Suppression Of Matrix Metalloproteinase-9 Research Articles

Effect of eucalyptol on matrix metalloproteinase-9 and its tissue inhibitor in hypertensive rats.

Effects of eucalyptol, a key component of eucalyptus globules, on matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor (TIMP-1), compared with lisinopril, were investigated in a model of hypertension induced by chronic intraperitoneal (IP) injection of low dose nicotine in rats. The hypertensive rats were randomly allocated to 4 groups (n=8): Positive control (PC, untreated), eucalyptol-treated group (1.0 mg/kg, IP), lisinopril-treated group (10 mg/kg, IP), and eucalyptol+lisinopril-treated group. Systolic blood pressure and plasma levels of MMP-9 and TIMP-1 were measured. All treatments decreased the elevated blood pressure and plasma levels of MMP-9 and TIMP-1 most significantly with the combination group which showed non-significant differences from the normal control group. Lisinopril reduced plasma levels of MMP-9 and TIMP-1 more significantly than eucalyptol. In conclusion, eucalyptol significantly decreased the increased plasma levels of MMP-9 and TIMP-1 in nicotine-induced hypertension in rats. Moreover, its combination with lisinopril exerted more significant effects compared to each drug alone. This makes this combination particularly useful in hypertension and related cardiovascular disorders where suppression of MMP-9 and TIMP-1 activities decreases the related complications and improves the overall morbidity and mortality. To our knowledge, the current data are novel, and may open the way for development of a co-delivery system of both drugs which could be beneficial in treatment of hypertension in chronic smokers.

PPARγ activation suppresses the expression of MMP9 by downregulating NF-κB post intracerebral hemorrhage

Peroxisome proliferator-activated receptor-gamma (PPARγ) is critical in protecting against inflammatory and oxidative stresses post brain injury. We have previously reported that rosiglitazone, an agonist of PPARγ, was effective to prevent microglia from apoptosis and ameliorate neuronal injuries post intracerebral hemorrhage (ICH) with suppression of matrix metalloproteinase-9 (MMP9) expression. However, molecular mechanisms linking how PPARγ decreases MMP9 remain unknown. Here, we hypothesize that PPARγ downregulates MMP9 expression post hemorrhage by inhibiting nuclear factor kappa B (NF-κB), an upstream regulator of MMPs gene and also key transcription factor involved in the control of immune and neuroinflammatory responses. We found both in vivo and in vitro that PPARγ was significantly downregulated post ICH with prominent increases of NF-κB and MMP9. Activation of PPARγ using rosiglitazone decreased the expression of both NF-κB and MMP9, while reversed effects were observed when administrating the PPARγ antagonist GW9662. Besides, inhibiting NF-κB by JSH-23 also suppressed the expression of MMP9, with only limited effect on PPARγ. Further studies revealed prominent colocalizations of NF-κB with PPARγ and MMP9, respectively. Finally, direct interactions of NF-κB with PPARγ and MMP9 gene were also confirmed, respectively, by protein and chromatin immunoprecipitations. These results suggested a role of NF-κB in mediating the reduction of MMP9 by PPARγ, potentially providing a new therapeutic target for brain hemorrhage.

High Estrogen Level Modifies Postoperative Hyperalgesia via GPR30 and MMP-9 in Dorsal Root Ganglia Neurons.

The cycling of sex hormones is one of the factors affecting pain in females, and the mechanisms are not fully understood. G-protein coupled estrogen receptor 30 (GPR30) is the estrogen receptor known to be involved in mechanical hyperalgesia. Studies have demonstrated that matrix metalloproteinase-9 (MMP-9) is a critical component in peripheral/central nervous system hypersensitivity and neuroinflammation, both of which participate in hyperalgesia. Here, ovariectomized rats were treated with low or high dose estrogen replacement, and then plantar incisions were made. Subsequently, mechanical allodynia was evaluated by determining the paw withdrawal mechanical threshold before and after the incision. In rats with incisions, high estrogen levels induced postoperative hyperalgesia and upregulation of GPR30 and MMP-9 in dorsal root ganglia (DRGs). MMP-9 was expressed primarily in DRG neurons co-expressing GPR30, and led to the activation of IL-1β. After intrathecal injection of the GPR30 agonist G1, female rats with low estrogen and plantar incisions continued to exhibit significant hyperalgesia until 48h post-incision. In high estrogen level rats with plantar incisions, intrathecal injection of GPR30 antagonist G15 significantly attenuated postoperative hyperalgesia. Intraperitoneal injection of N-acetyl-cysteine, a source of cysteine that prevents the oxidation of cysteine residues on MMP-9, significantly relieved high estrogen-induced postoperative hyperalgesia via suppression of MMP-9 and IL-1β activation in DRGs. These results demonstrate that high estrogen level in rats with incisions elicit GPR30 and MMP-9 upregulation in DRGs and subsequently activate IL-1β, leading to induced postoperative hyperalgesia.

Fentanyl inhibits the progression of gastric cancer through the suppression of MMP-9 via the PI3K/Akt signaling pathway.

Fentanyl is a drug commonly used for perioperative and postoperative analgesia. Previous studies have confirmed that fentanyl can affect the progression of gastric cancer; however, this effect has not yet been elucidated. The purpose of our study was thus to investigate the role of fentanyl in gastric cancer and clarify its potential mechanisms. A CCK-8 assay was used to determine the proliferation of MGC-803 cells, while Transwell assay and wound healing assay were used to determine the invasion and migration abilities, respectively. Apoptosis and the cell cycle were assessed by flow cytometry, and the ultrastructure of the cells was examined with a transmission electron microscope. The mRNA expression levels of serine-threonine protein kinase 1 (Akt-1), matrix metalloproteinase-9 (MMP-9), and death-associated protein kinase 1 (DAPK1) were evaluated by real-time (RT) quantitative PCR. The protein expression of p-Akt, MMP-9, and caspase-9 was detected by western blot analysis. To study the interaction of fentanyl with the phosphatidylinositol-3-kinase (PI3K)/Akt/MMP-9 pathway, PI3K inhibitor (LY294002) and MMP-9 inhibitor (SB-3CT) were used to treat the MGC-803 cells. Findings indicated that fentanyl inhibits the proliferation, invasion, and migration of MGC-803 cells. Specifically, fentanyl inhibits the expression of MMP-9 and enhances the expression of apoptosis-promoting factors such as caspase-9 and DAPK1 through the PI3K/Akt signaling pathway. Cell cycle arrest was observed in the G0/G1 phase. Furthermore, the inhibition of PI3K/Akt/MMP-9 by LY294002 and SB-3CT enhanced the anticancer effects of fentanyl. Fentanyl inhibits the proliferation, invasion and migration of gastric cancer cells by inhibiting the PI3K/Akt/MMP-9 pathway, which could be very useful for gastric cancer treatment.

ARHGAP6 regulates the proliferation, migration and invasion of lung cancer cells.

Lung cancer, a leading cause of cancer‑related deaths, is frequently diagnosed in both males and females worldwide. In the present study, the Ras homologue GTPase activation protein 6 (ARHGAP6), which belongs to the Rho GTPase‑activating protein (RhoGAP) family, was found to have low expression in tumor tissues from patients with lung cancer, accompanied by high expression of matrix metalloproteinase‑9 (MMP9) and vascular endothelial growth factor (VEGF). In A549 and H1299 cells, upregulation of ARHGAP6 inhibited tumor growth and metastasis and reduced the levels of MMP9, VEGF and p‑STAT3, while the levels STAT3 were unchanged, as demonstrated by CCK‑8, migration and invasion assays as well as western blot analysis. In addition, interleukin 6 (IL‑6)‑induced migration, invasion and MMP9 and VEGF expression, and STAT3 signaling activity were suppressed by ARHGAP6 upregulation. Based on these data, we concluded that ARHGAP6 is critically important in lung cancer progression and that upregulation of ARHGAP6 benefits the treatment and prevention of lung cancer, possibly through the suppression of MMP9, VEGF and STAT3 signaling activation.

Morin inhibits PDGF-induced proliferation, migration, and invasion of vascular smooth muscle cells via modulating p27KIP1, AKT, and MMP-9 activities.

Hyper-proliferation and migration of vascular smooth muscle cells (VSMCs) are closely associated with atherosclerosis. Recently, the flavonol morin has been reported to exhibit potent anti-oxidant and anti-inflammatory activities. Therefore, we investigated molecular mechanisms of morin in VSMCs stimulated by PDGF. Morin effectively inhibited PDGF-stimulated proliferation of VSMCs through a G1 cell-cycle arrest, leading to down-regulation of CDK2, CDK4, cyclin D1, and cyclin E proteins. Interestingly, PDGF markedly down-regulated p27KIP1 protein expression; however, morin treatment restored the p27KIP1expression to the basal level. Morin did not affect phosphorylation of MAPKs (ERK, p38, and JNK); however, phosphorylation of AKT was dramatically suppressed by morin in PDGF-stimulated VSMCs. Using the PI3K inhibitor, LY294002, we revealed that AKT is a key regulator in the inhibitory mechanism of morin against PDGF-induced proliferation of VSMCs. Morin disturbed migratory and invasive potential of VSMCs via suppression of matrix metalloproteinase-9 (MMP-9) activity. Using electrophoretic mobility shift assays, we verified that NF-κB, AP-1, and Sp-1 transcription factors are implicated in the mode of action of morin, which suppresses the MMP-9 activity in PDGF-induced VSMCs. Based on the results, we believe that morin may be a potential therapeutic agent for atherosclerosis without negative side effect.

Mild hypothermia alleviates brain oedema and blood-brain barrier disruption by attenuating tight junction and adherens junction breakdown in a swine model of cardiopulmonary resuscitation.

Mild hypothermia improves survival and neurological recovery after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). However, the mechanism underlying this phenomenon is not fully elucidated. The aim of this study was to determine whether mild hypothermia alleviates early blood–brain barrier (BBB) disruption. We investigated the effects of mild hypothermia on neurologic outcome, survival rate, brain water content, BBB permeability and changes in tight junctions (TJs) and adherens junctions (AJs) after CA and CPR. Pigs were subjected to 8 min of untreated ventricular fibrillation followed by CPR. Mild hypothermia (33°C) was intravascularly induced and maintained at this temperature for 12 h, followed by active rewarming. Mild hypothermia significantly reduced cortical water content, decreased BBB permeability and attenuated TJ ultrastructural and basement membrane breakdown in brain cortical microvessels. Mild hypothermia also attenuated the CPR-induced decreases in TJ (occludin, claudin-5, ZO-1) and AJ (VE-cadherin) protein and mRNA expression. Furthermore, mild hypothermia decreased the CA- and CPR-induced increases in matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) expression and increased angiogenin-1 (Ang-1) expression. Our findings suggest that mild hypothermia attenuates the CA- and resuscitation-induced early brain oedema and BBB disruption, and this improvement might be at least partially associated with attenuation of the breakdown of TJ and AJ, suppression of MMP-9 and VEGF expression, and upregulation of Ang-1 expression.

Suppression of MMP-9 and FAK expression by pomolic acid via blocking of NF-κB/ERK/mTOR signaling pathways in growth factor-stimulated human breast cancer cells.

The expression of matrix metalloproteinase-9 (MMP-9) and the phosphorylation of focal adhesion kinase (FAK) have been implicated in the invasion, metastasis and cell motility of cancer cells. It is considered that epidermal growth factor (EGF) may increase cell motility, an event involved in cancer cell invasion and metastasis. Pomolic acid(PA), an active triterpenoid from Euscaphis japonica, is known to inhibit the proliferation of a variety of cancer cells, but the effect of PA on the invasiveness of cancer cells is largely unknown. In this study, we first determined the molecular mechanism by which PA inhibits the migratory and invasive abilities of highly metastatic MDA-MB‑231 cells. Transwell invasion, wound-healing assay and F-actin reorganization showed that PA significantly inhibits the EGF-induced invasion, migration and cell motility by reducing expression of MMP-9 and FAK phosphorylation. In particular, PA potently suppressed the phosphorylation of nuclear factor (NF)-κB, extraceullar signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. Furthermore, PA treatment inhibited the DNA binding activity of NF-κB and activator protein (AP)-1, which is known to mediate the expression of EGFR and MMP-9. These results suggest that PA may be a potential therapeutic candidate for treatment of breast cancer metastasis.

Paeoniflorin inhibits human pancreatic cancer cell apoptosis via suppression of MMP-9 and ERK signaling.

Paeoniflorin exhibits anticancer, anti-inflammatory and antioxidation effects, as well as specific pharmacological effects on smooth muscle and the immune, cardiovascular and central nervous systems. The present study aimed to investigate the anticancer effects of paeoniflorin on pancreatic cancer cells and to elucidate the mechanisms by which these effects occur. In the present study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were performed to assess cell viability and cell cytotoxicity of BXPC-3 human pancreatic cancer cells, respectively. Cellular apoptosis and caspase-3/9 activities were analyzed using an Annexin V-fluorescein isothiocyanate/propidium iodide Apoptosis Detection kit, a DAPI staining assay and colorimetric kits, respectively. Matrix metalloproteinase-9 (MMP-9) and extracellular signal-regulated kinases (ERK) protein expression in BXPC-3 cells were also investigated using gelatin zymography assays and western blot analysis, respectively. In the present study, paeoniflorin was found to inhibit the cell viability and increase cell cytotoxicity of BXPC-3 cells in a dose- and time-dependent manner. In addition, cellular apoptosis, as well as caspase-3 and -9 activity of BXPC-3 cells was increased following paeoniflorin treatment. Notably, paeoniflorin reduced MMP-9 and ERK protein expression in BXPC-3 cells. These results indicate that paeoniflorin exhibits a potential anticancer effect by enhancing human pancreatic cancer cell apoptosis via the suppression of MMP-9 and ERK signaling.

Anti-angiogenic properties of coenzyme Q0 through downregulation of MMP-9/NF-κB and upregulation of HO-1 signaling in TNF-α-activated human endothelial cells

Various coenzyme Q (CoQ) analogs have been reported as anti-inflammatory and antioxidant substances. However, coenzyme Q0 (CoQ0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone), a novel quinone derivative, has not been well studied for its pharmacological efficacies, and its response to cytokine stimulation remains unclear. Therefore, we investigated the potential anti-angiogenic properties of CoQ0 in human endothelial (EA.hy 926) cells against tumor necrosis factor-α (TNF-α) stimulation. We found that the non-cytotoxic concentrations of CoQ0 (2.5–10μM) significantly suppressed the TNF-α-induced migration/invasion and tube formation abilities of endothelial cells. CoQ0 suppressed TNF-α-induced activity and protein expressions of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1) followed by an abridged adhesion of U937 leukocytes to endothelial cells. CoQ0 treatment remarkably downregulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) possibly through suppressed I-κBα degradation. Furthermore, CoQ0 triggered the expressions of heme oxygenase-1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCLC), followed by an increased nuclear accumulation of NF-E2 related factor-2 (Nrf2)/antioxidant response element (ARE) activity. In agreement with these, intracellular glutathione levels were significantly increased in CoQ0 treated cells. More interestingly, knockdown of HO-1 gene by specific shRNA showed diminished anti-angiogenic effects of CoQ0 against TNF-α-induced invasion, tube formation and adhesion of leukocyte to endothelial cells. Our findings reveal that CoQ0 protective effects against cytokine-stimulation are mediated through the suppression of MMP-9/NF-κB and/or activation of HO-1 signaling cascades. This novel finding emphasizes the pharmacological efficacies of CoQ0 to treat inflammation and angiogenesis.

Camptothecin suppresses expression of matrix metalloproteinase-9 and vascular endothelial growth factor in DU145 cells through PI3K/Akt-mediated inhibition of NF-κB activity and Nrf2-dependent induction of HO-1 expression

Though camptothecin (CPT) possesses potent anti-inflammatory, immunomodulatory, anticancerous, and antiproliferative effects, little is known about the mechanism by which CPT regulates the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). Therefore, the current study aimed to investigate the effects of CPT on the expression of MMP-9 and VEGF, which are important factors for the invasion of tumors. In vitro application of CPT resulted in a slight inhibition of cell proliferation and a significant reduction in the matrigel invasion of DU145 cells. Treatment with CPT also downregulated phorbol-12-myristate-13-acetate (PMA)- and tumor necrosis factor-α (TNF-α)-induced MMP-9 and VEGF expression by inhibiting nuclear factor-κB (NF-κB) activity. Downregulation of phosphoinositide 3-kinase (PI3K)/Akt phosphorylation in response to CPT was revealed as an upstream pathway regulating the expression of MMP-9 and VEGF accompanying the inhibition of NF-κB activity. We further confirmed that CPT inhibits PMA-induced MMP-9 and VEGF expression by upregulating nuclear factor-erythroid related factor-2 (Nrf2)-mediated heme oxygenase-1 (HO-1) induction. Taken together, these data indicate that CPT inhibits the invasion of cancer cells accompanied by suppression of MMP-9 and VEGF production by suppressing the PI3K/Akt-mediated NF-κB pathway and enhancing the Nrf2-dependent HO-1 pathway, suggesting that CPT may be a good candidate to inhibit MMP-9 and VEGF expression.

Bone morphogenetic protein 4 (BMP-4) and epidermal growth factor (EGF) inhibit metalloproteinase-9 (MMP-9) expression in cancer cells.

Matrix metalloproteinase-9 (MMP-9) plays a central role in the progression of the cancer. While a large number of studies have contributed to our understanding of the molecular mechanisms responsible for upregulating MMP-9 gene expression in normal and cancer cells, our knowledge on the signals that suppress MMP-9 expression is much more limited. Here, we report that EGF and BMP-4 cooperate to inhibit MMP-9 expression in cancer cells. Treatment with EGF reduces the expression of MMP-9 at both mRNA while augmenting BMP-4 expression. Interestingly, recombinant BMP-4 suppressed constitutive and PMA-induced MMP-9 expression in both fibrosarcoma and breast cancer cells. Addition of gremlin a natural inhibitor of BMP-4, inhibited the suppression of MMP-9 by EGF. The suppression of MMP-9 by BMP-4 likely occurs at the transcriptional level since BMP-4 suppressed MMP-9 mRNA expression and activation of a reporter vector encoding the human MMP-9 promoter. The suppressive effect of BMP-4 occurs via Smad1/5/8 and is specific since BMP-4 did not inhibit MMP-2 while BMP-2 was ineffective in suppressing MMP-9. Taken together, these results are consistent with a new paradigm for the role of EGF and BMPs in controlling MMP gene expression in cancer cells.

Hydrogen sulfide inhalation decreases early blood-brain barrier permeability and brain edema induced by cardiac arrest and resuscitation.

The effects of hydrogen sulfide (H2S) on blood-brain barrier (BBB) and brain edema after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) remain poorly understood. We investigated the effects of exogenous 80-p.p.m. H2S gas on BBB, brain water content, neurologic outcome, and survival rate after CA and CPR. Cardiopulmonary resuscitation followed CA induced in rats by ventricular fibrillation for 6 minutes. Results show that inhalation of 80-p.p.m. H2S significantly reduced the permeability of the BBB in both in the cortex and hippocampus at 24 hours after resuscitation. Hydrogen sulfide also lessened brain edema in the cortex and hippocampus, ameliorated neurologic outcome as evaluated by neurologic deficit score and tape removal test, and improved the 14-day survival rate. Hydrogen sulfide also attenuated CA and CPR-induced increases of matrix metalloproteinase-9 (MMP-9) activity and vascular endothelial growth factor (VEGF) expression, and increased the expression of angiogenin-1 (Ang-1). These results indicate that inhalation of 80-p.p.m. H2S immediately after CPR attenuated BBB permeability and brain edema, and improved neurologic outcome and 14-day survival of rats after CA. The therapeutic benefits of H2S could be associated with suppression of MMP-9 and VEGF expression and increased expression of Ang-1.

Essential Role of Program Death 1-Ligand 1 in Regulatory T-Cell–Afforded Protection Against Blood–Brain Barrier Damage After Stroke

Our recent research revealed that adoptively transferred regulatory T cells (Tregs) reduced acute ischemic brain injury by inhibiting neutrophil-derived matrix metalloproteinase-9 (MMP-9) and protecting against blood-brain barrier damage. The mechanisms underlying Treg interactions with neutrophils remain elusive. This study evaluates the contribution of program death 1-ligand 1 (PD-L1) to Treg-mediated neutrophil inhibition and neuroprotection after cerebral ischemia. In vitro experiments were performed using a transwell system or a coculture system allowing cell-to-cell contact. Focal cerebral ischemia was induced in mice for 60 minutes. Tregs (2×10(6)) isolated from donor animals (wild-type or PD-L1-/-) were intravenously injected into ischemic recipients 2 hours after middle cerebral artery occlusion (MCAO). MMP-9 production, blood-brain barrier permeability, and brain infarct were assessed at 1 or 3 days after MCAO. In vitro experiments reveal that Treg-mediated inhibition of neutrophil MMP-9 required direct cell-to-cell contact. The suppression of MMP-9 was abolished when Tregs were pretreated with PD-L1 neutralizing antibodies or when neutrophils were pretreated with PD-1 antibodies. In vivo studies confirmed that intravenous administration of Tregs pretreated with PD-L1 antibodies or Tregs isolated from PD-L1-deficient mice failed to inhibit MMP-9 production by blood neutrophils 1 day after 60 minutes MCAO. Furthermore, the blood-brain barrier damage after MCAO was greatly ameliorated in PD-L1-competent Treg-treated mice but not in PD-L1-compromised Treg-treated mice. Consequently, PD-L1 dysfunction abolished Treg-mediated brain protection and neurological improvements 3 days after MCAO. PD-L1 plays an essential role in the neuroprotection afforded by Tregs against cerebral ischemia by mediating the suppressive effect of Tregs on neutrophil-derived MMP-9.

Epigenetic regulation of mmp-9 gene expression.

Matrix metalloproteinase 9 (MMP-9) is one of the most studied enzymes in cancer. MMP-9 can cleave proteins of the extracellular matrix and a large number of receptors and growth factors. Accordingly, its expression must be tightly regulated to avoid excessive enzymatic activity, which is associated with disease progression. Although we know that epigenetic mechanisms play a central role in controlling mmp-9 gene expression, predicting how epigenetic drugs could be used to suppress mmp-9 gene expression is not trivial because epigenetic drugs also regulate the expression of key proteins that can tip the balance towards activation or suppression of MMP-9. Here, we review how our understanding of the biology and expression of MMP-9 could be exploited to augment clinical benefits, most notably in terms of the prevention and management of degenerative diseases and cancer.

MMP-9 silencing regulates hTERT expression via β1 integrin-mediated FAK signaling and induces senescence in glioma xenograft cells

In more than 90% of cancers including glioma, telomere elongation reverse transcriptase (hTERT) is overexpressed. In the present study, we sought to explore whether matrix metalloproteinase-9 (MMP-9) shRNA could alter hTERT-mediated proliferation in glioma cells. MMP-9 shRNA induced senescence and apoptosis in glioma cells by inhibiting hTERT expression and telomere activity. MMP-9 silencing decreased oncogenic c-Myc expression (hTERT activator), whereas the expression of the c-Myc antagonist MAD increased drastically (hTERT repressor); both c-Myc and MAD are transcription factors for hTERT. In addition, MMP-9 suppression turns the switch from c-Myc/MAX to MAD/MAX heterodimer binding to the hTERT promoter as determined by chromatin immunoprecipitation assay. We also show that silencing MAD via siRNA restored hTERT expression and inhibited senescence in glioma cells. MMP-9 transcriptional suppression decreased the expression of FAK, phospho FAK and β1 integrin in glioma xenograft cells. Further, MMP-9 suppression decreased the interaction of β1 integrin/FAK and also MMP-9/β1 integrin as confirmed by immunoprecipitation analysis. Studies with either function blocking β1 integrin or FAK shRNA indicate that suppression of MMP-9 decreased β1 integrin-mediated induction of FAK, which led to decreased hTERT expression. Moreover, 4910 and 5310 glioma xenograft tissue sections from mice treated with MMP-9 shRNA showed reduced expression of FAK/c-Myc and elevated MAD levels. Decreased co-localization of β1 integrin and MMP-9 was associated with MMP-9-suppressed tumor sections. Further, immunoprecipitation analysis showed decreased association of proteins involved in telomere end repair in MMP-9 shRNA-treated glioma cells. Elevated levels of p73 and TRAIL and the results of the FACS analysis show induction of apoptosis in MMP-9-silenced glioma cells. Taken together, these data provide new insights into the mechanisms underlying MMP-9-mediated hTERT expression in glioma proliferation.

Berberine inhibits human hepatoma cell invasion without cytotoxicity in healthy hepatocytes.

Conventional chemotherapy fails to cure metastatic hepatoma mainly due to its high hepatotoxicity. Many plant-derived agents have been accepted to effectively inhibit hepatoma cell invasion. However, the investigation that whether effectual plant-derived agents against invasive hepatoma cells exert unexpected cytotoxicity in healthy hepatocytes has been ignored. This study demonstrated that berberine exhibited significant cytotoxicity in HepG2 cells mainly through upregulation of reactive oxygen species (ROS) production but was ineffective in normal Chang liver cells. Berberine exerted anti-invasive effect on HepG2 cells through suppression of matrix metalloproteinase-9 (MMP-9) expression. Moreover, berberine could significantly inhibit the activity of PI3K-AKT and ERK pathways. Combination treatment of ERK pathway inhibitor PD98059 or AKT pathway inhibitor LY294002 and berberine could result in a synergistic reduction on MMP-9 expression along with an inhibition of cell invasion. Enhancement of ROS production by berberine had no influence on its suppressive effects on the activity of PI3K-AKT and ERK pathways, as well as MMP-9 expression and HepG2 cell invasion. In conclusion, our results suggest that berberine may be a potential alternative against invasive hepatoma cells through PI3K-AKT and ERK pathways-dependent downregulation of MMP-9 expression. This study also provides a previously neglected insight into the investigation of plant-derived agents-based therapy against tumor invasion with the consideration of damage to healthy cells.

Hyul-Tong-Ryung suppresses PMA-induced MMP-9 expression by inhibiting AP-1-mediated gene expression via ERK1/2 signaling pathway in MCF-7 human breast cancer cells

Our previous study has demonstrated that the methanol extract of Hyul-Tong-Ryung (HM) specifically suppresses the phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) production through the inhibition of MMP-9 mRNA expression in MCF-7 human breast carcinoma cells. However, the molecular mechanisms involved in transcriptional suppression of MMP-9 by HM in PMA-induced MCF-7 cells are not known. In this study, we aimed to elucidate the molecular mechanisms involved in the inhibition of MMP-9 expression by HM in PMA-induced MCF-7 cells. The results of promoter assay and EMSA showed that HM specifically inhibits MMP-9 gene expression by blocking PMA-stimulated activation of activator protein-1 (AP-1). In addition, PMA-stimulated phosphorylation of extracellular signal regulated kinase1/2 (ERK1/2) was suppressed by HM treatment, whereas the phosphorylation of either c-Jun N-terminal kinase (JNK) or p38 mitogen-activated protein kinase (MAPK) was not affected. HM could inhibit the PMA-induced MMP-9 expression through suppression of the transcriptional activity of MMP-9 gene in MCF-7 cells. These results indicate that HM inhibits PMA-induced MMP-9 expression by blocking the activation of activator protein-1 (AP-1) via extracellular signal regulated kinase1/2 (ERK 1/2) signaling pathway.

Endogenous brain protection by granulocyte-colony stimulating factor after ischemic stroke

Several lines of evidence have demonstrated beneficial effects of the hematopoietic factor G-CSF in experimental stroke. A conclusive demonstration of this effect in G-CSF deficient mice is, however, lacking. We therefore investigated the effect of G-CSF deficiency on infarct volumes, functional recovery, mRNA and protein expression of the matrix metalloproteinase 9 (MMP-9) after stroke. Furthermore we tested the efficacy of G-CSF substitution in G-CSF deficient animals to prevent the potential consequences of G-CSF deficiency. In the present study experimental stroke was induced in female non-treated wildtype (wt), G-CSF deficient mice and G-CSF substituted G-CSF deficient mice followed by assessment of infarct volumes, neurological outcome and sensorimotor function. In addition, immunohistochemistry and real-time PCR of the peri-ischemic area were performed. G-CSF deficient mice showed increased infarct volumes, whereas G-CSF substituted mice had a remarkable reduction in lesion size compared to wt mice. These findings are accompanied by an improvement in neurological and sensorimotor function. G-CSF deficiency resulted in an upregulation of MMP-9 in the direct peri-ischemic tissue. Treatment with G-CSF suppressed the upregulation of MMP-9. Taken together, G-CSF deficiency clearly resulted in enlarged infarct volumes, and worsened neurological outcome. G-CSF substitution abolished these negative effects, led to significant reduced lesion volumes, and improved neurological outcome. G-CSF mediated suppression of MMP-9 further demonstrates that endogenous G-CSF plays a significant role in brain protective mechanisms. We have shown for the first time that endogenous G-CSF is required for brain recovery mechanisms after stroke.

Rhein inhibits invasion and migration of human nasopharyngeal carcinoma cells in vitro by down-regulation of matrix metalloproteinases-9 and vascular endothelial growth factor

Progression of cancer invasion is believed to be dependent on the remodeling of extracellular matrix induced by tumor cells. Rhein has been shown to inhibit the growth and proliferation of human nasopharyngeal carcinoma (NPC) cells. However, the molecular mechanism underlying rhein-induced inhibition of cancer invasion has not been explored. Herein, we show that rhein could inhibit the invasion and migration of NPC cells in vitro. Rhein inhibits invasion by reducing the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). Moreover, we demonstrate that the pathway involved in rhein-inhibited invasion is presumably through the growth factor receptor bound protein 2/son of sevenless-Ras-mitogen-activated protein kinase (GRB2/SOS-Ras-MAPK) pathway, as shown by an decrease in the expression levels of GRB2, SOS-1 and Ras as well as led to suppression of the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK. Further study has shown that rhein also inhibited activation of transcription factor nuclear factor kappaB (NF-kappaB), which is known to implicate the regulation of MMP-9 and VEGF gene expression in cancer invasion. Our findings suggest that rhein inhibits the invasion of NPC cells may be mediated in part through the suppression of MMP-9 and VEGF expression via the modulation of NF-kappaB signaling pathway.