Inducible cyclooxygenase-2 (COX-2) has received much attention because of its role in neuro-inflammation and synaptic plasticity. Even though COX-2 levels are high in healthy animals, the function of this factor in adult neurogenesis has not been clearly demonstrated. Therefore, we performed the present study to compare the effects of pharmacological and genetic inhibition of COX-2 on adult hippocampal neurogenesis. Physiological saline or the same volume containing celecoxib was administered perorally every day for 5 weeks using a feeding needle. Compared to the control, pharmacological and genetic inhibition of COX-2 reduced the appearance of nestin-immunoreactive neural stem cells, Ki67-positive nuclei, and doublecortin-immunoreactive neuroblasts in the dentate gyrus. In addition, a decrease in phosphorylated cAMP response element binding protein (pCREB) at Ser133 was observed. Compared to pharmacological inhibition, genetic inhibition of COX-2 resulted in significant reduction of neural stem cells, cell proliferation, and neuroblast differentiation as well as pCREB levels. These results suggest that COX-2 is part of the molecular machinery that regulates neural stem cells, cell proliferation, and neuroblast differentiation during adult hippocampal neurogenesis via pCREB. Additionally, genetic inhibition of COX-2 strongly reduced neural stem cell populations, cell proliferation, and neuroblast differentiation in the dentate gyrus compared to pharmacological inhibition.
Animals ; Celecoxib/*pharmacology ; Cell Differentiation/drug effects/physiology ; Cell Proliferation/drug effects/physiology ; Cyclooxygenase 2/*genetics/metabolism ; Cyclooxygenase 2 Inhibitors/*pharmacology ; Dentate Gyrus/drug effects/*physiology ; Male ; Mice ; Mice, Knockout ; Neural Stem Cells/drug effects/physiology ; Neurogenesis/drug effects

Lining the inner surface of the walls of blood vessels, Endothelial cells (ECs) go beyond providing selective membrane to maintain the natural structure and function of vessels; they also synthesize varieties of vasoactive proteins to modify the pressure shift in the local flow field and hence they adapt the physiological activities of vessels. In this experiment, ELISA and RT-PCR technologies were adopted. We set up five different pressure loaded ECs groups,one non-activated cultured ECs group and one single shear stress loaded ECs group. Such a design was intended to demonstrate the effects of pressure shift on the expression of vasoactive protein synthesized by ECs [Endothelin-1(ET-1), endothelial Nitric Oxide Synthase (eNOS), Cyclooxygenase-2(COX-2) and Vascular Endothelial Growth Factor(VEGF)]. Our aim was to elucidate the mechanism of the pressure shift mediated dysfunction in ECs and the related dose-effect relationship. Based on these data, we suggest that ECs could modify the expression of vasoactive protein for adapting to the pressure shift in the local flow field; while in the process of--40 cmH2O induced ECs' dysfunction, the vasoactive proteins eNOS, COX-2 and VEGF play an important role in protecting ECs.
Cells, Cultured ; Cyclooxygenase 2 ; genetics ; metabolism ; Endothelial Cells ; metabolism ; physiology ; Endothelin-1 ; genetics ; metabolism ; Humans ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Pressure ; RNA, Messenger ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

BACKGROUNDExperimental evidence indicates that cyclooxygenase-2 (COX-2) plays a critical role in blastocyst implantation; however, little is known of the role of COX-2 in unexplained recurrent spontaneous abortion (URSA).

METHODSWe evaluated the expression level and potential signaling pathway of COX-2 in 30 cases of URSA who were excluded the abnormality of chromosomes, anatomy, endocrine, infectious, autoimmune diseases and in 30 normal pregnancies.

RESULTSThe mRNA and the protein expression level of COX-2 in the URSA group (-0.238 +/- 0.848, 0.368 +/- 0.089, respectively) were significantly lower than that in the control group (1.943 +/- 3.845, 1.046 +/- 0.108, respectively) (both, P < 0.01). The expression of prostaglandins PGF(2a), PGD(2), PGE(2), and PGI(2), in the URSA group ((2326.0 +/- 295.6) pg/ml, (2164.0 +/- 240.5) pg/ml, (238.7 +/- 26.4) pg/ml, (2337.0 +/- 263.0) pg/ml, respectively) were significantly lower than that in the control group ((3450.0 +/- 421.7) pg/ml, (3174.0 +/- 415.6) pg/ml, (323.5 +/- 43.8) pg/ml, (3623.0 +/- 460.4) pg/ml, respectively) (P < 0.05). The mRNA expression level of PPARbeta and RXRalpha (0.859 +/- 0.653, -0.172 +/- 0.752, respectively) in URSA group was significantly lower than that in the control group (1.554 +/- 1.735, 0.777 +/- 2.482, respectively) (both P< 0.05). The mRNA and protein expression levels of vascular endothelial growth factor-A (VEGF-A) in the URSA group (2.010 +/- 1.522, 0.35 +/- 0.46) was significantly lower than that in the control group (4.569 +/- 2.430, 0.750 +/- 0.350) (both P < 0.05).

CONCLUSIONSCOX-2 and the COX-2-derived PGI(2) signaling pathway possibly play an important role in successful embryo implantation, and their decreased expression may result in URSA. The decreased expression may influence the expression of VEGF-A which interferes with placental angiogenesis causing failure of embryo implantation, leading to spontaneous abortion.

Abortion, Habitual ; enzymology ; genetics ; Adult ; Blotting, Western ; Cyclooxygenase 2 ; genetics ; metabolism ; Dinoprost ; metabolism ; Dinoprostone ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Epoprostenol ; metabolism ; Female ; Humans ; Polymerase Chain Reaction ; Pregnancy ; Prostaglandin D2 ; metabolism ; Signal Transduction ; genetics ; physiology ; Vascular Endothelial Growth Factor A ; metabolism

It has been proposed that the pro-inflammatory catalytic activity of cyclooxygenase-2 (COX-2) plays a key role in the aging process. However, it remains unclear whether the COX-2 activity is a causal factor for aging and whether COX-2 inhibitors could prevent aging. We here examined the effect of COX-2 inhibitors on aging in the intrinsic skin aging model of hairless mice. We observed that among two selective COX-2 inhibitors and one non-selective COX inhibitor studied, only NS-398 inhibited skin aging, while celecoxib and aspirin accelerated skin aging. In addition, NS-398 reduced the expression of p53 and p16, whereas celecoxib and aspirin enhanced their expression. We also found that the aging-modulating effect of the inhibitors is closely associated with the expression of type I procollagen and caveolin-1. These results suggest that pro-inflammatory catalytic activity of COX-2 is not a causal factor for aging at least in skin and that COX-2 inhibitors might modulate skin aging by regulating the expression of type I procollagen and caveolin-1.
Animals ; Aspirin/administration & dosage ; Catalysis ; Caveolin 1/genetics/metabolism ; Collagen Type I/genetics/metabolism ; *Cyclooxygenase 2/metabolism/physiology ; Cyclooxygenase 2 Inhibitors/*administration & dosage ; Gene Expression Regulation ; Mice ; Nitrobenzenes/*administration & dosage ; Pyrazoles/administration & dosage ; Skin Aging/*drug effects/physiology ; Sulfonamides/*administration & dosage ; Tumor Suppressor Protein p53/genetics/metabolism

OBJECTIVETo extract tumor interstitial fluid (TIF) from MKN-45 gastric cancer which is similar to "muddy phlegm" in Chinese medicine and observe influences of MKN-45 tumor interstitial fluid (MKN-45 TIF) intervention on metastasis of gastric cancer and on the expressions of vascular endothelial growth factor (VEGF), kinase insert domain containing receptor (KDR), epithelial-cadherin (E-cad), cyclooxygenase-2 (COX-2), intercellular adhesion molecule-1 (ICAM-1) and telomerase genes and proteins in primary tumor tissue.

METHODSAn MKN-45 tumor-bearing model was established in 50 nude mice. The modeled animals were equally randomized to 5 groups: the simple tumor-bearing group (model group), the normal saline (NS) via tail vein injection (i.v.) group (NS i.v. group), MKN-45 TIF i.v. group (TIF i.v. group), NS intraperitoneal injection (i.p.) group (NS i.p. group), and MKN-45 TIF i.p. group (TIF i.p. group). The TIF and NS intervention groups received injection (i.p. or i.v.) of MKN-45 TIF or NS twice a week, 0.2 mL at a time. After 8 weeks, the primary tumors were removed, weighed and HE stained to observe tumor metastasis. The primary tumor tissues were analyzed by immunohistochemistry and real-time quantitative PCR to detect expressions of VEGF, KDR, E-cad, COX-2, ICAM-1, and telomerase genes and proteins in different groups.

RESULTSThere were significant differences in tumor weight between TIF intervention groups and the model and NS intervention groups. Tumor metastasis was observed in all 5 groups, but the tumor metastasis rate in TIF intervention groups was significantly higher than those in the model and NS intervention groups. The gene and protein expressions of gastric cancer-related factors VEGF, KDR, COX-2, ICAM-1 and telomerase were unregulated while the gene and protein expressions of E-cad were downregulated in TIF intervention groups.

CONCLUSIONSTIF promotes tumor growth, invasion and metastasis of gastric cancer. These findings provide preliminary experimental clues for verifying the hypothesis of "tumor-phlegm microenvironment".

Animals ; Cadherins ; genetics ; metabolism ; Cell Line, Tumor ; Cyclooxygenase 2 ; genetics ; metabolism ; Extracellular Fluid ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Male ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Stomach Neoplasms ; metabolism ; secondary ; Telomerase ; genetics ; metabolism ; Tumor Microenvironment ; physiology ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

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

OBJECTIVETo investigate the inhibition of COX-2 gene expression and its effects on malignant proliferation of human lung adenocarcinoma A549 cells after interfering at different target sites in vitro.

METHODSThe 3rd, 7th and 10th exon of COX-2 were selected as the targets and three COX-2 siRNA expression vectors with human U6 promoter were constructed. Three siRNA expression vectors and two vacant vectors were transfected into A549 cells expressing COX-2 with lipofectamine, respectively. The transfected cell strains were constructed and the change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of A549 cells after interfering at different target sites were studied by cell growth curve and colony formation assay in vitro.

RESULTSThe three siRNAs and U6 promoter were validated by PCR, restriction endonuclease digestion, DNA sequencing and BLAST alignment, and cloned into the pEGFP vector. The cell strains transfected were named as A549-3, A549-7, A549-10, A549-p and A549-pU6, respectively. A549-p cells showed expression of GFP and A549-3, A549-7, A549-10, A549-p and A549-pU6 cells did not show at 24, 48 and 72 hours after transfection. The results of RT-PCR and Western blot showed an inhibition of COX-2 expression after interfering at three target sites (3rd, 7th and 10th exons). In contrast to A549 cells, the levels of COX-2 mRNA of A549-3, A549-7 and A549-10 cells were reduced by 10.6%, 33.4% and 61.2%, respectively. The levels of COX-2 protein of A549-3, A549-7 and A549-10 cells were reduced by 26.7%, 44.7% and 56.2%, respectively. The results of cell growth curve and colony formation assay showed a slowing down of the growth of A549-10 cells and reduction of their colony formation rate. The other two targets had no apparent effect on the growth of A549 cells.

CONCLUSIONThere is a significant inhibiting effect of RNA interference on the malignant proliferation of A549 cells in vitro, and the most striking effect can be seen when the 10th exon of COX-2 is taken as the interference target.

Adenocarcinoma ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cyclooxygenase 2 ; genetics ; metabolism ; physiology ; Exons ; Genetic Vectors ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Promoter Regions, Genetic ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

BACKGROUNDEstrogen receptor (ER)-negative breast cancer cells are more aggressive than ER-positive cells. Elevated levels of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor-C (VEGF-C) expression have been detected in cultured human breast cancer cells and are associated with negative hormone receptor status. In this study, we created ERalpha stable transfectants in MDA-MB-231 cells to explore the effect of ERalpha on cell growth and COX-2 and VEGF-C expression.

METHODSThe green fluorescent protein (GFP)-ERalpha plasmids were stably transfected into ER-negative MDA-MB-231 cells. The proliferation and migration of untransfected MDA-MB-231 cells, ERalpha-transfected MDA-MB-231 cells and ER-positive MCF-7 cells were determined. The expression of COX-2, and the levels of VEGF-C mRNA and the VEGF-C secretion concentration were assayed in these cell lines.

RESULTSThe proliferation and migration capacities of ERalpha-tranfected MDA-MB-231 cells were significantly decreased (P < 0.05). The expression of COX-2 was significantly lower in ERalpha-tranfected MDA-MB-231 cells than in untranfected MDA-MB-231 cells. The mRNA and protein levels of VEGF-C were lower in ERalpha-tranfected MDA-MB-231 cells than in untransfected MDA-MB-231 cells (P < 0.05).

CONCLUSIONSERalpha stable transfection inhibits proliferation and migration capacities of MDA-MB-231 cells and decreases expression of COX-2 and VEGF-C. The decreases of proliferation and migration capacities may be related to suppression of COX-2 and VEGF-C expression.

Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; Cell Cycle ; genetics ; physiology ; Cell Line, Tumor ; Cell Movement ; genetics ; physiology ; Cell Proliferation ; Cyclooxygenase 2 ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Flow Cytometry ; Humans ; Polymerase Chain Reaction ; Transfection ; Vascular Endothelial Growth Factor C ; genetics ; metabolism

OBJECTIVETo study the effect of cyclooxygenase-2 (COX-2) on lymphangiogenesis in breast cancer.

METHODSBy the means of immunohistochemistry, COX-2, vascular endothelial growth factor-C (VEGF-C) and D2-40 were examined in the tissue samples of primary tumors from 94 patients underwent surgical resections for breast cancer from November 1998 to March 2002. Eighty-three patients were followed-up. The expressions of VEGF-C mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot in MDA-MB-231 cell lines by the treatment of selective COX-2 inhibitor Nimesulide at different doses. The expressions of VEGF-C protein were evaluated in MDA-MB-231 cells treated by PGE2 (1 microg/ml) and Trastuzumab (1 microg/ml), respectively.

RESULTSCOX-2 over-expression was observed in 46.8% of surgical specimens (44/94), while VEGF-C overexpression occurred in 51.1% of tumor samples (48/94). COX-2 was strongly correlated with VEGF-C expression (P < 0.01), micro-lymphatic vessels (P = 0.032) and metastatic lymph nodes (P = 0. 035). Patients with COX-2 positive tumors had a significant shorter survival time than those with negative tumors did, including disease-free survival (P = 0.010) and overall survival (P = 0.040). Nimesulide could down-regulate the expressions of VEGF-C mRNA and protein in a does-dependent manner, while PGE2 could up-regulate the expressions. The expression of VEGF-C protein up-regulated by PGE2 treatment was decreased by Trastuzumab.

CONCLUSIONSCOX-2 over-expression can up-regulate the expression of VEGF-C. VEGF-C might promote lymph node metastasis by a lymph-angiogenic pathway, then affect the prognosis of the patients with breast cancer.

Adolescent ; Adult ; Aged ; Breast Neoplasms ; metabolism ; pathology ; Cyclooxygenase 2 ; metabolism ; physiology ; Female ; Follow-Up Studies ; Humans ; Lymphangiogenesis ; Lymphatic Metastasis ; Middle Aged ; Prognosis ; Vascular Endothelial Growth Factor C ; genetics ; metabolism

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