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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., (© 2022. The Author(s).)

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., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 CCl 4 -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 CCl 4 . 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., (Copyright © 2021. Published by Elsevier Inc.)

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., (© 2021. The Author(s).)

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 (OR adj  = 1.31, p perm  = 0.016), haplotype AACG of loci rs17576-rs3787268-rs2250889-rs17577 of the MMP-9 gene decreased risk for PUD (OR adj  = 0.17, p perm  = 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 (OR adj  = 1.82, p perm  = 0.002; OR adj  = 1.53-1.95 p perm  = 0.001-0.013 and OR adj  = 1.49 p perm  = 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.

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-β, 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., (© 2020 Wiley Periodicals LLC.)

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Mutations in lysyl oxidase (LOX) genes cause severe vascular anomalies in mice and humans. LOX activity can be irreversibly inhibited by the administration of β-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-β 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-β 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.

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-β (TGF-β) 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-β, 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-β 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-β, and MMP-9 in endometrial stromal cells (ESCs).

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.

The aim of the present study was to investigate the new silicate cement mineral trioxide aggregate (MTA Repair HP) with respect to its effect on the inflammation process involving the tooth and periodontal tissues. The composition of MTA Repair HP was supplemented with plasticizer agents which can have a negative effect on the modulation of tooth inflammation. The silicate-based material in question is widely used in regeneration of the pulp-dentin complex, treatment of perforations of various locations in the tooth, as well as in surgical treatment of the complications of periapical tissue. The improved bioceramic restorative cement can affect the expression of metalloproteinases MMP-2 and MMP-9 in monocytes/macrophages involved in modulation of inflammation and regenerative processes of the tooth and periodontal tissues. The novel aspect of the present study lies in the application of the model of THP-1 monocyte/macrophage and applying the biomaterial in direct contact with the cells. Hence, it provides a representation of clinical conditions with respect to regenerative pulp and periodontal treatment with the use of MTA Repair HP. A lack of macrophage activation (as measured with flow cytometry) was found. Moreover, the study identified a lack of expression stimulation of the studied metalloproteinases (with the use of Western blotting and fluorescent microscopy). Similarly, no increase in MMP-2 and MMP-9 concentration was found (measured by ELISA method) in vitro when incubated with MTA Repair HP. Based on the results it can be concluded that new MTA Repair HP does not increase the inflammatory response in monocytes/macrophages associated with the activity of the described enzymes. It can also be speculated that they do not affect the process of dentin regeneration in which MMP-2 and MMP-9 play significant roles.

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 (IC 50 = 7.3 µM), colorectal HT-29 (IC 50 = 6.8 µM), and melanoma SK-MEL-28 (IC 50 = 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.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 (IC 50 ) 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.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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/m 2 ) 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/m 2 ) 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/m 2 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

Background: We aimed to assess whether a gene expression assay provided insights for understanding the heterogeneity among newborns affected by neonatal encephalopathy (NE)., Methods: Analysis by RT-qPCR of the mRNA expression of candidate genes in whole blood from controls (n = 34) and NE (n = 24) patients at <6, 12, 24, 48, 72 and 96 h of life, followed by determination of differences in gene expression between conditions and correlation with clinical variables., Results: During the first 4 days of life, MMP9, PPARG, IL8, HSPA1A and TLR8 were more expressed and CCR5 less expressed in NE patients compared to controls. MMP9 and PPARG increased and CCR5 decreased in moderate/severe NE patients compared to mild. At 6-12 h of life, increased IL8 correlated with severe NE and death, decreased CCR5 correlated with chorioamnionitis and increased HSPA1A correlated with expanded multiorgan dysfunction, severe NE and female sex., Conclusions: MMP9, PPARG and CCR5 mRNA expression within first days of life correlates with the severity of NE. At 6-12 h, IL8 and HSPA1A are good reporters of clinical variables in NE patients. HSPA1A may have a role in the sexual dimorphism observed in NE. CCR5 is potentially involved in the link between severe NE and chorioamnionitis.

Idiopathic pulmonary fibrosis (IPF), characterized by excessive collagen deposition, is a progressive and typically fatal lung disease without effective therapeutic strategies. Juglanin, as a natural product mainly isolated from green walnut husks of Juglans mandshuric, has various bioactivities, including anti-oxidative, anti-inflammatory and anti-fibrotic effects. Stimulator of interferon genes (Sting) is a signaling molecule and plays an essential role in meditating fibrosis. However, the effects of Jug and Sting on pulmonary fibrosis are not fully understood. In this study, we investigated the role of Jug in bleomycin (BLM)-induced inflammation and fibrosis mouse model, as well as the underlying molecular mechanism. The results here indicated that Jug-treated mice exhibited a definitively improved survival rate than that of the BLM-challenged mice. Jug administration significantly alleviated neutrophil alveolar infiltration, lung vascular permeability and pro-inflammatory response in BLM mice. Subsequently, the pulmonary fibrosis induced by BLM was markedly attenuated by Jug through reducing the expression of fibrotic hallmarks, including transforming growth factor-β1 (TGF-β1), fibronectin, matrix metallo-proteinase-9 (MMP-9), α-smooth muscle actin (α-SMA) and collagen I. Importantly, we found that BLM mice showed higher expression levels of Sting in lung tissues, which were notably restrained by Jug treatment. The role of Jug in suppressing Sting was confirmed in TGF-β-incubated cells. Notably, the in vitro analysis further showed that Sting knockdown could ameliorate TGF-β-triggered collagen accumulation. In contrast, TGF-β-induced fibrosis was accelerated by Sting over-expression. Therefore, BLM may induce lung fibrosis through activating Sting signaling, and Jug could be used therapeutically to improve tissue repair and attenuate the intractable disease., Competing Interests: Declaration of Competing Interest The authors see no conflict of interest., (Copyright © 2020. Published by Elsevier Masson SAS.)

Talin2 plays an important role in transduction of mechanical signals between extracellular matrix and actin cytoskeleton. Recent studies showed that talin2 is localized to invadopodia and regulates their maturation, subsequently cancer cell invasion and metastasis. However, the molecular mechanism whereby talin2 mediates invadopodium maturation is unknown. Here we show that ablation of talin2 in MDA-MB-231 cells inhibited the secretion of matrix metallopeptidase 9 (MMP9), a proteinase involved in extracellular matrix degradation in invadopodium maturation and metastasis. Furthermore, re-expression of talin2 WT in talin2-KO cells rescued MMP9 secretion, but talin2 S339C , a mutant with reduced β-integrin binding, did not, indicating that the talin2-β-integrin interaction is involved in the MMP9 secretion. Moreover, ablation of talin2 caused an accumulation of enlarged MMP9 vesicles. These vesicles co-localized with enlarged early, late endosomes and autophagosomes, suggesting talin2 controls MMP9 trafficking process. Therefore, these data suggest that talin2 regulates extracellular matrix degradation and invadopodium maturation by mediating MMP9 secretion., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Cigarette smoke (CS) is a major risk factor for the development of lung cancer and chronic obstructive pulmonary disease (COPD). Epithelial-mesenchymal transition (EMT) commonly coexists in lung cancer and COPD. CS triggers many factors including matrix metalloproteinases (MMPs) production, contributing to EMT progression in the lungs. Here, how Shp2 signaling regulates the CS-induced MMP-9 production and EMT progression were investigated in mouse lungs and in pulmonary epithelial cell cultures (NCI-H292) found CS induced MMP-9 production, EMT progression (increased vimentin and α-SMA; decreased E-cadherin) and collagen deposition in lung tissues; cigarette smoke extract (CSE) induced MMP-9 production and EMT-related phenotypes in NCI-H292 cells, which were partially prevented by Shp2 KO/KD or Shp2 inhibition. The CSE exposure induced EMT phenotypes were suppressed by MMP-9 inhibition. Recombinant MMP-9 induced EMT, which was prevented by MMP-9 inhibition or Shp2 KD/inhibition. Mechanistically, CS and CSE exposure resulted in ERK1/2, JNK and Smad2/3 phosphorylation, which were suppressed by Shp2 KO/KD/inhibition. Consequentially, the CSE exposure-induced MMP-9 production and EMT progression were suppressed by ERK1/2, JNK and Smad2/3 inhibitors. Thus, CS induced MMP-9 production and EMT resulted from activation of Shp2/ERK1/2/JNK/Smad2/3 signaling pathways. Our study contributes to the underlying mechanisms of pulmonary epithelial structural changes in response to CS, which may provide novel therapeutic solutions for treating associated diseases, such as COPD and lung cancer.

Loss of melanocytes is the pathological hallmark of vitiligo, a chronic inflammatory skin depigmenting disorder induced by exaggerated immune response, including autoreactive CD8 T cells producing high levels of type 1 cytokines. However, the interplay between this inflammatory response and melanocyte disappearance remains to be fully characterized. Here, we demonstrate that vitiligo skin contains a significant proportion of suprabasal melanocytes, associated with disruption of E-cadherin expression, a major protein involved in melanocyte adhesion. This phenomenon is also observed in lesional psoriatic skin. Importantly, apoptotic melanocytes were mainly observed once cells were detached from the basal layer of the epidermis, suggesting that additional mechanism(s) could be involved in melanocyte loss. The type 1 cytokines IFN-γ and TNF-α induce melanocyte detachment through E-cadherin disruption and the release of its soluble form, partly due to MMP-9. The levels of MMP-9 are increased in the skin and sera of patients with vitiligo, and MMP-9 is produced by keratinocytes in response to IFN-γ and TNF-α. Inhibition of MMP-9 or the JAK/STAT signaling pathway prevents melanocyte detachment in vitro and in vivo. Therefore, stabilization of melanocytes in the basal layer of the epidermis by preventing E-cadherin disruption appears promising for the prevention of depigmentation occurring in vitiligo and during chronic skin inflammation.

Tachykinins such as Substance P (SP) are a group of neuropeptides that are involved in cancer development. Neurokinin-1 receptor (NK-1R) is the main tachykinin receptor mediating the effects of SP, which is overexpressed in human esophageal squamous cell carcinoma (ESCC) and other malignant tissues. However, the effects of SP/NK-1R system on the migration of esophageal cancer cells and angiogenesis is not clear yet. This study seeks to obtain data to address these research gaps. In order to assess the effects of the FDA-approved aprepitant drug, a commercially available NK-1R antagonist, on the viability of KYSE-30 ESCC cells, resazurin assay was performed. The influence of SP/NK-1R system on the migration potential of these cells was examined using scratch assay. The effects of this system on the expression levels of metastatic factors were also examined by RT-PCR and western blot analyses. The half-maximal inhibitory concentration (IC 50 ) value for KYSE-30 cells treated with aprepitant found to be 29.88 μM. Treatment with SP significantly promoted KYSE-30 esophageal cancer cell migration, and aprepitant blocked this effect. In addition, SP significantly induced the expression of matrix metalloproteinase-2 (MMP-2), MMP-9, vascular endothelial growth factor-A (VEGF-A), and VEGF receptor1 (VEGFR1) in the cells, whereas aprepitant inhibited the up-regulation effects caused by SP. SP plays important roles in the development of human esophageal squamous cell carcinoma by promoting cancer cell invasion and enhancing the expression of factors involved in cellular migration and angiogenesis, which can be blocked by the NK-1R antagonist, aprepitant.

A Rho GTPase-activating protein (RhoGAP), deleted in liver cancer 1 (DLC1), is known to function as a tumor suppressor in various cancer types; however, whether DLC1 is a tumor-suppressor gene or an oncogene in melanoma remains to be clarified. Here we revealed that high DLC1 expression was detected in most of the melanoma tissues where it was localized in both the nuclei and the cytoplasm. Functional studies unveiled that DLC1 was both required and sufficient for melanoma growth and metastasis. These tumorigenic events were mediated by nuclear-localized DLC1 in a RhoGAP-independent manner. Mechanistically, mass spectrometry analysis identified a DLC1-associated protein, FOXK1 transcription factor, which mediated oncogenic events in melanoma by translocating and retaining DLC1 into the nucleus. RNA-sequencing profiling studies further revealed MMP9 as a direct target of FOXK1 through DLC1-regulated promoter occupancy for cooperative activation of MMP9 expression to promote melanoma invasion and metastasis. Concerted action of DLC1-FOXK1 in MMP9 gene regulation was further supported by their highly correlated expression in melanoma patients' samples and cell lines. Together, our results not only unravel a mechanism by which nuclear DLC1 functions as an oncogene in melanoma but also suggest an unexpected role of RhoGAP protein in transcriptional regulation.

CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a transmembrane glycoprotein that is highly expressed in tumor cells, particularly melanoma cells, and plays critical roles in tumor cell metastasis through the regulation of matrix metalloprotease (MMP) expression. In this study, we identified Fyn as a novel interacting protein of CD147. Fyn is a member of the Src family of nonreceptor tyrosine kinases that regulates diverse physiological processes, such as T lymphocyte differentiation, through the TCR signaling pathway. Our findings demonstrated that Fyn directly phosphorylates CD147 at Y140 and Y183. Two phosphospecific antibodies against Y140 and Y183 were developed to validate the phosphorylation of CD147 by Fyn. Moreover, the CD147-FF (Y140F/Y183F) mutation impaired the interaction between CD147 and GnT-V, leading to decreased CD147 glycosylation and membrane recruitment. In addition, CD147-FF significantly blocked MMP-9 expression as well as cell migration. Moreover, we found that Fyn is overexpressed in clinical melanoma tissues as well as in melanoma cell lines. Knockdown of Fyn expression markedly attenuated the malignant phenotype of melanoma cells in vitro and in vivo through downregulation of CD147 phosphorylation, indicating that Fyn/CD147 is a potential target molecule in melanoma treatment. Finally, through virtual screening, we identified amodiaquine as a potential inhibitor targeting the Fyn/CD147 axis. Amodiaquine treatment dramatically inhibited the phosphorylation of CD147 by Fyn, thus attenuating melanoma cell growth and invasion in vitro and in vivo, suggesting that amodiaquine is a promising inhibitor for melanoma treatment.

Activation of peroxisome proliferator-activated receptor γ (PPARγ) serves as a key factor in the proliferation and invasion of breast cancer cells and is a potential therapeutic target for breast cancer. However, the mechanisms underlying this effect remain largely unknown. Heme oxygenase-1 (HO-1) is induced and overexpressed in various cancers and is associated with features of tumor aggressiveness. Recent studies have shown that HO-1 is a major downstream target of PPARγ. In this study, we investigated the effects of induction of HO-1 by PPARγ on TPAinduced MMP-9 expression and cell invasion using MCF-7 breast cancer cells. TPA treatment increased NF-κB /AP-1 DNA binding as well as MMP-9 expression. These effects were significantly blocked by 15d-PGJ2, a natural PPARγ ligand. 15d-PGJ2 induced HO-1 expression in a dose-dependent manner. Interestingly, HO-1 siRNA significantly attenuated the inhibition of TPA-induced MMP-9 protein expression and cell invasion by 15d-PGJ2. These results suggest that 15d-PGJ2 inhibits TPA-induced MMP- 9 expression and invasion of MCF-7 cells by means of a heme oxygenase-1-dependent mechanism. Therefore, PPARγ/HO-1 signaling- pathway inhibition may be beneficial for prevention and treatment of breast cancer. [BMB Reports 2020; 53(4): 212-217].

Proline-, glutamic acid-, leucine-rich protein 1 (PELP1) is a novel estrogen receptor (ER) coregulator, demonstrated distinctive characters from other ERα coregulators, and has been suggested to be involved in metastasis of several cancers. In ERα-positive breast cancer, PELP1 overexpression enhanced ruffles and filopodium-like structure stimulated by estradiol (E 2 ) through extranuclear cell signaling transduction hereby increased cell motility. However, whether PELP1 is also involved in extracellular matrix remodeling of ERα-positive breast cancer cells is still unknown. In this study, we investigated the role of PELP1 in E 2 -induced MMP-9 expression and the underlined mechanism. The results demonstrated the following: E 2 -induced ERα-positive MCF-7 breast cancer cell MMP-9 mRNA and protein expression in a rapid response and concentration-dependent manner. Knocked down PELP1 significantly suppressed E 2 -induced MMP-9 expression. E 2 -bovine serum albumin (BSA), a large molecular membrane-impenetrable conjugate of E 2 , can also upregulate MMP-9 protein expression in MCF-7, and the action of E 2 -BSA can be abolished by PI3K inhibitor LY294002; treating MCF-7 simultaneously with PELP1-shRNA and LY294002 did not show synergetic inhibitory effect on E 2 -BSA-induced MMP-9 expression. Our results indicated that estrogen-induced MMP-9 expression in ER-positive breast cancer cells may be through PELP1-mediated PI3K/Akt signaling pathway.

Background: To investigate the expression and function of RSK4, MMP-9 and CD44 in primary clear cell renal cell carcinoma (primary ccRCC) and metastatic clear cell renal cell carcinoma (metastatic ccRCC), as well as the correlation with clinicopathological features of patients., Method: The expression levels of RSK4, CD44 and MMP-9 in 52 primary ccRCC samples and 48 metastatic ccRCC samples were detected by immunohistochemistry, and the relationship between RSK4, CD44 and MMP-9 expression and clinicopathological features as well as prognosis of metastatic ccRCC patients was statistically analysed. Ectopic RSK4 expression in ccRCC cell lines was performed to determine its effect on cell cycle regulation, tumour invasiveness, and metastatic capability., Results: The positive rates of RSK4, MMP-9 and CD44 expression in metastatic ccRCC tissues were 75, 68.75 and 91.7%, respectively, while the rates in primary ccRCC tissues were 44.2, 34.6 and 69.2%, respectively. Thus, the positive rates in metastatic ccRCC were higher than those in primary ccRCC (P RSK4  = 0. 002; P MMP-9  = 0. 002; P CD44  = 0. 001). However, the expression of RSK4, CD44 and MMP-9 was unrelated to age, gender, or metastatic sites (P > 0.05) but was related to WHO/ISUP nucleolar grade (P RSK4  = 0.019; P CD44  = 0.026; P MMP-9  = 0.049). In metastatic ccRCC, expression among the three proteins showed a positive correlation (P = 0.008). Moreover, expression between RSK4 and CD44 (P = 0.019) and MMP-9 and CD44 (P = 0.05) also showed positive correlations, whereas RSK4 and MMP-9 showed no significant correlation (P = 1.00). Molecular studies showed that overexpression of RSK4 could enhance the invasive and migratory abilities of ccRCC cell lines through the regulation of CD44 and MMP-9 expression and vice versa., Conclusions: The overexpression of RSK4, MMP-9 and CD44 is associated with the invasion and metastasis of ccRCC, indicating that they could be potential prognostic factors and serve as new potential therapeutic targets for ccRCC.

Purpose: To detect the expressions of CD74 and matrix metalloproteinase-9 (MMP-9) in colon adenocarcinomas, and to explore the relationship between the expressions and clinicopathological characteristics and prognosis., Methods: 98 cases of colon adenocarcinoma tissues from patients who underwent colon cancer resection in the Sixth Affiliated Hospital, Sun Yat-sen University from January 2013 to March 2015 comprised the experimental group, while 71 cases of colon mucosa tissues from patients who underwent colon polypectomy during the same period comprised the control group. qRT-PCR was used to detect the expressions of CD44 and MMP-9 mRNAs in the two groups, in order to analyze their correlation in colon adenocarcinomas, and to also analyze their relationship with clinicopathological characteristics and prognosis., Results: The expressions of CD74 and MMP-9 mRNAs in colon adenocarcinoma tissues were significantly higher than those in normal colon mucosa tissues (p<0.05). The expressions of CD74 and MMP-9 mRNAs had no significant relationship with the patient's gender, age, differentiation grade and tumor type in colon adenocarcinoma tissues (p>0.05), but had significant correlation with lymph node metastasis and pathological stage (p<0.05). According to the average expressions of CD74 and MMP-9 mRNAs, the patients were divided into low and high expression groups. The 3-year survival rate of patients in the low expression group was significantly higher than that in the high expression group (p<0.05). Moreover, the expressions of CD74 and MMP-9 were positively correlated (r = 0.853, p<0.001)., Conclusion: CD74 and MMP-9 are highly expressed in colon adenocarcinomas, and their expressions are closely related to the pathological stage, lymph node metastasis and prognosis of colon adenocarcinoma patients. Therefore, they can be used as important biological markers for diagnosis and prognosis prediction of colon adenocarcinoma.

Objective: To explore the role of estrogen and estrogen receptors in the migration of vascular smooth muscle cells in varicose lower-extremity veins., Patients and Methods: Tissue samples of normal lower extremity vein (56 cases) and varicose lower extremity vein (47 cases) were collected. Western blot and real-time fluorescent qPCR were performed to measure the expression of Estrogen receptor α (ERα) in tissues. Two cell co-culture systems were established for human umbilical vein endothelial cells (HUVECs) and human umbilical vein smooth muscle cells (HUVSMCs). One system was incubated under normal oxygen conditions (normal oxygen group), and the other was under oxygen-poor conditions (hypoxia group). The two systems were treated with 10-7 mM Estrogen E2, 10-7 mM BSA-conjugated Estrogen E2-BSA, 10-7 mM Estrogen E2+10-3 mM Tamoxifen (TAM), respectively for 24 h. The treated cells were subjected to cell scratch assay, transwell assay, and Western blot analysis of MMP2 and MMP9 protein expression., Results: The expression of ERα in varicose lower extremity vein was significantly up-regulated compared with that in normal lower extremity vein. The cell migration rate and the number of migrating cells in untreated hypoxia group and E2-treated normal oxygen group were comparable (p>0.05) to those in untreated normal oxygen group. The cell migration rate and the number of migrating cells were significantly increased (p<0.05) in E2-treated hypoxia group, compared with E2-treated normal oxygen group and untreated hypoxia group. The cell migration rate, the number of migrating cells, and expression levels of MMP2 and MMP9 were significantly decreased in E2/TAM-treated hypoxia group, compared with those in E2-treated hypoxia group., Conclusions: In summary, E2 can promote the migration of vascular smooth muscle cells and induce varicose veins of the lower extremities, which may be related to the promotion of MMP2 and MMP9 expression through the classical pathway of ER.

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.

Leucine aminopeptidase 3 is involved in the progression and metastasis of several cancers. This study aimed to screen anti-tumor lead compounds targeting leucine aminopeptidase 3. The compounds' suppression effect on enzyme activity and anti-tumor activity were evaluated through a series of assays. Leucine aminopeptidase 3 overexpression K562 cells were used as an enzyme source to screen 43 natural marine compounds. Compounds 5 and 6 exhibited high suppression effect on leucine aminopeptidase 3 activity. Cell activity tests indicated that both compounds have an anti-proliferative effect on triple-negative breast cancer cells. Wound healing assay and transwell invasion assay showed that both compounds could inhibit the migration and invasion of breast cancer cells. Immunoblot analysis exhibited that both compounds could downregulate the expression of metastasis-related proteins fascin and matrix metalloproteinase-2/9. A molecular dynamic simulation process was applied to discover the key features of compounds 5 and 6 in binding to leucine aminopeptidase 3 active site. This study described the anti-tumor effects of two leucine aminopeptidase 3 small molecule inhibitors. Taken together, compounds 5 and 6 could be used as anti-tumor lead compounds targeting leucine aminopeptidase 3.

Objective: To explore the mechanism underlying micro ribonucleic acid (miR)-21 in the invasion of rat aortic aneurysm cells in vitro by regulating matrix metalloproteinase (MMP)-2 and MMP-9., Materials and Methods: Rats were randomly divided into three groups: control group, model group, and miR-21 group. Real Time fluorescence quantitative Polymerase Chain Reaction (qRT-PCR) was adopted to detect the levels of miR-21 in each group of cells, transwell assay was performed to measure the effect of miR-21 on the invasion of aortic aneurysm cells. Western blotting was used to examine the expression of PTEN, which is the predicted target of miR-21 in aortic aneurysm cells, as well as the expressions of invasion-related proteases, MMP-2 and MMP-9., Results: The expression level of miR-21 in thoracic aortic aneurysm cells in model group was significantly higher than that in normal group (p<0.05), and that in miR-21 group was remarkably higher than that in model group (p<0.05). MiR-21 group had evidently more aortic aneurysm cells and stronger cell invasion ability than normal group and model group (p<0.05). In addition, the expression level of PTEN in model group was significantly higher than that in normal group (p<0.05), while that in miR-21 group notably declined compared to model group, (p<0.05). Compared with normal group and model group, the expressions of MMP-2 and MMP-9 were markedly increased in miR-21 group (p<0.05)., Conclusions: In aortic aneurysm cells of rats, miR-21 could suppress the expression of PTEN and activate MMP-2 and MMP-9 signals to promote the proliferation and migration of aortic aneurysm cells.

Chemokine (C-C Motif) ligand 21 (CCL21) plays an important role in tumor immunity. However, the molecular mechanisms by which CCL21 regulates tumor immunity remain largely unknown. In this study, we successfully generated a lentiviral vector expressing human CCL21 (Lenti-hCCL21), which was confirmed by biological assays. The Lenti-hCCL21 was transduced into PANC-1 cells, a chemokine (C-C motif) receptor 7 (CCR7)-positive human pancreatic cancer cell line. We used the scratch wound and transwell assays to measure cell migration of the CCL21-overexpressing PANC-1 cells. A DNA microarray assay was performed to determine gene expression profiles. The results showed that CCL21 lentiviral transduction significantly up- or down-regulated a panel of tumor-associated genes, although CCL21 appeared to have no effect on PANC-1 cell migration. Importantly, CCL21 promoted matrix metallopeptidase-9 (MMP-9) expression in PANC-1 cells. CCL21 regulates pancreatic cancer immunity possibly through governing the expression of a panel of tumor-associated genes, including MMP-9.

Inflammation and oxidative stress occurs in muscle of Duchenne muscular dystrophy (DMD). The relationship between a panel of biomarkers and the DMD outcome is necessary to indicate of disease progression and response to rehabilitation programs. The aim was to analyze the connective tissue of muscle of Mdx mice and immunoexpression of MMP-2, MMP-9, and 8-OHdG, which signalizes oxidative stress related to DNA damage. Biceps brachii of male C57BL/10 and C57BL/10-Dmd mdx mice was submitted to Hematoxylin-Eosin, Sirius red and immunohistochemistry (MMP-2, MMP-9 and 8-OHdG) analysis. Mdx showed focal lesions with intense inflammation and fibrosis related to immunoexpression of MMP-2 and MMP-9, proving the hypothesis that these MMPs are linked to muscular tissue degeneration, which can be regenerated by their inhibition, improving the treatment of DMD carriers. Histopathological findings related to centralized nuclei increase were related to higher 8-OHdG immunomarked nuclei in Mdx, which signalizes oxidative stress associated with DNA damage provoked by DMD. Such result shows that the evaluation of 8-OHdG during the evolution of the disease could be a method to evaluate DMD disease progression., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Perforating pilomatricoma (PP) is a rare clinical variant of pilomatricoma presenting as a crusted or ulcerated nodule. Previous reports have suggested that the tumor cells perforate the epidermis through a process of transepithelial elimination. Here, we report six cases of PP and examine the mechanism of transepithelial elimination in PP. Histologically, the dermis above or around the tumor nest exhibited edema, dilated vascular spaces, sparse collagen bundles and absence of elastic fibers, suggesting anetodermic changes in all cases. Immunohistochemistry demonstrated many CD68-positive macrophages around the tumor nests. Matrix metallopeptidase (MMP)-9 and MMP-12 were expressed in the inflammatory cells and tumor cells, and were also present in the epidermis and fibroblasts in all cases. We speculate that in PP anetodermic change caused by MMP and elastases including MMP-9 and MMP-12 may precede elimination of the tumor., (© 2019 Japanese Dermatological Association.)

Nonalcoholic fatty liver (NAFLD) may progress to nonalcoholic steatohepatitis (NASH) and ultimately to cirrhosis and hepatocellular carcinoma (HCC). Prognostic markers for these conditions are poorly defined. The aim of this study was to identify predictive gene markers for the transition from NAFL to NASH and then to poorer conditions. Gene expression omnibus datasets associated with a prediction analysis algorithm were used to create a matrix composed of control subject (n = 52), healthy obese (n = 51), obese with NAFL (n = 42) and NASH patients (n = 37) and 19,085 genes in order to identify specific genes predictive of the transition from steatosis to NASH and from NASH to cirrhosis and HCC and thus patients at high risk of complications. A validation cohort was used to validate these results. We identified two genes, fatty acid binding protein-4 (FABP4) and matrix metalloproteinase-9 (MMP9), which respectively allowed distinguishing patients at risk of progression from NAFL to NASH and from NASH to cirrhosis and HCC. Thus, NAFL patients expressing high hepatic levels of FABP4 and NASH patients expressing high hepatic levels of MMP9 are likely to experience disease progression. Therefore, using FABP4 and MMP9 as blood markers could help to predict poor outcomes and/or progression of NAFL during clinical trial follow-up.

Osteoarthritis (OA) is characterized by the progressive destruction of articular cartilage, which is involved in the imbalance between extracellular matrix (ECM) synthesis and degradation. MicroRNA-140-5p (miR-140) is specifically expressed in cartilage and plays an important role in OA-induced matrix degradation. The aim of this study was to investigate (1) whether intra-articular injection of melatonin could ameliorate surgically induced OA in mice and (2) whether melatonin could regulate matrix-degrading enzymes at the posttranscriptional level by targeting miR-140. In an in vitro OA environment induced by interleukin-1 beta (IL-1 β ), melatonin treatment improved cell proliferation of human chondrocytes, promoted the expression of cartilage ECM proteins (e.g., type II collagen and aggrecan), and inhibited the levels of IL-1 β -induced proteinases, such as matrix metalloproteinase 9 (MMP9), MMP13, ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4), and ADAMTS5. Both the microarray and polymerase chain reaction (PCR) experiments revealed that miR-140 was a melatonin-responsive microRNA and melatonin upregulated miR-140 expression, which was suppressed by IL-1 β stimulation. In vivo experiments demonstrated that intra-articular injection of melatonin prevented disruptions of cartilage matrix homeostasis and successfully alleviated the progression of surgery-induced OA in mice. Transfection of miR-140 antagomir completely counteracted the antiarthritic effects of melatonin by promoting matrix destruction. Our findings demonstrate that melatonin protects the articular cartilage from OA-induced degradation by targeting miR-140, and intra-articular administration of melatonin may benefit patients suffering from OA., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2019 Yijian Zhang et al.)

Among plant polyphenols, lemon peels extract (LPE) from the residues of the industrial processing of lemon ( Citrus limon ) shows anti-proliferative properties in cancer cells and anticholinesterase activity. In this study, we analyze the anti-cancer properties of LPE on migration and invasiveness in MKN-28 and AGS human gastric cancer cell lines either in the absence or presence of the pro-inflammatory cytokine IL-6. We find that the pretreatment with non-cytotoxic concentrations (0.5-1 μg/ml of gallic acid equivalent) of LPE inhibits interleukin-6 (IL-6)-induced cell migration and invasiveness in MKN-28 and AGS cells, as analyzed by wound and matrigel assays. Pretreatment with LPE is able to prevent either IL-6-induced matrix metalloproteinases (MMP)-9/2 activity, as assessed by gel zymography, or mRNA and protein MMP-9/2 expression, as evaluated by qPCR and Western blotting analysis, respectively. These LPE effects are associated with an IL-6-dependent STAT3 signaling pathway in MKN-28 and AGS cells. Furthermore, LPE shows acetylcholinesterase inhibitory activity when assayed by the Ellman method. In conclusion, our results demonstrate that LPE reduces the invasiveness of gastric MKN-28 and AGS cancer cells through the reduction of IL-6-induced MMP-9/2 up-regulation. Therefore, these data suggest that LPE exerts a protective role against the metastatic process in gastric cancer., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Objective: Granulomatosis with polyangiitis (GPA) is a multi-organ vasculitic syndrome typically associated with neutrophil extracellular trap (NET) formation and aggressive tissue inflammation. Manifestations in head and neck (H&N) GPA include septal perforations, saddle-nose deformities, bony erosions of the orbital and sinus walls, middle ear damage and epiglottitis, indicative of bone, cartilage, and connective tissue destruction. Whether H&N-centric lesions engage disease pathways distinctive from the ischemic tissue damage in the lungs, kidneys, skin, and peripheral nerves is unknown. We have compared inflammatory responses triggered by neutrophilic NETs in patients with H&N GPA and systemic GPA (sGPA). Methods: Neutrophils and monocytes were isolated from the peripheral blood of patients with H&N GPA, sGPA, and age/gender matched healthy individuals. Neutrophil NETosis was induced. NETs were isolated and cocultured with monocytes. Gene induction was quantified by RT-PCR, protein upregulation by flow cytometry. Tissue invasiveness of monocytes was measured in a 3D collagen matrix system. Expression of MMP-9 in tissue-residing macrophages was assessed by immunohistochemistry in tissue biopsies. Results: Neutrophils from H&N GPA patients showed more intense NETosis with higher frequencies of netting neutrophils ( P < 0.001) and release of higher amounts of NETs ( P < 0.001). Isolated NETs from H&N GPA functioned as an inducer of danger-associated molecular patterns in monocytes; specifically, alarmin S100A9. NET-induced upregulation of monocyte S100A9 required recognition of DNA. S100A9 release resulted in the induction of metalloproteinases, including MMP-9, and enabled monocytes to invade into extracellular matrix. Anti-MMP-9 treatment attenuated the tissue invasiveness of monocytes primed with NETs from H&N GPA patients. MMP-9-producing macrophages dominated the tissue infiltrates in naso-sinal biopsies from H&N GPA patients. Conclusion: Distinct disease patterns in GPA are associated with differences in NET formation and NET content. H&N GPA patients with midline cartilaginous and bony lesions are highly efficient in generating NETs. H&N GPA neutrophils trigger the induction of the alarmin S100A9, followed by production of MMP-9, endowing monocytes with tissue-invasive capabilities., (Copyright © 2019 Akiyama, Zeisbrich, Ibrahim, Ohtsuki, Berry, Hwang, Goronzy and Weyand.)

Arylamine N -acetyltransferase 1 (NAT1) is a drug-metabolizing enzyme that influences cancer cell proliferation and survival. However, the mechanism for these effects is unknown. Because of previous observations that NAT1 inhibition decreases invasiveness, we investigated the expression of the metalloproteinase matrix metalloproteinase 9 (MMP9) in human breast cancer samples and in cancer cells. We found a negative correlation between the expression of NAT1 and MMP9 in 1904 breast cancer samples. Moreover, when NAT1 was deleted in highly invasive breast cancer cells, MMP9 mRNA and protein significantly increased, both of which were reversed by reintroducing NAT1 into the knockout cells. After NAT1 deletion, there was an increased association of acetylated histone H3 with the SET and MYND-domain containing 3 (SMYD3) element in the MMP9 promoter, consistent with an increase in MMP9 transcription. NAT1 deletion also up-regulated hypoxia-inducible factor 1-α (HIF1- α ). Treatment of the NAT1 knockout cells with small interfering RNA directed toward HIF1- α mRNA inhibited the increased expression of MMP9. Taken together, these results show a direct inverse relationship between NAT1 and MMP9 and suggest that HIF1- α may be essential for the regulation of MMP9 expression by NAT1. SIGNIFICANCE STATEMENT: The expression of the enzyme NAT1 was found to be negatively correlated with MMP9 expression in tumor tissue from breast cancer patients. In cells, NAT1 regulated MMP9 expression at a transcriptional level via HIF1- α . This finding is important as it may explain some of the pathological features associated with changes in NAT1 expression in cancer., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

To investigate the matrix metalloproteinase (MMP)-9, (MMP)-13, E-Cadherin and Ki-67 expressions in road pavers' skin chronically exposed to bitumen products in order to contribute to a better understanding of the earlier tissue alteration. Skin punch biopsies from 16 daily exposed workers and a control group were studied by immunohistochemistry. Morphometric and densitometric analyses were also conducted. Morphological specimen evaluation of skin of road pavers showed epidermal thinning, flattening and loss of intercellular junction with a decreased expression of E-cadherin confined to the basal skin layer, together with MMP-9 and MMP-13 overexpressions in all epidermis layers, vascular structures and adnexa. No immunohistochemical alteration was reported for Ki-67 vs normal skin. Results from this study show that overexpression of MMP-9 and MMP-13 may represent an early response of the first human barrier to exposure to bitumen products. Regulation of MMPs could be one of the strategies to prevent primary skin disease.

Sulfur mustard (SM), a potent vesicating chemical warfare agent, and its analog nitrogen mustard (NM), are both strong bi-functional alkylating agents. Eyes, skin, and the respiratory system are the main targets of SM and NM exposure; however, ocular tissue is most sensitive, resulting in severe ocular injury. The mechanism of ocular injury from vesicating agents' exposure is not completely understood. To understand the injury mechanism from exposure to vesicating agents, NM has been previously employed in our toxicity studies on primary human corneal epithelial cells and ex vivo rabbit cornea organ culture model. In the current study, corneal toxicity from NM ocular exposure (1%) was analyzed for up to 28 days post-exposure in New Zealand White male rabbits to develop an acute corneal injury model. NM exposure led to conjunctival and eyelid swelling within a few hours after exposure, in addition to significant corneal opacity and ulceration. An increase in total corneal thickness and epithelial degradation was observed starting at day 3 post-NM exposure, which was maximal at day 14 post-exposure and did not resolve until 28 days post-exposure. There was an NM-induced increase in the number of blood vessels and inflammatory cells, and a decrease in keratocytes in the corneal stroma. NM exposure resulted in increased expression levels of cyclooxygenase-2, Interleukin-8, vascular endothelial growth factor and Matrix Metalloproteinase 9 indicating their involvement in NM-induced corneal injury. These clinical, biological, and molecular markers could be useful for the evaluation of acute corneal injury and to screen for therapies against NM- and SM-induced ocular injury., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: The aim of our study was to investigate the clinicopathologic values of the expression of CD44, matrix metalloproteinase (MMP)2, and MMP9 in renal cell carcinoma (RCC)., Patients and Methods: A total of 107 clear cell RCCs (ccRCCs) and 32 nonclear cell RCCs (non-ccRCCs) were examined for CD44, MMP2, and MMP9 expression by immunohistochemistry. The membrane and cytoplasmic expression levels of the 3 proteins were scored by semiquantitative methods, and the correlations of the 3 proteins with clinicopathological parameters were verified., Results: The expression levels of CD44, MMP2, and MMP9 were positively correlated with nuclear grade (grade 1-2 vs. grade 3-4) (P = 0.003, P < 0.001 and P < 0.001, respectively) in the ccRCCs, while in the non-ccRCCs, only CD44 expression was correlated with higher nuclear grade (grade 1-3 vs. grade 4) (P = 0.001). Furthermore, CD44 expression in ccRCCs and non-ccRCCs was correlated with shorter overall survival in the univariate analyses (P < 0.001 and P = 0.015, respectively). In the multivariate analysis, which accounted for age, sex, nuclear grade, and pathologic stage, CD44 expression was an independent predictor of shorter overall survival only in ccRCCs. Correlations among the 3 proteins were all positive in ccRCCs, but in non-ccRCCs, only MMP2 and MMP9 were positively correlated., Conclusion: CD44 expression may play an important role in the progression of both ccRCC and non-ccRCC. CD44 expression in ccRCC may be associated with elevated MMP2 and MMP9 expression levels, which is in contrast to non-ccRCC. The different correlations between CD44, MMP2, and MMP9 in ccRCC and non-ccRCC can be useful in understanding the mechanisms of carcinogenesis and stratifying patients for therapeutic purposes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Skin acts as a barrier, which protects internal tissues and promotes moisture retention. Atopic dermatitis (AD) is an inflammatory skin disease associated with a variety of genetic and environmental factors that involve helper T cells. β-Carotene (provitamin A) exhibits antioxidant activity and activates the immune system. However, it is not clear whether inflammation in AD skin is improved by posttreatment with β-carotene. In the current study, we investigated the effects of β-carotene on the skin of hairless mice with oxazolone-induced inflammation/oedema (Ox-AD mice). We found that skin inflammation was significantly reduced by oral administration of β-carotene. In addition, treatment with β-carotene suppressed protein levels of TNF-α, IL-1β and MCP-1, as well as mRNA expression associated with IL-1β, IL-6, IL-4 and Par-2 in skin tissues. Furthermore, the mRNA and protein levels of filaggrin, a structural protein in the epidermal stratum corneum, were elevated by β-carotene administration as compared with Ox-AD mice. β-Carotene significantly reduced the activity of proMMP-9, but not proMMP-2. These results suggest that in Ox-AD mice, β-carotene improves skin inflammation by suppressing the expression of inflammatory factors, promoting filaggrin expression and reducing MMP-9 activity. β-Carotene is a potent anti-inflammatory agent that improves the barrier functions of AD skin., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Atherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease., Design and Methods: The aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen., Results: Apo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites., Conclusions: These results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis.

Metastasis accounts for the majority of cancer-associated mortality and renders the targeted therapy fruitless in the patients of breast cancer. Matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF-C) are thought to be involved in tumor progression and metastasis. The aim of this study was to investigate the expression of MMP-9 and VEGF-C at both mRNA and protein levels in breast cancer and to correlate with lymph node metastasis and other clinicopathological characteristics. Biopsy specimens (N = 100) of breast cancer & benign breast disease (N = 100) were investigated for the mRNA expression of MMP-9 and VEGF-C by Real-time PCR and Protein expression by Western blot. Elevated levels of MMP-9 (p < 0.001) and VEGF-C (p < 0.001) expression were detected in breast cancer with corresponding to benign breast disease. Additionally, we found significantly increased levels of MMP-9 and VEGF-C in node-positive group with respect to node-negative group. Moreover, the levels of MMP-9 were significantly increased in larger tumor size (T3/T4) (p < 0.05) as compared to smaller size (T1/T2), which suggests that MMP-9 plays an important role in the progression of breast cancer. VEGF-C expression was associated with the TNM stage of tumor (p < 0.05). Further, a significant positive correlation was established between the mRNA levels of these two genes (p < 0.001). However, we could not obtain any significant correlation between expression of these genes with other clinicopathological parameters like tumor grade, age, menopausal status, and receptor status like ER, PR, and Her2. This study suggests that the high expression of MMP-9 and VEGF-C could act as markers for the tumor presence in breast cancer. In addition, this study recommends that expression of MMP-9 and VEGF-C was significantly associated with lymph node status and may provide valuable diagnosis of lymph node metastasis in breast cancer patients. Further, MMP-9 expression was associated with the tumor size and VEGF-C expression was correlated with the staging of the tumor, although no association was observed with other clinicopathological variables.