MMP-9 Enzymatic Activity Research Articles

Unexplored features of Ru(ii) polypyridyl complexes - towards combined cytotoxic and antimetastatic activity.

The well-documented cytotoxic activity of coordinatively saturated and substitutionally inert polypyridyl Ru(ii) complexes substantiates their high potency as antiproliferative agents against primary tumors. However, the primary cause of cancer morbidity and mortality responsible for about 90% of cancer deaths is the occurrence of metastasis. Therefore, scientists have to concentrate their efforts on designing compounds affecting not only the primary tumor, but also efficiently inhibiting metastasis. Herein, we report two families of Ru(ii) polypyridyl complexes bearing 2,2'-bipyridine substituted by a semicarbazone 2-formylopyridine moiety as one of the ligands and 4,4'-di-tert-butyl-2,2'-dipyridyl or 4,7-diphenyl-1,10-phenanthroline as auxiliary ligands. These complexes strengthen cells' adherent properties and inhibit the activity of metalloproteinases (MMPs) in vitro, which is relevant in anti-metastatic treatment. The in vitro studies were performed on human lung adenocarcinoma (A549) and human pancreatic cancer (PANC-1) cells, which have a well-documented invasive potential. The induced alteration of the tumor cells' adhesion properties correlated with the high cytotoxic effect exerted by the complexes and their excellent cellular uptake. It was also proved that both complexes directly inhibit M-MP2 and M-MP9 enzyme activities, which are essential for the development of tumor metastasis. The results of this study indicate that the biological properties of polypyridyl Ru(ii) complexes extend beyond the standard cytotoxic activity and represent an important step towards designing new anti-metastatic agents.

Inhibitory Activity of Extract, Fractions, and Compounds from Zingiber montanum Rhizomes on the Migration of Breast Cancer Cells.

Zingiber montanum rhizomes are traditionally used for the treatment of numerous human ailments. The present study was carried out to investigate the inhibitory activity of the crude extract, chromatographic fractions, and purified compounds from Z. montanum rhizomes on the migration of MDA-MB-231 cells. The effect of the extract on cell migration was investigated by a scratch assay, which showed significant inhibition in a concentration-dependent manner. Vacuum liquid chromatography on silica gel afforded four fractions (Frs. 1 - 4), which were tested on cell migration in the scratch assay. Frs. 1 and 2 showed the most significant inhibition of MDA-MB-231 cell migration. The effect of the most potent fraction (Fr. 2) was further confirmed in a transwell migration assay. The study of Frs. 1 and 2 by gelatin zymography showed significant inhibition of MMP-9 enzyme activity. Chromatographic separation of Frs. 1 and 2 afforded buddledone A (1: ), zerumbone (2: ), (2E,9E)-6-methoxy-2,9-humuradien-8-one (3: ), zerumbone epoxide (4: ), stigmasterol (5: ), and daucosterol (6: ). In a cell viability assay, compounds 1: - 4: inhibited the viability of MDA-MB-231 cells in a concentration-dependent manner. The study of buddledone A (1: ) and zerumbone epoxide (4: ) on cell migration revealed that 4: significantly inhibited the migration of MDA-MB-231 cells in both scratch and transwell migration assays. The results of the present study may lead to further molecular studies behind the inhibitory activity of zerumbone epoxide (4: ) on cell migration and support the traditional use of Z. montanum rhizomes for the treatment of cancer.

Effects of 12-Week Exercise Program on Enzyme Activity of Serum Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 in Female Patients with Postmenopausal Osteoporosis: A Randomized Control Study.

Background Osteoporosis is a disease characterized by decreased bone density and destruction of bone microarchitecture. Indicators for altered bone homeostasis are changes in the serum level of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The purpose of the current study was to evaluate the effect of a 12-week exercise program on enzyme activity of serum MMP-9 and TIMP-1 in postmenopausal osteoporotic patients. Materials and methods. Participants were randomized in two groups: exercise (EG) (N = 37) and control (CG) (N = 37) and control (CG) (Results Significant differences between pretreatment and posttreatment enzyme activities of serum MMP-9 (p=0.009), TIMP-1 (p=0.009), TIMP-1 (p=0.009), TIMP-1 (Conclusion Our results suggest that a 12-week exercise program has an influence on enzyme activity of serum MMP-9, revealing a possible role of MMPs in initiating training-specific adaptation. Although measurements of circulating MMP-9 and TIMP-1 allowed us to detect effects of exercise, as of today, they have no real role in the diagnosis of osteoporosis and/or follow-up of osteoporotic patient's response to treatment. MMP-9 might be used as an important prognostic marker for the evaluation of patient's response to exercise. Larger-randomized controlled studies need to be performed to expand this area of knowledge. This trial is registered with trial registration number: NCT03816449).

Maternal Embryonic Leucine Zipper Kinase Promotes Tumor Growth and Metastasis via Stimulating FOXM1 Signaling in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignancy and is one of the most important cause of cancer related mortalities in the world. However, there is no clinically effective targeted therapeutic drugs for ESCC due to lack of valuable molecular therapeutic targets. In the present study, we investigated the biological function and molecular mechanisms of maternal embryonic leucine zipper kinase (MELK) in ESCC. The expression of MELK mRNA and protein was determined in cell lines and clinical samples of ESCC. MTT, focus formation and soft agar assays were carried out to measure cell proliferation and colony formation. Wound healing and transwell assays were used to assess the capacity of tumor cell migration and invasion. Nude mice models of subcutaneous tumor growth and lung metastasis were performed to examine the function of MELK in tumorigenecity and metastasis of ESCC cells. High expression of MELK was observed in ESCC cell line and human samples, especially in the metastatic tumor tissues. Moreover, overexpression of MELK promoted cell proliferation, colony formation, migration and invasion, and increased the expression and enzyme activity of MMP-2 and MMP-9 in ESCC cells. More importantly, enhanced expression of MELK greatly accelerated tumor growth and lung metastasis of ESCC cells in vivo. In contrast, knockdown of MELK by lentiviral shRNA resulted in an opposite effect both in vitro and in animal models. Mechanistically, MELK facilitated the phosphorylation of FOXM1, leading to activation of its downstream targets (PLK1, Cyclin B1, and Aurora B), and thereby promoted tumorigenesis and metastasis of ESCC cells. In conclusion, MELK enhances tumorigenesis, migration, invasion and metastasis of ESCC cells via activation of FOXM1 signaling pathway, suggesting MELK is a potential therapeutic target for ESCC patients, even those in an advanced stage.

Resveratrol inhibits ACHN cells via regulation of histone acetylation

Context The relationship between resveratrol and histone acetylation in renal cell carcinoma (RCC) has not yet been reported. Objective To explore the functional role of resveratrol in RCC. Materials and methods Functional experiments were performed to determine proliferatio n of ACHN cells with treatment of resveratrol (0, 7.8125, 15.625, 31.25 and 62.5 μg/mL, for 12, 24 and 48 h of culture) or 0.1 μM SAHA. The enzyme activities of MMP-2/-9 were measured by gelatine zymography and histone acetylation by Western blot. Results When the cells were treated with 15.625, 31.25 and 62.5 μg/mL resveratrol, ACHN cells viability was 73.2 ± 3.5%, 61.4 ± 3.1%, 50.2 ± 4.7% for 12 h, 62.7 ± 4.5%, 52.4 ± 5.5%, 40.2 ± 3.8% for 24 h, and 60.8 ± 3.7%, 39.4 ± 5.1%, 37.6 ± 2.7% for 48 h, and the wound closure (%) of migration was increased from 0.6 to 0.7, 0.85, 0.9 for 12 h and from 0.23 to 0.3, 0.48, 0.59 for 24 h. The invasion rate was 8.5 ± 0.9%, 7.4 ± 0.3% and 5.8 ± 0.6%, and cell cycle was arrested at G1 from 42.5 ± 2.9% to 55.3 ± 5.7%, 59.8 ± 3.4%, 68.7 ± 4.6%. MMP-2/-9 expression (p < 0.05) was inhibited by resveratrol. The protein levels of histone acetylation (p < 0.01) was increased by resveratrol. Discussion and conclusions Our results suggest that these effects might be related to a high level of histone acetylation, and resveratrol can be considered as an alternative treatment for RCC.

Therapeutic Potential of Allogenic Bone Marrow-Derived Mesenchymal Stem Cells for Aortic Aneurysm

Introduction - We have previously reported effectiveness of autologous bone marrow derived mesenchymal stem cells (BM-MSCs) for regression of aortic aneurysm (AA). However, they have various disadvantage in terms of cell quality and quantity. Meanwhile, it is known that the stem cell has ability of immunological tolerance. The purpose of this study were to investigate whether treatment of allogeneic MSCs is effective for angiotensin II (AT II)-induced apolipoprotein E-deficient (apoE−/−) already-formed AA mouse. Methods - To identify characterization of each MSCs, the surface markers were checked by flow cytometry. AA was induced in apoE−/− mice by Ang II-infusion for 28 days through subcutaneously implanted osmotic mini-pump. The mice were randomly divided into 3 groups: (ⅰ) 0.2ml saline was intra-veinously injected (n=10, group S), (ⅱ) 1 × 106 autologous BM-MSCs(isolated from C57BL/6) in 0.2 ml saline (n=10, group Au), (ⅲ) 1 × 106 allogeneic BM-MSCs (isolated from BALB/C) (n=10, group Al) via tail vein. Mice were sacrificed at 2weeks after injection. Aortic diameter, matrix metalloproteinase (MMP)-2 and -9 enzymatic activity and cytokine concentrations were measured in obtained aortic tissue. Results - Flow cytometric analysis demonstrated that the both MSCs were similar in surface markers .The Aortic diameters of group Au and Al were significantly decreased compared to group S (group S vs Au vs Al; 2.29 vs 1.40 vs 1.36 (mm), p<0.01). The enzymatic activities of MMP-2 and -9 were significantly reduced in both MSCs groups (active-MMP2: group S vs Au vs Al; 0.45 vs 0.28 vs 0.24 (unit/ml), p<0.05 amd active-MMP9: group S vs Au vs Al; 0.34 vs 0.16 vs 0.14 (unit/ml), p<0.05). Inflammatory cytokines were down-regulated in the both MSC groups (interleukin-6: group S vs Au vs Al; 3399.5 vs 1475.6 vs 937.5 (pg/ml), p<0.01 and monocyte chemotactic protein-1: group S vs Au vs Al; 352.7 vs 208.0 vs 165.1 (pg/m), p<0.05).Insulin-like growth factor (IGF)-1 and tissue inhibitor of metalloproteinase (TIMP)-2 were up-regulated in the MSC groups (IGF-1: group S vs Au vs Al; 2.0 vs 4.7 vs 5.7 (pg/ml), p<0.01; TIMP-2: group S vs Au vs Al; 4.0 vs 9.5 vs 11.1 (pg/ml), p<0.01). Both MSCs injection inhibited infiltration of M1 macrophages in the aortic tissue. Conclusion - Our results suggest that the therapeutic effect of allogeneic MSCs might be similar to that of autologous MSCs for treatment of AA. Allogeneic MSCs injection could provide an alternative therapeutic strategy for the treatment of AA.

The Role of PAK1 in the Maturation of Invadopodia During Transient Mechanical Stimulation.

Cancer cells are affected by a wide range of mechanical forces within their extracellular environment. It has been widely shown that these forces can lead to increased metastatic activity of these cells. One such force is a transient tugging-like force that results from contractile forces generated by cells within the tumor microenvironment. When this force is simulated in vitro with a mechano-invasion assay, human fibrosarcoma cells exhibit enhanced cell invasion in a 3D collagen-fibronectin matrix by downregulating the expression of integrin β3. Furthermore, this force stimulates the maturation of invadopodia in an integrin β3-dependent manner that includes an increase in the active form of cofilin and MMP-2 secretion. In the present study we discovered that the decrease in integrin β3 signaling in response to mechanical stimulation is coupled to the activity of p21-activated kinase 1 (PAK1). It was found that PAK1 has decreased activity, as detected by a decrease in Ser144 phosphorylation, with mechanical stimulation. However, this loss in phosphorylation can be reversed if integrin β3 is overexpressed. Furthermore, PAK1 mutants show a correlated response in MMP-2 enzyme expression and activity, in addition to the lengthening of invadopodia, in response to stimulation. These results identify a novel mechano-sensitive response in human fibrosarcoma that utilizes PAK1 as a signaling player positioned downstream of integrin β3.

CD147 self-regulates matrix metalloproteinase-2 release in gingival fibroblasts after coculturing with U937 monocytic cells.

Cluster of differentiation 147 (CD147) is a multifunctional glycoprotein that functions as an inducer of matrix metalloproteinase (MMP) expression in fibroblasts. Synergistically enhanced MMP-2 expression was recently observed in the coculture of human gingival fibroblasts (HGFs) and U937 human monocytic cells; however, the responsible mechanisms have not yet been fully established. The aim of this study was to evaluate the release of soluble CD147 in HGFs after coculturing with U937 cells and its functional effect on the enhancement of MMP-2 expression in HGFs. Enzyme-linked immunosorbent assay was used to determine the amount of CD147 protein in media, whereas real-time polymerase chain reaction was performed to evaluate the mRNA levels of CD147 and MMP-2 in HGFs and U937 cells. The enzyme activities of MMP-2 released from cells were examined by zymography. Transwell coculturing and conditioned media treatments were selected to rule out the effect of direct contact of HGFs and U937 cells. The protein and mRNA expression of CD147 in HGFs were enhanced after transwell coculturing with U937 cells and exposure to U937-conditioned medium. MMP-2 enzyme activities in HGFs were also significantly increased by the coculturing methods. Administration of exogenous CD147 enhanced MMP-2 expression in HGFs, whereas treatment with cyclosporine-A, which inhibited CD147 expression, reduced U937-enhanced MMP-2 expression in HGFs. CD147 can interact with fibroblasts to stimulate the expression of MMPs associated with periodontal extracellular matrix degradation. This study has demonstrated that CD147 released from fibroblasts might play a role in monocyte-enhanced MMP-2 expression in HGFs.

Protective Effects of Ginseng Proteins on Photoaging of Mouse Fibroblasts Induced by UVA.

UVA can penetrate dermis and cause functional damage of dermal fibroblasts leading photoaging. Ginseng is a widely used traditional Chinese medicine for skin aging. However, its effects on skin photoaging induced by UVA are not clear. In this study, we isolated ginseng proteins (GP), with molecular weights of 27 kDa and 13 kDa, and found that they alleviated the inhibitory effects of UVA on cell viability and increased percentage of NIH-3T3 fibroblasts in the S phase of cells cycle. GP also improved cell contraction ability, increased the expression and secretion of CoL-I, similar to MAPK phosphorylation inhibitors and reduced expression and secretion of MMP-1, MMP-2 and MMP-9 as well as the enzyme activities of MMP-2 and MMP-9. They reduced ROS content, DNA damage and 8-OHdG content, as well as the protein expression of p53, p21 and p16. The levels of p-ERK, p-p38 and p-JNK, p-c-Fos and p-c-Jun proteins were decreased by GP. Inactivated GP did not inhibit the cellular activity and expression and secretion of CoL-I irradiated by UVA. The results showed that GP can improve cell viability and contractile function by inhibiting DNA damage and collagen degradation to inhibit the photoaging effects of skin dermal cells caused by UVA.

P3112The oral administration of colchicine prevents the progression of aortic aneurysm

Abstract Objective The pathogenesis of aortic aneurysm (AA) is characterized by the chronic inflammation of the aortic wall with the accumulation of macrophages and the degradation of the extracellular matrix (ECM) including elastin. Colchicine (COL) is an alkaloid derived from the plant Lily family Colchicum autumnale, and it is known for anti-inflammatory effects. Plant extracts containing COL have been used in the treatment of gout from ancient period. Currently, pseudogout, familial Mediterranean fever, Behçet's disease and pericarditis are also treated by COL. Furthermore, recent evidence suggests the use of COL for secondary prevention of cardiovascular disease, and the phase 3 clinical trial for it has begun. The objective of this study is to investigate whether COL could prevent the progression of aortic aneurysms. Methods In vitro: Macrophages (J774A.1 cell line) stimulated TNF-α 24 hours before and smooth muscle cell (SMC) were cultured with 10 ng/mL COL, and the gene expression of inflammatory cytokines involved in the AA formation was measured 24 hours later. In vivo: Male apolipoprotein E-deficient mice (30–35 weeks of age) were infused with angiotensin II for 28 days. COL (20 μg/kg/d) or saline (NS, as a control) was administered orally to the mice every day (COL group, n=8; NS group, n=8). Aortic diameter was measured by echography every week and all mice were sacrificed and their thoracoabdominal aorta was harvested at the last day of the administration period and elastin content, MMP activitis, and levels of inflammatory cytokines involved in the AA formation were measured. Results In vitro: The gene expression of IL-1β, TNF-α, MCP-1, NF-κB, MMP-9 in the macrophages was significantly decreased in the COL group. The gene expression of Lox, TIMP-2 in the SMC were significantly increased in COL group. In vivo: Aortic diameter measured by echography every week was significantly suppressed in the COL group (2.25 vs 2.81 mm, p&lt;0.05). The incidence of AA was decreased in the COL group (62.5% vs 100%). COL significantly suppressed the degeneration of aortic elastin in EVG staining (p&lt;0.05). There is no significant difference in the enzyme activities of MMP-2 and MMP-9 between COL and NS groups, but IL-1β (54.4 vs 81.4, p&lt;0.05), TNF-α (31.0 vs 60.6, p&lt;0.05), MCP-1 (258.2 vs 411.2, p&lt;0.05), NLRP3 inflammasome (7.1 vs 8.6, p&lt;0.05), NE (1.5 vs 2.4, p&lt;0.05), MPO (44.9 vs 48.1, p&lt;0.05) were decreased in the COL group. Discussion In AA model mice, COL seems to suppress the progression of AA by anti-infammation and preservation of the ECM structure through the inhibition of NLRP3 inflammasome. That NLRP3 inflammasome activation leads to the progression of AA in AA model mice was previously reported and this supports out results. Methods and Results (in vivo) Conclusions This results suggest that the oral administration of COL prevents the progression of AA in AA model mice and it is expected as a novel therapeutic agent for AA. Acknowledgement/Funding JSPS KAKENHI Grant

Cytotoxic Effects of Ferula Latisecta on Human Glioma U87 Cells.

Glioblastoma multiforme (GBM) is the fatal type of astrocytic tumors with a survival rate of 12 months. The present study, for the first time, evaluated the cytotoxic impacts of Ferula latisecta (F. latisecta) hydroalcoholic extract on U87 GBM cell line. The MTT assay measured the cellular toxicity following 24- and 48 h treatment with various doses of F. latisecta (0-800 μg/mL). Apoptosis was evaluated by an Annexin V/propidium iodide (PI) staining 24 h after treatment by F. latisecta. Moreover, to determine the cellular metastasis of U87 cells, we used a gelatin zymography assay (matrix metalloproteinase [MMP]-2/-9 enzymatic activity). The outcomes showed that F. latisecta mitigated the viability of U87 cells in a concentration- and time-dependent manner with IC50 values of 145.3 and 192.3 μg/mL obtained for 24- and 48 h treatments, respectively. F. latisecta induced apoptosis in a concentration-dependent manner after 24 h. Also, MMP-9 activity was significantly decreased following 24 h after treatment concentration-dependently with no change in MMP-2 enzymatic activity. This study showed that F. latisecta induced cytotoxicity and apoptosis, and mitigated metastasis of U87 GBM cells. Hence, F. latisecta could be beneficial as a promising natural herb against GBM after further studies.

Matrix metalloproteinase activity in the lung is increased in Hermansky-Pudlak syndrome

BackgroundHermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by oculocutaneous albinism and platelet dysfunction and can sometimes lead to a highly aggressive form of pulmonary fibrosis that mimics the fatal lung condition called idiopathic pulmonary fibrosis (IPF). Although the activities of various matrix metalloproteinases (MMPs) are known to be dysregulated in IPF, it remains to be determined whether similar changes in these enzymes can be detected in HPS.ResultsHere, we show that transcript and protein levels as well as enzymatic activities of MMP-2 and -9 are markedly increased in the lungs of mice carrying the HPS Ap3b1 gene mutation. Moreover, immunohistochemical staining localized this increase in MMP expression to the distal pulmonary epithelium, and shRNA knockdown of the Ap3b1 gene in cultured lung epithelial cells resulted in a similar upregulation in MMP-2 and -9 expression. Mechanistically, we found that upregulation in MMP expression associated with increased activity of the serine/threonine kinase Akt, and pharmacological inhibition of this enzyme resulted in a dramatic suppression of MMP expression in Ap3b1 deficient lung epithelial cells. Similarly, levels and activity of different MMPs were also found to be increased in the lungs of mice carrying the Bloc3 HPS gene mutation and in the bronchoalveolar lavage fluid of subjects with HPS. However, an association between MMP activity and disease severity was not detected in these individuals.ConclusionsIn summary, our findings indicate that MMP activity is dysregulated in the HPS lung, suggesting a role for these proteases as biological markers or pathogenic players in HPS lung disease.

Spectroscopic method for estimation of MMP-9 enzyme concentration and activity

Two highly sensitive fluorescent dye-labelled peptide indicators for metalloproteinase 9 (MMP-9) activity were designed and tested in vitro. Excellent sensitivity of these indicators is achieved by careful selection of the amino acid sequence corresponding to the substrate recognition preference of MMP-9. The phenomenon of Förster resonance energy transfer (FRET) is applied for detection of the peptide cleavage by MMP-9. FRET occurs between fluorescent dyes AMCA (donor) and TAMRA (acceptor) bound to the flexible peptide fragments differing in the donor acceptor separation distance. Enzymatic hydrolysis of the peptide leads to a significant weakening of energy transfer and increased donor fluorescence intensity. The enzyme action efficiency is higher for the longer proline-rich peptide which is reflected in the results of steady-state fluorescence studies. In particular, the use of longer peptide allows shorter detection time of MMP-9 (about 20–30 min). The possible reasons for this effect are discussed.

Effects of Catechin-rich Green Tea Extract on the MMP-1 Activity of HaCaT Keratinocyte Cells and on UVB-induced Skin Damage in Hairless Mice

Background: Skin is an organ that protects the human body from various environmental stimuli that can induce immune system activation. Skin aging can be largely divided into two categories: physiological aging, which is caused by the a decreased physiological function of the skin and structural changes with aging, and photoaging, which is caused by the chemical stress induced by external stimuli such as ultraviolet (UV) radiation.BR Methods and Results: The objective of this study was to investigate the anti-wrinkle and UV protective effect of catechin-rich green tea extract (CGTE) in activated keratinocyte (HaCaT cells) and UV-induced skin damage in hairless mice. The results showed that CGTE inhibits the tumor necrosis factor-alpha interferon-gamma (TNF-α + IFN-γ)-induced expression of matrix metalloproteinase (MMP)-1 in HaCaT cells. In addition, the CGTE treatment significantly reduced wrinkle formation, epidermal thickness, collagen deposition, and transepidermal water loss in dorsal skin irradiated with UVB. However, the β-glucosidase activity was significantly increased. The CGTE treatment inhibits mRNA expression and enzyme activity of MMP-2 and MMP-9 in the dorsal skin irradiated with UVB.BR Conclusions: It is expected that CGTE can be effectively used as a functional food and cosmetic ingredient to improve skin moisture retention and reduce wrinkle formation.

DNMT and HDAC inhibitors modulate MMP-9-dependent H3\xa0N-terminal tail proteolysis and osteoclastogenesis

BackgroundMMP-9-dependent proteolysis of histone H3 N-terminal tail (H3NT) is an important mechanism for activation of gene expression during osteoclast differentiation. Like other enzymes targeting their substrates within chromatin structure, MMP-9 enzymatic activity toward H3NT is tightly controlled by histone modifications such as H3K18 acetylation (H3K18ac) and H3K27 monomethylation (H3K27me1). Growing evidence indicates that DNA methylation is another epigenetic mechanism controlling osteoclastogenesis, but whether DNA methylation is also critical for regulating MMP-9-dependent H3NT proteolysis and gene expression remains unknown.ResultsWe show here that treating RANKL-induced osteoclast progenitor (OCP) cells with the DNMT inhibitor 5-Aza-2′-deoxycytidine (5-Aza-CdR) induces CpG island hypomethylation and facilitates MMP-9 transcription. This increase in MMP-9 expression results in a significant enhancement of H3NT proteolysis and OCP cell differentiation. On the other hand, despite an increase in levels of H3K18ac, treatment with the HDAC inhibitor trichostatin A (TSA) leads to impairment of osteoclastogenic gene expression. Mechanistically, TSA treatment of OCP-induced cells stimulates H3K27ac with accompanying reduction in H3K27me1, which is a key modification to facilitate stable interaction of MMP-9 with nucleosomes for H3NT proteolysis. Moreover, hypomethylated osteoclastogenic genes in 5-Aza-CdR-treated cells remain transcriptionally inactive after TSA treatment, because H3K27 is highly acetylated and cannot be modified by G9a.ConclusionsThese findings clearly indicate that DNA methylation and histone modification are important mechanisms in regulating osteoclastogenic gene expression and that their inhibitors can be used as potential therapeutic tools for treating bone disorders.

Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level

The extracellular matrix (ECM) is a highly dynamic network constantly remodeled by a fine-tuned protein formation and degradation balance. Matrix metalloproteinases (MMPs) constitute key orchestrators of ECM degradation. Their activity is controlled by tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAG). Here, we investigated the molecular interplay of MMP2 with different GAG (chondroitin sulfate, hyaluronan (HA), sulfated hyaluronan (SH) and heparin (HE)) and the impact of GAG on MMP2/TIMP3 complex formation using in vitro-experiments with human bone marrow stromal cells, in silico docking and molecular dynamics simulations. SH and HE influenced MMP2 and TIMP3 protein levels and MMP2 activity. Only SH supported the alignment of both proteins in fibrillar-like structures, which, based on our molecular models, would be due to a stabilization of the interactions between MMP2-hemopexin domain and TIMP3-C-terminal tail. Dependent on the temporal sequential order in which the final ternary complex was formed, our models indicated that SH and HA can affect TIMP3-induced MMP2 inhibition through precluding or supporting their interactions, respectively. Our combined experimental and theoretical approach provides valuable new insights on how GAG interfere with MMP2 activity and MMP2/TIMP3 complex formation. The results obtained evidence GAG as promising molecules for fine-balanced intervention of ECM remodeling.

Sea cucumber extract TBL-12 inhibits the proliferation, migration, and invasion of human prostate cancer cells through the p38 mitogen-activated protein kinase and intrinsic caspase apoptosis pathway.

Sea cucumber is a kind of nutritious echinoderm that has multiple biological activities, including antioxidant, antibacterial, and antitumor activities. However, there is no extensive study on the antitumor effect of sea cucumber extract on prostate cancer (PCa). TBL-12 is a new sea cucumber extract. In this study, we investigated the in vivo anti-PCa effect of TBL-12 and its in vitro effects on the proliferation, apoptosis, migration, and invasion of the human PCa cell lines LNCaP, 22RV1, PC-3, and DU145, and evaluated its possible mechanisms. Cell proliferation was analyzed by cell counting kit-8 and colony formation assays. Scratch migration assay and transwell invasiveness assay were used to observe TBL-12 effect on the migration and invasion of PCa cells. Matrix metalloproteinase 2 (MMP-2) and MMP-9 expression and enzymatic activity was determined by Western blot analysis, quantitative reverse-transcription polymerase chain reaction, and gelatin zymography. Apoptosis level was detected by flow cytometry analysis. Western blot analysis was used to analyze p38 mitogen-activated protein kinase (MAPK) and apoptosis pathways. Angiogenic array analysis was used to explore autocrine and paracrine growth factors in PCa cell lines. Xenograft tumor model was built to observe the in vivo anticancer effect. TBL-12 could significantly inhibit tumor growth in xenograft PCa mice in vivo, and dramatically inhibit the proliferation, colony formation, migration, and invasiveness of PCa cells in vitro (P < 0.05 and P < 0.001). The expression and enzyme activity of MMP-2 and MMP-9 were significantly suppressed by TBL-12 ( P < 0.01), and decreased phosphorylation level of p38 in PCa cells was detected ( P < 0.001). Furthermore, TBL-12 could reinforce the MMP-2/MMP-9 inhibitory effect of SB203580, a specific inhibitor of the p38 MAPK pathway ( P < 0.05). Besides, TBL-12 could induce the apoptosis of PCa cells by activating caspase-9, caspase-7, and poly(ADP-ribose) polymerase and suppressing survivin, and inhibit the secretion of angiogenin, angiopoietin-2, and vascular endothelial growth factor in PCa cells. Sea cucumber extract TBL-12 could suppress the proliferation and metastasis of human PCa cells by inhibiting MMP-2 and MMP-9 via blocking the p38 MAPK pathway, inducing apoptosis through intrinsic caspase apoptosis pathway and inhibiting the secretion of angiogenic factors. Our findings may be of importance and significance for the research and clinical applications of sea cucumber extract in PCa treatment.

Abstract WP151: Ischemic Injury in Diabetes Augments Cerebral Microvascular Matrix Metalloproteinase Activation in Female Rats

Introduction: Diabetic female patients are at greater risk of stroke and worsened recovery. We have previously shown that matrix metalloprotease 3 (MMP3) exacerbates hemorrhagic transformation (HT) in hyperglycemic stroke in male rats while high glucose and hypoxia increases MMP3 activity in brain microvascular endothelial cells (BMVECs). Furthermore, diabetic female rats develop greater HT. Current study was designed to test the hypothesis that ischemic stroke increases the cerebral microvascular MMP3 activity in diabetic female animals. Methods: Female rats after 7-8 weeks of diabetes (high fat diet and low dose of streptozotocin) were subjected to 60 min MCAO. After 3 days, adhesive removal time (ART) and behavioral composite score were measured and cerebral microvessels were isolated. Human female BMVECs were grown under normal (5.5mM glucose) or diabetic condition (25mM glucose plus 100μM sodium palmitate) for 48 hours, followed by hypoxia insult for 12 hours (200μM cobalt chloride). Cell lysate, media and microvascular homogenates were analyzed for MMP profiling by immunoblotting, zymography and MMP3 enzyme activity by fluorescence resonance energy transfer (FRET) assay. Results: MMP3 protein expression and activity were significantly increased in the ischemic microvasculature and this was accompanied by a worse neurobehavioral score (Table 1). In vitro, expression of MMP3, 2 and 9 proteins in cell lysate were not different between the groups. However, MMP3 and MMP9 enzyme activities were significantly increased in the diabetes condition while, there was no additional increase in enzyme activity with hypoxia. MMP2 activity was significantly increased with hypoxia in diabetes condition (Table 1). Conclusions: In female rats, diabetes and hypoxia increase MMP activity in the microvasculature and endothelial cells. These observations provide initial evidence to further explore the role of MMPs in the increased HT seen in females with diabetes.

In vitro effects of interleukin (IL)-1 beta inhibition on the epithelial-to-mesenchymal transition (EMT) of renal tubular and hepatic stellate cells

BackgroundThe epithelial to mesenchymal transition (EMT) is a multi-factorial biological mechanism involved in renal and hepatic fibrosis and the IL-1 beta has been assumed as a mediator of this process although data are not exhaustive. Therefore, the aim of our study was to evaluate the role of this cytokine in the EMT of renal proximal tubular epithelial cells (HK-2) and stellate cells (LX-2) and the protective/anti-fibrotic effect of its inhibition by Canakinumab (a specific human monoclonal antibody targeted against IL-1beta).MethodsBoth cell types were treated with IL-1 beta (10 ng/ml) for 6 and 24 h with and without Canakinumab (5 μg/ml). As control we used TGF-beta (10 ng/ml). Expression of EMT markers (vimentin, alpha-SMA, fibronectin) were evaluated through western blotting and immunofluorescence. Genes expression for matrix metalloproteinases (MMP)-2 was measured by Real-Time PCR and enzymatic activity by zymography. Cellular motility was assessed by scratch assay.ResultsIL-1 beta induced a significant up-regulation of EMT markers in both cell types and increased the MMP-2 protein expression and enzymatic activity, similarly to TGF-beta. Moreover, IL-1 beta induced a higher rate of motility in HK-2. Canakinumab prevented all these modifications in both cell types.ConclusionsOur results clearly demonstrate the role of IL-1 beta in the EMT of renal/stellate cells and it underlines, for the first time, the therapeutic potential of its specific inhibition on the prevention/minimization of organ fibrosis.

LOX inhibition downregulates MMP-2 and MMP-9 in gastric cancer tissues and cells.

Objective: The objective of this study was to analyze the effects of lysyl oxidase (LOX) on the expression and enzyme activity of the matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) and to study its preliminary effect mechanisms.Methods: We collected fresh cancer specimens from 49 gastric cancer patients who underwent surgery. Immunohistochemistry was used to quantitate the protein expression levels of LOX and MMP-9 in gastric cancer tissues and to analyze their correlation. Also, six-week old nude mice were divided into a control group and a LOX inhibition group. SGC-7901 gastric cancer cells were inoculated subcutaneously into the backs of the two groups of these mice to construct a gastric cancer-bearing nude mouse model. In the LOX inhibition group, β-aminopropionitrile (BAPN) was used to inhibit LOX. Western blotting was used to quantitate the relative expression levels of MMP-2 and MMP-9 in mouse tumor tissues, and gelatin zymography was used to quantitate their enzyme activity levels. In addition, BGC-823 gastric cancer cells were cultured, then 0.1 mM, 0.2 mM, and 0.3 mM BAPN and 2.5 nM, 5 nM, and 10 nM LOX were added to treat BGC-823 cells. ELISA and gelatin zymography were used to quantitate the protein concentrations and changes in enzyme activity of MMP-2 and MMP-9 in the culture supernatant. Western blotting was used to quantitate the relative expression levels of platelet derived growth factor receptor (PDGFR) in the BGC-823 gastric cancer cells after LOX inhibition and exogenous LOX addition.Results: In the tissues from the gastric cancer patients, the relative expression levels of LOX and MMP-9 were positively correlated (r = 0.326, P < 0.05). Compared with the control group, the tumor tissues from mice in the LOX inhibition group had reduced relative expression levels and enzyme activities of MMP-2 and MMP-9 (P < 0.05). After LOX were inhibited with different concentrations of BAPN in BGC-823 gastric cancer cells, the protein concentrations and enzyme activity levels of MMP-2 and MMP-9 in the culture supernatants were decreased (P < 0.05). In addition, the relative expression level of PDGFR in gastric cancer was decreased when BAPN concentrations increased, showing a negative dose-dependent manner (rPDGFR-α = -0.964, rPDGFR-β = -0.988, P < 0.05). After exogenous LOX treating BGC-823 cells, the concentrations and enzyme activity levels of MMP-2 and MMP-9 in the cell supernatant were increased (P < 0.05). Further, the relative expression of PDGFR in gastric cancer cells was increased with the increase of exogenous LOX, showing a positive dose-dependent manner (rPDGFR-α=0.952, rPDGFR-β=0.953, P<0.05).Conclusions: LOX inhibition can inhibit the expression and enzyme activity of MMP-2 and MMP-9 in gastric cancer tissues and cells, and the probable mechanism is through its effects on the PDGF-PDGFR signaling pathway.