OBJECTIVETo investigate the effect and mechanism of signal transducers and activators of transcription 3 (STAT-3) modulates human tongue squamous cell carcinoma invasion ability via targeting mircoRNA-21.

METHODSTscca and Tca8113P160 human tongue squamous cell carcinoma cell lines were used.WP1066 (STAT-3 inhibitor) , the small molecule inhibitor of STAT-3 was used to suppress the STAT-3 expression. The half maximal inhibitory concentration (IC50 value) of WP1066 in the two cell lines was determined by methyl thiazolyl tetrazolium (MTT) assay. The expression level of STAT-3 and phosphorylation of STAT-3 (pSTAT-3) was examined by Western blotting. Real-time PCR was used to detect the mircoRNA-21 expression after treated with WP1066. Matrigel matrix and transwell assay were used to determine cancer cell colony formation and invasion ability after treated with WP1066. Tumor invasion related proteins in Tscca and Tca8113P160 cell lines were measured by Western blotting. Luciferase reporter gene assay was conducted to detect the relationship between STAT-3 and mircoRNA-21.

RESULTSThe IC50 to WP1066 in Tscca cell was 3.1 and 3.5 µmol/L for Tca8113P160 cell respectively. STAT-3/pSTAT-3 protein level was suppressed significantly (Tscca: STAT-3: F = 887.154, P = 0.000; pSTAT-3: F = 332.212, P = 0.000; Tca8113P160: STAT-3: F = 322.895, P = 0.000; pSTAT-3:F = 788.357, P = 0.000). mircoRNA-21 expression was down-regulated (Tscca:F = 32.157, P = 0.000; Tca8113P160: F = 11.349, P = 0.007). The diameters of culture clone in cell treated with WP1066 were less than control groups (Tscca:F = 15.751, P = 0.004; Tca8113P160: F = 12.964, P = 0.007). The number of tongue cancer cell migrating through the transwell membrane in WP1066 treated group was less than in control groups (Tscca: F = 1688.926, P = 0.000; Tca8113P160: F = 327.528, P = 0.000). In addition, MMP-2/9 protein expression was decreased in both of the cell lines treated with WP1066, while TIMP-3 was up regulated dramatically. STAT-3 could modulate mircoRNA-21 directly.

CONCLUSIONSReduction of STAT-3 can inhibit tongue cancer cell invasion ability via targeting mircoRNA-21.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; MicroRNAs ; genetics ; metabolism ; Phosphorylation ; Pyridines ; pharmacology ; STAT3 Transcription Factor ; antagonists & inhibitors ; metabolism ; Signal Transduction ; Tissue Inhibitor of Metalloproteinase-3 ; metabolism ; Tongue Neoplasms ; genetics ; metabolism ; pathology ; Tyrphostins ; pharmacology

OBJECTIVETo observe the effect of intra-articular injection of SB203580, a selective p38 mitogen-activated protein kinase inhibitor, on the expression of matrix metalloproteinase (MMP)-3, MMP-13 in a rat model of osteoarthritis (OA) and to explore the relationship between the MMP-3/MMP-13 expressions and the severity of OA.

METHODSFourty SD rats underwent unilateral anterior cruciate ligament transection (ACLT) and then randomly divided into four groups, with 10 rats in each group. Group A received 0.1 ml intra-articular injection of SB203580 at a high concentration of 100 micromol/L (once a week) immediately after surgery, and group B were treated under the same condition using SB203580 with a low concentration of 10 micromol/ L Group C received 0.1 ml intra-articular normal saline, and group D were not injected as controls after ACLT. All rats were sacrificed seven weeks after the surgery. Macroscopic and immunohistochemical studies were performed on the cartilage. Protein expressions of MMP-3 and MMP-13 were determined by Western blot. RESULTS Cartilage degradation was significantly milder in group A and group B than in the control groups, as shown by morphological studies (P < 0. 05) and immunohistochemical studies (P < 0. 05). The protein expressions of MMP-3 and MMP-13 in cartilage were significantly lower in groups A and B than in groups C and D (P < 0.01).

CONCLUSIONSB203580 can inhibit the expressions of MMP-3 and MMP-13 and thus protect the cartilage.

Animals ; Cartilage, Articular ; drug effects ; enzymology ; Imidazoles ; administration & dosage ; pharmacology ; therapeutic use ; Injections, Intra-Articular ; Matrix Metalloproteinase 13 ; metabolism ; Matrix Metalloproteinase 3 ; metabolism ; Matrix Metalloproteinases, Secreted ; metabolism ; Osteoarthritis ; drug therapy ; enzymology ; Protein Kinase Inhibitors ; administration & dosage ; pharmacology ; therapeutic use ; Pyridines ; administration & dosage ; pharmacology ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

This study investigated effects of Ginsenoside Ro (Ro) on interleukin-1β (IL-1β)-induced apoptosis and inflammation in rat chondrocytes. The rat chondrocytes were co-treated with IL-1β (10 ng·kg(-1)) and Ro (50, 100 and 200 μmol·L(-1)) for 48 h. Chondrocytes viability was detected by the MTT assay and Annexin V-FITC/PI dual staining assay. Caspase 3 activity was measured by using caspase 3 colorimetric assay kit. Apoptosis related proteins Bax, Bad, Bcl-xL, PCNA, p53 and phospho-p53, along with inflammation related protein MMP 3, MMP 9 and COX-2, and the expression of phospho-NF-κB p65 were assayed by western blotting analyses. Ro could improve IL-1β-induced chondrocytes viability. Ro could suppress IL-1β-induced apoptosis by inhibiting levels of Bax and Bad, decreasing p53 phosphorylation and promoting the expression of Bcl-xL and PCNA. Ro inhibited caspase 3 activity. IL-1β-induced inflammation and matrix degration were also alleviated by Ro with down-regulating the expression of MMP 3, MMP 9 and COX-2. Moreover, Ro inhibited NF-κB p65 phosphorylation induced by IL-1β. In conclusion, these results suggested Ro exerted anti-apoptosis and anti-inflammation in IL-1β-induced rat chondrocytes, which might be related to NF-κB signal pathway. Therefore, we propose that Ro might be a potential novel drug for the treatment of osteoarthritis.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; drug effects ; metabolism ; Caspase 3 ; metabolism ; Cell Survival ; drug effects ; Chondrocytes ; cytology ; drug effects ; Cyclooxygenase 2 ; drug effects ; metabolism ; Down-Regulation ; drug effects ; Drug Evaluation, Preclinical ; Ginsenosides ; pharmacology ; Inflammation ; chemically induced ; drug therapy ; Interleukin-1beta ; antagonists & inhibitors ; pharmacology ; Matrix Metalloproteinase 3 ; drug effects ; metabolism ; Matrix Metalloproteinase 9 ; drug effects ; NF-kappa B ; antagonists & inhibitors ; drug effects ; metabolism ; Phosphorylation ; drug effects ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune cells to produce immune mediators. This study demonstrates that in murine macrophage RAW 264.7 cells, CpG ODN-mediated matrix metalloproteinase-9 (MMP-9) expression is regulated at transcriptional level and requires de novo protein synthesis. Inhibition of ERK and p38 MAPK, but not JNK, results in significant decrease of CpG ODN-induced MMP-9 expression. We found that endosomal maturation inhibitors, chloroquine and bafilomycin A, block CpG ODN-induced ERK and p38 MAPK activation and the subsequent MMP-9 expression. We also observed that CpG ODN induces NF-kappa B activation and NF-kappa B is a downstream target of p38 MAPK. Taken together, our data demonstrate that CpG ODN triggers MMP-9 expression via TLR-9 dependent ERK and p38 MAPK activation followed by NF-kappa B activation.
Animals ; Cell Line ; Enzyme Activation/drug effects ; Enzyme Induction/drug effects ; Matrix Metalloproteinase 9/*biosynthesis ; Mice ; Mitogen-Activated Protein Kinase 1/antagonists & inhibitors/metabolism ; Mitogen-Activated Protein Kinase 3/antagonists & inhibitors/metabolism ; Mitogen-Activated Protein Kinases/*metabolism ; NF-kappa B/*metabolism ; Oligodeoxyribonucleotides/*pharmacology ; Signal Transduction/drug effects ; Toll-Like Receptor 9/antagonists & inhibitors/*metabolism ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism

Cordycepin (3'-deoxyadenosine) has been shown to exhibit many pharmacological activities, including anti-cancer, anti-inflammatory, and anti-infection activities. However, the anti-skin photoaging effects of cordycepin have not yet been reported. In the present study, we investigated the inhibitory effects of cordycepin on matrix metalloproteinase-1 (MMP-1) and -3 expressions of the human dermal fibroblast cells. Western blot analysis and real-time PCR revealed cordycepin inhibited UVB-induced MMP-1 and -3 expressions in a dose-dependent manner. UVB strongly activated NF-kappa B activity, which was determined by I kappa B alpha degradation, nuclear localization of p50 and p65 subunit, and NF-kappa B binding activity. However, UVB-induced NF-kappa B activation and MMP expression were completely blocked by cordycepin pretreatment. These findings suggest that cordycepin could prevent UVB-induced MMPs expressions through inhibition of NF-kappa B activation. In conclusion, cordycepin might be used as a potential agent for the prevention and treatment of skin photoaging.
Aging/physiology ; Cells, Cultured ; Deoxyadenosines/*pharmacology ; *Dermis/cytology/drug effects/physiology/radiation effects ; Dose-Response Relationship, Drug ; Enzyme Induction/drug effects ; Fibroblasts/drug effects/metabolism/radiation effects ; Gene Expression Regulation, Enzymologic ; Humans ; Infant, Newborn ; Male ; *Matrix Metalloproteinase 1/antagonists & inhibitors/biosynthesis/genetics/radiation effects ; Matrix Metalloproteinase 3/antagonists & inhibitors/*biosynthesis/genetics/radiation effects ; NF-kappa B/*antagonists & inhibitors/genetics/metabolism ; Skin/physiopathology/radiation effects ; *Ultraviolet Rays

One of the 14-3-3 protein isoforms, 14-3-3epsilon, was previously shown to be increased during skin aging. We suggest here a possible role for the 14-3-3epsilon protein in skin aging by providing evidence that 14-3-3epsilon increases the expression of the matrix-metalloproteinase (MMP)-2 gene in NIH3T3 fibroblast cells. Expression of the 14-3-3epsilon gene in NIH3T3 cells primarily up-regulated the expression of the MMP-2 gene at the transcriptional level by inducing specific DNA binding proteins bound to an upstream region of the MMP-2 promoter from -1,629 to -1,612. Inhibition of endogenous 14-3-3epsilon gene expression by RNA interference also decreased endogenous MMP-2 gene expression. Furthermore, up-regulation of the MMP-2 gene by 14-3-3epsilon was suppressed by expression of a dominant-negative mutant of p38 MAP kinase. These findings strongly suggest that increased expression of 14-3-3epsilon contributes to remodeling of extracellular matrix in skin through increasing MMP-2 gene expression via p38 MAP kinase signaling.
14-3-3 Proteins/*physiology ; Animals ; Electrophoretic Mobility Shift Assay ; Gene Expression Regulation, Enzymologic/*physiology ; Matrix Metalloproteinase 2/antagonists & inhibitors/*genetics/metabolism ; Mice ; NIH 3T3 Cells ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering/pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; *Signal Transduction ; Transfection ; p38 Mitogen-Activated Protein Kinases/*metabolism

OBJECTIVETo investigate P2Y purinergic receptor activated PI-3K/Akt signaling pathway in the regulation of growth and invasion of prostate cancer in vitro.

METHODSWestern blot was used to detect phosphorylation of Akt (a downstream target molecule of PI-3K) by P2Y receptor agonist in 1E8 cells (a highly metastatic subclone derived from PC-3 prostatic cancer cell line). Cell counts, flow cytometry, Matrigel invasion assay, wound healing assay and gelatin zymography were used to detect changes of biological behaviors of 1E8 cells after P2Y receptor activation.

RESULTSAMP-PNP, one non-hydrolysis ATP analogue and P2Y receptor agonist, induced significant phosphorylation of Akt in a time- and dose-dependent manner in IE8 cells. LY294002, a specific inhibitor of PI-3K, effectively blocked Akt phosphorylation induced by AMP-PNP. Continuous exposure to AMP-PNP induced significant growth inhibition of 1E8 cells (inhibition rate at 50.2% at the 8th day), and this inhibition was mainly due to an arrest at S phase of the cell cycle (the S phase fraction of AMP-PNP treated cells was 22.3% higher than that of the control). Application of LY294002 did not reverse the growth inhibition effect of AMP-PNP. Matrigel invasion assay showed that AMP-PNP stimulation increased invasive ability of 1E8 cells, and this effect was effectively blocked by LY294002. No significant changes in the activation of MMP-2 and MMP-9 were detected by gelatin zymography, although wound healing assay showed 21.2% increase in cell migration after AMP-PNP treatment.

CONCLUSIONSPI-3K/Akt signaling pathway participates in P2Y receptor-stimulated prostate cancer invasion by enhancing cell motility, rather than up-regulating MMP-2 and MMP-9 activities. PI-3K signaling pathway plays an important role in prostate cancer proliferation, but is not involved in P2Y receptor mediated growth inhibition.

Adenylyl Imidodiphosphate ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Chromones ; pharmacology ; Humans ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Nude ; Morpholines ; pharmacology ; Neoplasm Invasiveness ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Phosphorylation ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Purinergic P2 Receptor Agonists ; S Phase ; drug effects ; Signal Transduction ; drug effects

OBJECTIVETo evaluate the efficacy and safety of etanercept, a tumor necrosis factor (TNF)-alpha inhibitor, in the treatment of ankylosing spondylitis (AS), and investigate its effect on serum levels of matrix metalloproteinase-3 (MMP-3).

METHODSForty-eight patients with AS received etanercept 25 mg twice a week for a treatment course of 12 weeks. The patients' symptoms, signs, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) levels and side effects were observed before and after the treatment. The serum levels of MMP-3 was determined using enzyme-linked immunosorbent assay (ELISA).

RESULTSAll the patients completed the treatment. The degree of spinal pain and pain at night, the duration of morning stiffness, the finger-to-floor distance, BASDAI and BASFI were significantly improved after the treatment (P<0.05). Etanercept treatment resulted in a significant reduction in serum MMP-3 level in the AS patients to 31.22-/+10.26 ng/ml as compared with the level before treatment (46.17-/+25.74 ng/ml, P<0.05). The reduction of serum MMP-3 was positively correlated to decrement of ESR and CRP (r=0.397 and 0.474, respectively, P<0.05). The most common adverse events of etanercept included injection site reaction and upper respiratory infection.

CONCLUSIONEtanercept treatment has obvious therapeutic effects on AS without serious adverse effects. MMP-3 may be a potentially useful indicator to assess the effect of anti-TNF-alpha treatment in AS patients.

Adolescent ; Adult ; Antirheumatic Agents ; therapeutic use ; C-Reactive Protein ; metabolism ; Etanercept ; Female ; Humans ; Immunoglobulin G ; therapeutic use ; Male ; Matrix Metalloproteinase 3 ; blood ; Middle Aged ; Receptors, Tumor Necrosis Factor ; therapeutic use ; Spondylitis, Ankylosing ; blood ; drug therapy ; pathology ; Treatment Outcome ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors ; Young Adult

The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distribution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIF1-α, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 μmol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIF1-α, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the expression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, S6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib (IC50:1.385 vs. 15.09 μmol/L respectively), whereas combination of NVP-BKM120 and gefitinib (1 μmol/L) did not show more obvious effect than NVP-BKM120 used alone on inhibition of cell growth (P>0.05). NVP-BKM120 (1 μmol/L) increased the proportion of H1975 cells in G0-G1 phase and the effect was concentration-dependent, and 2 μmol/L NVP-BKM120 promoted apoptosis of H1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib (1 μmol/L)-treated group or between DMSO-treated control group and gefitinib (1 μmol/L)-treated alone group (P>0.05 for all). It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 (1 μmol/L), and NVP-BKM120 (1 μmol/L) or NVP-BKM120 (1 μmol/L) plus gefitinib (1 μmol/L) obviously inhibited the activation of Akt, S6 and 4E-BP1 as compared with control group, but single use of gefitinib (1 μmol/L) exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H1975 cells to gefitinib.
Adenocarcinoma ; metabolism ; Aminopyridines ; pharmacology ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Lung Neoplasms ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; Quinazolines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IkappaB, and nuclear AP-1 or NF-kappaB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IkappaB-NF-kappaB are involved.
1-Phosphatidylinositol 3-Kinase/metabolism ; Alkyl and Aryl Transferases/metabolism ; Animals ; Anticholesteremic Agents/pharmacology ; Cells, Cultured ; Enzyme Inhibitors/metabolism/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Gene Expression Regulation, Enzymologic/*drug effects ; I-kappa B Kinase/antagonists & inhibitors/metabolism ; Macrophages, Alveolar/cytology/*drug effects/*enzymology ; Matrix Metalloproteinase 9/genetics/*metabolism ; Mitogen-Activated Protein Kinase Kinases/metabolism ; Polyisoprenyl Phosphates/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Sesquiterpenes/metabolism ; Signal Transduction/physiology ; Simvastatin/*pharmacology ; Smoke/*adverse effects ; *Tobacco/adverse effects/chemistry