A Disintegrin And Metalloproteinase 8 Research Articles

A Disintegrin and Metalloproteinase Protein 8 (ADAM 8) in Autism Spectrum Disorder: Links to Neuroinflammation.

Converging lines of evidence confirmed neuroinflammation's role in autism spectrum disorder (ASD) etiological pathway. A disintegrin and metalloproteinase 8 (ADAM8) play major roles in inflammatory and allergic processes in various diseases. This study aimed to investigate ADAM8 plasma levels in autistic children compared to healthy controls. Also, to discover the association between ADAM8, disease severity, and neuroinflammation in ASD. This case-control study included children with ASD (n=40) and aged-matched healthy controls (n=40). The plasma levels of the ADAM 8 were determined using enzyme-linked immunosorbent assay (ELISA). The assessment of ASD severity and social and sensory behaviors were categorized as mild, moderate and severe. Correlations among ADAM8 plasma levels and ASD severity scores [Childhood Autism Rating Scale (CARS), Social Responsiveness Scale (SRS) and Short Sensory Profile (SSP)] were obtained by Spearman correlation coefficient (r). ASD children (n=40), including severe autism (n=21) and mild-to-moderate autism (n=19), showed significantly (p ≤ 0.05) lower plasma levels of ADAM8 [4683 (2885-5229); 4663 (4060-5000); 4632 (2885-5229)], respectively, than those of healthy controls [5000 (4047-5000)] [median (IQR) pg/mL]. However, there was no significant difference between the ADAM8 levels of children with severe and mild-to-moderate autism (p = 0.71). Moreover, ADAM8 plasma levels were not significantly correlated with the severity of ASD measured by behavioral scales [CARS (r= -0.11, p=0.55), SRS (r=0.11, p= 0.95), SSP (r=-0.23, p=0.23)]. The low ADAM8 plasma levels in children with ASD possibly indicated that ADAM8 might be implicated in the pathogenesis of ASD but not in the severity of the disease. These results should be interpreted with caution until additional studies are carried out with larger populations to decide whether the reduction in plasma ADAM8 levels is a mere consequence of ASD or if it plays a pathogenic role in the disease.

ALG3 Promotes Peritoneal Metastasis of Ovarian Cancer through Increasing Interaction of α1,3-mannosylated uPAR and ADAM8.

Peritoneal metastasis is the main cause of poor prognoses and high mortality in ovarian cancer patients. Abnormal protein glycosylation modification is associated with cancer malignancy. Elevated α1,3-mannosyltransferase 3 (ALG3), which catalyzes the α1,3-mannosylation of glycoproteins, has been found in some malignant tumors. However, the pathological significance of ALG3 and its regulatory mechanism in ovarian cancer metastasis is unclear. The results showed that the level of ALG3/α1,3-mannosylation was higher in human ovarian cancer tissues compared with normal ovarian tissues, as measured by Lectin chip, Western blot and Lectin blot analyses, as well as ovarian tissue microarray analysis. ALG3 was also correlated with the poor prognosis of ovarian cancer patients, according to survival analysis. The downregulation of ALG3 decreased the proliferation, stemness and peritoneal metastasis of ovarian cancer cells. The increase in urokinase plasminogen activator receptor (uPAR) α1,3-mannosylation catalyzed by ALG3 enhanced urokinase plasminogen activator (uPA)/uPAR activation and the interaction of uPAR with a disintegrin and metalloproteinase 8 (ADAM8), which promoted ovarian cancer peritoneal metastasis via the ADAM8/Ras/ERK pathway. Furthermore, decreased ALG3 suppressed ascites formation and the peritoneal metastasis of ovarian cancer cells in mice. This study highlights ALG3 as a potential diagnostic biomarker and prospective therapeutic target for ovarian cancer.

Correlation between gingival crevicular fluid levels of a disintegrin and metalloproteinase 8 and periodontal disease

Background: Periodontal disease is an inflammation caused by host parasite interaction leading to the destruction of tooth supporting structures. During inflammation there is an elevated levels of biochemical signalling molecules. A disintegrin and metalloproteinase 8 (ADAM8) molecule has been implicated in various physiologic functions and pathologies. The expression of ADAM8 is upregulated in inflammatory conditions, and with upregulated expression they play a pivotal role in inflammation, immunity and osteoclastogenesis. Objective: To evaluate ADAM8 levels in gingival crevicular fluid (GCF) collected from healthy individuals and individuals with periodontal disease at baseline and in the periodontal disease group after nonsurgical periodontal therapy. Methods: In total, 48 subjects aged 20–65 years were divided into separate groups: a periodontally healthy group (group 1: males, n = 7, females, n = 9), a chronic gingivitis group (group 2: males, n = 6, females, n = 10), and a chronic periodontitis group (group 3: males, n = 8, females, n = 8). GCF samples were collected from all the groups at baseline and 3 months after nonsurgical periodontal therapy in the chronic gingivitis (group 4) and chronic periodontitis (group 5) cases. Statistical analysis was performed using Shapiro-wilk normality test, One way ANOVA test, Kruskal-Wallis test followed by Mann-Whitney U test, Spearman’s rank correlation test and multiple linear regression analysis was done. The level of significance was determined at P < 0.05. Results: ADAM8 levels in the gingival crevicular fluid was significantly higher in group 3 (26,416.25 ± 7,817.59) than groups 1 and 2 at baseline at P < 0.001. After non-surgical periodontal therapy, ADAM8 levels in the gingival crevicular fluid was significantly reduced for group 2 (13,186.88 ± 3,247.62) and group 3 (18,375.63 ± 3,339.07) at P < 0.001. Conclusion: ADAM8 levels were increased in chronic gingivitis (group 2) and chronic periodontitis group (group 3) and reduced after non-surgical periodontal therapy in groups 2 and 3.

Suppression of ADAM8 attenuates angiotensin II-induced cardiac fibrosis and endothelial-mesenchymal transition via inhibiting TGF-β1/Smad2/Smad3 pathways

Endothelial-to-mesenchymal transition (EndMT) is involved in cardiac fibrosis induced by angiotensin II (Ang II). A disintegrin and metalloproteinase 8 (ADAM8), a member of ADAMs family, participates in cell adhesion, proteolysis and various signaling. However, its effects on the development of cardiac fibrosis remain completely unknown. This study aimed to reveal whether ADAM8 aggravates cardiac fibrosis induced by Ang II in vivo and in vitro. The C57BL/6J mice or cardiac endothelial cells were subjected to Ang II infusion to induce fibrosis. The results showed that systolic blood pressure and diastolic blood pressure were significantly increased under Ang II infusion, and ADAM8 was up-regulated. ADAM8 inhibition attenuated Ang II-induced cardiac dysfunction. ADAM8 knockdown suppressed Ang II-induced cardiac fibrosis as evidenced by the down-regulation of CTGF, collagen I, and collagen III. In addition, the endothelial marker (VE-cadherin) was decreased, whilst mesenchymal markers (α-SMA and FSP1) were increased following Ang II infusion. However, ADAM8 repression inhibited Ang II-induced EndMT. Moreover, ADAM8 silencing repressed the activation of TGF-β1/Smad2/Smad3 pathways. Consistent with the results in vivo, we also found the inhibitory effects of ADAM8 inhibition on EndMT in vitro. All data suggest that ADAM8 promotes Ang II-induced cardiac fibrosis and EndMT via activating TGF-β1/Smad2/Smad3 pathways.

Propofol mediates pancreatic cancer cell activity through the repression of ADAM8 via SP1.

Propofol is a commonly used anesthetic with controversial effects on cancer cells. A growing number of studies have demonstrated that low concentrations of propofol are associated with tumor suppression and when used as an intravenous anesthesia improved recurrence‑free survival rates for many cancers, but deeper insights into its underlying mechanism are needed. The study detailed herein focused upon the effect of propofol on pancreatic cancer cells and the mechanism by which propofol reduces A disintegrin and metalloproteinase8(ADAM8) expression. The ability of propofol to impact the proliferation, migration and cell cycle of pancreatic cancer cell lines was assessed invitro. This was mechanistically explored following the identification of SP1 binding sites within ADAM8, which enabled the regulatory effects of specificity protein1(SP1) on ADAM8 following propofol treatment to be further explored. Ultimately, this study was able to show that propofol significantly inhibited the proliferation, migration and invasion of pancreatic cancer cells and decreased the percentage of cells in S‑phase. Propofol treatment was also shown to repress ADAM8 and SP1 expression, but was unable to affect ADAM8 expression following knockdown of SP1. Moreover, a direct physical interaction between SP1 and ADAM8 was verified using co‑immunoprecipitation and dual‑luciferase reporter assays. Cumulatively, these results suggest that propofol represses pathological biological behaviors associated with pancreatic cancer cells through the suppression of SP1, which in turn results in lower ADAM8 mRNA expression and protein levels.

A disintegrin and metalloproteinase 8 induced epithelial‐mesenchymal transition to promote the invasion of colon cancer cells via TGF‐β/Smad2/3 signalling pathway

A disintegrin and metalloproteinase 8 (ADAM8) protein is a multi‐domain transmembrane glycoprotein which involves in extracellular matrix remodelling, cell adhesion, invasion and migration. ADAM8 and epithelial‐mesenchymal transition (EMT) play an important role in tumour invasion has been well established. However, the interaction between ADAM8 and EMT has remained unclear. The data of colon cancer patients obtained from TCGA (The Cancer Genome Atlas) and GTEx (Genotype‐Tissue Expression Project) were analysed by the bioinformatics research method. The expression of ADAM8 in colon cancer cells was up‐regulated and down‐regulated by transfecting with the expression plasmid and small interfering RNA, respectively. Transwell invasion assay, immunohistochemistry, immunocytochemistry, Western blotting and qRT‐PCR were utilized to study the effect of ADAM8 on colon cancer cell's EMT and its related mechanisms. Analysis of TCGA and GTEx data revealed that ADAM8 was linked to poor overall survival in colon cancer patients. Besides, ADAM8 was correlated with multiple EMT biomarkers (E‐cadherin, N‐cadherin, Vimentin, Snail2 and ZEB2). In vitro, we also proved that the up‐regulation of ADAM8 could promote EMT effect and enhance the invasive ability of colon cancer cells. On the contrary, the down‐regulation of ADAM8 in colon cancer cells attenuated these effects above. Further studies suggested that ADAM8 modulated EMT on colon cancer cells through TGF‐β/Smad2/3 signalling pathway. Our research suggested that ADAM8 could be a potential biomarker for the prognosis of colon cancer and induced EMT to promote the invasion of colon cancer cells via activating TGF‐β/Smad2/3 signalling pathway.

Propofol inhibits pancreatic cancer proliferation and metastasis by up-regulating miR-328 and down-regulating ADAM8.

Propofol is commonly used for anaesthesia during surgery, and accumulating evidence has demonstrated that propofol is associated with tumour suppression. For example, propofol down-regulates the expression of vascular endothelial growth factor to inhibit pancreatic cancer malignancy. However, deeper insights into its underlying mechanism are needed. In this study, we treated pancreatic cell lines Panc1 and Bxpc3 with or without propofol. Cell proliferation, migration and invasion were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and transwell assays. Real-time polymerase chain reaction was used to measure RNA expression levels. Luciferase assay was performed to determine the transcriptional activity of microRNAs (miRNAs). We found that propofol significantly reduced the proliferation, migration and invasion of pancreatic cancer cells compared to untreated cells. By testing the changes in miRNAs after propofol treatment, propofol was shown to strikingly enhance the expression of miR-328. Luciferase assays demonstrated that propofol repressed the transcriptional activity of miR-328, while a disintegrin and metalloproteinase 8 (ADAM8) was a direct target of miR-328. Knockdown of miR-328 or ADAM8 led to significantly decreased cell growth and viability. Our results implicate that propofol inhibits pancreatic cancer growth and metastasis by enhancing miR-328 which targets ADAM8.

Reduced ADAM8 levels upon non-surgical periodontal therapy in patients with chronic periodontitis

ObjectiveTo determine effect of non-surgical periodontal treatment on a disintegrin and metalloproteinase 8 (ADAM8) levels in gingival crevicular fluid (GCF) of patients with chronic periodontitis (CP) in comparison with those of patients with gingivitis and to find correlations between ADAM8 levels and clinical parameters. DesignTwenty-two and eleven patients with CP and gingivitis, respectively, were examined for four clinical parameters, probing depth, clinical attachment level, gingival and plaque indices. GCF from the selected gingivitis or periodontitis sites with distinct severities was sampled by Periopaper strips. The non-surgical treatments, including scaling and/or root planing and oral hygiene instruction, were provided for all patients. Clinical measurements and GCF sampling were repeated at three months after the treatments. ADAM8 concentrations were analyzed by ELISA and normalized by GCF volumes or total protein amounts. ResultsAll patients exhibited significant improvement of almost every clinical parameter after treatment, whereas the median ADAM8 concentrations were significantly decreased at the moderate and severe periodontitis sites of patients with CP (p < 0.05). Moreover, the significantly positive correlations between ADAM8 concentrations and four clinical parameters were found in both moderate and severe groups (p < 0.05). ConclusionADAM8 concentrations were decreased by non-surgical periodontal therapy in patients with chronic periodontitis at the moderate and severe sites and were correlated with four clinical parameters, implying that GCF ADAM8 levels reflect inflammatory and bone-resorbing activities in the periodontal pocket.

ADAM8 localizes to extravillous trophoblasts within the maternal-fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism.

Does A Disintegrin And Metalloproteinase 8 (ADAM8) control extravillous trophoblast (EVT) differentiation and migration in early human placental development? ADAM8 mRNA preferentially localizes to invasive HLA-G-positive trophoblasts, associates with the acquirement of an EVT phenotype and promotes trophoblast migration through a mechanism requiring β1-integrin. Placental establishment in the first trimester of pregnancy requires the differentiation of progenitor trophoblasts into invasive EVTs that produce a diverse repertoire of proteases that facilitate matrix remodeling and activation of signaling pathways important in controlling cell migration. While multiple ADAM proteases, including ADAM8, are highly expressed by invasive trophoblasts, the role of ADAM8 in controlling EVT-related processes is unknown. First trimester placental villi and decidua (6-12 weeks' gestation), primary trophoblasts and trophoblastic cell lines (JEG3, JAR, Bewo, HTR8/SVNeo) were used to examine ADAM8 expression, localization and function. All experiments were performed on at least three independent occasions (n = 3). Placental villi and primary trophoblasts derived from IRB approved first trimester placental (n = 24) and decidual (n = 4) were used to examine ADAM8 localization and expression by in situ RNAScope hybridization, flow cytometry, quantitative PCR and immunoblot analyses. Primary trophoblasts were differentiated into EVT-like cells by plating on fibronectin and were assessed by immunofluorescence microscopy and immunoblot analysis of keratin-7, vimentin, epidermal growth factor receptor (EGFR), HLA-G and ADAM8. ADAM8 function was examined in primary EVTs and trophoblastic cell lines utilizing siRNA-directed silencing and over-expression strategies. Trophoblast migration was assessed using Transwell chambers, cell-matrix binding was tested using fibronectin-adhesion assays, and ADAM8-β1-integrin interactions were determined by immunofluorescence microscopy, co-immunoprecipitation experiments and function-promoting/inhibiting antibodies. Within first trimester placental tissues, ADAM8 preferentially localized to HLA-G+ trophoblasts residing within anchoring columns and decidua. Functional experiments in primary trophoblasts and trophoblastic cell lines show that ADAM8 promotes trophoblast migration through a mechanism independent of intrinsic protease activity. We show that ADAM8 localizes to peri-nuclear and cell-membrane actin-rich structures during cell-matrix attachment and promotes trophoblast binding to fibronectin matrix. Moreover, ADAM8 potentiates β1-integrin activation and promotes cell migration through a mechanism dependent on β1-integrin function. The primary limitation of this study was the use of in vitro experiments in examining ADAM8 function, as well as the implementation of immortalized trophoblastic cell lines. Histological localization of ADAM8 within placental and decidual tissue sections was limited to mRNA level analysis. Further, patient information corresponding to tissues obtained by elective terminations was not available. The novel non-proteolytic pro-migratory role for ADAM8 in controlling trophoblast migration revealed by this study sheds insight into the importance of ADAM8 in EVT biology and placental development. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC-Discovery Grant) and the Canadian Institutes of Health Research (CIHR-Open Operating Grant). There are no conflicts or competing interests. NA.

Inducible expression of A Disintegrin and Metalloproteinase 8 in chronic periodontitis and gingival epithelial cells.

The expression of A Disintegrin and Metalloproteinase 8 (ADAM8) is associated with several inflammatory diseases. Elevated ADAM8 levels have been shown in gingival crevicular fluid of patients with chronic periodontitis. The objective of this study was to investigate ADAM8 expression in chronic periodontitis tissues compared with that in normal tissues. ADAM8 expression and its inductive mechanism were examined in human gingival epithelial cells (HGECs) and human gingival fibroblasts. Total RNA and protein were extracted from gingival biopsies of 33 patients with chronic periodontitis and those of 23 healthy volunteers. ADAM8 mRNA and protein expression was analyzed by real-time polymerase chain reaction, immunoblotting and immunohistochemistry. ADAM8 expression in control and stimulated cells in the presence or absence of specific inhibitors for mitogen-activated protein kinase pathways was assayed by real-time polymerase chain reaction, immunoblotting, flow cytometry and immunofluorescence. ADAM8 mRNA and protein expression in chronic periodontitis tissues was significantly greater than that in normal tissues (p < 0.01). Significantly increased ADAM8 expression was detected in the gingival epithelium of chronic periodontitis tissues (p < 0.001). ADAM8 mRNA expression in HGECs, but not in human gingival fibroblasts, was significantly induced by stimulation with Fusobacterium nucleatum (p < 0.05), partially via the p44/42 mitogen-activated protein kinase pathway. ADAM8 expression in the cell lysates and on the surface of HGECs was induced by stimulation with F. nucleatum. ADAM8 expression is increased in inflamed chronic periodontitis tissues and localized within gingival epithelium, consistent with an upregulation of ADAM8 expression in F. nucleatum-stimulated HGECs, suggesting a possible role of ADAM8 in innate immunity of periodontal tissue.

A novel peptide ADAM8 inhibitor attenuates bronchial hyperresponsiveness and Th2 cytokine mediated inflammation of murine asthmatic models.

A disintegrin and metalloproteinase 8 (ADAM8) has been identified as a signature gene associated with moderate and severe asthma. Studies in mice have demonstrated that the severity of asthma can be reduced by either transgenic knock-out or by antibodies blocking ADAM8 function, highlighting ADAM8 as potential drug target for asthma therapy. Here, we examined the therapeutic effect of an ADAM8 inhibitor peptide (BK-1361) that specifically blocks cellular ADAM8 activity in ovalbumin-sensitized and challenged Balb/c mice. We found that BK-1361 (25 μg/g body weight) attenuated airway responsiveness to methacholine stimulation by up to 42%, concomitantly reduced tissue remodeling by 50%, and decreased inflammatory cells (e.g. eosinophils down by 54%)/inflammatory factors (e.g. sCD23 down by 50%)/TH2 cytokines (e.g. IL-5 down by 70%)/ADAM8-positive eosinophils (down by 60%) in the lung. We further verified that BK-1361 specifically targets ADAM8 in vivo as the peptide caused significantly reduced levels of soluble CD23 in wild-type but not in ADAM8-deficient mice. These findings suggest that BK-1361 blocks ADAM8-dependent asthma effects in vivo by inhibiting infiltration of eosinophils and TH2 lymphocytes, thus leading to reduction of TH2-mediated inflammation, tissue remodeling and bronchial hyperresponsiveness. Taken together, pharmacological ADAM8 inhibition appears as promising novel therapeutic strategy for the treatment of asthma.

Targeted inhibition of GATA-6 attenuates airway inflammation and remodeling by regulating caveolin-1 through TLR2/MyD88/NF-κB in murine model of asthma.

The purpose of this study was to evaluate the effects of GATA-6 on airway inflammation and remodeling and the underlying mechanisms in a murine model of chronic asthma. Female BALB/c mice were randomly divided into four groups: phosphate-buffered saline control (PBS), ovalbumin (OVA)-induced asthma group (OVA), OVA+ siNC and OVA+ siGATA-6. In this mice model, GATA-6 expression level was significantly elevated and the expression in Caveolin-1 (Cav-1) inversely correlated with the abundance of GATA-6 in OVA-induced asthma of mice. Silencing of GATA-6 gene expression upregulated Cav-1 expression. Additionally, downregulation of GATA-6 dramatically decreased OVA-challenged inflammation, infiltration, and mucus production. Moreover, silencing of GATA-6 resulted in decreased levels of immunoglobulin E (IgE) and inflammatory mediators and reduced inflammatory cell accumulation, as well as inhibiting the expression of important mediators including matrix metalloproteinase (MMP)-2 and MMP-9, TGF-β1, and a disintegrin and metalloproteinase 8 (ADAM8) and ADAM33, which is related to airway remodeling. Further analysis confirmed that silencing of GATA-6 attenuated OVA-induced airway inflammation and remodeling through the TLR2/MyD88 and NF-κB pathway. In conclusion, these findings indicated that the downregulation of GATA-6 effectively inhibited airway inflammation and reversed airway remodeling via Cav-1, at least in part through downregulation of TLR2/MyD88/NF-κB, which suggests that GATA-6 represents a promising therapeutic strategy for human allergic asthma.

MiR-720 is a downstream target of an ADAM8-induced ERK signaling cascade that promotes the migratory and invasive phenotype of triple-negative breast cancer cells.

BackgroundADAM8 (a disintegrin and metalloproteinase 8) protein promotes the invasive and metastatic phenotype of triple-negative breast cancer (TNBC) cells. High ADAM8 expression in breast cancer patients is an independent predictor of poor prognosis. Here, we investigated whether ADAM8 regulates specific miRNAs, their roles in aggressive phenotype, and potential use as biomarkers of disease.MethodsMicroarray analysis was performed on RNA from MDA-MB-231 cells after transient ADAM8 knockdown using TaqMan miRNA cards. Changes in miRNA levels were confirmed using two ADAM8 siRNAs in TNBC cell lines. Kinase inhibitors, β1-integrin antagonist antibody, and different forms of ADAM8 were employed to elucidate the signaling pathway required for miR-720 expression. miR-720 levels were modulated using a specific antagomiR or a mimic, and effects on aggressive phenotype of TNBC cells were determined using Boyden chamber and 3D-Matrigel outgrowth assays. Plasma was isolated from mice before and after implantation of MDA-MB-231 cells and analyzed for miR-720 levels. Serum samples of TNBC patients were evaluated for their ADAM8 and miR-720 levels.ResultsWe identified 68 miRNAs differentially regulated upon ADAM8 knockdown, including decreased levels of secreted miR-720. Ectopic overexpression of wild-type ADAM8 or forms that lack metalloproteinase activity similarly induced miR-720 levels. The disintegrin and cysteine-rich domains of ADAM8 were shown to induce miR-720 via activation of a β1-integrin to ERK signaling cascade. Knockdown of miR-720 led to a significant decrease in migratory and invasive abilities of TNBC cells. Conversely, miR-720 overexpression rescued these properties. A profound increase in plasma levels of miR-720 was detected 7 days after TNBC cell inoculation into mouse mammary fat pads when tumors were barely palpable. Concordantly, miR-720 levels were found to be significantly higher in serum samples of TNBC patients with high ADAM8 expression.ConclusionsWe have shown for the first time that miR-720 is induced by ADAM8 signaling via ERK and plays an essential role in promoting the aggressive phenotype of TNBCs. miR-720 is elevated in serum of patients with ADAM8-high TNBC and, in a group with other miRNAs downstream of ADAM8, holds promise as a biomarker for early detection of or treatment response of ADAM8-positive TNBCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0699-z) contains supplementary material, which is available to authorized users.

A colorimetric-based amplification system for proteinases including MMP2 and ADAM8

We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity.

The important role of ADAM8 in the progression of hepatocellular carcinoma induced by diethylnitrosamine in mice.

This study focuses on investigating the concrete role of a disintegrin and metalloproteinase 8 (ADAM8) in the progression of hepatocellular carcinoma (HCC). Mice received anti-ADAM8 monoclonal antibody (mAb) of 100 μg/100 μl, 200 μg/100 μl or 300 μg/100 μl, respectively, in phosphate-buffered saline (PBS) or PBS intervention during the progression of HCC induced by diethylnitrosamine. The survival rate, body weight, and relative liver weight were determined in the mice. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and α-fetoprotein (AFP) level, hematoxylin-eosin staining, the expression level of vascular endothelial growth factor A (VEGF-A), proliferating cell nuclear antigen (PCNA), caspase 3 (Casp3), B cell leukemia 2 (Bcl2), B cell leukemia 2-associated X protein (Bax), protein p53 (P53), and ADAM8 were detected in the mice at the end of the 24th week. Our results showed that anti-ADAM8 mAb intervention effectively improved the survival rate, reduced the body weight loss and increased the relative liver weight in mice in a dose-dependent manner (p < 0.05 or p < 0.01). Anti-ADAM8 mAb intervention also significantly lowered serum AST, ALT, and AFP levels (p < 0.05 or p < 0.01), slowed the progression of HCC (p < 0.05 or p < 0.01), induced the expression of Casp3, Bax, and P53 (p < 0.05 or p < 0.01), and inhibited the expression of VEGF-A, PCNA, and Bcl2 in the liver of mice (p < 0.05 or p < 0.01) in a dose-dependent manner compared with the mice receiving PBS intervention. Our study suggested that ADAM8 might promote the progression of HCC by regulating the expression of these factors. Anti-ADAM8 mAb intervention might be suitable as a potential method for HCC therapy.

Comparative analysis of gingival crevicular fluid a disintegrin and metalloproteinase 8 levels in health and periodontal disease: A clinic-biochemical study.

Aim and Background:A disintegrin and metalloproteinase 8 (ADAM8) is a marker belonging to the class of ADAM family of metalloproteinase which is found to be involved in inflammation and bone resorption in periodontal disease by acting as osteoclast stimulating factor. In several systemic inflammatory diseases, elevated levels of ADAM8 are detected in human serum and other body fluids. Recently, ADAM8 was even detected in gingival crevicular fluid (GCF) of patients with periodontal diseases. Hence, the aim of the study was to estimate the levels of ADAM8 in GCF of healthy and chronic periodontitis subjects.Materials and Methods:Periodontal examination and collection of GCF by the extracrevicular method was performed in 30 subjects selected randomly and categorized into two groups. Group I (healthy, n = 15) and Group II (chronic periodontitis, n = 15). ADAM8 levels in GCF were estimated by enzyme-linked immunosorbent assay.Results:ADAM8 was detected in both Group I and II. Highest mean ADAM8 concentration was obtained for Group II, whereas the lowest concentration was seen in Group I. This suggests that ADAM8 levels increase proportionally with the progression of periodontal disease. There was a significant correlation between ADAM8 levels and clinical parameters in the study group.Conclusion:The results of our study indicate that the ADAM8 levels in GCF are positively associated with periodontal disease, which may provide a useful tool in monitoring its progression. Nevertheless, further longitudinal studies are required with larger sample sizes in which ADAM8 levels are progressively estimated and compared to baseline values.

N-Glycosylation Regulates ADAM8 Processing and Activation

The transmembrane ADAM8 (A Disintegrin And Metalloproteinase 8) protein is abundantly expressed in human breast tumors and derived metastases compared with normal breast tissue, and plays critical roles in aggressive Triple-Negative breast cancers (TNBCs). During ADAM8 maturation, the inactive proform dimerizes or multimerizes and autocatalytically removes the prodomain leading to the formation of the active, processed form. ADAM8 is a glycoprotein; however, little was known about the structure or functional role of these sugar moieties. Here, we report that in estrogen receptor (ER)α-negative, but not -positive, breast cancer cells ADAM8 contains N-glycosylation, which is required for its correct processing and activation. Consistently ADAM8 dimers were detected on the surface of ERα-negative breast cancer cells but not on ERα-positive ones. Site-directed mutagenesis confirmed four N-glycosylazhytion sites (Asn-67, Asn-91, Asn-436, and Asn-612) in human ADAM8. The Asn-67 and Asn-91 prodomain sites contained high mannose, whereas complex type N-glycosylation was observed on Asn-436 and Asn-612 in the active and remnant forms. The Asn-91 and Asn-612 sites were essential for its correct processing and cell surface localization, in particular its exit from the Golgi and endoplasmic reticulum, respectively. The N436Q mutation led to decreased ADAM8 stability due to enhanced lysosomal degradation. In contrast, mutation of the Asn-67 site had only modest effects on enzyme stability and processing. Thus, N-glycosylation is essential for processing, localization, stability, and activity of ADAM8.

Anti-inflammatory macrophages activate invasion in pancreatic adenocarcinoma by increasing the MMP9 and ADAM8 expression

Patients with chronic pancreatitis with local inflammation have high risk for pancreatic cancer. The aim of this study was to examine the role of the inflammatory cells in the invasion of pancreatic cancer cells, focusing on the involvement of a disintegrin and metalloproteinase 8 (ADAM8) and matrix metalloproteinase 9 (MMP9) proteins. ADAM8 expression is associated with worse survival of pancreatic cancer patients. Monocytes from healthy donors were differentiated into macrophages. Pancreatic adenocarcinoma cells were cultured either alone or with differentiated macrophages. The cancer cell migration rate in Matrigel was measured by imaging fluorescently stained cells for 24 h. After invasion, cells were sorted into CD14 positive/negative macrophages and cancer cells with magnetic separation. The expression of ADAM8 and MMP9 was measured by the real-time PCR. Protein-level expression of ADAM8 and MMP9 was analyzed by Western blotting. In two series, siRNA technique was used to reduce either ADAM8 or MMP9 expression in the cancer cells. The coculture with macrophages increased cancer cell migration rate in Matrigel, and increased ADAM8 and MMP9 mRNA expression and protein level in the cancer cells. Reduction of ADAM8 expression with siRNA in the cancer cells decreased macrophage-induced migration rate of the cancer cells from 11.7±0.3 μm/h to 9.0±0.2 μm/h (p<0.01), and reduction of MMP9 expression decreased the migration rate to 10.1±0.2 μm/h (p<0.01). Anti-inflammatory macrophages increase pancreatic cancer cell migration rate in basement membrane matrix by inducing ADAM8 and MMP9 expression in cancer cells, thereby possibly enhancing the invasiveness of cancer.

Abstract B166: MicroRNAs downstream of ADAM8 as therapeutic targets and non-invasive biomarkers for triple-negative breast cancer.

Abstract Triple-negative breast cancer (TNBC) represents ∼20% of breast cancers, is highly aggressive and lacks targeted therapies due to the absence of hormone and HER2 receptors. TNBCs respond only poorly to chemotherapy and recur more frequently than other breast cancers. Their recurrence often involves distant metastasis, especially to the brain and lungs with a poor prognosis, which lead to approximately 25% of the half million yearly deaths worldwide from breast cancer. Thus, there is a critical unmet need for early detection and new therapeutic targets for TNBC treatment, which was the focus of our current study. Recently, our lab has shown that the transmembrane protein ADAM8 (A Disintegrin And Metalloproteinase 8) plays a key role in invasion and metastasis of TNBCs and high ADAM8 levels correlate with a poor prognosis in breast cancer patients. MicroRNAs (miRNAs) play important functions in both normal and disease development. It is estimated that more than 60% of the protein-coding genes in the human genome are regulated by miRNAs. Furthermore, circulating tumor cell-associated miRNAs and free circulating miRNAs have been identified in whole blood, plasma and serum in various cancer subtypes. Here we tested two related hypotheses, that: (1) miRNAs regulated by the ADAM8 pathway are important mediators promoting invasion and migration of TNBC cells; and (2) these miRNAs represent potential therapeutic targets and/or noninvasive biomarkers for detection of TNBC recurrence. A microarray analysis was performed using TaqMan microRNA cards on RNA isolated from MDA-MB-231 cells after 72 h of transient siRNA-mediated knockdown of ADAM8. We identified 68 miRNAs that were differentially regulated upon ADAM8 knockdown. Thus far we have confirmed 13 miRNAs using real-time PCR. Of the miRNAs confirmed, miR-720 was selected as the initial candidate of study, since it has been previously identified as an oncomiR and also detected in blood from breast cancer patients. Transient ectopic ADAM8 overexpression in MDA-MB-231 and SUM149 TNBC cell lines induced levels of miR-720 whereas ADAM8 siRNA reduced them. Furthermore, knockdown of miR-720 using an antagomiR led to a significant decrease of the migratory and invasive phenotype of TNBC cells as judged by Boyden chamber and 3D Matrigel outgrowth assays, respectively. Consistently, we used a miRNA mimic to overexpress miR-720 activity and observed increased migration and invasion of TNBC cells. Importantly, a profound increase in levels of miR-720 was detected in the plasma of mice bearing orthotopic mammary tumors derived from control shRNA-MDA-MB-231 cells in contrast to shADAM8-MDA-MB-231 cells and non-tumor bearing mice, which suggests that miR-720 levels in blood correlate with ADAM8 expression in tumors. Taken together our data indicates that the miR-720 is induced by ADAM8 and miRNAs regulated downstream of ADAM8 have a strong potential as both biomarkers and therapeutic targets for the treatment of TNBC. Research was funded by grants from the DOD (W81XWH-11-1-0814) and the NIH (R01 CA129129) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B166. Citation Format: Sonia G. Das, Mathilde Romagnoli, Nora D. Mineva, Gail E. Sonenshein. MicroRNAs downstream of ADAM8 as therapeutic targets and non-invasive biomarkers for triple-negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B166.

Elevated Levels of a Disintegrin and Metalloproteinase 8 in Gingival Crevicular Fluid of Patients With Periodontal Diseases

A disintegrin and metalloproteinase 8 (ADAM8) is involved in inflammation and is essential for osteoclastogenesis. Elevated ADAM8 levels are detected in human serum and other body fluids in several inflammatory conditions. Therefore, we hypothesized that ADAM8 levels are also raised in gingival crevicular fluid (GCF) of patients with periodontal diseases. Forty-five patients with periodontal diseases (n = 15 for each group: the group of patients with gingivitis, the group with aggressive periodontitis [AgP], and the group with chronic periodontitis [CP]) and 15 volunteers who exhibited healthy gingiva were recruited. Four periodontal parameters, gingival index, plaque index, probing depth, and clinical attachment level, were recorded before GCF collection. The presence of ADAM8 in GCF was shown by immunoblotting using anti-human ADAM8 polyclonal antibody against its prodomain, and the ADAM8 levels were measured by an enzyme-linked immunosorbent assay. Four immunoreactive bands at 120, 70, 50, and <30 kDa were detected in the groups of patients with periodontitis, whose intensities were stronger than those in the group of patients with gingivitis, consistent with significantly greater ADAM8 levels in both groups of patients, with either CP or AgP, than those in the group of patients with gingivitis and in the group that was healthy (P <0.001). Moreover, the ADAM8 levels correlated significantly with the four periodontal parameters (P <0.001), indicating that ADAM8 levels are positively associated with the degree of periodontal tissue inflammation and destruction. The ADAM8 levels are elevated in the GCF of patients with periodontal diseases, including gingivitis, CP, and AgP, in comparison to control participants who are healthy, and they correlate with four clinical parameters that reflect the degree of disease severity.