OBJECTIVETo explore the effect of the Hsp90 inhibitor anacardic acid on cell proliferation, invasion and migration of breast cancer MDA-MB-231 cells.

METHODSThe inhibitory effect of anacardic acid on Hsp90 was assessed with in vitro ATPase inhibition assay and ATP-sepharose binding assay. MTT assay was used to detect the growth inhibition induced by anacardic acid in MDA-MB-231 cells. Transwell assays were used to evaluate MDA-MB-231 cell invasion and migration. Western blotting was performed to assess the effect of anacardic acid in triggering the degradation of MMP-9, TIMP-1, Hsp90, and Hsp70.

RESULTSAnacardic acid exhibited a modest activity of ATPase inhibition with an IC50 value of 82.5 µmol/L. Anacardic acid significantly suppressed the proliferation of MDA-MB-231 cells in a dose-dependent manner (IC50 value of 29.3 µmol/L). Treatment with 12.5, 25, and 50 µmol/L anacardic acid for 36 h caused inhibition of cell invasion by 23.6%, 56.6%, and 67.0% in MDA-MB-231 cells, respectively (P<0.05), and anacardic acid treatment for 24 h inhibited the cell migration by 30.0%, 45.5%, and 77.5%, respectively (P<0.05). Anacardic acid dose-dependently induced MMP-9 degradation, but did not obviously affect Hsp90 or Hsp70 expressions.

CONCLUSIONAnacardic acid can significantly inhibit the proliferation, invasion, and migration of MDA-MB-231 cells, the mechanism of which may involve the inhibition of Hsp90 ATPse activity and down-regulation of MMP-9 expression.

Anacardic Acids ; pharmacology ; Breast Neoplasms ; pathology ; Cell Line, Tumor ; drug effects ; Cell Movement ; Cell Proliferation ; Down-Regulation ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; metabolism ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β (IL-1β)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1β restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.
3T3 Cells ; Animals ; Cartilage, Articular ; cytology ; Cell Survival ; physiology ; Cells, Cultured ; Chondrocytes ; drug effects ; enzymology ; Coculture Techniques ; Culture Media, Conditioned ; Gelatinases ; drug effects ; Interleukin-1beta ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; physiology ; Matrix Metalloproteinase 2 ; drug effects ; Matrix Metalloproteinase 9 ; drug effects ; Mice ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; drug effects ; Monocytes ; cytology ; NF-kappa B ; antagonists & inhibitors ; Osteoclasts ; physiology ; Protease Inhibitors ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; Tissue Inhibitor of Metalloproteinase-2 ; drug effects ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

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

BACKGROUNDThe decreased degradation of extra-cellular matrix proteins plays an important role in the onset of diabetic nephropathy. Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1), which are members of the matrix metalloproteinase family, are associated with this process. Angiotensin II (AII) plays an important role in the development of diabetic nephropathy also. This research aimed to investigate the effect of angiotensin II receptor blocker on glucose-induced mRNA expressions of MMP-9 and TIMP-1 in rat mesangial cells.

METHODSRat mesangial cells were cultured and divided into 5 groups: normal glucose (group NG), high glucose (group HG), group NG + AII, NG + AII + saralasin (group NG + AII + S, saralasin is the AII receptor blocker) and HG + saralasin (group HG + S). After the cells were incubated for 24 hours, AII concentrations in the supernatant were measured by radioimmunoassay and the expression of MMP-9 and TIMP-1 mRNA was assayed by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSAII concentrations were higher in group HG ((56.90 +/- 13.54) pg/ml) and group HG + S ((51.30 +/- 5.96) pg/ml) than in group NG ((37.89 +/- 8.62) pg/ml, P < 0.05), whereas there was no significant difference between group HG and group HG + S. The expression of MMP-9 mRNA and MMP-9/TIMP-1 mRNA ratio in group NG + AII (MMP-9, 0.33 +/- 0.04; MMP-9/TIMP-1, 0.40 +/- 0.06) and group HG (MMP-9, 0.36 +/- 0.02; MMP-9/TIMP-1, 0.45 +/- 0.03) were decreased more significantly than those in group NG (MMP-9, 0.72 +/- 0.02; MMP-9/TIMP-1, 1.21 +/- 0.07). These values in group NG + AII + S (MMP-9, 0.71 +/- 0.02; MMP-9/TIMP-1, 1.18 +/- 0.05) were higher than those in group NG + AII, and the values in group HG + S (MMP-9, 0.71 +/- 0.02; MMP-9/TIMP-1, 1.16 +/- 0.05) were higher than those in group HG (all were P < 0.05). TIMP-1 mRNA expression was increased more significantly in group NG + AII (0.81 +/- 0.03) and group HG (0.80 +/- 0.03) than in group NG (0.59 +/- 0.02), but it was lower in group NG + AII + S (0.60 +/- 0.01) than in group NG + AII and also lower in group HG + S (0.61 +/- 0.01) than in group HG (all were P < 0.05).

CONCLUSIONSHigh glucose stimulates AII production. Both high glucose and AII induce a decrease in MMP-9 mRNA expression and MMP-9/TIMP-1 mRNA ratio as well as an increase in TIMP-1 mRNA expression, which can be reversed by saralasin, suggesting that high glucose can aggravate impaired matrix degradation by altering gene expression of MMP-9 and TIMP-1 and that the effect of high glucose may be mediated by AII.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Angiotensin Receptor Antagonists ; Animals ; Cells, Cultured ; Gene Expression ; drug effects ; Glucose ; pharmacology ; Matrix Metalloproteinase 9 ; genetics ; Mesangial Cells ; cytology ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Saralasin ; pharmacology ; Tissue Inhibitor of Metalloproteinase-1 ; genetics

Cardiac fibrosis occurs after pathological stimuli to the cardiovascular system. One of the most important factors that contribute to cardiac fibrosis is angiotensin II (Ang II). Accumulating studies have suggested that reactive oxygen species (ROS) plays an important role in cardiac fibrosis and sodium tanshinone IIA sulfonate (STS) possesses antioxidant action. We therefore examined whether STS depresses Ang II-induced collagen type I expression in cardiac fibroblasts. In this study, Ang II significantly enhanced collagen type I expression and collagen synthesis. Meanwhile, Ang II depressed matrix metalloproteinase-1 (MMP-1) expression and activity. These responses were attenuated by STS. Furthermore, STS depressed the intracellular generation of ROS, NADPH oxidase activity and subunit p47(phox) expression. In addition, N-acetylcysteine the ROS scavenger, depressed effects of Ang II in a manner similar to STS. In conclusion, the current studies demonstrate that anti-fibrotic effects of STS are mediated by interfering with the modulation of ROS.
Acetylcysteine/pharmacology ; Angiotensin II/*antagonists & inhibitors/pharmacology ; Animals ; Blotting, Western ; Cells, Cultured ; Collagen Type I/*metabolism ; Drugs, Chinese Herbal/*pharmacology ; Fibroblasts/*drug effects/metabolism ; Free Radical Scavengers/pharmacology ; Matrix Metalloproteinase 1/metabolism ; Myocardium/*cytology ; NADPH Oxidase/metabolism ; Oxidative Stress/drug effects ; Phenanthrenes/*pharmacology ; Rats ; Rats, Wistar ; Reactive Oxygen Species/metabolism

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

OBJECTIVETo study whether yishen ruanjian san contained serum (S-YRS) could intervene the action of aristolochic acid (AA) in antagonizing human renal interstitial fibroblasts (hRIFs) to induce extracellular matrix (ECM) accumulation.

METHODSAA-Na 40 microg/ml, with or without 10% S-YRS, was co-cultured with hRIFs, then the hRIFs mRNA of transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type I collagen (Col I) in the cultured cells were detected by RT-PCR, and their protein expression monitored with ELISA and Western blot respectively.

RESULTSThe mRNA and protein expression of all the above-mentioned factors were significantly up-regulated by AA-Na (P < 0.05). Excepting PAI-1, the enhanced mRNA and protein expression were significantly down-regulated by S-YRS (P < 0.05).

CONCLUSIONS-YRS could down-regulate the hRIF to promote the expression of ECM synthesis factors and inhibit the ECM degradation factors in hRIFs, so as to antagonize the AA stimulated accumulation of ECM such as Col I.

Animals ; Aristolochic Acids ; antagonists & inhibitors ; toxicity ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacokinetics ; pharmacology ; toxicity ; Extracellular Matrix ; metabolism ; Fibroblasts ; drug effects ; pathology ; Kidney ; pathology ; Male ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serum ; Tissue Inhibitor of Metalloproteinase-1 ; analysis ; metabolism ; Transforming Growth Factor beta ; analysis ; metabolism ; Transforming Growth Factor beta1

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