Structural insights into the binding mode of flavonols with the active site of matrix metalloproteinase-9 through molecular docking and molecular dynamic simulations studies.

Cartilage degradation in rheumatoid arthritis is mediated principally by the collagenases and gelatinases. Gelatinase B (also called matrix metalloproteinase 9 - MMP-9), is a valid target molecule which is known to participate in cartilage degradation as well as angiogenesis associated with the disease and inhibition of its activity shall prevent cartilage damage and angiogenesis. The focus of this study is to investigate the possibilities of MMP-9 inhibition by flavonol class of bioflavonoids by studying their crucial binding interactions at the active site of MMP 9 using molecular docking (Glide XP and QPLD) and further improvisation by post-docking MM-GBSA and molecular dynamic (MD) simulations. The results show that flavonols can convincingly bind to active site of MMP-9 as demonstrated by their stable interactions at the S1' specificity pocket and favourable binding energies. Gossypin has emerged as a promising candidate with a docking score of -14.618 kcal/mol, binding energy of -79.97 kcal/mol and a stable MD pattern over 15 ns. In addition, interaction mechanisms with respect to catalytic site zinc are also discussed. Further, the drug-like characters of the ligands were also analysed using ADME analysis.

Lactate dehydrogenase downregulation mediates the inhibitory effect of diallyl trisulfide on proliferation, metastasis, and invasion in triple-negative breast cancer.

The Warburg effect plays a critical role in tumorigenesis, suggesting that specific agents targeting Warburg effect key proteins may be a promising strategy for cancer therapy. Previous studies have shown that diallyl trisulfide (DATS) inhibits proliferation of breast cancer cells by inducing apoptosis in vitro and in vivo. However, whether the Warburg effect is involved with the apoptosis-promoting action of DATS is unclear. Here, we show that the action of DATS is associated with downregulation of lactate dehydrogenase A (LDHA), an essential protein of the Warburg effect whose upregulation is closely related to tumorigenesis. Interestingly, inhibition of the Warburg effect by DATS in breast cancer cells did not greatly affect normal cells. Furthermore, DATS inhibited growth of breast cancer cells, particularly in MDA-MB-231, a triple-negative breast cancer (TNBC) cell, and reduced proliferation and migration; invasion was reversed by over-expression of LDHA. These data suggest that DATS inhibits breast cancer growth and aggressiveness through a novel pathway targeting the key enzyme of the Warburg effect. Our study shows that LDHA downregulation is involved in the apoptotic effect of DATS on TNBC. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1390-1398, 2017.

Inhibition of hybrid layer degradation by cavity pretreatment: Meta- and trial sequential analysis.

OBJECTIVES: Inhibition of hybrid layer degradation, for example via inhibition of matrix-metallo-proteinases (MMP) could reduce risk of retention loss and failure of adhesively placed restorations. This systematic review investigated such inhibitory pretreatment qualitatively and via meta- and trial-sequential-analysis. DATA SOURCES: We included randomized clinical trials comparing degradation inhibitory cavity pretreatment versus no, placebo or alternative treatments prior adhesive placement of resin-based restorations. Trials reporting retention loss or failure (graded bravo-delta in USPHS or similar criteria) were included. Trial selection, data extraction, and risk of bias assessment were conducted independently by two reviewers. Fixed- or random-effects intention-to-treat, per-protocol, and scenario meta-analyses were performed, and trial-sequential-analysis used to control for risk of random errors. Electronic databases (PubMed, Embase, Cochrane CENTRAL) were systematically screened, and hand searches and cross-referencing performed. STUDY SELECTION: The ten included trials involved 208 patients (695 cavities) and used chlorhexidine (seven trials), ethanol-wet-bonding (two trials), and quaternary ammonium compounds for degradation inhibition. All but one trial had high risk of bias. Follow-up ranged from 6 to 36 months. Risk of retention loss was not significantly decreased after pretreatment (per-protocol OR [95% CI] 1.37 [0.68/2.77], intention-to-treat: 1.25 [0.76/2.04]). This was found for risk of restoration failure as well (per-protocol: 0.86 [0.56/1.34], intention-to-treat: 1.22 [0.83/1.80]). Scenario analyses found great uncertainty introduced by attrition. According to trial sequential analysis, no firm evidence was reached. CONCLUSIONS: There is insufficient evidence to recommend or refute degradation inhibitory cavity pretreatment prior adhesively placing resin-based restorations. This may change if teeth are followed-up for longer. CLINICAL SIGNIFICANCE: Dentists can perform cavity pretreatments for inhibition of hybrid layer degradation, but a beneficial effect is not supported by sufficient evidence. The impact of further effects (e.g. disinfection, pulp-irritation) remains unclear.

Correlation between peri-implant sulcular fluid rate and expression of collagenase2 (MMP8).

OBJECTIVES: The rapidly increasing numbers of inserted dental implants and the growing incidence of peri-implant mucositis and peri-implantitis with the current absence of reliable disease risk prediction highlight the importance of early and sensitive diagnosis of possible disease progression. The aim of this study is to assess quantitative and qualitative analysis of peri-implant sulcular fluid (PISF) during implant maintenance control and to identify whether there is a positive correlation and statistical significance between peri-implant sulcular fluid volume results and collagenase2 level obtained from both superficial and fundus area of peri-implant sulcus. MATERIAL AND METHOD: Twenty-seven implants from patients under recall provided peri-implant sulcular fluid volume samples, which were collected with the Periotron 8000 micro-moisture meter, and collagenase2 levels, which were assessed using dentoTest aMMP8. Statistical analysis was obtained using Spearman's correlation. RESULTS: Positive correlation was found between collagenase2 collected from sulcular and fundus areas on both mesial and distal sides. There was correlation between peri-implant sulcular fluid volume and collagenase2 level from fundus and distal area, but not from the mesial and superficial area. CONCLUSIONS: Examination of collagenase2 is a sensitive method when examining early inflammatory changes but depends from the depth of the sample collection in the gingival pocket. CLINICAL RELEVANCE: The examination of MMP8 seems to be a more sensitive method than the analysis of peri-implant sulcular fluid to detect peri-implant mucositis and peri-implantitis.

Biocompatible nanoparticles sensing the matrix metallo-proteinase 2 for the on-demand release of anticancer drugs in 3D tumor spheroids.

The balance between dose-dependent tolerability, effectiveness and toxicity of systemically administered antitumor drugs is extremely delicate. This issue highlights the striking need for targeted release of chemotherapeutic drugs within tumors. In this work, a smart strategy of drug targeting to tumors relying upon biodegradable/biocompatible nanoparticles releasing cytotoxic drugs after sensing physiological variations intrinsic to the very nature of tumor tissues is exploited. Here, the well-known over-expression of matrix metallo-proteinase 2 (MMP2) enzyme in tumors has been chosen as a trigger for the release of a cytotoxic drug. Nanoparticles made up of a biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA)--block--polyethylene glycol (PEG) copolymer (namely PELGA), blended with a tumor-activated prodrug (TAP) composed of a MMP2-sensitive peptide bound to doxorubicin (Dox) and to PLGA chain have been produced. The obtained devices are able to release Dox specifically upon MMP2 cleavage of the TAP. More interestingly, they can sense the differences in the expression levels of endogenous MMP2 protein, thus modulating drug penetration within a three-dimensional (3D) tumor spheroid matrix, accordingly. Therefore, the proposed nanoparticles hold promise as a useful tool for in vivo investigations aimed at an improved therapeutic efficacy of the conjugated drug payload.

Complete sequencing and characterization of equine aggrecan.

OBJECTIVES: To fully sequence and characterize equine aggrecan and confirm conservation of major aggrecanase, calpain and matrix metalloproteinase (MMP) cleavage sites. METHODS: Reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends were used to generate clones that encompassed the complete equine aggrecan sequence. Clones were sequenced and compared with the equine genome database to determine intron-exon boundaries. RESULTS: The aggrecan gene spans over 61 kb on chromosome 1 and is encoded by 17 exons. Two major variants of aggrecan were cloned; one containing 8187 bp (2728 amino acids) and a second sequence of 8061 nucleotides (2686 amino acids). The variation was due to a CS1 domain polymorphism. Both sequences are substantially larger than predicted by the genomic database; 11 CS1 repeat elements are absent in the database sequence. The equine amino acid sequence was compared with human, bovine and murine sequences. Globular domains 1, 2 and 3 are highly conserved (overall identity over 80%). Equine CS1 is considerably larger than in other species and, therefore, is the least conserved domain (an overall amino acid identity of 22%). Previously defined aggrecanase, calpain and MMP cleavage sites were identified. Western blotting of chondrocyte culture samples showed complex post-secretion processing. CLINICAL SIGNIFICANCE: The complete equine aggrecan sequence will support more in-depth research on aggrecan processing and degradation in equine articular cartilage and other musculoskeletal tissues.

Potential role of proteasome on c-jun related signaling in hypercholesterolemia induced atherosclerosis.

Atherosclerosis and its complications are major causes of death all over the world. One of the major risks of atherosclerosis is hypercholesterolemia. During atherosclerosis, oxidized low density lipoprotein (oxLDL) regulates CD36-mediated activation of c-jun amino terminal kinase-1 (JNK1) and modulates matrix metalloproteinase (MMP) induction which stimulates inflammation with an invasion of monocytes. Additionally, inhibition of proteasome leads to an accumulation of c-jun and phosphorylated c-jun and activation of activator protein-1 (AP-1) related increase of MMP expression. We have previously reported a significant increase in cluster of differentiation 36 (CD36) mRNA levels in hypercholesterolemic rabbits and shown that vitamin E treatment prevented the cholesterol induced increase in CD36 mRNA expression. In the present study, our aim is to identify the signaling molecules/transcription factors involved in the progression of atherosclerosis following CD36 activation in an in vivo model of hypercholesterolemic (induced by 2% cholesterol containing diet) rabbits. In this direction, proteasomal activities by fluorometry and c-jun, phospo c-jun, JNK1, MMP-9 expressions by quantitative RT-PCR and immunoblotting were tested in aortic tissues. The effects of vitamin E on these changes were also investigated in this model. As a result, c-jun was phosphorylated following decreased proteasomal degradation in hypercholesterolemic group. MMP-9 expression was also increased in cholesterol group rabbits contributing to the development of atherosclerosis. In addition, vitamin E showed its effect by decreasing MMP-9 levels and phosphorylation of c-jun.

Calcitonin-induced NF-κB activation up-regulates fibronectin expression in MG63 osteosarcoma cells.

Salmon calcitonin has been used extensively as a therapeutic tool in the regulation of bone remodeling. However, there is a growing body of evidence indicating that the calcitonin peptides are involved in regulation of cell growth, differentiation, survival and tissue development. In the present study, we investigated the effect of calcitonin in cell matrix interactions in MG63 cell line. Our results demonstrated that calcitonin increases cell growth of MG63 osteosarcoma cells in parallel with serine/threonine protein kinase B (AKT/PKB) activation. Moreover, calcitonin induced up-regulation of fibronectin expression in a nuclear factor-kappa B (NF-κB)-dependent manner, accompanied by enhanced enzymatic activity of matrix metalloproteinase-9 (MMP-9) and increased expression of tissue inhibitors of MMP-1 and -2. MMP-9 stimulation with calcitonin was accompanied by an increase in protein expression of the α5ß1 integrin receptor. To our knowledge, our results demonstrate, for the first time, that calcitonin is a potent inducer of fibronectin, an extacellular matrix component that is suggested to have a pro-oncogenic and healing effect, in a NF-κB-dependent manner.

The predictive value of serum soluble E-cadherin levels in breast cancer patients undergoing preoperative systemic chemotherapy.

OBJECTIVES: To date, no reliable markers are available to predict response to or to assess prognosis after preoperative systemic chemotherapy (PST) in patients with locally advanced breast cancer. Previous studies demonstrated that elevated levels of soluble E-cadherin (sE-cadherin), a product of proteolytic cleavage of cell surface E-cadherin, are associated with higher risk for metastatic disease and poor prognosis in various tumor types. We, therefore, hypothesized that serum sE-cadherin levels measured before PST may correlate with pathological response. DESIGN AND METHODS: In a retrospective analysis, sE-cadherin levels were measured in sera of 108 female patients with histologically proven breast cancer before initiation of PST by using a commercially available quantitative sandwich enzyme immunoassay technique. Patients received a median number of 4 (range 3-6) cycles of anthracyline-based chemotherapy. The median patient age was 51.5 (range 21-71) years. Tumor size was measured clinically and translated into the tumor-node-metastasis (TNM)-system before the start of chemotherapy. Histopathological response in surgically removed specimens was evaluated using a modified Sinn regression score. In univariate analyses the correlations between levels of sE-cadherin and pathological response to PST were calculated. RESULTS: The histopathological regression scores correlated significantly with tumor grading (p=0.045), clinical lymph node status before PST (p=0.031) and sE-cadherin levels (p=0.039). No correlation was seen between histopathological regression scores and hormone receptor and menopausal status as well as Her2-neu status. CONCLUSION: sE-cadherin may be a marker predicting response to PST for patients with breast cancer. Our findings warrant further evaluation of sE-cadherin in a prospective trial.

Cell type-specific release of matrix-metallo-proteinase-9 by bacterial chemoattractant in human blood phagocytic leukocytes.

Stimulation of phagocytic leukocytes with bacterial chemoattractant resulted in the release of matrix metal-loproteinases (MMPs). Little is known about the mechanisms of bacterial chemoattractant regulation of MMP in phagocytic leukocytes. We report here that the mechanisms of the bacterial chemotactic peptidefMLP-induced MMP -9 release in monocytes appeared to be different from fMLP-stimulated MMP-9 release in neutrophils. In freshly prepared peripheral blood monocytes, fMLP induces MMP-9 release, starting at 8 h after stimulation. These functions of fMLP is accompanied by an increase in TNFα expression, and mediated through the phosphorylation of ERK1/2 in monocytes. However, neutrophil preparations that responded to fMLP with MMP-9 release did not require activation of ERK1/2 and TNFα expression. These results suggest a different role of fMLP in MMP-9 expression in neutrophils and monocytes, and the signal molecules involved in mediating this effect in human blood monocytes stimulated by bacterial chemoattractant.