Matrix Metalloproteinase-9 Protein Levels Research Articles

Increased expression of MMP9 is correlated with poor prognosis of nasopharyngeal carcinoma

IntroductionThe aim of the present study was to analyze the expression of matrix metalloproteinase 9 (MMP9) in nasopharyngeal carcinoma (NPC) and its correlation with clinicopathologic features, including the survival of patients with NPC.MethodsUsing real-time PCR, we detected the mRNA expression of MMP9 in normal nasopharyngeal tissues and nasopharyngeal carcinoma (NPC) tissues. Using immunohistochemistry analysis, we analyzed MMP9 protein expression in clinicopathologically characterized 164 NPC cases (116 male and 48 female) with age ranging from 17 to 80 years (median = 48.4 years) and 32 normal nasopharyngeal tissues. Cases with greater than or equal to 6 and less than 6 of the score value of cytoplasmic MMP9 immunostaining were regarded as high expression and low expression, respectively. The relationship between the expression levels of MMP9 and clinical features was analyzed.ResultsThe expression level of MMP9 mRNA was markedly greater in NPC tissues than that in the nasopharyngeal tissues. Immunohistochemical analysis revealed that the protein expression of MMP9 detected in NPC tissues was higher than that in the nasopharyngeal tissues (P = 0.004). In addition, high levels of MMP9 protein were positively correlated with the status of lymph node metastasis (N classification) (P = 0.002) and clinical stage (P < 0.001) of NPC patients. Patients with higher MMP9 expression had a significantly shorter overall survival time than did patients with low MMP9 expression. Multivariate analysis suggested that the level of MMP9 expression was an independent prognostic indicator (P = 0.008) for the survival of patients with NPC.ConclusionHigh level of MMP9 expression is a potential unfavorable prognostic factor for patients with NPC.

Matrix Metalloproteinase-9 Expression and Protein Levels after Fluid Percussion Injury in Rats: The Effect of Injury Severity and Brain Temperature

The temporal and regional expression profiles of matrix metalloproteinase-9 (MMP-9), after moderate or severe traumatic brain injury (TBI) were measured to investigate the effects of post-traumatic hypothermia (33 degrees C) or hyperthermia (39 degrees C). In the first phase of this study, adult male Sprague-Dawley rats were randomly assigned to groups of moderate TBI (1.8-2.2 atm), severe TBI (2.4-2.7 atm), and sham-injured control. The rats were killed at 4, 6, 12, 24, 48, and 72 h, or 1 week after TBI, for mRNA and protein analysis. In the second phase, rats underwent moderate fluid percussion brain injury, followed immediately by 4 h of post-traumatic normothermia (37 degrees C), hyperthermia (39 degrees C), or hypothermia (32 degrees C). The rats were killed at 12 and 48 h after TBI for mRNA expression analyses, or killed at 24 and 72 h after TBI for protein expression analyses. Brain samples, including the cerebral cortex and hippocampus (both ipsilateral and contralateral hemispheres of each group), were assayed using RT-PCR and Western blot techniques. MMP-9 levels in both the ipsilateral and contralateral hemispheres were significantly increased after TBI compared with those of sham injured animals (p < 0.01). Two expression peaks of MMP-9 were observed in the ipsilateral cortex and hippocampus. An increase in injury severity was associated with an increase in mRNA (12 and 48 h), and protein (24 and 72 h) levels of MMP-9. Post-traumatic hypothermia attenuated the increase in both the mRNA and protein levels of MMP-9, compared with normothermia and hyperthermia (p < 0.01). In contrast, hyperthermia had no significant effect on mRNA (at 12 h) and protein levels (at 24 h) of MMP-9, compared with normothermic values (p > 0.05), but resulted in a significant increase in the levels of MMP-9 mRNA and protein at 24 and 72 h, respectively (p < 0.01). Increases in MMP-9 mRNA and protein after TBI were proportional to injury severity in this model. The effects of post-traumatic hypothermia on the expression of MMP-9 may partially explain the observed effects of post-traumatic temperature on secondary injury after TBI.

Effects of exogenous urotensin II on vascular remodelling after balloon injury

1. Urotensin II (U-II) is a powerful vasoconstrictor peptide that stimulates cell proliferation. However, the systemic effects of U-II on cellular and extracellular matrix responses of vessel walls have not been investigated. The aim of the present study was to determine the effect of exogenous U-II on arterial neointimal hyperplasia after balloon injury. 2. A stenosis model of the thoracic aorta after balloon injury was established in male Wistar rats. Rats were divided into three groups (n = 5 in each): (i) uninjured; (ii) injured for 21 days; and (iii) injured and then treated with U-II (1 nmol/kg per h) via an osmotic minipump for 21 days. Another group of rats were killed on Days 7 and 14 after balloon injury for the analysis of in vitro collagen synthesis and secretion with U-II treated by [(3)H]-proline incorporation and determination of [(3)H]-hydroxyproline radioactivity, respectively. 3. Urotensin II immunoreactivity was 1.74-fold higher in vessels injured for 21 days than in uninjured vessels and mRNA levels of the urotensin UT receptor were upregulated by 55% following injury. After U-II treatment, the mRNA levels of the UT receptor were further upregulated (by 40%). In addition, U-II treatment increased the intimal area of injured aortas (13 +/- 5 vs 7 +/- 2% in group iii and ii, respectively), as well as increasing collagen content and cell proliferation. Protein levels of matrix metalloproteinase 1 were decreased in U-II-treated rats. In vitro, U-II treatment increased collagen synthesis and secretion in uninjured vessels in a concentration-dependent manner (10(-10), 10(-9) and 10(-8) mol/L U-II), especially in injured aortas on Day 7 after injury. 4. In conclusion, exogenous U-II may upregulate mRNA levels of the UT receptor, as well as increase collagen and cell proliferation, all of which would contribute to intimal hyperplasia after angioplasty.

Single minded 2-s (SIM2-s) gene is expressed in human GBM Cells and involved in GBM invasion

The human Single Minded 2 (SIM2) gene is mapped within the Down Syndrome Critical Region (DSCR) of chromosome 21 and its short splice variant SIM2-s is suggested to be a molecular target for cancer therapy. It has been shown that SIM2-s is expressed in colon, pancreatic and prostate tumors but not in their corresponding normal tissues. In present study, we found that SIM2-s was expressed in glioma tissues as well as in glioblastoma (GBM) cell lines but not in other brain tumors or normal cortex. The invasive potential of GBM cells was significantly decreased by RNA interference targeting SIM2-s, while the proliferation was not affected. Further investigation showed that the mRNA and protein levels of matrix metalloproteinase 2 (MMP-2) were down-regulated in cells treated with SIM2-s siRNA, concomitance with the up-regulation of tissue inhibitor of matrix metalloproteinase 2 (TIMP-2). Moreover, the enzymatic activity of MMP-2 was clearly decreased. Our results collectively suggested that SIM2-s expressed in gliomas selectively and played a role in GBM cells invasion, which may be partly associated with the expression of MMP-2 and TIMP-2.

Matrix metalloproteinase: investigation from gene to protein as effective factor in myocardial infarction

Current evidence indicates that extracellular matrix (ECM) remodeling is a component of acute myocardial infarction (AMI) and matrix metalloproteinase (MMP) has a role in early atherosclerosis, plaque rupture and myocardial infarction (MI). The necessity of inhibition of ECM remodeling and subsequent injuries in patients with AMI suggests that MMP might be involved in this task. Therefore, we investigated the activities of MMP-1, -2, -3, and -9 which play an important role in AMI. Plasma and peripheral blood mononuclear cells (PBMCs) of 50 patients with AMI were isolated from peripheral blood after the onset of AMI within 24h, comparing with 50 control subjects. The active form of MMPs was measured by enzyme linked immunosorbent assay (ELISA); MMP proteins presence and expression by immunoblotting and zymography analysis; and mRNA expression of MMPs by real time reverse transcriptase polymerase chain reaction. Plasma concentrations of MMPs increase in patients rather than control subjects. Gel zymography revealed 43, 66, 45, and 83kDa molecular weight bands which consistent with active MMP-1, -2, -3, and -9, respectively, exhibiting gelatin-degrading activity in both patient and control subjects. No up-regulation of mRNA expression was found. To our knowledge, it is the first monitoring of MMP gene and protein expression and also circulating active MMPs in Iranian patients with AMI and normal subjects. Up-regulation of MMPs activity is common in the falling myocardium and missing up-regulation of transcription indicates that protein levels of MMPs were regulated at the post transcriptional level.

Benzyl Isothiocyanate (BITC) Inhibits Migration and Invasion of Human Colon Cancer HT29 Cells by Inhibiting Matrix Metalloproteinase-2/-9 and Urokinase Plasminogen (uPA) through PKC and MAPK Signaling Pathway

Benzyl isothiocyanate (BITC), a component of dietary cruciferous vegetables, has antioxidant and anticancer properties. In this study, we show for the first time the antimetastatic effects of BITC in human colon cancer HT29 cells. BITC had an inhibitory effect on cell migration and invasion. Protein levels of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-plasminogen activator (u-PA) were reduced by BITC in a concentration-dependent manner. BITC also exerted an inhibitory effect on phosphorylation of c-Jun N-terminal kinase 1 and 2 (JNK1/2), extracellular signal-regulated kinases 1 and 2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) that are upstream of nuclear factor kappa B (NF-kappaB). BITC inhibited DNA binding activity of NF-kappaB. Moreover, BITC decreased the levels of c-Fos, c-Jun, Ras, FAK, PI3K and GRB2 in HT29 cells. Reductions in the enzyme activity, protein and mRNA (mRNA) levels of MMP-2 were observed in BITC-treated HT29 cells. BITC also inhibited mRNA levels of MMP-2, -7, and -9 in HT29 cells. Results from zymography showed that BITC treatment decreased MMP-2 expression in a concentration-dependent manner. BITC inhibited PKCdelta activity in HT29 cells. Furthermore, inhibitors specific for JNK (SP600125) reduced expression of MMP-2, MMP-9, and u-PA. These results demonstrated that BITC could alter HT29 cell metastasis by reduction of MMP-2, MMP-9, and u-PA expression through the suppression of a PKC, MAPK signaling pathway and inhibition of NF-kappaB levels. These findings suggest that BITC has potential as an antimetastatic agent.

Prognostic significance of matrix metalloproteinase-1 levels in peripheral plasma and tumour tissues of lung cancer patients

Matrix metalloproteinase-1 (MMP-1) participates in a variety of physiological and pathological processes. We previously found that MMP-1 was one of the lung cancer-related proteins detectable in peripheral blood. To validate our preliminary observations and explore the clinical significance of MMP-1 for lung cancer further, we carried out the present study. The concentrations of MMP-1 in circulating plasma specimens of 170 lung cancer patients and 70 healthy individuals were measured by an enzyme-linked immunosorbance assay. The expression status of the MMP-1 in archival tissue samples from 122 lung cancer patients was examined by immunohistochemical analysis. The correlation between the MMP-1 levels and prognosis of the lung cancer patients was then assessed statistically. Protein levels of MMP-1 were considerably raised in the plasma from lung cancer patients relative to those in healthy controls. The high plasma MMP-1 levels were associated with advanced-stage of the disease and significantly lower overall survival rate of the patients. Coincidently, MMP-1 protein extraordinarily overexpressed in the tumour tissues of lung cancer; and the up-regulated MMP-1 was associated with the progression (including tumour size, staging and lymphatic invasion), especially with decreased survival rate of the patients. Statistic analysis revealed that MMP-1 protein levels had an independent influence on survival. MMP-1 levels were elevated in both tumour tissue and blood; the latter may serve as an independent predictor for survival of lung cancer patients. MMP-1 protein levels in plasma/serum thus represent a potential and clinically relevant biomarker for the prognosis of patients with lung cancers.

Interleukin-lβ Induces Migration of Rat Arterial Smooth Muscle Cells Through a Mechanism Involving Increased Matrix Metalloproteinase-2 Activity

Interleukin-lβ (IL-lβ) is associated with vascular smooth muscle cell (VSMC) migration during neointimal formation following arterial injury, of which matrix metalloproteinase-2 (MMP-2) may have an important role. We investigated whether IL-lβ stimulated migration and MMP-2 production in VSMC, and, if so, whether migration correlated with MMP-2 activity. Modified Boyden chamber assay quantified cultured rat aorta VSMC migration. Methyl-thiazolyl-tetrazolium assay assessed cell growth. Gelatin zymography and Western blotting determined MMP-2 activity and protein levels, respectively. IL-lβ (0.1 - 10 ng/mL) induced migration of VSMC in a concentration-dependent manner without cell proliferation. VSMC released increasing levels of active MMP-2 in a dose-response fashion at IL-1β 1-10 ng/mL (P < 0.05) while significantly increased levels of latent MMP-2 (pro-MMP-2) were attained more gradually (10 ng/mL, P < 0.05). There was a dose-dependent increase in the ratio of active MMP-2 to pro-MMP-2 in response to IL-1β (1-10 ng/mL, P < 0.05), suggesting extracellular activation of pro-MMP-2. Protein levels on Western blot paralleled enzyme activity, with the synthesis of more active MMP-2 than pro-MMP-2 in response to IL-1β. IL-lβ-stimulated VSMC migration was significantly attenuated by both the pan-selective MMP inhibitor GM6001 and cis-9-octadecenoyl-N-hydroxylamide, a MMP-2-selective inhibitor. IL-lβ increases MMP-2 activity in VSMC through increased protein synthesis and activation of pro-MMP-2. VSMC migration induced by IL-lβ requires active MMP-2. IL-lβ may play a role in arterial remodeling following injury.

α‐Mangostin, A Novel Dietary Xanthone, Suppresses TPA‐Mediated MMP‐2 and MMP‐9 Expressions through the ERK Signaling Pathway in MCF‐7 Human Breast Adenocarcinoma Cells

This study first investigates the anti-metastatic effect of alpha-mangostin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expressions in human breast adenocarcinoma cells, MCF-7. First, the result demonstrated alpha-mangostin could inhibit TPA-induced abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay and Boyden chamber assay. Data also showed alpha-mangostin could inhibit the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) involved in the downregulation the enzyme activities, protein, and messenger RNA levels of MMP-2 and MMP-9 induced by TPA. Next, alpha-mangostin also strongly inhibited TPA-induced degradation of inhibitor of kappaBalpha (IkappaBalpha) and the nuclear levels of nuclear factor kappa B (NF-kappaB), c-Fos, and c-Jun. Also, a dose-dependent inhibition on the binding abilities of NF-kappaB and activator protein-1 (AP-1) by alpha-mangostin treatment was further observed. Further, the treatment of specific inhibitor for ERK (U0126) to MCF-7 cells could inhibit TPA-induced MMP-2 and MMP-9 expressions along with an inhibition on cell invasion and migration. Presented data reveal that alpha-mangostin is a novel, effective, antimetastatic agent that functions by downregulating MMP-2 and MMP-9 gene expressions.

Disruption of Nrf2 Enhances the Upregulation of Nuclear Factor-kappaB Activity, Tumor Necrosis Factor-α, and Matrix Metalloproteinase-9 after Spinal Cord Injury in Mice

Matrix metalloproteinase-9 (MMP-9) plays an important role in the acute periods of spinal cord injury (SCI), and its expression is related to the inflammation which could cause the disruption of the blood-spinal barrier (BBB). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that plays a crucial role in cytoprotection against inflammation. The present study investigated the role of Nrf2 in upregulating of nuclear factor kappa B (NF-κB) activity, tumor necrosis factor-α (TNF-α), and MMP-9 after SCI. Wild-type Nrf2 (+/+) and Nrf2-deficient (Nrf (−/−)) mice were subjected to an SCI model induced by the application of vascular clips (force of 10 g) to the dura after a three-level T8-T10 laminectomy. We detected the wet/dry weight ratio of impaired spinal cord tissue, the activation of NF-κB, the mRNA and protein levels of TNF-α and MMP-9, and the enzyme activity of MMP-9. Nrf2 (−/−) mice were demonstrated to have more spinal cord edema, NF-κB activation, TNF-α production, and MMP-9 expression after SCI compared with the wild-type controls. The results suggest that Nrf2 may play an important role in limiting the upregulation of NF-κB activity, TNF-α, and MMP-9 in spinal cord after SCI.

Suppression of PMA-induced tumor cell invasion and metastasis by aqueous extract isolated from Prunella vulgaris via the inhibition of NF-κB-dependent MMP-9 expression

Matrix metalloproteinase-9 (MMP-9) plays an important role in the invasion and metastasis of cancer cells. In this study, we examined the inhibitory effects of tumor cell migration and invasion by aqueous extract isolated from Prunella vulgaris (PVAE) using in vitro and in vivo assays. PVAE reduced PMA-induced activation of MMP-9 and further inhibited cell invasion and migration. PVAE suppressed PMA-enhanced expression of MMP-9 protein, mRNA and transcription activity levels through suppression of NF-κB activation without changing the tissue inhibitor of metalloproteinase level. PVAE inhibited PMA-induced NF-κB nuclear translocation, which is upstream of PMA-induced MMP-9 expression and invasion. Furthermore, pretreatment with NF-κB activation inhibitor inhibited the PMA-induced MMP-9 expression and activity. PVAE repressed the PMA-induced phosphorylation of ERK1/2, which is upstream signaling molecules in MMP-9 expression. We confirmed that the inhibitory effect of PVAE on lung metastasis and tumor cell growth using B16-F10 melanoma cells or B16-F1 melanoma cells in C57BL/6 mice. The oral administrations of PVAE reduced the lung metastasis and tumor cell growth by B16-F10 or B16-F1 melanoma cells. These results suggested that the anti-metastatic effect of PVAE is mediated through the suppression of MMP-9 expression by the inhibition of NF-κB via ERK1/2 signaling pathway as well as MMP-9 activity.

Induction of matrix metalloproteinase-9 by galectin-7 through p38 MAPK signaling in HeLa human cervical epithelial adenocarcinoma cells

The expression of matrix metalloproteinase-9 (MMP-9) has been reported in various cancers. Its expression is associated with tumorigenesis and tumor metastasis. Studies have shown that galectin-7, a beta-galactoside-binding animal lectin, is involved in various processes including the suppression of tumor growth. However, a recent study reported that the development of thymic lymphoma is accelerated by galectin-7 expression. In this report, we demonstrate that the expression of MMP-9 was increased by galectin-7 in human cervix epithelial cells (HeLa). When the galectin-7 gene was transfected to HeLa cells with the ECFP vector, the expression of MMP-9 mRNA increased, as compared to non-transfected cells. As a result, MMP-9 protein levels also increased, as indicated by Western blot and gelatin zymography. In addition, galectin-7-transfected cells exhibited increased invasion in the matrigel invasion system. Expression of MMP-9 is involved in several signaling pathways by various stimulation factors. Therefore, we investigated how the signaling pathway in galectin-7-transfected cells differs from that of non-transfected cells. Upon transfection of galectin-7, p38 MAPK was activated and SB203580, a chemical inhibitor of p38 MAPK, reversed the effects of galectin-7. These results indicate that galectin-7 increases the expression of MMP-9 through the p38 MAPK signaling pathway.

Glioma-astrocyte interaction modifies the astrocyte phenotype in a co-culture experimental model

As the majority of gliomas arise through malignant transformation of astrocytes, we aimed at investigating the interaction between malignant glioma cells and astrocytes in a co-culture experimental model. For this purpose we analyzed the expression of genes and proteins involved in tumor promotion and invasion, such as glial fibrillary acidic protein (GFAP), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of MMP-2 (TIMP-2), transforming growth factor-beta1 (TGF-beta1), secreted protein acidic and rich in cysteine (SPARC), and connexin 43 (CX43). Co-cultures of human neural stem cell-derived astrocytes and U87 MG astrocytoma cells were performed in a transwell system. Gene expression was evaluated by real-time RT-PCR, and protein analysis was performed by Western blotting, SDS-zymography, and immunofluorescence. GFAP tended to be up-regulated in astrocytes co-cultivated with U87, suggesting a reactive response induced by glioma cells. CX43 mRNA tended to be down- regulated in co-cultured astrocytes, as well as the non-phosphorylated isoform at the protein level. MMP-2 mRNA tended to be up-regulated, and MMP-2 protein levels were significantly increased in astrocytes co-cultivated with U87. TIMP-2 and SPARC mRNA decreased in astrocytes co-cultivated with U87, showing lower expression in glioma cells. By contrast, SPARC protein expression was strongly induced in supernatants of co-cultured astrocytes. TGF-beta1 was not modified. Our results suggest that U87 cells elicit phenotype modifications in the neighbouring resident astrocytes very likely mediated by soluble factors. Glioma/astrocyte interaction could possibly trigger an astrocyte phenotype modification consistent with a malignant transformation, and favouring a more permissive environment for glioma cells invasion.

Plumbagin inhibits invasion and migration of liver cancer HepG2 cells by decreasing productions of matrix metalloproteinase‐2 and urokinase‐ plasminogen activator

To investigate the inhibitory effects of plumbagin (5-hydroxy-2 methyl-1,4-naphthoquinone) on the invasion and migration and its correlation with matrix metalloproteinase-2 (MMP-2) and urokinase-plasminogen activator (u-PA) in liver cancer HepG2 cells under non-cytotoxic concentrations. The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The adhesion, migration and invasion were measured by cell-matrix adhesion assay and Boyden chamber assay. The MMP-2 and u-PA activities were estimated by gelatin and casein-plasminogen zymography. The mRNA and protein levels of MMP-2, u-PA, urokinase-plasminogen activator receptor (u-PAR), tissue inhibitor of metalloproteinase-2 (TIMP-2), plasminogen activator inhibitor-1 (PAI-1), nuclear factor kappa B (NF-kappaB), c-Fos and c-Jun were evaluated by semi-quantitative reverse transcription polymerase chain reaction and western blotting. Also, the binding abilities of NF-kappaB and activator protein-1 (AP-1) were analyzed by electrophoretic mobility shift assay (EMSA). In this study, plumbagin had exhibited an inhibitory effect on the abilities of adhesion, migration and invasion. The results from zymography showed plumbagin treatment may decrease the activities of MMP-2 and u-PA. Further, the mRNA and protein levels of MMP-2, u-PA and u-PAR were significantly reduced, while TIMP-2 and PAI-1 were elevated by plumbagin treatment. Next, plumbagin significantly decreased the nuclear levels of NF-kappaB, c-Fos and c-Jun. Also, treating HepG2 cells with plumbagin leads to dose-dependent inhibition on the binding abilities of NF-kappaB and AP-1. We demonstrated the inhibitory effects of plumbagin on the invasion, migration and adhesion of HepG2 cells, while plumbagin treatment may decrease the expressions of MMP-2 and u-PA and enhance the expressions of TIMP-2 and PAI-1.

Fucoidan increases TNF-α-induced MMP-9 secretion in monocytic cell line U937

To investigate the effect of fucoidan on the expression of matrix metalloproteinase-9 (MMP-9) from monocytes. Human monocytic cell line U937 was purchased from ATCC. During the experiment, FBS-free 1640 was used and U937 was cultivated with 20 ng/ml TNF-alpha and/or different concentrations of fucoidan for 24 h. RT-PCR experiments were used to determine the MMP-9 mRNA expression. ELISA and gelatin zymography detected MMP-9 amounts and activity in the supernatant. The intracellular level of MMP-9 was assayed by Western blot, and the level of CD44 on the surface was assayed by FACS. In this study, we showed that pro-inflammatory cytokine TNF-alpha up-regulated U937 MMP-9 mRNA and protein levels (P < 0.05). Fucoidan can increase the TNF-alpha-induced MMP-9 secretion from U937 (P < 0.05), but no significant difference was observed in MMP-9 mRNA. The intracellular level of MMP-9 treated with TNF-alpha and fucoidan was lower (P < 0.05) than that treated with TNF-alpha alone. In addition, we demonstrated that fucoidan downregulated the surface level of CD44, the main molecule to which MMP-9 attaches. We demonstrated that fucoidan post-translationally regulated MMP-9 secretion from U937. Reduced intracellular level and decreased membrane attachment may contribute to the increase in MMP-9 secretion.

Danthron inhibits the migration and invasion of human brain glioblastoma multiforme cells through the inhibition of mRNA expression of focal adhesion kinase, Rho kinases-1 and metalloproteinase-9

In this study, we investigated the effect of danthron on the cell migration and invasion of human brain glioblastoma multiforme GBM 8401 cells in vitro. The changes of migration and invasion of GBM 8401 cells after treatment with danthron were detected by cell migration assay and cell invasion assay. The levels of mRNA gene expression associated with cell migration and invasion were detected by real-time PCR. Results indicated that human brain glioblastoma multiforme GBM 8401 cells treated with danthron in vitro migrated and invaded less than cells treated with phosphate-buffered saline (PBS) (control). Western blotting showed that danthron inhibited the protein levels of FAK, MMP-7, MMP-9 and uPA in GBM 8401 cells. Real-time PCR assay also showed that danthron inhibited the mRNA expression of matrix metalloproteinase-9 (MMP-9), FAK and ROCK-1 of GBM 8401 cells. These results showed that danthron inhibited invasion and migration of GBM 8401 cells by downregulating mRNA expression associated with these processes, resulting in reduced metastasis. Thus, danthron may be considered a therapeutic agent that can inhibit primary tumor growth and prevent metastasis.

High glucose and tea polyphenols on expression of matrix metalloproteinase-2 and extracellular matrix metalloproteinase inducer in human umbilical vein endothelial cells

To investigate the effects of high glucose on expression of matrix metalloproteinase-2 (MMP-2) and extracellular matrix metalloproteinase inducer (EMMPRIN) in human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in DMEM media containing high glucose with or without polyphenols for 24 hours respectively. The activity of MMP-2 in the supernatant was detected by zymography. The expression of MMP-2 mRNA and protein in HUVECs were detected by RT-PCR and Western blot respectively. The expression of EMMPRIN mRNA and protein in the cells were determined by RT-PCR as well as immunocytochemistry and Western blot respectively. The expression of MMP-2 and EMMPRIN mRNA were suppressed by a high concentration of glucose. Both the MMP-2 activity and protein level of EMMPRIN expression were also significantly decreased. Polyphenols abolished all the above changes of HUVECs induced by a high glucose level (P < 0.05). An acute high glucose stimulation down-regulates the activity of MMP-2, the expressions of MMP-2 and EMMPRIN at RNA and protein levels in the endothelial cells, which may play an important roles in diabetic vascular complications in the early phase. Polyphenols treatment can diminish the detrimental effects of high glucose on HUVECs.

Antimetastatic potential of fisetin involves inactivation of the PI3K/Akt and JNK signaling pathways with downregulation of MMP-2/9 expressions in prostate cancer PC-3 cells

Fisetin (3,3',4',7-tetrahydroxyflavone), a naturally occurring flavonoid, has been reported to possess some anti-cancer and anti-inflammation capabilities. In this study, fisetin has exhibited inhibitory effects on the adhesion, migration, and invasion ability of a highly metastatic PC-3 cells under non-cytotoxic concentrations. Gelatin zymography assay showed that fisetin inhibited the matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activities. Our result also showed that fisetin could inhibit the phosphorylation of c-Jun N-terminal kinase 1 and 2 (JNK1/2) and Akt. Moreover, fisetin significantly decreased the nuclear levels of nuclear factor kappa B (NF-kappaB), c-Fos, and c-Jun, and the binding abilities of NF-kappaB and activator protein-1 (AP-1). Also, the results showed that the protein and mRNA levels of MMP-2 and MMP-9 were significantly reduced by Western blot and semi-quantitative RT-PCR. Further, treating specific inhibitors for PI3K (Wortmannin) or JNK (SP600125) to PC-3 cells could reduce the protein expressions of MMP-2 and MMP-9. These results showed fisetin could inhibit the metastatic ability of PC-3 by reducing MMP-2 and MMP-9 expressions through suppressing phosphoinositide 3-kinase/Akt (PI3K/Akt) and JNK signaling pathways. This suggested fisetin can serve as a potential candidate for treating cancer metastasis.

P38 MAPK and MMP-9 cooperatively regulate mucus overproduction in mice exposed to acrolein fog

Objective To evaluate the role of p38 mitogen-activated protein kinase (MAPK) on mice airway inflammation, mucus production and the possible cross-talk between p38 MAPK and matrix metalloproteinase-9 (MMP-9) in mucin protein synthesis. Methods Mice were exposed to 4.0 ppm of acrolein for 21 days with daily intraperitoneal injection of SB203580, a specific inhibitor of p38 MAPK. In control mice, sterile saline was administered instead. On days 7 and 21, mice were sacrificed to examine airway inflammation and mucus production by BALF cell counts, cytokine ELISA, and H&E and AB-PAS staining. The mRNA and protein levels of Muc5ac, p38 MAPK and MMP-9 in the lung were determined by RT-PCR, immunohistochemistry and Western blotting analysis. MMP-9 activity was measured by gelatin zymography. Results Both the numbers of inflammatory cells and mucus-secreting goblet cells were significantly increased in the airways of mice exposed to acrolein as compared to the control mice. Acrolein-increased phosphorylation of p38 MAPK was significantly reduced by SB203580. The airway inflammation and goblet cell hyperplasia after acrolein challenge were also attenuated by SB203580 administration. Moreover, SB203580 treatment decreased the acrolein-induced increase of Muc5ac and MMP-9 expression and MMP-9 activity in airway epithelium. Conclusions The results indicate an important role of p38 MAPK in acrolein-induced airway inflammation and mucus hypersecretion in mice. The cooperation of p38 and MMP-9 may contribute to the mucin overproduction after inflammatory challenge.

Glomerular Protein Levels of Matrix Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinase-1 Are Lower in Diabetic Subjects

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix turnover throughout the body, including in renal glomeruli. We investigated protein levels of multiple MMPs (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 in glomeruli and investigated whether disease phenotypes were associated with levels of these proteins. Renal cortex was collected from 100 adult autopsy subjects arrayed across 17 tissue microarrays. Immunohistochemical staining intensity for each MMP and TIMP-1 was determined using quantitative color deconvolution techniques. We observed significantly decreased glomerular MMP-1 and TIMP-1 staining in subjects with diabetes, hypertension, and an estimated glomerular filtration rate <30 ml/min/1.73 m(2) in univariate analyses. MMP-1 staining, but not TIMP-1 staining, was inversely correlated with increased glomerular fibrosis (r = -0.40). In multivariable analysis, diabetes was robustly associated with decreased staining intensity. This study indicates that in human subjects, the long-term sequelae of diseases such as diabetes that cause chronic renal failure result in decreased TIMP-1 and MMP-1 proteins in renal glomeruli. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.