Inhibition Of MMP-9 Activity Research Articles

Curcumin Prevents Renal Damage of l-NAME Induced Hypertension in by Reducing MMP-2 and MMP-9.

In the present study, we investigated whether curcumin administration would interfere with the main renal features of l-NAME-induced hypertension model. For this purpose, we conducted both in vitro and in vivo experiments to evaluate renal indicators of inflammation, oxidative stress, and metalloproteinases (MMPs) expression/activity. Hypertension was induced by l-NAME (70 mg/kg/day), and Wistar rats from both control and hypertensive groups were treated with curcumin (50 or 100 mg/kg/day; gavage) or vehicle for 14 days. Blood and kidneys were collected to determine serum creatinine levels, histological alterations, oxidative stress, MMPs expression and activity, and ED1 expression. l-NAME increased blood pressure, but both doses of curcumin treatment reduced these values. l-NAME treatment increased creatinine levels, glomeruli area, Bowman's space, kidney MMP-2 activity, as well as MMP-9 and ED1 expression, and reduced the number of glomeruli. Curcumin treatment prevented the increase in creatinine levels, MMP-2 activity, and reduced MMP-2, MMP-9, ED1, and superoxide levels, as well as increased superoxide dismutase activity and partially prevented glomeruli alterations. Moreover, curcumin directly inhibited MMP-2 activity in vitro. Thus, our main findings demonstrate that curcumin reduced l-NAME-induced hypertension and renal glomerular alterations, inhibited MMP-2 and MMP-9 expression/activity, and reduced oxidative stress and inflammatory processes, which may indirectly impact hypertension-induced renal outcomes.

Abstract Tu129: Collagen Peptide modulates Enzymatic Activity post-Myocardial Infarction

Matrix metalloproteinases (MMPs) play critical roles post-myocardial infarction (MI) and directly correlate with adverse LV remodeling. Of the MMPs, MMP-9 shows the highest correlation with MI patient mortality. Clinical trials trying to inhibit MMP-9 were unsuccessful or detrimental, mostly because MMP-9 has critical roles post-MI and inhibition results in exacerbation of adverse remodeling. Previously, we reported a collagen peptide – p1159 – to reduce LV dilation and adverse LV remodeling post-MI. Recently, we discovered p1159 acts as an MMP-9 competitive substrate and reduces substrate proteolysis in vitro . Herein, we investigated whether p1159 can act as a competitive substrate in vivo to reduce proteolysis of endogenous substrates post-MI without inhibiting MMP-9 activity. Also, we compared p1159 actions to those of a MMP-9 inhibitor (MMP-9i). We hypothesized that animals that receive p1159 would show reduced inflammation and adverse remodeling compared to those that received MMP-9i. Adult male and female C57Bl/6 mice (n = 6-8/group) were grouped as follows: saline (control); p1159; MMP-9i, and p1159 + MMP-9i. We performed baseline echocardiography followed by surgery to induce a permanent occlusion MI model. Three hours later, mice received a randomized osmotic mini-pump to continuously deliver p1159 (14μg/day/kg body weight) or vehicle (saline, 0.9%) and/or MMP-9i (10mg/kg body weight, JNJ 0966 by gavage). The LV and plasma were harvested on day 3 (D3) and D5 for downstream proteomics analysis following a final echocardiographic assessment. Infarct sizes were similar between groups, indicating same level of initial injury. Compared to control, p1159 did not inhibit activity or levels of MMP-9, while animals treated with MMP-9i showed significantly reduced MMP-9 activity. As expected, p1159 reduced (p < 0.05) substrate cleavage, as seen by analysis of the LV peptidome, particularly at D3. Substrates that displayed a reduction in proteolysis included fibrinogen, MIF, myosin, titin, and tubulin. These data support p1159 as a competitive substrate of MMP-9 that can modulate its activity without inhibition. Moreover, these results may explain the previously reported effect of p1159 at reducing LV dilation and improving systolic function.

Parecoxib and 5-Fluorouracil Synergistically Inhibit EMT and Subsequent Metastasis in Colorectal Cancer by Targeting PI3K/Akt/NF-κB Signaling.

Colorectal cancer is one of the most common causes of cancer mortality worldwide, and innovative drugs for the treatment of colorectal cancer are continually being developed. 5-Fluorouracil (5-FU) is a common clinical chemotherapeutic drug. Acquired resistance to 5-FU is a clinical challenge in colorectal cancer treatment. Parecoxib is a selective COX-2-specific inhibitor that was demonstrated to inhibit metastasis in colorectal cancers in our previous study. This study aimed to investigate the synergistic antimetastatic activities of parecoxib to 5-FU in human colorectal cancer cells and determine the underlying mechanisms. Parecoxib and 5-FU synergistically suppressed metastasis in colorectal cancer cells. Treatment with the parecoxib/5-FU combination induced an increase in E-cadherin and decrease in β-catenin expression. The parecoxib/5-FU combination inhibited MMP-9 activity, and the NF-κB pathway was suppressed as well. Mechanistic analysis denoted that the parecoxib/5-FU combination hindered the essential molecules of the PI3K/Akt route to obstruct metastatic colorectal cancer. Furthermore, the parecoxib/5-FU combination could inhibit reactive oxygen species. Our work showed the antimetastatic capacity of the parecoxib/5-FU combination for treating colorectal cancers via the targeting of the PI3K/Akt/NF-κB pathway.

A Novel Strategy for the Treatment of Aneurysms: Inhibition of MMP-9 Activity through the Delivery of TIMP-1 Encoding Synthetic mRNA into Arteries.

Aneurysms pose life-threatening risks due to the dilatation of the arteries and carry a high risk of rupture. Despite continuous research efforts, there are still no satisfactory or clinically effective pharmaceutical treatments for this condition. Accelerated inflammatory processes during aneurysm development lead to increased levels of matrix metalloproteinases (MMPs) and destabilization of the vessel wall through the degradation of the structural components of the extracellular matrix (ECM), mainly collagen and elastin. Tissue inhibitors of metalloproteinases (TIMPs) directly regulate MMP activity and consequently inhibit ECM proteolysis. In this work, the synthesis of TIMP-1 protein was increased by the exogenous delivery of synthetic TIMP-1 encoding mRNA into aortic vessel tissue in an attempt to inhibit MMP-9. In vitro, TIMP-1 mRNA transfection resulted in significantly increased TIMP-1 protein expression in various cells. The functionality of the expressed protein was evaluated in an appropriate ex vivo aortic vessel model. Decreased MMP-9 activity was detected using in situ zymography 24 h and 48 h post microinjection of 5 µg TIMP-1 mRNA into the aortic vessel wall. These results suggest that TIMP-1 mRNA administration is a promising approach for the treatment of aneurysms.

Role of MMP-9 and NF-κB in Diabetic Retinopathy: Progression and Potential Role of Bioflavonoids in the Mitigation of Diabetic Retinopathy.

"Diabetes mellitus" is a chronic metabolic disorder manifested by elevated blood glucose levels, primarily due to insufficient insulin production or resistance to insulin. Long-term diabetes results in persistent complications like retinopathy, cardiomyopathy, nephropathy, and neuropathy, causing significant health risks. The most alarming microvascular consequence allied with diabetes is "diabetic retinopathy," distinguished by the proliferation of anomalous blood vessels in the eye, mainly in the retina, resulting in visual impairment, diabetic macular edema, and retinal detachment if left untreated. According to estimates, 27.0% of people with diabetes worldwide have retinopathy, which leads to 0.4 million blindness cases. It is believed that mitochondrial damage and the production of inflammatory mediators are the early indicators of diabetic retinopathy before any histological changes occur in the retina. Moreover, it is evident that augmented oxidative stress in the retina further initiates the NF-κB/MMP-9 downstream signaling pathway. Interestingly, these downstream regulators, Nuclear Factor Kappa B [NF- kB] and matrix metalloproteinases 9 [MMP-9], have been recognized as important regulators of the inception and advancement of diabetic retinopathy. This diabetes and oxidative stress-induced MMP-9 are believed to regulate various cellular functions, including angiogenesis and apoptosis, causing blood-retinal barrier breakdown and tight junction protein degradation that further leads to diabetic retinopathy. Thus, there is an emergency need for the treatment of diabetic retinopathy. Emerging treatment options include anti-VEGF, laser treatment, and eye surgery, but these have certain limitations. This comprehensive review explores the mechanisms of MMP-9 and NF-kB involvement in diabetic retinopathy and bioflavonoids' therapeutic potential and mechanisms of action in inhibiting MMP-9 activity and suppressing NF-kB-mediated inflammation. Clinical evidence supporting the use of bioflavonoids in mitigating diabetic complications and future perspectives are also examined.

Probing Anticancer Molecules from Onion Peels Waste; First Synthesis and Biological Studies of a Rare Quercetin Derivative-Pachypodol

Background: The constant increase in global onion production escalates the generation of onion peel waste. For instance, globally, >50 lakh tons of onion waste are generated annually. Methods: Its objectionable odor precludes its use in agriculture or disposal as landfilling presents environmental issues. Previous studies show that two major flavonoids, quercetin and its glycosides (spiraeoside), have been identified in abundance in onion waste. By utilizing the spiraeoside (quercetin-4ʹ-glucoside), a rapid synthesis of pachypodol (quercetin-3,3′,7-trimethyl ether, and a rare flavonol), an essential Ayurvedic product, has been developed and achieved. Pachypodol and analogs were studied for their ability to inhibit matrix metalloproteinase-2 and -9 (MMP-2 & 9) activity. Amongst the compounds tested, pachypodol significantly inhibited MMP-2 activity. Results: In-silico docking studies suggest that, unlike most known MMP inhibitors, pachypodol interacts selectively with MMP-2 through the residues Ile222, Tyr223, and Thr227 in a zincindependent manner. Conclusion: The experimental studies also prove that pachypodol inhibits the MMP-2 enzyme in a zinc-independent way.

Moraea sisyrinchium inhibits proliferation, cell cycle, and migration of cancerous cells, and decreases angiogenesis in chick chorioallantoic membrane.

Experimental studies reported that some plants in the genus of Moraea (Iridaceae family) show anticancer potential. This study aimed to evaluate the effects of Moraea sisyrinchium on U87 glioblastoma multiforme and HepG2 liver cancer cells. The cells were incubated for 24 hr with hydroalcoholic extract of the stem, flower, and bulb of M. sisyrinchium. Then, the cell proliferation (MTT) assay, cell cycle analysis (propidium iodide staining), cell migration test (scratch), Western blotting (Bax and Bcl-2 expression), and gelatin zymography (for matrix metalloproteinases, MMPs) were performed. Oxidative stress was evaluated by determining the levels of reactive oxygen species and lipid peroxidation. Angiogenesis was evaluated on chick embryo chorioallantoic membrane. The extracts of the flower, stem, and bulb significantly decreased the proliferation of HepG2 and U87 cells. This effect was more for U87 than HepG2 and for the bulb and stem than the flower. In U87 cells, the bulb extract increased oxidative stress, cell cycle arrest, and the Bax/Bcl-2 ratio. Also, this extract suppressed the migration ability of HepG2 and U87 cells, which was associated with the inhibition of MMP2 activity. In addition, it significantly reduced the number and diameter of vessels in the chorioallantoic membrane. Liquid chromatography-mass spectrometry revealed the presence of xanthones (bellidifolin and mangiferin), flavonoids (quercetin and luteolin), isoflavones (iridin and tectorigenin), and phytosterols (e.g., stigmasterol) in the bulb. M. sisyrinchium bulb decreased the proliferation and survival of cancer cells by inducing oxidative stress. It also reduced the migration ability of the cells and inhibited angiogenesis.

Remote ischemic preconditioning prevents sarcolemmal-associated proteolysis by MMP-2 inhibition.

The death of myocytes occurs through different pathways, but the rupture of the plasma membrane is the key point in the transition from reversible to irreversible injury. In the myocytes, three major groups of structural proteins that link the extracellular and intracellular milieus and confer structural stability to the cell membrane: the dystrophin-associated protein complex, the vinculin-integrin link, and the spectrin-based submembranous cytoskeleton. The objective was to determine if remote ischemic preconditioning (rIPC) preserves membrane-associated cytoskeletal proteins (dystrophin and β-dystroglycan) through the inhibition of metalloproteinase type 2 (MMP-2) activity. A second objective was to describe some of the intracellular signals of the rIPC, that modify mitochondrial function at the early reperfusion. Isolated rat hearts were subjected to 30min of global ischemia and 120min of reperfusion (I/R). rIPC was performed by 3 cycles of ischemia/reperfusion in the lower limb (rIPC). rIPC significantly decreased the infarct size, induced Akt/GSK-3 β phosphorylation and inhibition of the MPTP opening. rIPC improved mitochondrial function, increasing membrane potential, ATP production and respiratory control. I/R increased ONOO- production, which activates MMP-2. This enzyme degrades β-dystroglycan and dystrophin and collaborates to sarcolemmal disruption. rIPC attenuates the breakdown of β-dystroglycan and dystrophin through the inhibition of MMP-2 activity. Furthermore, we confirm that rIPC activates different intracellular pathway that involves the an Akt/Gsk3β and MPTP pore with preservation of mitochondrial function.

A structural and functional comparison between two recombinant human lubricin proteins: Recombinant human proteoglycan-4 (rhPRG4) vs ECF843

Proteoglycan 4 (PRG4, lubricin) is a mucin-like glycoprotein present on the ocular surface that has both boundary lubricating and anti-inflammatory properties. Full-length recombinant human PRG4 (rhPRG4) has been shown to be clinically effective in improving signs and symptoms of dry eye disease (DED). In vitro, rhPRG4 has been shown to reduce inflammation-induced cytokine production and NFκB activity in corneal epithelial cells, as well as to bind to and inhibit MMP-9 activity. A different form of recombinant human lubricin (ECF843), produced from the same cell line as rhPRG4 but manufactured using a different process, was recently assessed in a DED clinical trial. However, ECF843 did not significantly improve signs or symptoms of DED compared to vehicle. Initial published characterization of ECF843 showed it had a smaller hydrodynamic diameter and was less negatively charged than native PRG4. Further examination of the structural and functional properties of ECF843 and rhPRG4 could contribute to the understanding of what led to their disparate clinical efficacy. Therefore, the objective of this study was to characterize and compare rhPRG4 and ECF843 in vitro, both biophysically and functionally.Hydrodynamic diameter and charge were measured by dynamic light scattering (DLS) and zeta potential, respectively. Size and molecular weight was determined for individual species by size exclusion chromatography (SEC) with in-line DLS and multi-angle light scattering (MALS). Bond structure was measured by Raman spectroscopy, and sedimentation properties were measured by analytical ultracentrifugation (AUC). Functionally, MMP-9 inhibition was measured using a commercial MMP-9 activity kit, coefficient of friction was measured using an established boundary lubrication test at a latex-glass interface, and collagen 1-binding ability was measured by quart crystal microbalance with dissipation (QCMD). Additionally, the ability of rhPRG4 and ECF843 to inhibit urate acid crystal formation and cell adhesion was assessed.ECF843 had a significantly smaller hydrodynamic diameter and was less negatively charged than rhPRG4, as assessed by DLS and zeta potential. Size was further explored with SEC-DLS-MALS, which indicated that while rhPRG4 had 3 main peaks, corresponding to monomer, dimer, and multimer as expected, ECF843 had 2 peaks that were similar in size and molecular weight compared to rhPRG4's monomer peak and a third peak that was significantly smaller in both size and molar mass than the corresponding peak of rhPRG4. Raman spectroscopy demonstrated that ECF843 had significantly more disulfide bonds, which are functionally determinant structures, relative to the carbon-carbon backbone compared to rhPRG4, and AUC indicated that ECF843 was more compact than rhPRG4. Functionally, ECF843 was significantly less effective at inhibiting MMP-9 activity and functioning as a boundary lubricant compared to rhPRG4, as well as being slower to bind to collagen 1. Additionally, ECF843 was significantly less effective at inhibiting urate acid crystal formation and at preventing cell adhesion.Collectively, these data demonstrate ECF843 and rhPRG4 are significantly different in both structure and function. Given that a protein's structure sets the foundation for its interactions with other molecules and tissues in vivo, which ultimately determine its function, these differences most likely contributed to the disparate DED clinical trial results.

Machine learning and biological evaluation-based identification of a potential MMP-9 inhibitor, effective against ovarian cancer cells SKOV3

MMP-9, also known as gelatinase B, is a zinc-metalloproteinase family protein that plays a key role in the degradation of the extracellular matrix (ECM). The normal function of MMP-9 includes the breakdown of ECM, a process that aids in normal physiological processes such as embryonic development, angiogenesis, etc. Interruptions in these processes due to the over-expression or downregulation of MMP-9 are reported to cause some pathological conditions like neurodegenerative diseases and cancer. In the present study, an integrated approach for ML-based virtual screening of the Maybridge library was carried out and their biological activity was tested in an attempt to identify novel small molecule scaffolds that can inhibit the activity of MMP-9. The top hits were identified and selected for target-based activity against MMP-9 protein using the kit (Biovision K844). Further, MTT assay was performed in various cancer cell lines such as breast (MCF-7, MDA-MB-231), colorectal (HCT119, DL-D-1), cervical (HeLa), lung (A549) and ovarian cancer (SKOV3). Interestingly, one compound viz., RJF02215 exhibited anti-cancer activity selectively in SKOV3. Wound healing assay and colony formation assay performed on SKOV3 cell line in the presence of RJF02215 confirmed that the compound had a significant inhibitory effect on this cell line. Thus, we have identified a novel molecule that can inhibit MMP-9 activity in vitro and inhibits the proliferation of SKOV3 cells. Novel molecules based on the structure of RJF02215 may become a good value addition for the treatment of ovarian cancer by exhibiting selective MMP-9 activity. Communicated by Ramaswamy H. Sarma

Influence of N-arylsulfonamido d-valine N-substituents on the selectivity and potency of matrix metalloproteinase inhibitors

To develop matrix metalloproteinase inhibitors (MMPIs) for both therapy and medicinal imaging by fluorescence-based techniques or positron-emission tomography (PET), a small library of eighteen N-substituted N-arylsulfonamido d-valines were synthesized and their potency to inhibit two gelatinases (MMP-2, and MMP-9), two collagenases (MMP-8, and MMP-13) and macrophage elastase (MMP-12) was determined in a Structure-Activity-Relation study with ({4-[3-(5-methylthiophen-2-yl)-1,2,4-oxadiazol-5-yl]phenyl}sulfonyl)-d-valine (1) as a lead. All compounds were shown to be more potent MMP-2/-9 inhibitors (nanomolar range) compared to other tested MMPs. This is a remarkable result considering that a carboxylic acid group is the zinc binding moiety. The compound with a terminal fluoropropyltriazole group at the furan ring (P1′ substituent) was only four times less potent in inhibiting MMP-2 activity than the lead compound 1, making this compound a promising probe for PET application (after using a prosthetic group approach to introduce fluorine-18). Compounds with a TEG spacer and a terminal azide or even a fluorescein moiety at the sulfonylamide N atom (P2′ substituent) were almost as active as the lead structure 1, making the latter derivative a suitable fluorescence imaging tool.

Newly Synthesized 3-Indolyl Furanoid Inhibits Matrix Metalloproteinase-9 Activity and Prevents Nonsteroidal Anti-inflammatory Drug-Induced Gastric Ulceration.

Indomethacin, a known nonsteroidal anti-inflammatory drug (NSAID) induces gastric inflammation, causing degradation of the extracellular matrix by specific matrix metalloproteinases (MMPs). We investigated the antiulcer efficacy of 3-indolyl furanoids (3g and 3c, i.e., methoxy substitution at 4- and 5-positions of the indole ring, respectively), derived from indomethacin. Interestingly, 3g protected against indomethacin-induced gastropathy in vivo by inhibiting MMP-9. Our work established a chemical modification strategy for the development of safer NSAIDs. Moreover, in vitro and in silico studies confirmed that 3g inhibited MMP-9 activity with an IC50 value of 50 μM by binding to the catalytic cleft of MMP-9, leading to ulcer prevention. Pharmacokinetics was presented as the mean concentration-time profile in the rat plasma, and the extraction efficiency was greater than 70%, showing a Cmax of 104.48 μg/mL after 6.0 h (tmax) treatment with half-life and area under the curve being 7.0 h and 1273.8 h μg/mL, respectively, indicating the higher antiulcer potency of 3g.

Abstract 3530: Mechanisms underlying Ewing sarcoma-induced osteolysis

Abstract Ewing Sarcoma (ES) is an aggressive pediatric tumor, which often arises in bones and forms osseous metastases, both of which present as osteolytic lesions. Our previous studies indicated that neuropeptide Y (NPY), which is secreted from ES tumors, stimulates tumor-induced bone degradation via its effect on macrophage recruitment and their subsequent osteoclastic differentiation. This effect was dependent on the autocrine activation of the NPY Y5 receptor (Y5R) expressed by ES cells. However, the exact mechanisms of the osteolytic activity induced by the NPY/Y5R axis remained unknown. To identify the osteolytic factors released from ES cells due to the NPY/Y5R autocrine loop activity, we used shRNA to silence NPY or Y5R in SK-ES-1 ES cells and compared concentrations of known factors involved in the regulation of bone homeostasis in their conditioned media by an antibody array. Four secreted factors were found to be significantly decreased after silencing of NPY signaling: interleukin 8 (IL-8; p<0.005), matrix metalloproteinase 9 (MMP-9; p<0.05), intercellular adhesion molecule 1 (ICAM-1; p<0.005), and monocyte chemoattractant protein 1 (MCP-1; p<0.05). Among these proteins, IL-8 and MMP-9 were selected as the main candidates for mediators of NPY-induced osteolysis. To test their role in ES-mediated stimulation of osteolysis, we co-cultured mouse RAW 264.7 cells, that represent monocyte/macrophage lineage, with SK-ES-1 cells in the presence or absence of the MMP-9 inhibitor (JNJ-0966; 10-7M-10-5M) or anti-IL-8 neutralizing antibody (0.1-0.4µg/ml) and tested their effect on osteoclast differentiation detected by TRAP staining. Inhibiting MMP-9 activity significantly decreased osteoclastogenesis of RAW264.7 cells in dose-dependent manner, while blocking IL-8 had no significant effect on the number of formed osteoclasts. These findings implicate MMP-9 as the mediator of NPY osteolytic activity. Further studies are required to understand the exact mechanisms underlying these effects, as well as potential role of NPY-induced release of MCP-1 in macrophage recruitment to ES tumors. Citation Format: James J. Williams, Joanna B. Kitlinska. Mechanisms underlying Ewing sarcoma-induced osteolysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3530.

The ethanol extract of Cyperus exaltatus var. iwasakii exhibits cell cycle dysregulation, ERK1/2/p38 MAPK/AKT phosphorylation, and reduced MMP-9-mediated metastatic capacity in prostate cancer models in vitro and in vivo

BackgroundProstate cancer is the second most common cause of cancer death worldwide in men. The development of novel and highly efficient therapeutic strategies is strongly recommended to treat prostate cancer. Cyperaceae are an ecologically and economically important family of plants with several pharmacological effects. However, the biological efficacy of Cyperus exaltatus var. iwasakii (CE) is unknown. PurposeThis study aimed to investigate the antitumor effect of the ethanol extract of CE against prostate cancer. MethodsIn vitro antitumor efficacy of CE was explored by the MTT assay, cell counting assay, FACS analysis, immunoblot, wound-healing migration, invasion assay, zymographic assay, and EMSA in prostate cancer cells, DU145 and LNCaP. For in vivo experiments, xenograft mice were injected with LNCaP cells. Histology (H&E and Ki-67) and biochemical enzyme assay were then performed. The toxicity test was evaluated by an acute toxicity assay. The phytochemical constituents of CE were identified by spectrometric and chromatographic analyses. ResultsCE exerted a significant antiproliferative effect against prostate cancer cells. CE-induced antiproliferative cells were associated with cell cycle arrest at G0/G1 (cyclin D1/CDK4, cyclin E/CDK2, p21Waf1) in DU145 cells, but G2/M (ATR, CHK1, Cdc2, Cdc25c, p21Waf1, and p53) in LNCaP cells. CE stimulated the phosphorylation of ERK1/2, p38 MAPK, and AKT in DU145 cells, but only p38 MAPK phosphorylation was increased in LNCaP cells. CE treatment suppressed migration and invasion in the two types of prostate cancer cells by inhibiting MMP-9 activity through the regulation of transcription factors, such as AP-1 and NF-κB. In vivo experiments showed a reduction in tumor weight and size following oral CE administration. Histochemistry confirmed that CE inhibited tumor growth in the mouse LNCaP xenograft model. The administration of CE had no adverse effects on body weight, behavioral patterns, blood biochemistry, and histopathology findings of vital organs in mice. Finally, a total of 13 phytochemical constituents were identified and quantified in CE. The most abundant secondary metabolites in CE were astragalin, tricin, and p-coumaric acid. ConclusionOur results demonstrated the antitumor efficacy of CE against prostate cancer. These findings suggest that CE might be a potential candidate for prostate cancer prevention or treatment.

Gold Nanorods Inhibit Tumor Metastasis by Regulating MMP-9 Activity: Implications for Radiotherapy.

Dysregulation of matrix metalloproteinase (MMP) is strongly implicated in tumor invasion and metastasis. Nanomaterials can interact with proteins and have impacts on protein activity, which provides a potential strategy for inhibiting tumor invasion and metastasis. However, the regulation of MMP activity by nanomaterials has not been fully determined. Herein, we have found that gold nanorods (Au NRs) are able to induce the change of the secondary structure of MMP-9 and thereby inhibit their activity. Interestingly, the inhibition of MMP-9 activity is highly dependent on the aspect ratio of Au NRs, and an aspect ratio of 3.3 shows the maximum inhibition efficiency. Molecular dynamics simulations combined with mathematical statistics algorithm reveal the binding behaviors and interaction modes of MMP-9 with Au NRs in atomic details and disclose the mechanism of aspect ratio-dependent inhibition effect of Au NRs on MMP-9 activity. Au NRs with an aspect ratio of 3.3 successfully suppress the X-ray-activated invasion and metastasis of tumor by inhibiting MMP-9 activity. Our findings provide important guidance for the modulation of MMP-9 activity by tuning key parameters of nanomaterials and demonstrate that gold nanorods could be developed as potential MMP inhibitors.

Inhibition of matrix metalloproteinase-9 by 10-MDP and its calcium salt contributes to improved dentin-bonding durability

The activation of matrix metalloproteinases (MMPs) is responsible for degradation of the hybrid layer. The effect of 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) on biodegradation of the hybrid layer remains unknown. This study aimed to evaluate the effects of 10-MDP and its Ca salt on MMP-9 activity and dentin-bonding durability. 10-MDP-Ca salt was synthesized and characterized by X-ray diffraction (XRD) and nuclear magnetic resonance (NMR). The effects of chlorhexidine (CHX), 10-MDP, and 10-MDP-Ca salt on MMP activity were evaluated by in-situ zymography, MMP-9 inhibition assay, and hydroxyproline (HYP) assay. Dentin-resin bonded specimens were fabricated with using CHX or 10-MDP, including Single Bond 2 (Ctr), CHX + Single Bond 2 (CHX), 10-MDP + Single Bond 2 (10-MDP), and SBU (SBU). Micro-tensile bond strength (μTBS) and nanoleakage tests were performed after 24-h or two-years water storage. XRD and NMR confirmed the presence of 10-MDP-Ca salt. The relative inhibition percentages of recombinant MMP-9 for 10-MDP and 10-MDP-Ca salt were 8.04 ± 0.83% and 55.96 ± 1.82% respectively. HYP assay showed that 10-MDP, CHX, and 10-MDP-Ca salt conditioning decreased collagen degradation. Water storage weakened μTBS of all the groups, with the Ctr and CHX groups showing more significant decreases (P < 0.05). After aging, the Ctr and CHX groups also presented more nanoleakage, and the CHX group showed more in-situ zymography fluorescence than the 10-MDP and SBU groups. The capacity to inhibit MMP-9 activity, decrease HYP release, and reduce nanoleakage of 10-MDP and its salt contribute to improvement of dentin-bonding durability.

Downregulation of ASF1B inhibits tumor progression and enhances efficacy of cisplatin in pancreatic cancer.

Pancreatic cancer is an aggressive and lethal cancer with the highest mortality rate. Hence, the development of new targeting and innovative treatment strategies is needed. Recent studies reported that the histone chaperone anti-silencing function 1B (ASF1B) can be used as a diagnosis and prognosis cancer biomarker. However, functional studies of ASF1B in pancreatic cancer have not been performed. This study compared expression levels of ASF1B in pancreatic cancer specimens with those of normal tissues using publicly available online databases. We found that ASF1B was commonly overexpressed in pancreatic cancer specimens, which is associated with poor prognosis. ASF1B downregulation in pancreatic cancer cells reduced their colony formation, proliferation, migration, and invasion abilities, and inhibited MMP9 activity. Furthermore, ASF1B expression downregulation increased cell cycle S-phase arrest and DNA damage though activation of the checkpoint kinases Chk1 and Chk2 pathways. Additionally, increased caspase (caspases-3 and -9) activation and PARP cleavage led to enhanced caspase-dependent apoptosis and improved cisplatin sensitivity. Collectively, our results indicate that ASF1B may serve as a potential biomarker of pancreatic cancer and a novel therapeutic target.

Fluoride and trimetaphosphate association as a novel approach for remineralization and antiproteolytic activity in dentin tissue

ObjectiveThe study evaluated the effect of solutions containing fluoride (F) and/or sodium trimetaphosphate (TMP) and F/TMP on the inhibition of MMP-2 and MMP-9, and on dentin remineralization in vitro. DesignBovine root dentin blocks were prepared, and caries-like lesions were induced in two thirds of the surface. Blocks were then randomly divided into 13 groups/solutions (n = 10): Placebo; 0.3 %, 1 % and 3 % NaOH-hydrolyzed TMP; 0.3 %, 1 % and 3 % TMP; 250, 500 and 1100 ppm F; 250 ppm F + 0.3 % TMP; 500 ppm F + 1 % TMP and 1100 ppm F + 3 % TMP. One third of each specimen was treated with the respective solutions in pH-cycling. The mineral concentration (gHAp × cm−3 × µm) was determined by computed X-ray microtomography, and data submitted to ANOVA and Student-Newman-Keuls’ test (p < 0.05). The ability of the solutions to inhibit MMP-2 and MMP-9 activity was assessed by zymography. ResultsF/TMP association led to less mineral loss in the deeper region of the lesion and reduced the depth of lesions when compared to its counterpart without TMP (p < 0.001). 3 % TMP (hydrolyzed or not), 500 ppm F and 1100 ppm F completely inhibited MMP-2 activity, while for MMP-9 such effects were only achieved by treatment with 1100 ppm F + 3 % TMP. ConclusionTreatment with 1100 ppm F + 3 % TMP fully inhibits the gelatinolytic activity of MMPs-2 and − 9 and shows greater remineralizing capacity in artificial caries lesions in dentin. However, hydrolyzing TMP does not improve its anti-proteolytic activity and its remineralizing capacity.

Abstract 3837: The physiological relevance of TIMP dimer formation

Abstract Tissue inhibitors of metalloproteinases (TIMPs) are a family of four paralogous genes that modulate the activity of matrix metalloproteinases (MMPs) and are vital for tissue homeostasis. Additionally, TIMPs display activities that are independent of their MMP inhibitory activities. Dysregulation and perturbations within this system can lead to complex tissue-specific pathologies. TIMP2 is the most abundantly expressed member of the TIMP family and has been shown to display potential therapeutic uses in cancer and other diseases. We have noted the persistent presence of SDS-stable TIMP2 dimers/multimers in SDS-PAGE at a level of ~5% total protein. The goal of this study is to determine the stability and biological relevance of SDS-stable dimer formation of TIMP2 in in vitro and in vivo. We found that TIMP2 dimers display an enhanced ability to inhibit MMP2 activity in reverse zymography assays. Immunoblotting revealed gel extracted TIMP2 dimer entered an equilibrium with monomeric TIMP2, while the extracted monomer did not form new dimer. In addition, MMP2 activity assays do not corroborate the enhanced MMP2 inhibitory activity observed in reverse zymograms but are consistent with the observation that isolated TIMP2 dimers re-equilibrate with monomers. This implies that structural or sequence difference mediates the formation of dimers. Dimer/multimer formation is observed across the TIMP family. Mass spectrometry analysis of gel extracted TIMP monomers and dimers show that monomer and dimer forms of TIMPs display unique post-translation modification (PTM) profiles. We propose that understanding the interplay between TIMP multimers and PTMs will reveal new biological functions within this intriguing family of proteins. We are currently working to resolve the functional differences between monomer and dimer and reveal the underlying mechanisms at play. Citation Format: Carolyn Lazaroff, David Peeney, Sadeechya Gurung, William G. Stetler-Stevenson. The physiological relevance of TIMP dimer formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3837.

Lupin Protein Concentrate as a Novel Functional Food Additive That Can Reduce Colitis-Induced Inflammation and Oxidative Stress.

Food fortification with bioactive compounds may constitute a way to ameliorate inflammatory bowel diseases (IBDs). Lupin seeds contain an oligomer named deflamin that can reduce IBD’s symptoms via MMP-9 inhibition. Here, our goal was to develop a lupin protein concentrate (LPC) enriched in deflamin and to test its application as a food additive to be used as a functional food against colitis. The nutritional profile of the LPC was evaluated, and its efficacy in vivo was tested, either alone or as added to wheat cookies. The LPC presented high protein and carbohydrate contents (20.09 g/100 g and 62.05/100 g, respectively), as well as antioxidant activity (FRAP: 351.19 mg AAE/10 mg and DPPH: 273.9 mg AAE/10 mg). It was also effective against TNBS-induced colitis in a dose dependent-manner, reducing DAI scores by more than 50% and concomitantly inhibiting MMP-9 activity. When added to cookies, the LPC activities were maintained after baking, and a 4-day diet with LPC cookies induced a significant protective effect against acetic acid-induced colitis, overall bringing lesions, oxidative stress and DNA damage levels to values significantly similar to controls (p < 0.001). The results show that the LPC is an efficient way to deliver deflamin in IBD-targeted diets.