Proteases are important molecules that are involved in many physiological and pathological processes of the human body, such as growth, apoptosis and metastasis cancer cells. They are potential targets in cancer diagnosis and biotherapy. In this study, we analyzed the salivary protease spectrum of patients with oral squamous cell carcinoma (OSCC), oral benign masses and chronic periodontitis, as well as that of health, using human protease array kits, enzyme-linked immunosorbent assay, western blot and immunofluorescence. The salivary protease spectrum was found to be associated with oral diseases. For example, the saliva of patients with OSCC contained increased numbers of proteases than those of other oral diseases and health. The levels of matrix metalloproteinase (MMP)-1, MMP-2, MMP-10, MMP-12, A disintegrin and metalloprotease (ADAM)9, A disintegrin and metalloprotease with thrombospondin type 13 motifs (ADAMST13), cathepsin V and kallikrein 5 in the saliva of patients with OSCC were significantly increased compared with those of other groups. Taking MMP-1, cathepsin V, kallikrein 5 and ADAM9 as biomarkers of OSCC, cutoff values were199, 11.34, 9.29 and 202.55 pg·mL, respectively. From the area under the curve, sensitivity and specificity, the combination of cathepsin V/kallikrein5/ADAM9 was an optimal biomarker for diagnosing OSCC. Thus, analysis of the salivary protease spectrum may be an innovative and cost-efficient approach to evaluating the health status of the oral cavity. Specifically, increases in cathepsin V, kallikrein 5 and ADAM9 may be useful biomarkers in the screening and diagnosis of OSCC.
ADAM Proteins ; Biomarkers, Tumor ; analysis ; Carcinoma, Squamous Cell ; diagnosis ; metabolism ; Humans ; Matrix Metalloproteinase 9 ; analysis ; Membrane Proteins ; Mouth Neoplasms ; diagnosis ; metabolism ; Saliva ; chemistry

OBJECTIVETo explore the influence of precondintioning on ADAMTS-13 activity and vWF level, and its clinical significance by measuring the alterations of ADAMTS-13 activity and vWF antigen levels before and after preconditioning of hematopoietic stem cell transplantation (HSCT) in the patients.

METHODA total of 113 patients received HSCT in the First Hospital affiliated to Soochow University were investigated, 20 healthy volunteers were used as the control. The ADAMTS-13 activity and vWF antigen level were measured by FRETS-vWF73 and ELISA respectively. Modified BU/CY, TBI/CY or BEAM were used as preconditioning regimen.

RESULTS(1) out of all the patients enrolled in this study, 8 patients were diagnosed as thrombotic disorders, and 49 patients were with occurence of acute graft-versus-host disease (aGVHD); (2) comparing with the control group, the ADAMTS-13 activity were lower and vWF antigen was higher in the patients after preconditioning (P < 0.01). Among all the patients, 69 (59.3%) cases showed the decrease of ADAMTS-13 activity after preconditioning, including 9 patients with more than 60% (9/113, 8.0%) decrease, in the meantime, the average plasma vWF antigen level of these 69 patients was significantly increased after preconditioning (P < 0.05); (3) the plasma ADAMTS-13 activity in 8 patients with thrombotic complications decreased after preconditioning, and there was significant difference in comparision with that of patients without thrombotic complication (P < 0.01). The patients with decrease of ADAMTS-13 activity more than 60% accounted for 37.5% (3/8), at the same time, the the level of vWF antigen increased (P < 0.01); (4) The medium level of ADAMTS-13 activity was dropped in 49 patients with aGVHD after preconditoning, but there was no abvious difference in comparision with that of patients without aGVHD. Among them 25 patients were with significant drop actvity of ADAMTS-13 at that time when aGVHD occurred (P < 0.01). Out of them, the patients with drop more than 60% before preconditioning accounted for 60% (2/35). The logistic regression analyse showed that the drop of ADAMTS-13 activity more than 60% before preconditioning was the risk factor for occurence of thrombosis at the later stage (P < 0.01), but the drop of ADAMTS-13 activity after preconditioning did not was the risk factor for occurence of aGVHD.

CONCLUSIONThe decrease of ADAMTS-13 activity after preconditioning of HSCT confirmed to be lower than that befor preconditioning of HSCT, and the level of vWF antigen after preconditioning of HSCT could be higher than that before precondifioning, especially in patients with thrombosis. The decrease of ADAMTS-13 activity after preconditioning has been found to be mone than 60%, then this decrease can be considered as an independent risk factor for later thrombogenesis, but the decrease of ADAMTS-13 after precoditioning activity did not associate with occurence of aGVHD, so the decrease of ADAMTS-13 activity can be used as an important predictor of thrombosis after HSCT.

ADAM Proteins ; metabolism ; ADAMTS13 Protein ; Case-Control Studies ; Graft vs Host Disease ; diagnosis ; pathology ; Hematopoietic Stem Cell Transplantation ; Humans ; Risk Factors ; Thrombosis ; diagnosis ; pathology ; Transplantation Conditioning ; von Willebrand Factor ; metabolism

OBJECTIVETo investigate the expression of miR-140-5p and ADAM10 in hypopharyngeal carcinoma tissues and their effects on the migration and invasion of FaDu cells and underlying mechanism.

METHODSThe miR-140-5p and ADAM10 mRNA levels in 33 cases of hypopharyngeal carcinoma tissues and adjacent normal tissues were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Transwell migration assay and transwell invasion assay were used to test the metastasis ability of FaDu cells after upregulation or downregulation of miR-140-5p and downregulation of ADAM10. The protein expression levels of ADAM10 in hypopharyngeal carcinoma tissues and the FaDu cells after transfection were determined by Western blot assays.

RESULTSThe expression level of miR-140-5p was significantly downregulated in hypopharyngeal carcinoma tissues compared with adjacent tissues (t=-4.016, P<0.01), which was significantly correlated with tumor classification and lymph node metastasis (P<0.05). Conversely, mRNA and protein expressions of ADAM10 were significantly upregulated in tumor tissues (t=3.960, P<0.01; t=12.089, P<0.01), and were significantly downregulated in the FaDu cells after tranfected with si-ADAM10 (t=8.653, P<0.05; t=5.191, P<0.05). Transwell assay showed that compare with control group, the migration and invasive cells decreased significantly in hsa-mir-140-5p group (t=3.255, P<0.05; t=2.942, P<0.05), while increased significantly in anti-hsa-mir-140-5p group, (t=-13.521, P<0.05; t=-6.223, P<0.05). The migration and invasive cells in si-ADAM10 group were less than those in control group (t=4.759, P<0.05; t=3.663, P<0.05). The downregulation of ADAM10 attenuated the effect of anti-mir-140-5p in FaDu cells. Western blot assay showed that ADAM10 expression was apparently decreased in hsa-mir-140-5p group and increased in anti-mir-140-5p group compared with control group.

CONCLUSIONSThe expression of miR-140-5p was significantly downregulated in hypopharyngeal carcinoma tissues and correlated with tumor classification and lymph node metastasis. ADAM10 was upregulated in tumor tissues. MiR-140-5p suppresses the migration and invasion abilities of FaDu cells, possibly through downregulation of ADAM10.

ADAM Proteins ; metabolism ; ADAM10 Protein ; Amyloid Precursor Protein Secretases ; metabolism ; Cell Line, Tumor ; Cell Movement ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; Hypopharyngeal Neoplasms ; pathology ; Lymphatic Metastasis ; Membrane Proteins ; metabolism ; MicroRNAs ; metabolism ; RNA, Messenger ; metabolism ; Transfection

OBJECTIVETo investigate the protective effect of LR-90 on articular cartilage in rabbit model of osteoarthritis.

METHODSThe cultured rabbits chondrocytes were assigned to be treated with IL-1β (10ng/ml) or IL-1β (10ng/ml)+LR-90 (50 mg/L). The mRNA expression of MMP-13, ADAMTS-5, aggrecan and collagen II in chondrocytes were assessed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Twenty male New Zealand white rabbits underwent bilateral anterior cruciate ligament transection (ACLT) to establish a animal model of osteoarthritis. Four weeks after model established, on the basis of randomization one knee of each rabbit was treated with 50 mg/L LR-90 in normal saline (NS) (experimental group) and the other knee was treated with same volume of NS (control group), 1/week × 5. Nine weeks after ACLT all rabbits were sacrificed and the knee joints were evaluated by gross morphology and histology. The mRNA expression of IL-1β, MMP-13, ADAMTS-5, aggrecan and collagen Ⅱ in articular cartilage was analyzed by RT-PCR.

RESULTSGross morphology and Mankin histological evaluation showed that the extent and grade of cartilage damage in the experimental group were less severe than those in the control group.Compared to IL-1β group, LR-90 treatment suppressed the mRNA expression of MMP-13 and ADAMTS-5, and enhanced aggrecan and collagen Ⅱ mRNA expression. Consistent with the in vitro results, the intraarticular LR-90 administration suppressed the mRNA expression of IL-1β,MMP-13 and ADAMTS-5 (all P<0.01), while enhanced mRNA expression of aggrecan and collagen Ⅱ in cartilage (all P<0.01).

CONCLUSIONLR-90 protects against cartilage degradation and inhibits the progression of osteoarthritis in rabbit mode1 of osteoarthritis, which is associated with the suppressing IL-1β, MMP-13, ADAMTS-5 and promoting aggrecan and collagen Ⅱ mRNA expression in cartilage.

ADAM Proteins ; metabolism ; Aggrecans ; metabolism ; Animals ; Anterior Cruciate Ligament ; surgery ; Butyrates ; pharmacology ; Cartilage, Articular ; metabolism ; pathology ; Cells, Cultured ; Chondrocytes ; metabolism ; Collagen Type II ; metabolism ; Disease Models, Animal ; Injections, Intra-Articular ; Interleukin-1beta ; pharmacology ; Male ; Matrix Metalloproteinase 13 ; metabolism ; Osteoarthritis ; drug therapy ; Rabbits

OBJECTIVETo assess the association of a disintegrin and metallo-proteinase with thrombospondin type 1 motifs (ADAMTS-1) gene polymorphism and ischemic stroke caused by large artery atherosclerosis (LAA).

METHODSIn total 767 patients and 506 controls were recruited. Single nucleotide polymorphisms (SNPs) rs416905 (T/C) and rs402007 (G/C) of the ADAMTS-1 gene were genotyped by polymerase chain reaction and DNA sequencing.

RESULTSFrequencies of the rs402007 GC+CC genotype and the C allele were significantly different between the two groups (68.84% vs. 60.67%, χ2=9.012, P=0.003, OR=1.432; 45.24% vs. 38.54%, χ2=11.208, P=0.001, OR=1.318). Binary logistic regression has confirmed that the above difference was significant (P=0.001, OR=1.521, 95%CI: 1.183-1.955). The frequencies of TC+CC and GC+CC genotypes were similar between the two groups, and so was it with the C allele. The two SNPs had been in complete linkage disequilibrium (D'=1.0, r2=1.0).

CONCLUSIONThe rs416905 and rs402007 polymorphisms of the ADAMTS-1 gene may be associated with ischemic stroke caused by LAA. The C allele of the rs402007 locus may be a susceptibility factor for this subtype of stroke.

ADAM Proteins ; genetics ; ADAMTS1 Protein ; Aged ; Alleles ; Atherosclerosis ; complications ; Base Sequence ; Blood Glucose ; metabolism ; Brain Ischemia ; complications ; Fasting ; blood ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Logistic Models ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Risk Factors ; Sequence Analysis, DNA ; Smoking ; Stroke ; blood ; etiology ; genetics

OBJECTIVEAlthough chondroprotective activities have been documented for polysaccharides, the potential target of different polysaccharide may differ. The study was aimed to explore the effect of glucan HBP-A in chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs in vivo, especially on the expression of type II collagen.

METHODSChondrocytes isolated from rabbit articular cartilage were cultured and verified by immunocytochemical staining of type II collagen. Chondrocyte viability was assessed after being treated with HBP-A in different concentrations. Morphological status of chondrocytes-alginate hydrogel constructs in vitro was observed by scanning electron microscope (SEM). The constructs were treated with HBP-A and then injected to nude mice subcutaneously. Six weeks after transplantation, the specimens were observed through transmission electron microscopy (TEM). The mRNA expressions of disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTs-5), aggrecan and type II collagen in both monolayer culture and constructs were determined by real time polymerase chain reaction (PCR). The expression of type II collagen and matrix metalloproteinases-3 (MMP-3) in chondrocyte monolayer culture was also tested through Western blot and enzyme linked immunosorbent assay (ELISA), respectively.

RESULTSMMP-3 secretion and ADAMTs-5 mRNA expression in vitro were inhibited by HBP-A at 0.3 mg/mL concentration. In morphological study, there were significant appearance of collagen in those constructs treated by HBP-A. Accordingly, in both chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs, the expression of type II collagen was increased significantly in HBP-A group when compared with control group (P<0.001).

CONCLUSIONSThe study documented that the potential pharmacological target of glucan HBP-A in chondrocytes monolayer culture and tissue engineered cartilage in vivo may be concerned with the inhibition of catabolic enzymes MMP-3, ADAMTs-5, and increasing of type II collagen expression.

ADAM Proteins ; genetics ; metabolism ; Aggrecans ; genetics ; metabolism ; Alginates ; pharmacology ; Animals ; Cartilage, Articular ; drug effects ; physiology ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Chondrocytes ; cytology ; drug effects ; metabolism ; ultrastructure ; Collagen Type II ; genetics ; metabolism ; Female ; Glucans ; pharmacology ; Glucuronic Acid ; pharmacology ; Hexuronic Acids ; pharmacology ; Hydrogel, Polyethylene Glycol Dimethacrylate ; pharmacology ; Immunohistochemistry ; Matrix Metalloproteinase 3 ; metabolism ; Mice, Nude ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Tissue Engineering ; methods

OBJECTIVETo establish a model of chondrocyte degeneration in vitro.

METHODSChondrocytes were isolated from articular cartilages of newly born SD rats by digestion with typeⅡ collagenase. The chondrocytes were cultured with H-DMEM medium containing 10%FBS, 50 ng/mL IL-1β+10%FBS, 2.5% rat serum and 5% rat serum, respectively; and the chondrocytes at passage one were used in the experiments. The morphology changes were investigated under phase contrast microscope after chondrocytes were treated with rat serum and IL-1β. Proliferation of chondrocytes was detected by MTT method. The protein expression levels of PCNA, typeⅡ collagen and MMP-13 were examined by Western blotting. The levels of ADAMTS5, MMP-9, Aggrecan and SOX-9 mRNA were detected by real-time PCR.

RESULTSThe cell morphology was changed from polygon to spindle in both rat serum groups and IL-1β group, and the proliferation of chondrocytes in these groups was much higher than that in control group. The results showed that the expression levels of typeⅡ collagen, Aggrecan and SOX-9 decreased while the expression levels of MMP-13, MMP-9 and ADMATS5 were up-regulated in rat serum and IL-1β-treated groups compared with control group.

CONCLUSIONThe results indicate that rat serum can induce chondrocyte degeneration and may be used for osteoarthritis model in vitro.

ADAM Proteins ; metabolism ; ADAMTS5 Protein ; Aggrecans ; metabolism ; Animals ; Cartilage, Articular ; cytology ; Cells, Cultured ; Chondrocytes ; pathology ; Collagen Type II ; metabolism ; Disease Models, Animal ; Interleukin-1beta ; pharmacology ; Matrix Metalloproteinase 13 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Osteoarthritis ; pathology ; Proliferating Cell Nuclear Antigen ; metabolism ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; SOX9 Transcription Factor ; metabolism ; Serum ; Up-Regulation

BACKGROUND/AIMS: SKI306X, a mixed extract of three herbs, Clematis mandshurica (CM), Prunella vulgaris (PV), and Trichosanthes kirilowii (TK), is chondroprotective in animal models of osteoarthritis (OA). The objectives of this study were to investigate its effect on interleukin (IL)-1beta-induced degradation of glycosaminoglycan (GAG) and the basis of its action in human OA cartilage, as well as to screen for the presence of inhibitors of matrix metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 in SKI306X and its component herbs, as well as in fractions from SKI306X. METHODS: Human OA chondrocytes and cartilage explants were obtained during total knee replacements and incubated with IL-1beta +/- oncostatin M with or without SKI306X or its component herb extracts. GAG degradation was assayed in cartilage explants using a commercial kit. Expression of genes involved in cartilage destruction was measured by real-time polymerase chain reaction using chondrocyte RNA. SKI306X was fractionated by preparative liquid chromatography to test for the presence of inhibitors of MMP-13 and ADAMTS-4. RESULTS: SKI306X and PV inhibited IL-1beta-induced GAG release from cartilage explants, and SKI306X, CM, PV, and TK inhibited IL-1beta-induced MMP gene expression. Unexpectedly, SKI306X greatly stimulated IL-1beta + oncostatin M-induced ADAMTS-4 gene expression, probably due to its TK component. Some fractions of SKI306X also inhibited ADAMTS-4 activity. CONCLUSIONS: SKI306X and its herbal components inhibit GAG degradation and catabolic gene expression in human OA chondrocytes and cartilage explants. SKI306X likely also contains one or more ADAMTS-4 inhibitor.
ADAM Proteins/antagonists & inhibitors ; Cartilage, Articular/*drug effects/*metabolism ; Cells, Cultured ; Chondrocytes/drug effects/metabolism ; Down-Regulation/drug effects ; Drugs, Chinese Herbal/*pharmacology ; Glycosaminoglycans/*metabolism ; Humans ; Interleukin-1beta/metabolism ; Matrix Metalloproteinase 13/metabolism ; Matrix Metalloproteinase Inhibitors/pharmacology ; Oncostatin M/metabolism ; Osteoarthritis, Knee/drug therapy/genetics/metabolism ; Procollagen N-Endopeptidase/antagonists & inhibitors

ADAMTS13, a plasma metalloprotease, specifically cleaves von Willebrand factor (vWF). Severe deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP). In this review, the structure and function of ADAMTS13 protease and its relationship with TTP are summarized.
ADAM Proteins ; metabolism ; ADAMTS13 Protein ; Humans ; Purpura, Thrombotic Thrombocytopenic ; diagnosis ; pathology ; therapy

A disintegrin-metalloproteinase 28 (ADAM28) is one of important members of ADAM family, that is involved in various biological events including cell adhesion, proteolysis, growth and metastasis of solid tumors and hematological malignancies. Studies have shown that ADAM28 is highly expressed in several human tumors, such as lung, breast and bladder cancers, and chronic lymphocytic leukemia, and its tissue expression levels correlate with cancer metastasis. ADAM28-mediated cancer cell metastasis may be related with the cleavage of von Willebrand's factor (vWF), insulin-like growth factor binding protein-3 (IGFBP-3) and connective tissue growth factor (CTGF), as well as the promoting PSGL-1/P-selectin-mediated cell adhesion. This review summarizes the basic and translational aspects of ADAM28 biology that might stimulate the interest in ADAM28 research and discovery of novel ADAM28 targets, providing potential novel therapies for metastatic cancers.
ADAM Proteins ; metabolism ; Cell Adhesion ; Connective Tissue Growth Factor ; metabolism ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; metabolism ; Neoplasm Metastasis ; Neoplasms ; pathology ; von Willebrand Factor ; metabolism