Auraptene decreases the activity of matrix metalloproteinases in dextran sulfate sodium-induced ulcerative colitis in ICR mice

Previously we reported that auraptene was a potent suppressant for matrix metalloproteinase (MMP)-7 expression in HT-29 human colon cancer cells. In the present study, we examined the effects of auraptene on MMP-2, -7, and -9 expression in colonic mucosa from dextran sulfate sodium (DSS)-induced ulcerative colitis mice. Auraptene remarkably suppressed the DSS-induced gelatinolytic activity of MMP-7 as well as the expression of MMP-2 and -9, suggesting that it might be useful in anti-metastatic therapies via the targeting of MMPs.     

Citrus auraptene targets translation of MMP-7 (matrilysin) via ERK1/2-dependent and mTOR-independent mechanism

Matrix metalloproteinase (MMP)-7 is considered to play essential roles in cancer progression. We examined the efficacy of auraptene, a citrus coumarin derivative, for suppressing MMP-7 expression in the human colorectal adenocarcinoma cell line HT-29. Auraptene remarkably inhibited the production of proMMP-7 protein, without affecting its mRNA expression level. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), showed similar results, suggesting that auraptene suppresses mTOR-dependent proMMP-7 translation. Interestingly, however, auraptene showed no effects on the activation of Akt/mTOR signaling, whereas the phosphorylation levels of 4E binding protein (4EBP)1 and eukaryotic translation initiation factor (eIF)4B were substantially decreased. In addition, auraptene remarkably dephosphorylated constitutively activated extracellular signal-regulated kinase (ERK)1/2. Transfection of ERK1/2 siRNA led to a significant reduction of proMMP-7 protein production as well as of the phosphorylation of eIF4B. These results demonstrate that auraptene targets the translation step for proMMP-7 protein synthesis by disrupting ERK1/2-mediated phosphorylation of 4EBP1 and eIF4B.     

Silibinin prevents TPA-induced MMP-9 expression and VEGF secretion by inactivation of the Raf/MEK/ERK pathway in MCF-7 human breast cancer cells

Matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) expression are pivotal steps in cancer metastasis. Herein, we investigated the effect of silibinin, a major constituent (flavanolignan) of the fruits of Silybum marianum, on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 and VEGF expression in MCF-7 human breast cancer cells. The expression of MMP-9 and VEGF in response to TPA was increased, whereas TPA-induced MMP-9 and VEGF expression was decreased by silibinin. To investigate the regulatory mechanism of silibinin on TPA-induced MMP-9 and VEGF expression, we pretreated cells with various inhibitors, such as UO126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 inhibitor). Interestingly, TPA-induced MMP-9 expression was significantly inhibited by UO126, but not by SP600125 and SB203580. In addition, we pretreated cells with 100 μM silibinin prior to TPA treatment. TPA-induced MEK and ERK phosphorylation was significantly decreased by silibinin in MCF7 cells. TPA-induced VEGF expression was also suppressed by UO126. On the other hand, we found that adenoviral constitutive active-MEK (Ad-CA-MEK) significantly increased MMP-9 and VEGF expression. Taken together, we suggest that the inhibition of TPA-induced MMP-9 and VEGF expression by silibinin is mediated by the suppression of the Raf/MEK/ERK pathway in MCF-7 breast cancer cells.     

骨关节炎患者血清中基质金属蛋白酶-2、-9的明胶酶谱分析

为探讨基质金属蛋白酶-2、-9在骨关节炎发病中的作用,应用明胶酶谱分析方法研究其在骨关节炎患者血清中的表达水平。实验对象为27例膝骨关节炎患者,对照组为7例外伤骨折患者。结果发现病例组血清中基质金属蛋白酶-2和-9的表达水平均明显高于对照组(P<0.05)。该结果显示基质金属蛋白酶-2和-9可能在骨关节炎的发病过程中均起着重要的作用。     

Metalloproteinases 2 and -9 activity during promotion and progression stages of rat liver carcinogenesis

Activity of metalloproteinases 2 and 9 (MMP-2 and 9) during promotion and progression of rat liver carcinogenesis was investigated in a modified resistant hepatocyte model. Development of preneoplastic liver lesions positive for glutathione S-transferase 7-7-(GST-P 7-7-positive PNL) and tumors besides hepatocytes positive for proliferating cell nuclear antigen (PCNA) were quantified and compared to MMP-2 and-9 activity using gelatin zymography. Marked increases in GST-P 7-7-positive PNL development, PCNA labeling indices, MMP-2 (pro, intermediate and active forms) and pro-MMP-9 activity were observed after proliferative stimulus induced by 2-acetylaminofluorene (2-AAF) exposure cycles. After 2-AAF withdrawal, increase in MMP-2 activity was detected only in neoplastic mixed lesions, whereas active MMP-9 was increased in both PLN and neoplastic tissues. Our findings suggest that MMP-2 may be associated with proliferative events induced by 2-AAF rather than with selective growth of PNL and that MMP-9 could be associated with progression of PNL and neoplastic mixed lesions.     

Haishengsu, a Protein from Shellfish Tegillarca L. granosa, Inhibits the Growth and the Activity of Matrix Metalloproteinases-2 and -9 in Human Lung Carcinoma

Haishengsu (HSS) is a seashell protein extracted from Tegillarca L. granosa, a type of Malaysian shellfish. Previous in vitro studies showed that HSS might possess biological anticancer activity. In this combined in vitro and in vivo study, we investigated the inhibitory effects of HSS on tumor growth, invasion, and metastasis using human lung carcinoma cell lines A549 and NCI-H292, both intensely positive for matrix metalloproteinases-2 (MMP-2) and MMP-9. HSS significantly inhibited the proliferation of A549 and NCI-H292 as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The transwell chamber assay showed that HSS effectively blocked the invasion and migration of the carcinoma cells through the reconstituted extracellular matrix (Matrigel). Gelatin zymography analysis revealed that the secretion and activity of MMP-2 and MMP-9 in the supernatants of the cultured cells A549 and NCI-H292 were decreased after treatment with HSS. The levels of MMP-2 and MMP-9 in these cancer cells were further examined by Western blot assay in which a significant decrease of MMP-2 and MMP-9 was observed in A549 and NCI-H292 cells after 24 h of exposure to HSS. The anticancer activity of HSS was verified in a mouse model in which HSS delayed the growth of A549 xenografts after 3 weeks of oral administration. Inhibition of MMP-2 and MMP-9 expression was also demonstrated in the A549 xenografts as determined by Western blot analysis. These results suggest that HSS is a novel seashell protein that cannot only inhibit tumor growth but also prevent tumor invasion and metastasis through suppressing the activity of MMP-2 and MMP-9.     

Ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions increase levels of nitric oxide and matrix metalloproteinase-9 without a direct association between them

Inflammatory and infectious conditions were simulated in cultures of ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells, using interferon-gamma (IFN-γ, 100 units/ml) and lipopolysaccharide (LPS, 0.5 μg/ml) co-treatment for 24 h, to investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA and the production of NO. Aminoguanidine (AG, 1 mM; an NOS inhibitor) along with IFN-γ and LPS, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 100 μM; an NO donor) and/or (±)-N-[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4, 100 μM; an NO donor), were also added to analyze the possible association of NO with matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Co-treatment of cells with IFN-γ and LPS increased iNOS mRNA expression, NO production, MMP-9 mRNA expression, and 105 kDa MMP-9 production. Additional treatment with the NOS inhibitor AG inhibited NO production, but did not down-regulate the expression of MMP-9 mRNA or 105 kDa MMP-9. The NO donors SNAP and NOR4 did not affect the expression of MMP-9 mRNA, 105 kDa MMP-9 or TIMP-1 mRNA. These results suggest that ras/myc SFME cells respond to infectious and inflammatory conditions and can enhance malignancy as cancer cells due to their increased levels of NO and MMP-9 production, but that NO is not directly associated with MMP-9 in these cells. H. Yamaguchi and Y. Kidachi contributed equally to this work     

Infection with human cytomegalovirus alters the MMP-9/TIMP-1 balance in human macrophages

Human cytomegalovirus (HCMV) has been suggested to contribute to the development of vascular diseases. Since matrix metalloproteinases (MMPs) have been implicated in atherosclerosis and plaque rupture, we investigated the effect of HCMV infection on MMP expression in human macrophages. We used quantitative real-time PCR, Western blotting, and gelatin zymography to study the expression and activity of MMP-2, -3, -7, -9, -12, -13, and -14 and of tissue inhibitor of metalloproteinase 1 (TIMP-1), -2, -3, and -4. HCMV infection reduced MMP-9 mRNA, protein, and activity levels but increased TIMP-1 mRNA and protein levels. Furthermore, a decrease in MMP-12, MMP-14, TIMP-2, and TIMP-3 mRNA levels could be detected. The MMP-9 and TIMP-1 mRNA alterations required viral replication. MMP-9 mRNA expression was affected by an immediate-early or early viral gene product, whereas TIMP-1 mRNA expression was affected by late viral gene products. We conclude that HCMV infection specifically alters the MMP-9/TIMP-1 balance in human macrophages, which in turn reduces MMP-9 activity in infected cells. Since MMP-9 prevents atherosclerotic plaque development in mice, these results suggest that HCMV may contribute to atherogenesis through specific effects on MMP-9 activity.     

Differential regulation of matrix metalloproteinase-2 and -9 expression and activity in adult rat cardiac fibroblasts in response to interleukin-1beta

Matrix metalloproteinases (MMPs), a family of endoproteinases, are implicated in cardiac remodeling. Interleukin-1beta (IL-1beta), which is increased in the heart following myocardial infarction, increases expression and activity of MMP-2 (gelatinase A) and -9 (gelatinase B) in cardiac fibroblasts. Previously, we have shown that IL-1beta activates ERK1/2, JNKs, and protein kinase C (PKC). However, signaling pathways involved in the regulation of MMP-2 and -9 expression and activity are not yet well understood. Using adult rat cardiac fibroblasts, we show that inhibition of ERK1/2 and JNKs inhibits IL-1beta-stimulated increases in MMP-9, not MMP-2, expression and activity. Chelerythrine, an inhibitor of PKC, inhibited activation of ERK1/2 and JNKs and expression and activity of both MMPs. Selective inhibition of PKC-alpha/beta1 using G?6976 inhibited JNKs activation and the expression and activity of MMP-9, not MMP-2. Inhibition of PKC-theta and PKC-zeta using pseudosubstrates inhibited IL-1beta-stimulated activation of ERK1/2 and JNKs and the expression and activity of MMP-2 and -9. Inhibition of PKC-epsilon had no effect. IL-1beta activated NF-kappaB pathway as measured by increased phosphorylation of IKKalpha/beta and IkappaB-alpha. Inhibition of ERK1/2, JNKs, and PKC-alpha/beta1 had no effect on NF-kappaB activation, whereas inhibition of PKC-theta and PKC-zeta inhibited IL-1beta-stimulated activation of NF-kappaB. SN50, NF-kappaB inhibitor peptide, inhibited IL-1beta-stimulated increases in MMP-2 and -9 expression and activity. These observations suggest that 1) activation of ERK1/2 and JNKs plays a critical role in the regulation of MMP-9, not MMP-2, expression and activity; 2) PKC-alpha/beta1 act upstream of JNKs, not ERK1/2; 3) PKC-zeta and -theta, not PKC-epsilon, act upstream of JNKs, ERK1/2, and NF-kappaB; and 4) activation of NF-kappaB stimulates expression and activity of MMP-2 and -9.     

Mixed lineage kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells.     

Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes

Citrus fruit compounds have many health-enhancing effects. In this study, using a luciferase ligand assay system, we showed that citrus auraptene activates peroxisome proliferator-activated receptor (PPAR)-α and PPARγ. Auraptene induced up-regulation of adiponectin expression and increased the ratio of the amount of high-molecular-weight multimers of adiponectin to the total adiponectin. In contrast, auraptene suppressed monocyte chemoattractant protein (MCP)-1 expression in 3T3-L1 adipocytes. Experiments using PPARγ antagonist demonstrated that these effects on regulation of adiponectin and MCP-1 expression were caused by PPARγ activations. The results indicate that auraptene activates PPARγ in adipocytes to control adipocytekines such as adiponectin and MCP-1 and suggest that the consumption of citrus fruits, which contain auraptene can lead to a partial prevention of lipid and glucose metabolism abnormalities.     

Expression of matrix metalloproteinase -2, -9 and tissue inhibitors of metalloproteinase -1, -2, -3 mRNAs in rat uterus during early pregnancy

Zymography and in situ hybridizition were used to investigate matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and expression of mRNAs for MMP-2, -9 and tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3) in the rat uterus during early pregnancy (day 1-7). The zymography results showed two forms of MMP-2 (64 and 67 kDa) in the rat uteri during early pregnancy. The 64-kDa MMP-2 activity was the highest on day 2 (P < 0.01) and higher on day 5 and 6 (P < 0.05). The 67-kDa MMP-2 activity reached the highest on day 5 and 6 (P < 0.01). The 64-kDa MMP-2 activity at the implantation sites was higher than those at interimplantation sites (P < 0.05). Furthermore, the 67 kDa MMP-2 can be converted to 64 kDa forms by incubation with p-aminophenylmercuric acetate (APMA) and trypsin in vitro. The 92-kDa MMP-9 activity was only detected on day 5 and 6 of pregnancy (P < 0.01). In situ hybridization showed that on day 1-4 of pregnancy, both MMP-2 and TIMP-2 mRNAs were evidently localized in the basal stromal cells. On day 5, MMP-2 mRNA signals were decreased in the basal stromal cells and mRNA for TIMP-2 was expressed in the epithelial cells and subepithelial stromal cells. The mRNAs for MMP-9, TIMP-1, and -3 were mainly expressed in epithelial cells on day 1-5. At the implantation site on day 6, the mRNAs for MMP-2, -9, TIMP-1, -2, and -3 were highly expressed in the primary decidual zone surrounding the implanting embryo, and in the whole decidualized stromal cells (the primary and secondary decidual zones) at the implantation site on day 7. The intensities of mRNAs for the TIMPs in decidualized stromal cells at the implantation site on day 6 and 7 were stronger than those for the MMPs. The weak mRNAs for MMP-2, -9, TIMP-1, and -3 but not TIMP-2 were also observed in the ectoplacental cone/trophoblastic cells of the implanting embryos. However, at the interimplantation sites on day 6 and 7, MMP-2, -9, TIMP-1, -2, and -3 mRNAs were weakly expressed in the epithelial cells, subepithelial stromal cells, and myometrium. The results suggested that the implanting rat embryo strongly induced MMP-2 and -9 proteins and gene expression for decidulization and embryo invasion, which were strictly controlled and balanced by the simultaneous expression of TIMP-1, -2 and -3.     

Quercetin downregulates matrix metalloproteinases 2 and 9 proteins expression in prostate cancer cells (PC-3)

Background: Cancer metastasis, involving multiple processes and various cytophysiological changes, is a primary cause of cancer death and may complicate the clinical management, even lead to death. Quercetin is a flavonoid and widely used as an antioxidant and recent studies have revealed its pleiotropic anticancer and antiproliferative capabilities. Gelatinases A and B (matrixmetalloproteinases 2 and 9) are enzymes known to involve in tumor invasion and metastases. In this study, we observed the precise involvement of quercetin role on these proteinases expression and activity. Design and methods: PC-3 cells were treated with quercetin at various concentrations (50 and 100 μM), for 24 h period and then subjected to western blot analysis to investigate the impact of quercetin on matrix metalloproteinase-2 (MMP-2) and 9 (MMP-9) expressions. Conditioned medium and cell lysate of quercetin-treated PC-3 cells were subjected to western blot analysis for proteins expression of MMP-2 and MMP-9. Gelatin zymography was also performed in quercetin treated PC-3 cells. Results: The results showed that quercetin treatment decreased the expressions of MMP-2 and MMP-9 in dose-dependent manner. The level of pro-MMP-9 was found to be high in the 100 μM quercetin-treated cell lysate of PC-3 cells, suggesting inhibitory role of quercetin on pro-MMP-9 activation. Gelatin zymography study also showed the decreased activities of MMP-2 and MMP-9 in quercetin treated cells. Conclusion: Hence, we speculated that inhibition of metastasis-specific MMPs in cancer cells may be one of the targets for anticancer function of quercetin, and thus provides the molecular basis for the development of quercetin as a novel chemopreventive agent for metastatic prostate cancer.     

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (A review of own results)

Own results of long-term studies of expression of matrix metalloproteinases (MMPs) and their endogenous regulators examined in fibroblasts transformed by oncogene E7 HPV16 (TF), immortalized fibroblasts (IF), cell lines associated with HPV16 and HPV18, and tumor tissue samples from patients with squamous cervical carcinoma (SCC) associated with HPV16 have been summarized. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. MMP expression correlated with the tumorigenic of transformed clones. Expression of MMP-9 was found only in TF. In TF expression mRNA TIMP-1 decreased, while expression of the genatinase inhibitor, TIMP-2, increased. Collagenase activity and expression of the MMP-14 (collagenase) mRNA increased, while gelatinase activity remained unchanged. The destructive potential of TF is associated with induction of collagenases, gelatinase MMP-9 and decreased levels of MMP inhibitors. MMP-9 may serve as a TF marker. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-2 activity significantly increased in Caski and HeLa cell lines, while MMP-9 expression in these cell lines was not detected. The comparative study of expression MMP of and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously associated with increased expression of collagenases MMP-1, MMP-14 and gelatinase MMP-9, as well as decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent with increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. The morphologically normal tissue adjacent to the tumor tissue is characterized by significant expression of MMP-1, MMP-2, and MMP-9. This also contributes to the increased destructive potential of the tumor.     

Matrix metalloproteinase-2 and -9 expression increases in Mycoplasma-infected airways but is not required for microvascular remodeling

Murine Mycoplasma pulmonis infection induces chronic lung and airway inflammation accompanied by profound and persistent microvascular remodeling in tracheobronchial mucosa. Because matrix metalloproteinase (MMP)-2 and -9 are important for angiogenesis associated with placental and long bone development and skin cancer, we hypothesized that they contribute to microvascular remodeling in airways infected with M. pulmonis. To test this hypothesis, we compared microvascular changes in airways after M. pulmonis infection of wild-type FVB/N mice with those of MMP-9(-/-) and MMP-2(-/-)/MMP-9(-/-) double-null mice and mice treated with the broad-spectrum MMP inhibitor AG3340 (Prinomastat). Using zymography and immunohistochemistry, we find that MMP-2 and MMP-9 rise strikingly in lungs and airways of infected wild-type FVB/N and C57BL/6 mice, with no zymographic activity or immunoreactivity in MMP-2(-/-)/MMP-9(-/-) animals. However, microvascular remodeling as assessed by Lycopersicon esculentum lectin staining of whole-mounted tracheae is as severe in infected MMP-9(-/-), MMP-2(-/-)/MMP-9(-/-) and AG3340-treated mice as in wild-type mice. Furthermore, all groups of infected mice develop similar inflammatory infiltrates and exhibit similar overall disease severity as indicated by decrease in body weight and increase in lung weight. Uninfected wild-type tracheae show negligible MMP-2 immunoreactivity, with scant MMP-9 immunoreactivity in and around growing cartilage. By contrast, MMP-2 appears in epithelial cells of infected, wild-type tracheae, and MMP-9 localizes to a large population of infiltrating leukocytes. We conclude that despite major increases in expression, MMP-2 and MMP-9 are not essential for microvascular remodeling in M. pulmonis-induced chronic airway inflammation.     

Monocyte-astrocyte networks regulate matrix metalloproteinase gene expression and secretion in central nervous system tuberculosis in vitro and in vivo

CNS tuberculosis (CNS-TB) is the most deadly form of tuberculous disease accounting for 10% of clinical cases. CNS-TB is characterized by extensive tissue destruction, in which matrix metalloproteinases (MMPs) may play a critical role. We investigated the hypothesis that Mycobacterium tuberculosis activates monocyte-astrocyte networks increasing the activity of key MMPs. We examined the expression of all human MMPs and the tissue inhibitors of metalloproteinases (TIMPs) in human astrocytes stimulated by conditioned medium from M. tuberculosis-infected monocytes (CoMTB). Real-time RT-PCR showed that gene expression of MMP-1, -2, -3, -7, and -9 was increased (p < 0.05). MMP-9 secretion was significantly up-regulated at 24 h and increased over 120 h (p < 0.01). MMP-1, -3, and -7 secretion was not detected. Secretion of MMP-2 was constitutive and unaffected by CoMTB. Astrocyte gene expression and secretion of TIMP-1 was not affected by CoMTB although TIMP-2 secretion increased 3-fold at 120 h. Immunohistochemical analysis of human brain biopsies confirmed that astrocyte MMP-9 secretion is a predominant feature in CNS-TB in vivo. Dexamethasone inhibited astrocyte MMP-9, but not TIMP-1/2 secretion in response to CoMTB. CoMTB stimulated the nuclear translocation of NF-kappaB, inducing a 6-fold increase in nuclear p65 and a 2-fold increase in nuclear p50. This was associated with degradation of IkappaBalpha and beta within 30 min, persisting for 24 h. In summary, networks active between monocytes and astrocytes regulate MMP-9 activity in tuberculosis and astrocytes are a major source of MMP-9 in CNS-TB. Astrocytes may contribute to a matrix degrading environment within the CNS and subsequent morbidity and mortality.