Inhibition of cell invasion and migration by targeting matrix metalloproteinase-9 expression via sirtuin 6 silencing in human breast cancer cells.

Sirtuin 6 (SIRT6) regulation is involved in carcinogenesis. However, its role in breast cancer (BC) metastasis remains unclear. We investigated the effects of SIRT6 on protein kinase C activator- and cytokine-mediated cancer cell invasion and migration in MCF-7 and MDA-MB-231 cells and the association between SIRT6 and matrix metalloproteinase-9 (MMP-9) expression. To assess MMP-9 and SIRT6 expression in patients, protein levels in BC tissues were analyzed. MCF-7 and MDA-MB-231 cell viability was analyzed using MTT assays. SIRT6 was silenced in both cell lines and protein secretion, expression, and mRNA levels were analyzed. Transcription factor DNA activity was investigated using luciferase assays. Matrigel invasion assays were used to assess the effects of SIRT6 in both cell lines. SIRT6 and MMP-9 expression in cancer tissues was significantly higher than in paired normal breast tissues. 12-O-tetradecanoylphorbol-13-acetate (TPA) or tumor necrosis factor-α (TNF-α) increased MMP-9 expression and cell invasion and migration, but SIRT6 knockdown abolished these effects. SIRT6 overexpression additively increased TPA- and TNF-α-induced MMP-9 expression. SIRT6 knockdown suppressed the mitogen-activated protein kinase (MAPK) signaling pathway and thus TPA- and TNF-α-induced MMP-9 expression. SIRT6 silencing suppressed TPA- and TNF-α-induced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) expressions in both cell lines, and treatment with MAPK, NF-κB, and AP-1 inhibitors reduced MMP-9 expression. The anti-invasive effects of SIRT6 in BC cells might be mediated by suppression of MAPK phosphorylation and reduction in NF-κB and AP-1 DNA activities, leading to MMP-9 downregulation, suggesting that SIRT6 modulation has the potential to target BC metastasis.

Comparative matrix metalloproteinase-2 and -9 expression and activity during endotheliochorial and hemochorial trophoblastic invasiveness.

To establish a functional placenta, its development needs adequate trophoblastic invasiveness. The intricate and complex morphological and molecular aspects regulating trophoblastic invasion during endotheliochorial placentation of domestic carnivores and their similarities and differences with the hemochorial placenta are still poorly understood. During placentation processes, from the time of implantation, trophoblast cells invade the uterine endometrium where they achieve extensive degradation and remodeling of extracellular matrix components; in this process, matrix metalloproteinases (MMPs), particularly MMP-2 and 9, have an essential role in rebuilding, cell migration, and invasiveness. This review provides an overview of comparative trophoblast invasive events and the expression and activity of MMP-2 and 9 during endotheliochorial and hemochorial placentation, emphasizing dog and mouse placental models. Understanding of trophoblastic invasiveness in two models of placentation, the intermediately invasive domestic carnivore endotheliochorial placenta, and the more highly invasive mouse hemochorial placenta, contributes to deepen knowledge of the trophoblast invasive processes and their diverse and complex human placental alterations, such as preeclampsia.

Targeting cIAPs attenuates CCl4-induced liver fibrosis by increasing MMP9 expression derived from neutrophils.

AIMS: Liver fibrosis is a growing public health concern without effective medical treatment. Recent reports have indicated that inhibitors of apoptosis proteins (IAPs) were potential targets for idiopathic pulmonary fibrosis therapy. However, their roles have not been well identified in liver fibrosis. METHODS: The expression of IAPs were examined in human liver tissue and experimental mouse models. Liver fibrosis in CCl4-induced mouse models were investigated by Sirius red staining, RT-PCR, Western blotting after hepatocytes-specific cIAP2 knockout or IAPs inhibitor APG-1387 treatment. The underlying molecular mechanism of APG-1387 action was explored by apoptosis analysis, matrix metalloprotein 9 (MMP9) inhibition, neutrophils depletion, and CC Motif Chemokine Ligand 5 (CCL5) gene knockout in vitro and in vivo. FINDINGS: Our study showed that increased expression of cIAP2 was associated with liver fibrosis severity in liver tissues. Deletion of cIAP2 from hepatocytes or degrading cIAPs by APG-1387 ameliorated liver fibrosis induced by CCl4. APG-1387 treatment exhibited increased expression of MMP9 and resulted in higher ratio of MMP9 to tissue inhibitor of metalloproteinase-1. MMP9 was mainly derived from CCL5 chemotactic neutrophils. Further, MMP9 inhibition by CTT peptide, neutrophil depletion by Ly6G antibody or CCL5 deficiency blocked the anti-fibrotic effects of APG-1387 in vivo. SIGNIFICANCE: These results suggested that cIAPs, especially cIAP2, might play a novel role in the pathogenesis of liver fibrosis, and targeting cIAPs represented a promising therapeutic strategy for liver fibrosis by increasing MMP9 expression induced by CCL5 chemotactic neutrophils.

Bilateral Increase of Matrix Metalloproteinase-9 Expression in Tears After the Unilateral Decrease of Corneal Sensitivity.

PURPOSE: The purpose of this study is to analyze bilateral changes of matrix metalloproteinase-9 (MMP-9) expression in the tear film in patients with unilateral neurotrophic keratitis. METHODS: We included 24 eyes from 24 subjects with normal corneal sensitivity according to Cochet-Bonnet corneal esthesiometry in the control group and 25 subjects with the unilateral neurotrophic keratitis in the unilateral decrease of corneal sensitivity (UDCS) group. The expression of MMP-9 in tears was assessed using point-of-care immunoassay. The clinical parameters including meibomian gland plugging, quality of the secreted meibum, tear secretion using Schirmer I without anesthesia, and corneal erosions were evaluated among the control, affected, and contralateral unaffected eyes in the UDCS group. RESULTS: Tear MMP-9 grades in affected (2.5 ± 1.0) and contralateral eyes (2.6 ± 0.9) in the UDCS group were higher than those in control eyes (1.5 ± 0.7, P = 0.0003 and P = 0.0006, respectively). The lacrimal secretion decreased in the affected eyes but preserved in the contralateral eyes. There was no difference in corneal erosion scores between control eyes and contralateral eyes in the UDCS group. Tear MMP-9 grades in the contralateral eyes were significantly correlated with the meibum quality grades in the contralateral eyes (r = 0.525 and P = 0.025). CONCLUSIONS: Patients with the unilateral neurotrophic keratitis demonstrated a significant bilateral increase of the MMP-9 expression in the tear film as compared with controls. Despite the tearing reflex and corneal surface barrier being preserved in the unaffected contralateral eyes, it is necessary to pay attention to the possible attenuation of meibomian gland function in the opposite eyes as well.

The Elk1/MMP-9 axis regulates E-cadherin and occludin in ventilator-induced lung injury.

BACKGROUND: Ventilator-induced lung injury (VILI) is a common complication in the treatment of respiratory diseases with high morbidity and mortality. ETS-domain containing protein (Elk1) and Matrix metalloproteinase (MMP) 9 are involved in VILI, but the roles have not been fully elucidated. This study examined the mechanisms of the activation of MMP-9 and Elk1 regulating barrier function in VILI in vitro and in vivo. METHODS: For the in vitro study, Mouse lung epithelial cells (MLE-12) were pre-treated with Elk1 siRNA or MMP-9 siRNA for 48 h prior to cyclic stretch at 20% for 4 h. For the in vivo study, C57BL/6 mice were pre-treated with Elk1 siRNA or MMP-9 siRNA for 72 h prior to 4 h of mechanical ventilation. The expressions of Elk1, MMP-9, Tissue inhibitor of metalloproteinase 1 (TIMP-1), E-cadherin, and occludin were measured by Western blotting. The intracellular distribution of E-cadherin and occludin was shown by immunofluorescence. The degree of pulmonary edema and lung injury were evaluated by Hematoxylin-eosin (HE) staining, lung injury scores, Wet/Dry (W/D) weight ratio, total cell counts, and Evans blue dye. RESULTS: 20% cyclic stretch and high tidal volume increases the expressions of Elk1, MMP-9, and TIMP-1, increases the ratio of MMP-9/TIMP-1, decreases the E-cadherin and occludin level. Elk1 siRNA or MMP-9 siRNA reverses the degradations of E-cadherin, occludin, and the ratio of MMP-9/TIMP-1 caused by cyclic stretch. Elk1 siRNA decreases the MMP-9 level with or not 20% cyclic stretch and high tidal volume. CONCLUSIONS: The results demonstrate mechanical stretch damages the tight junctions and aggravates the permeability in VILI, Elk1 plays an important role in affecting the tight junctions and permeability by regulating the balance of MMP-9 and TIMP-1, thus indicating the therapeutic potential of Elk1 to treat VILI.

Functionally significant polymorphisms of the MMP-9 gene are associated with peptic ulcer disease in the Caucasian population of Central Russia.

This study analyzed the association of functionally significant SNPs of matrix metalloproteinase (MMP) genes in the development of peptic ulcer disease (PUD) in Caucasians from Central Russia. Ten SNPs of the MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 genes were analyzed for association with PUD in a cohort of 798 patients with PUD (including 404 H. pylori-positive and 394 H. pylori-negative) and 347 H. pylori-negative controls using logistic regression and assuming the additive, recessive, and dominant genetic models. The variants of MMP-1, MMP-2, MMP-3, and MMP-8 did not manifest any significant associations with the diseases. Five SNPs of the MMP-9 gene demonstrated such association. Allele G of the rs17576 MMP-9 locus conferred a higher risk for PUD (ORadj = 1.31, pperm = 0.016), haplotype AACG of loci rs17576-rs3787268-rs2250889-rs17577 of the MMP-9 gene decreased risk for PUD (ORadj = 0.17, pperm = 0.003). Also, allele C of rs3918249, allele G of rs17576 and haplotype CG of rs3918249-rs17576 of the MMP-9 gene increased risk for H. pylori-positive PUD (ORadj = 1.82, pperm = 0.002; ORadj = 1.53-1.95 pperm = 0.001-0.013 and ORadj = 1.49 pperm = 0.009 respectively). The above loci and 50 linked to them possess significant regulatory effects and may affect the alternative splicing of four genes and the expression of 17 genes in various organs and tissues related to the PUD pathogenesis.

The effects of resveratrol on the expression of VEGF, TGF-ß, and MMP-9 in endometrial stromal cells of women with endometriosis.

Resveratrol is a phytochemical with anti-angiogenic, anti-inflammatory, and antioxidant properties. The present study has evaluated the effect of resveratrol on the expression of vascular endothelial growth factor (VEGF), transforming growth factor-ß (TGF-ß) and matrix metalloproteinase-9 (MMP-9) as factors related to endometriosis progression. Thirteen eutopic (EuESCs) and 8 ectopic (EESCs) endometrial stromal cells from women with endometriosis and 11 control endometrial stromal cells (CESCs) were treated with resveratrol (100 µM) for 6, 24 and 48 h. The gene and protein expression levels of VEGF, TGF-ß, and MMP-9 were measured using real-time PCR and ELISA methods, respectively. Results showed that the basal gene and protein expression of VEGF and MMP-9 were higher in EESCs compared to EuESCs and CESCs (P < 0.01 to < 0.001 and P < 0.05 to < 0.01 respectively). Also, resveratrol treatment decreased the gene and protein expression of VEGF and MMP-9 in EuESCs, EESCs and CESCs (P < 0.05 to < 0.01 and P < 0.05 to < 0.01 respectively) and gene and protein expression of TGF-ß in EESCs and EuESCs (P < 0.05 to < 0.01). The effect of resveratrol in reduction of VEGF gene expression was statistically more noticeable in EESCs compared to EuESCs and CESCs (P < 0.05). According to the findings, resveratrol may ameliorate endometriosis progression through reducing the expression of VEGF, TGF-ß, and MMP-9 in endometrial stromal cells (ESCs).

Immunohistochemical Expression of MMP-9, TIMP-1, and Vimentin and its Correlation With Inflammatory Reaction and Clinical Parameters in Oral Epithelial Dysplasia.

The aim of this study was to investigate the immunoexpression of matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), and vimentin (VIM) and its association with the inflammatory reaction (IR) and clinical parameters in oral epithelial dysplasia (ED). The sample was composed of 66 cases of ED, 27 oral squamous cell carcinoma (OSCC), and 28 non-neoplastic epithelium (NNE). ED was graded according to the binary system as low-risk ED (n=42) and high-risk epithelial dysplasia (HRED: n=24). The IR was defined as the median number of inflammatory cells present on the connective tissue in 5 consecutive fields. Tissue sections of paraffin-embedded samples were immunohistochemically stained; MMP-9 and TIMP-1 expression was analyzed separately in the epithelium and the connective tissue; VIM was analyzed in the epithelium. Clinical parameters such as age, sex, lesion site and clinical presentation, alcohol/tobacco use, and malignant transformation of ED were retrospectively obtained from medical records. Nonhomogeneous leukoplakia presented higher odds (3.857; 95% confidence interval: 1.16-12.85) of being graded as HRED than did homogeneous lesions. The IR was higher in OSCC and ED than in NNE, and correlated with the epithelial expression of VIM. HRED and nonhomogeneous leukoplakias presented higher IR than did low-risk ED and homogeneous leukoplakias. Alcohol users had higher IR than nonalcohol users. Smokers had higher epithelial expression of MMP-9 and VIM. High IR in OSCC and HRED, and its positive correlation with VIM expression suggest a contribution of the IR in the progression of OSCC. Moreover, the high expression of MMP-9 and VIM in smokers implies its involvement in tobacco carcinogenesis.

Expression Pattern of iNOS, BCL-2 and MMP-9 in the Hip Synovium Tissue of Patients with Osteoarthritis.

Hip osteoarthritis (HOA) is characterized by degradation of the cartilage and synovitis. However, the pathohistological effects of synovial tissue inflammation on HOA are not clear. The aim of this study was to evaluate the expression of iNOS, BCL-2 and MMP-9 markers in different synovial cell populations. A total of 32 patients were evaluated retrospectively. Age, sex, height, weight, body mass index were recorded and lymphocyte, fibrocytes and macrophages were analysed in tissue sections. Osteoarthritis cartilage histopathology assessment system (OARSI), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Krenn score, Harris Hip Score (HHS) and Kellgren-Lawrence (K-L) grading of the hip joints were performed. Total hip arthroplasty was performed on 32 patients and controls. Patients were divided into two groups according to their disease severity. The tissues were immunohistochemically analysed. K-L grade and Krenn score differ between all three groups, but also between moderate and severe OA. Synovial lining cell layer, resident cells in stroma and especially inflammatory infiltration were increasing with severity of OA. iNOS expression in both intima and subintima was positively correlated with Krenn score in moderate and severe osteoarthritis (OA) groups. Expression of BCL-2 in intima of severe OA patients was positively correlated with Krenn score. In conclusion, iNOS, BCL-2 and MMP-9 are involved in the regulation of HOA. Our study indicates a relationship between the pathohistological features, the synovial inflammation and the cartilage condition at the time of hip replacement due to OA or femoral neck fracture.

Inhibition of lysyl oxidase stimulates TGF-ß signaling and metalloproteinases-2 and -9 expression and contributes to the disruption of ascending aorta in rats: protection by propylthiouracil.

Mutations in lysyl oxidase (LOX) genes cause severe vascular anomalies in mice and humans. LOX activity can be irreversibly inhibited by the administration of ß-aminoproprionitrile (BAPN). We investigated the mechanisms underlying the damage to the ascending thoracic aorta induced by LOX deficiency and evaluated whether 6-propylthiouracil (PTU) can afford protection in rats. BAPN administration caused disruption of the ascending aortic wall, increased the number of apoptotic cells, stimulated TGF-ß signaling (increase of nuclear p-SMAD2 staining), and up-regulated the expression of metalloproteinases-2 and -9. In BAPN-treated animals, PTU reduced apoptosis, p-SMAD2 staining, MMP-2, and -9 expression, and markedly decreased the damage to the aortic wall. Our results suggest that, as in some heritable vascular diseases, enhanced TGF-ß signaling and upregulation of MMP-2 and -9 can contribute to the pathogenesis of ascending aorta damage caused by LOX deficiency. We have also shown that PTU, a drug already in clinical use, protects against the effects of LOX inhibition. MMP-2 and -9 might be potential targets of new therapeutic strategies for the treatment of vascular diseases caused by LOX deficiency.

High molecular weight form of hyaluronic acid reduces neuroinflammatory response in injured sciatic nerve via the intracellular domain of CD44.

Inflammatory response after peripheral nerve injury is required for clearance of tissue debris and effective regeneration. Studies have revealed that hyaluronic acid (HA) may exert different properties depending on their molecular size. High molecular weight HA (>>1,000 kDa; HMW-HA) displays immunosuppressive properties, whereas low molecular weight HA (<800 kDa; LMW-HA) induces proinflammatory responses. The role of HMW-HA interaction with CD44, a major HA receptor, in neuroinflammatory responses has not been fully elucidated. The purpose of this experimental study was to investigate the effects of topical applications of HMW-HA on the sciatic nerve injury in an adult rat model. At the crush site on the sciatic nerve, the recordings of compound muscle action potential (CMAP) and the levels of several proteins related to inflammatory response were assessed at time intervals of 2, 4, and 6 weeks postsurgery. Here, we show that the recovery effect of HMW-HA treatment had significantly shortened latency and increased amplitude of CMAP compared with crushed alone, crushed plus γ-secretase inhibitor with or without HA treatment at 6 weeks after surgery. Our data reveal that HMW-HA could downregulate the expression of IL1-ß, TLR4, and MMP-9, whereas these proteins expression were increased when the CD44-ICD activity was inhibited using γ-secretase inhibitor. Our findings demonstrated a novel role of CD44-ICD in HA-mediated recovery of peripheral nerve injury. Clinical relevance: an alternative for the regeneration of peripheral nerve injury.

Sea Anemone Heteractis crispa Actinoporin Demonstrates In Vitro Anticancer Activities and Prevents HT-29 Colorectal Cancer Cell Migration.

Actinoporins are the most abundant group of sea anemone cytolytic toxins. Their membranolytic activity is of high interest for the development of novel anticancer drugs. However, to date the activity of actinoporins in malignant cells has been poorly studied. Here, we report on recombinant analog of Hct-S3 (rHct-S3), belonging to the combinatory library of Heteractis crispa actinoporins. rHct-S3 exhibited cytotoxic activity against breast MDA-MB-231 (IC50 = 7.3 µM), colorectal HT-29 (IC50 = 6.8 µM), and melanoma SK-MEL-28 (IC50 = 8.3 µM) cancer cells. The actinoporin effectively prevented epidermal growth factor -induced neoplastic transformation of JB6 Cl41 cells by 34% ± 0.2 and decreased colony formation of HT-29 cells by 47% ± 0.9, MDA-MB-231 cells by 37% ± 1.2, and SK-MEL-28 cells by 34% ± 3.6. Moreover, rHct-S3 decreased proliferation and suppressed migration of colorectal carcinoma cells by 31% ± 5.0 and 99% ± 6.4, respectively. The potent anti-migratory activity was proposed to mediate by decreased matrix metalloproteinases-2 and -9 expression. In addition, rHct-S3 induced programmed cell death by cleavage of caspase-3 and poly (ADP-ribose) polymerase, as well as regulation of Bax and Bcl-2. Our results indicate rHct-S3 to be a promising anticancer drug with a high anti-migratory potential.

Drupin, a cysteine protease from Ficus drupacea latex accelerates excision wound healing in mice.

Wound healing is a tightly regulated physiological process that restores tissue integrity after injury. Plant latex proteases (PLPs) are considered an integral part in herbal wound care as it interferes at different phases of the wound healing process. Although many studies have reported the involvement of PLPs in healing process, an in-depth investigation is required to understand the molecular mechanism. Hence, the effect of PLPs with fibrinolytic activity on wound healing was investigated systematically using mouse excision wound model. Among 29 latices from Ficus genus tested, Ficus drupacea exhibited potent fibrinolytic activity. Cysteine protease responsible for fibrinolysis was purified from the F. drupacea latex named it as drupin, tested for its wound healing efficacy. The accelerated wound healing was mediated by downregulation of matrix metalloprotease (MMP)-9 without altering MMP-8 expression. Besides, drupin enhanced the rate of collagen synthesis at the wound site by increasing arginase 1 activity. And also, drupin increased the expression of arginase 1 in macrophages and involved in cell proliferation, and migration via MAP kinase and PI3K/Akt pathways. Overall, the present study highlights the interference of drupin in wound healing by increased arginase 1 activity and collagen synthesis, and cell proliferation and migration.

SMAGP knockdown inhibits the malignant phenotypes of glioblastoma cells by inactivating the PI3K/Akt pathway.

Small trans-membrane and glycosylated protein (SMAGP), a novel small trans-membrane glycoprotein, is reported to be upregulated in multiple cancers and involved in tumor development. However, little is known about its role in the development of glioblastoma (GBM). GEPIA database was used to analyze SMAGP expression and evaluate the prognostic value of SMAGP in GBM. GO and KEGG pathway enrichment analyses were used to predict the biological functions and pathways of SMAGP and 948 SMAGP-correlated genes using DAVID database. Cell viability, colony formation ability, apoptosis, and invasion were evaluated by MTT, colony formation assay, flow cytometry analysis, and Transwell invasion assay, respectively. Western blot was applied to detect the expression of SMAGP, matrix metalloproteinase (MMP)-2, and MMP-9 and analyze the changes of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling. Results showed that SMAGP was upregulated and correlated with poor prognosis in GBM. Functional annotation analysis revealed that SMAGP and 948 SMAGP-correlated genes were primarily associated with cell adhesion and PI3K/Akt pathway. SMAGP interference inhibited cell viability and colony formation ability and promoted apoptosis in GBM cells. Moreover, SMAGP interference inhibited GBM cell invasion and suppressed MMP-2 and MMP-9 expression. Additionally, SMAGP silencing inhibited the PI3K/Akt pathway in GBM cells. Overexpression of Akt abolished the effects of SMAGP knockdown on the malignant phenotypes of GBM cells. In conclusion, SMAGP silencing inhibited the malignant phenotypes of GBM cells by inactivating the PI3K/Akt pathway.

Expression of Matrix Metalloproteinase-2, Matrix Metalloproteinase-9, Tissue Inhibitor of Metalloproteinase-1, and Changes in Alveolar Septa in Patients with Chronic Obstructive Pulmonary Disease.

BACKGROUND This study investigated the relationship between the pathological alteration of alveolar septa and (1) pulmonary function and (2) matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor matrix metalloproteinase 1 (TIMP-1) expression in chronic obstructive pulmonary disease (COPD). MATERIAL AND METHODS Sixty patients with pulmonary disease were divided into control (n=20) and COPD (n=40) groups. Postoperative lung tissue specimens were examined. Hematoxylin and eosin and elastin van Gieson staining detected pathological alterations of pulmonary alveolar septa. Septa thickness was measured. MMP-2, MMP-9, and TIMP-1 expression levels were detected by immunohistochemical staining. Correlations were determined by Pearson analysis. RESULTS Forced expiratory volume in 1 s (FEV1), forced vital capacity, FEV1 percent predicted (FEV1%pre), and diffusion capacity of carbon monoxide percent predicted (DLCO%pre) in COPD patients were significantly lower than in those of the control group (P<0.05). MMP-2, MMP-9, and TIMP-1 expression levels were significantly higher in the COPD group than in control, especially the severe group (P<0.05). Septa thickness was negatively correlated with FEV1%pre (r=-0.335; P<0.05) and positively correlated with MMP-2 and TIMP-1 expression (P<0.05). Proportion of collagenous fiber was negatively correlated with FEV1%pre and DLCO%pre (P<0.01), and positively correlated with MMP-2, MMP-9, and TIMP-1 expression (P<0.01). Proportion of elastic fibers was negatively correlated with collagenous fiber. CONCLUSIONS The pathological alteration of alveolar septa was correlated with pulmonary function and expression levels of MMP-2, MMP-9, and TIMP-1, which can play vital roles in COPD progression.

Combination of ACY-241 and JQ1 Synergistically Suppresses Metastasis of HNSCC via Regulation of MMP-2 and MMP-9.

Overexpression of histone deacetylase 6 (HDAC6) and bromodomain-containing protein 4 (BRD4) is related to aggressiveness of head and neck squamous carcinoma (HNSCC). Based on studies that HDAC6 and BRD4 are potential therapeutic targets of HNSCC, we hypothesized that the combination treatment of BET inhibitor JQ1 and HDAC6-selective inhibitor ACY-241 could exhibit synergistic anticancer effects in human papillomavirus (HPV)-positive and HPV-negative HNSCC cells. In this study, HNSCC cell growth and viability were measured by CCK-8 assay, apoptosis was analyzed by flow cytometry, and metastasis was studied by wound healing and transwell assays. Furthermore, immunoblotting is conducted to investigate proteins that modulate apoptosis or metastasis. Here, we report that the combination of ACY-241 and JQ1 shows synergistic cell growth inhibition, viability reduction, and apoptosis induction in HNSCC cells through inactivation of AKT and NF-κB signaling. Importantly, we demonstrate that combined treatment of ACY-241 and JQ1 synergistically suppresses TNF-α-induced migration and invasion via dysregulating matrix metalloproteinase (MMP)-2, MMP-9, and MT1-MMP. Overall, the combination of ACY-241 and JQ1 significantly suppresses proliferation and metastasis in HPV-positive and HPV-negative HNSCC. Collectively, these findings suggest that the co-inhibition of BET and HDAC6 can be a new therapeutic strategy in HNSCC.

Immunohistochemical Profiles of Matrix Metalloproteinases and Vascular Endothelial Growth Factor Overexpression in the Antoni B Area of Vestibular Schwannomas.

OBJECTIVE: To evaluate the clinical manifestations of cystic vestibular schwannomas (VSs), investigate the immunohistochemical profiles of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) expression in Antoni A and B areas, and speculate the pathogenesis of cystic formation and intratumoral hemorrhage. METHODS: Clinical features and outcomes of 24 cases of cystic VSs and 38 cases of solid VSs were retrospectively compared. Immunohistochemical studies were conducted to evaluate the characteristics of MMPs and VEGF in cystic and solid VSs. RESULTS: The tumor size was 38.92 ± 1.86 mm and 31.95 ± 1.74 mm in the cystic and solid VSs group, respectively (P = 0.011). Cystic VSs were rich in the Antoni B area. MMP-9 expression was low in the Antoni A and B areas. MMP-2 was moderately expressed. No significant difference in MMP-2 expression existed between the Antoni A and B areas (P > 0.05). VEGF and MMP-14 expression were moderate in the Antoni A area and intense in the Antoni B area, and the expression of both was significantly greater in the Antoni B area than in the Antoni A area (P < 0.001). CONCLUSIONS: MMP-14 and VEGF expression were significantly greater in the Antoni B area than in the Antoni A area. Upregulated MMP-14 may degrade loose collagen in the Antoni B area and contribute to cystic formation. MMP-14 can enhance VEGF activity, which may induce extravasation of a plasma ultrafiltrate, cystic expansion, and intratumoral hemorrhage. Therefore, MMP-14 inhibition may be a therapeutic strategy for treating cystic VSs.

Relative gene expression analysis of human pterygium tissues and UV radiation-evoked gene expression patterns in corneal and conjunctival cells.

A sight threatening, pterygium is a common ocular surface disorders identified by fibrovascular growth of the cornea and induced by variety of stress factors, like ultraviolet (UV) exposure. However, the genes involved in the etiopathogenesis of this disease is not well studied. Herein, we identified the gene expression pattern of pterygium and examined the expression of pterygium-related genes in UV-B-induced human primary cultured corneal epithelial cells (HCEpCs), telomerase immortalized human corneal epithelial (hTCEpi), primary conjunctival fibroblast (HConFs) and primary pterygium fibroblast cells (HPFCs). A careful analysis revealed that the expression of 10 genes was significantly modulated (by > 10-fold). Keratin 24 (KRT24) and matrix metalloproteinase 9 (MMP-9) were dramatically upregulated by 49.446- and 24.214-fold, respectively. Intriguingly, UV-B exposure (50 J/m2) induced the upregulation of the expressions of MMP-9 in corneal epithelial cells such as HCEpCs and hTCEpi. Furthermore, UV-B exposure (100 and/or 200 J/m2) induced the upregulation of the expressions of MMP-9 in fibroblast such as HConFs and HPFCs. The exposure of HCEpCs to 100 and 200 J/m2 UV-B induced significant expressions of KRT24 mRNA. Nevertheless, no expression of KRT24 mRNA was detected in HConFs and HPFCs. The findings provide evidence that the progression of pterygium may involve the modulation of extracellular matrix-related genes and vasculature development and the up-regulation of KRT24 and MMP-9 by UV stress. UV radiation may promote the modulation of these pterygium-related genes and induce the initiation and progression of human pterygium.

The Effect of Sialic Acid on the Expression of miR-218, NF-kB, MMP-9, and TIMP-1.

Sialic acid (N-acetylneuraminic acid, NANA) is found at all cell surfaces of vertebrates. Although it is widely accepted that sialic acid is an essential substrate for brain development via a significant role in nerve transfers, structure of glycosides, and synaptogenesis phenomena, there are some reports on the elevated levels of sialic acid and prevalence of neurodegeneration. Matrix metalloproteases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are involved in neuroinflammation disorders and produced by many cell types, including activated T cells, macrophages, neurons, astrocytes, and microglial cells. It can be hypothesized that sialic acid may have a potentially critical role in regulation of a wide range of uncovered neurodegeneration factors as its downstream targets. In this study, for the first time, we aimed to analyze the possible effect of the sialic acid solution exposure in the human C118 cell line, which was derived from a human brain astrocytoma (glial cells), on the expression patterns of miR-218, NF-kB, MMP-9, and TIMP-1. For MMP-9, protein levels were studied too. Half maximal inhibitory concentration (IC50) value of NANA was obtained by MTT assay. Glial cell line was treated with sialic acid (300, 500, and 1000 µg/ml) for 24 h to investigate the effects of this ligand on the expression of miR-218, NF-kB, MMP-9, and TIMP-1 genes. Protein levels were checked by Western blotting, and by using zymography, the gelatinolytic activity of MMP-9 secreted into conditioned media was assayed. At 300 µM, 500 µM, and 1000 µM sialic acid treatments, the expression of miR-218 was downregulated; subsequently, the NF-kB, MMP-9, and TIMP-1 genes as well as their protein expressions were upregulated. More interestingly, the enzyme activity of secreted MMP-9 was upregulated too (p-values ≤ 0.05). This study could demonstrate the significant effect of sialic acid on miR-218, NF-kB, MMP-9 , and TIMP-1 expressions in gene and protein levels and also the levels of enzyme activity of secreted MMP-9. Therefore, provided information indicates the novel idea of a possible linkage between sialic acid species and regulation of these neuroinflammation genes in Glial cell line.

Analysis of the Relationship Between Metalloprotease-9 and Tau Protein in Alzheimer's Disease.

BACKGROUND: Neurofibrillary tangles (NFTs) and amyloid plaques are the neuropathological hallmarks in brains with Alzheimer's disease (AD). Post-translational modifications of tau, such as phosphorylation and truncation, have been proposed as initiators in the assembly of the abnormal paired helical filaments that constitute the NFTs. Neurons and NFTs are sites of matrix metalloproteinases (MMPs). OBJECTIVE: The aim of this study was to analyze the relationship of MMP-9 and tau protein in brain samples with AD. METHODS: This study was performed on brain tissue samples from patients with early, moderate, and late AD. MMPs and tau levels were analyzed by western blot and gelatin-substrate zymography. Immunofluorescence techniques and confocal microscopy were used to analyze the presence of both proteins in NFTs. Further, molecular dynamics simulations (MDS) and protein-protein docking were conducted to predict interaction between MMP-9 and tau protein. RESULTS: MMP-9 expression was greatest in moderate and late AD, whereas MMP-2 expression was only increased in late-stage AD. Interestingly, confocal microscopy revealed NFTs in which there was co-localization of MMP-9 and tau protein. MDS and protein-protein docking predictions indicate that a high-affinity complex can be formed between MMP-9 and full-length tau protein. CONCLUSION: These observations provide preliminary evidence of an interaction between these two proteins. Post-translational modifications of tau protein, such as C-terminal truncation or phosphorylation of amino acid residues in the MMP-9 recognition site and conformational changes in the protein, such as folding of the N-terminal sequence over the three-repeat domain, could preclude the interaction between MMP-9 and tau protein during stages of NFT development.