits ; p38 Mitogen-Activated Protein Kinases/*physiology

OBJECTIVETo transfect antisense vector of human cyclooxygenase-2 (COX-2) gene into COX-2 highly expressing cholangiocarcinoma cell line QBC939 and explore its biological activities and role in carcinogenesis.

METHODSQBC939 cells were transfected with antisense vector of human COX-2 gene using LipoVec transfecting technique. Transfected cells were selected with G418; COX-2 mRNA was examined using reverse transcription polymerase chain reaction (RT-PCR) and COX-2 protein expression was detected by immunocytochemistry using isozyme selective antibodies. The proliferative status of transfected cells was measured by using methabenzthiazuron (MTT) assay; Cell cycle and apoptosis were analyzed by using flow cytometry.

RESULTSRT-PCR showed a lower COX-2 mRNA level in antisense vector transfected cells and immunocytochemistry showed a weaker COX-2 protein expression in antisense vector transfected cells. The antisense vector transfected cells proliferative index decreased significantly (P < 0.01), the percentage of S phase decreased remarkably (P < 0.05) in antisense vector transfected cells (9.27% +/- 1.91%) compared with that in QBC939 cells without transfection(16.35% +/- 2.87%), and the percentage of G0/G1 phase increased remarkably (P < 0.05) in antisense vector transfected cells (75.16% +/- 4.13%) compared with that in QBC939 cells without transfection (57.31% +/- 10.16%). Transfection with antisense vector of human COX-2 gene had no significant influence on the apoptosis in QBC939 cells (P > 0.05).

CONCLUSIONTransfection with antisense vector of human COX-2 gene could inhibit the proliferation of human cholangiocarcinoma QBC939 cells.

Apoptosis ; Bile Duct Neoplasms ; metabolism ; pathology ; Bile Ducts, Intrahepatic ; Cell Cycle ; Cell Division ; Cell Line, Tumor ; Cholangiocarcinoma ; metabolism ; pathology ; Cyclooxygenase 2 ; DNA, Antisense ; genetics ; Humans ; Isoenzymes ; biosynthesis ; genetics ; physiology ; Membrane Proteins ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; genetics ; physiology ; RNA, Messenger ; genetics ; Transfection

Antineoplastic Agents ; pharmacology ; Bile ; physiology ; Bile Duct Neoplasms ; metabolism ; pathology ; Bile Ducts, Intrahepatic ; Celecoxib ; Cell Division ; drug effects ; Cell Line, Tumor ; Cholangiocarcinoma ; metabolism ; pathology ; Cyclooxygenase 2 ; Dinoprostone ; metabolism ; Humans ; Isoenzymes ; biosynthesis ; genetics ; Membrane Proteins ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; genetics ; Pyrazoles ; RNA, Messenger ; genetics ; Sphincterotomy, Transduodenal ; adverse effects ; Sulfonamides ; pharmacology ; Up-Regulation

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA- induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.
Butadienes/pharmacology ; Cell Line, Tumor ; Cell Movement/drug effects/*physiology ; Cyclooxygenase 2/*biosynthesis ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors/metabolism ; Female ; Flavonoids/pharmacology ; GTP-Binding Protein alpha Subunits, Gi-Go/antagonists & inhibitors/*metabolism ; Humans ; Lysophospholipids/pharmacology ; Nitriles/pharmacology ; Ovarian Neoplasms/metabolism/*pathology ; Pertussis Toxin/pharmacology ; Protein-Tyrosine Kinases/antagonists & inhibitors/*metabolism ; Proto-Oncogene Proteins/antagonists & inhibitors/*metabolism ; Pyrimidines/pharmacology ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptors, Lysophosphatidic Acid/*metabolism ; Receptors, Prostaglandin E/metabolism ; Signal Transduction ; Transcriptional Activation ; Tyrphostins/pharmacology