Physical Properties and Biochemical Composition of Extracellular Matrix-Derived Hydrogels Dictate Vascularization Potential in an Organ-Dependent Fashion.

The inherent extracellular matrix (ECM) originating from a specific tissue impacts the process of vascularization, specifically vascular network formation (VNF) orchestrated by endothelial cells (ECs). The specific contribution toward these processes of ECM from highly disparate organs such as the skin and lungs remains a relatively unexplored area. In this study, we compared VNF and ECM remodeling mediated by microvascular ECs within gel, lung, and combinations thereof (hybrid) ECM hydrogels. Irrespective of the EC source, the skin-derived ECM hydrogel exhibited a higher propensity to drive and support VNF compared to both lung and hybrid ECM hydrogels. There were distinct disparities in the physical properties of the three types of hydrogels, including viscoelastic properties and complex architectural configurations, including fiber diameter, pore area, and numbers among the fibers. The hybrid ECM hydrogel properties were unique and not the sum of the component ECM parts. Furthermore, cellular ECM remodeling responses varied with skin ECM hydrogels promoting matrix metalloproteinase 1 (MMP1) secretion, while hybrid ECM hydrogels exhibited increased MMP9, fibronectin, and collagen IV deposition. Principal component analysis (PCA) indicated that the influence of a gel's mechanical properties on VNF was stronger than the biochemical composition. These data indicate that the organ-specific properties of an ECM dictate its capacity to support VNF, while intriguingly showing that ECs respond to more than just the biochemical constituents of an ECM. The study suggests potential applications in regenerative medicine by strategically selecting ECM origin or combinations to manipulate vascularization, offering promising prospects for enhancing wound healing through pro-regenerative interventions.

In silico evaluation of new mangiferin-based Positron Emission Tomography radiopharmaceuticals through the inhibition of metalloproteinase-9.

Metalloproteinase-9 (MMP-9) is a key protein in cancer advancement and metastasis owing to its ability to degrade some extracellular matrix components. Mangiferin, a natural polyphenolic compound, has demonstrated through experimental and theoretical studies to be a great anticancer agent for the selective inhibition of MMP-9. This work aimed to evaluate the utility of several fluorinated compounds obtained from MF as possible Positron Emission Tomography (PET) radiopharmaceuticals oriented to MMP-9. Density Functional Theory calculations of MF were made to obtain the most active sites toward electrophilic and nucleophilic reactions and propose a synthetic route to produce its fluorinated derivatives. The reactivity study allowed us to propose a late-stage synthetic route based on click chemistry to obtain three fluorinated MF-based derivatives. Molecular docking calculations suggested that the derivative F-propyl-MF could be suitable as PET radiopharmaceutical owing to the establishment of a five-coordinated complex with the catalytic Zn atom belonging to the active site of MMP-9, crucial factor in the inhibition of MMP-9.

Quest for selective MMP9 inhibitors: a computational approach.

Matrix Metalloproteinases-9 (MMP-9) is one of the important targets that play a vital role in various diseases such as cancer, Alzheimer's, arthritis, etc. Traditionally, MMP-9 inhibitors have been unable to achieve selectivity to get around this target; thereby, novel mechanisms such as inhibition of activated MMP-9 zymogen (pro-MMP-9) have been discovered. The JNJ0966 was one of the few compounds that attained the requisite selectivity by inhibiting the activation of MMP-9 zymogen (pro-MMP-9). Since JNJ0966, no other small molecules have been identified. Herein, extensive in silico studies were called upon to bolster the prospect of exploring potential candidates. The key objective of this research is to identify the potential hits from the ChEMBL database via molecular docking and dynamics approach. Protein with PDB ID: 5UE4, having a unique inhibitor in an allosteric binding pocket of MMP-9, was chosen for the study. Structure-based virtual screening and MMGBSA binding affinity calculations were performed, and five potential hits were finalized. Detailed analysis of the best-scoring molecules was performed with ADMET analysis and molecular dynamics (MD) simulation. All five hits outperformed JNJ0966 in the docking assessment, ADMET analysis, and molecular dynamics simulation. Accordingly, our research findings imply that these hits can be investigated for in vitro and in vivo studies against proMMP9 and might be explored as potential anticancer drugs. The outcome of our research might contribute in expediting the exploration of drugs that inhibits proMMP-9.Communicated by Ramaswamy H. Sarma.

Longitudinal Changes in BDNF and MMP-9 Protein Plasma Levels in Children after Cochlear Implantation.

Congenitally deaf children who undergo cochlear implantation before 1 year of age develop their auditory skills faster than children who are implanted later. In this longitudinal study, a cohort of 59 implanted children were divided into two subgroups according to their ages at implantation-below or above 1 year old-and the plasma levels of matrix metalloproteinase-9 (MMP-9), brain-derived neurotrophic factor (BDNF), and pro-BDNF were measured at 0, 8, and 18 months after cochlear implant activation, while auditory development was simultaneously evaluated using the LittlEARs Questionnaire (LEAQ). A control group consisted of 49 age-matched healthy children. We identified statistically higher BDNF levels at 0 months and at the 18-month follow-ups in the younger subgroup compared to the older one and lower LEAQ scores at 0 months in the younger subgroup. Between the subgroups, there were significant differences in the changes in BDNF levels from 0 to 8 months and in LEAQ scores from 0 to 18 months. The MMP-9 levels significantly decreased from 0 to 18 months and from 0 to 8 months in both subgroups and from 8 to 18 months only in the older one. For all measured protein concentrations, significant differences were identified between the older study subgroup and the age-matched control group.

2-Nucleobase-substituted 4,6-Diaminotriazine Analogs: Synthesis and Anti-cancer Activity in 5-Fluorouracil-sensitive and Resistant Colorectal Cancer Cells.

BACKGROUND: Cancer continues to be the second leading cause of death worldwide, with colorectal cancer (CRC) being the third most common type. Despite significant advances in cancer therapies, the current treatment of CRC remains suboptimal. In addition, the effectiveness of available chemotherapeutic drugs such as 5-Fluorouracil (5-FU) is limited by CRC-acquired resistance. METHODS: In this study, we provide innovative approaches employed in synthesizing four novel nucleobase analogs. Equally, we describe the effects of these compounds on proliferation, migration, aggregation, and adhesion of 5-FU-sensitive (HCT116) and -resistant (5-FU-R-HCT116) human CRC cells. In either cell type, our synthesized novel analogs significantly inhibited cell viability in a concentration- and time-dependent manner. This highlights the higher potency of these novel analogs. In addition, these compounds attenuated migration and adhesion of both cell types while they promoted homotypic cell-cell interaction. RESULTS: These changes were reflected by the downregulation of matrix metalloproteases (MMP-2 and MMP-9). Furthermore, our analogs exhibited potent anti-angiogenic activity in vivo. CONCLUSION: These novel nucleobase analogs reduced the level of secreted vascular endothelial growth factor (VEGF) and nitric oxide (NO) production in both 5-FU-sensitive and -resistant CRC cells. Taken together, our data highlight the potential chemotherapeutic properties of our novel analogs against CRC, including the 5-FU-resistant form.

Insight into the structural requirements of gelatinases (MMP-2 and MMP-9) inhibitors by multiple validated molecular modelling approaches: Part II.

Inhibition of the matrix metalloproteinases (MMPs) is effective against metastasis of secondary tumours. Previous MMP inhibitors have failed in clinical trials due to their off-target toxicity in solid tumours. Thus, newer MMP inhibitors now have paramount importance. Here, different molecular modelling techniques were applied on a dataset of 110 gelatinase (MMP-2 and MMP-9) inhibitors. The objectives of the present study were to identify structural fingerprints for gelatinase inhibition and also to develop statistically validated QSAR models for the screening and prediction of different derivatives as MMP-2 (gelatinase A) and MMP-9 (gelatinase B) inhibitors. The Bayesian classification study provided the ROC values for the training set of 0.837 and 0.815 for MMP-2 and MMP-9, respectively. The linear model also produced the leave-one-out cross-validated Q2 of 0.805 (eq. 1, MMP-2) and 0.724 (eq. 2, MMP-9), an r2 of 0.845 (eq. 1, MMP-2) and 0.782 (eq. 2, MMP-9), an r2Pred of 0.806 (eq. 1, MMP-2) and 0.732 (eq. 2, MMP-9). Similarly, non-linear learning models were also statistically significant and reliable. Overall, this study may help in the rational design of newer compounds with higher gelatinase inhibition to fight against both primary and secondary cancers in future.

In Silico Analysis of Cissus rotundifolia Constituents as Human Neutrophil Elastase (HNE), Matrix Metalloproteinases (MMP 2 and MMP 9), and Tyrosinase Inhibitors.

Cissus rotundifolia has been reported to possess various biological activities such as anti-diabetic, anti-fertility, anti-hyperlipidemic, anti-malarial, anti-osteoporotic, and anti-parasitic activities. Therefore in the present study, eleven selected constituents of Cissus rotundifolia which includes aconitic acid, astragalin, acteoside, aliospiroside A, beta amyrin, bergenin, formononetin, gallic acid, isovitexin, isoorientin, and isoquercitrin were studied on the docking behavior of human neutrophil elastase (HNE), matrix metalloproteinases (MMP 2 and MMP 9), and tyrosinase by using PatchDock method. Furthermore, molecular physicochemical, bioactivity score/drug-likeness, ADME (absorption, distribution, metabolism, and excretion), and toxicity analyses were also carried out using Molinspiration, Swiss ADME, and ProTox-II methods, respectively. The molecular physicochemical investigation showed that three ligands such as acteoside, aliospiroside A, and isoorientin have three violations for Lipinski's rule of five. Similarly, ADME analysis one ligand (formononetin) predicated to have high blood-brain barrier (BBB) permeability effect. The docking studies showed that isovitexin exhibited the highest atomic contact energy (-341.61 kcal/mol) for human neutrophil elastase (HNE), more over alliospiroside A has shown maximum atomic contact energy for both matrix metalloproteinases (MMP 2 [-618.00 kcal/mol] and MMP 9 [-634.73 kcal/mol]). Furthermore, isoquercitrin has exhibited the highest atomic contact energy (-145.70 kcal/mol) for tyrosinase. Thus, the present investigation outcome provides new knowledge in understanding eleven Cissus rotundifolia constituents as possible novel inhibitors against HNE, MMP 2, MMP 9, and tyrosinase.

Downregulation of MMP-9 Enhances the Anti-Migratory Effect of Cyclophosphamide in MDA-MB-231 and MCF-7 Breast Cancer Cell Lines.

Metastasis is one of the most urgent issues in breast cancer patients. One of the factors necessary in the migration process is the remodeling of the extracellular matrix (ECM). Metalloproteinases (MMPs) can break down the elements of the ECM, which facilitates cell movement. Many highly aggressive tumors are characterized by high levels of MMPs. In the case of breast cancer, the association between MMP-9 and the migration potential and invasiveness of cells has been demonstrated. In addition, reports indicating increased migration of breast cancer cells after the administration of the commonly used cytostatic cyclophosphamide (CP) are particularly disturbing. Hence, our research aimed to assess the effect of CP treatment on MDA-MB-231 and MCF-7 cells and how this response is influenced by the downregulation of the MMP-9 level. The obtained results suggest that CP causes a decrease in the survival of breast cancer cells of various invasiveness, and the downregulation of MMP-9 enhances this effect, mainly by inducing apoptosis. Moreover, in the group of MMP-9 siRNA-transfected CP-treated cells, a more severe reduction in invasion and migration of cells of both lines was observed, as indicated by the migration and invasion transwell assays and Wound healing assay. Hence, we suggest that CP alone may not result in satisfactory therapeutic effects. On the other hand, the use of combination therapy targeting MMP-9, together with the CP, could improve the effectiveness of the treatment. Additionally, we confirmed a relationship between the levels of MMP-9 and cytokeratin 19 (CK19).

Lidocaine Alleviates Sepsis-Induced Acute Lung Injury in Mice by Suppressing Tissue Factor and Matrix Metalloproteinase-2/9.

Acute lung injury (ALI) is one of the fatal symptoms of sepsis. However, there were no effective clinical treatments. TF accumulation-induced fibrin deposit formations and coagulation abnormalities in pulmonary vessels contribute to the lethality of ALI. Suppressor of cytokine signaling 3 (SOCS3) acts as an endogenous negative regulator of the TLR4/TF pathway. We hypothesized that inducing SOCS3 expression using lidocaine to suppress the TLR4/TF pathway may alleviate ALI. Hematoxylin and eosin (H&E), B-mode ultrasound, and flow cytometry were used to measure the pathological damage of mice. Gelatin zymography was used to measure matrix metalloproteinase-2/9 (MMP-2/9) activities. Western blot was used to assay the expression of protein levels. Here, we show that lidocaine could increase the survival rate of ALI mice and ameliorate the lung injury of ALI mice including reducing the edema, neutrophil infiltration, and pulmonary thrombosis formation and increasing blood flow velocity. Moreover, in vitro and in vivo, lidocaine could increase the expression of p-AMPK and SOCS3 and subsequently decrease the expression of p-ASK1, p-p38, TF, and the activity of MMP-2/9. Taken together, our study demonstrated that lidocaine could inhibit the TLR4/ASK1/TF pathway to alleviate ALI via activating AMPK-SOCS3 axis.

A nutrient mixture reduced tumor growth of SK-UT-1 human leiomyosarcoma cells in vivo and in vitro by inhibiting MMPs and inducing apoptosis.

BACKGROUND: Uterine leiomyosarcoma is a rare malignant smooth muscle tumor originating in the uterine wall that generally responds poorly to chemotherapy and radiation. AIM: We investigated the in vitro effects of a novel nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract on the human leiomyosarcoma cell line SK-UT-1 by measuring cell proliferation, invasiveness, apoptosis, and expression of matrix metalloproteinases (MMP). We also tested the effects of nutrient mixture in vivo using nude mice. MATERIALS AND METHODS: Human leiomyosarcoma SK-UT-1 cells were treated with different concentrations of nutrient mixture. Cell proliferation was determined by MTT assay; MMP expression by gelatinase zymography; invasion by Matrigel assay; migration by scratch test; apoptosis using Live Green caspase kit. In vivo studies were conducted on 5-6 weeks old female nude mice inoculated subcutaneously with 3 â€¢ 106 SK-UT-1 cells. The mice were fed a regular diet or a diet supplemented with 0.5% nutrient mixture. After four weeks, the mice were sacrificed and the tumors were weighed and processed for histology. RESULTS: In vitro, nutrient mixture treatment was not toxic to SK-UT-1 cells at 250 µg/ml but exhibited 20% and 40% cytotoxicity at 500 and 1000 µg/ml respectively. Zymography did not show bands for either MMP-2 or MMP-9 in SK-UT-1 cells. However, treatment with phorbol myristate acetate stimulated the expression of MMP-9, both active and inactive forms in equal proportion. Nutrient mixture inhibited the secretion of both active and inactive forms in a dose dependent manner. Invasion through Matrigel and migration by scratch test were inhibited in a dose dependent fashion, with both invasion and migration inhibited at 250 µg/ml. Live Green Caspase apoptosis assay demonstrated slight apoptosis at 100 µg/ml and significant apoptosis at 250 to 1000 µg/ml. The results of in vitro studies were further confirmed in vivo by showing 50% decrease in tumor weight, 40% reduction in tumor burden compared to the tumors from mice fed regular diet. CONCLUSION: The results suggest a therapeutic potential for nutrient mixture in uterine leiomyosarcoma treatment.

Interleukin-33 is a Novel Immunosuppressor that Protects Cancer Cells from TIL Killing by a Macrophage-Mediated Shedding Mechanism.

Recognition of specific antigens expressed in cancer cells is the initial process of cytolytic T cell-mediated cancer killing. However, this process can be affected by other non-cancerous cellular components in the tumor microenvironment. Here, it is shown that interleukin-33 (IL-33)-activated macrophages protect melanoma cells from tumor-infiltrating lymphocyte-mediated killing. Mechanistically, IL-33 markedly upregulates metalloprotease 9 (MMP-9) expression in macrophages, which acts as a sheddase to trim NKG2D, an activating receptor expressed on the surface of natural killer (NK) cells, CD8+ T cells, subsets of CD4+ T cells, iNKT cells, and γδ T cells. Further, MMP-9 also cleaves the MHC class I molecule, cell surface antigen-presenting complex molecules, expressed in melanoma cells. Consequently, IL-33-induced macrophage MMP-9 robustly mitigates the tumor killing-effect by T cells. Genetic and pharmacological loss-of-function of MMP-9 sheddase restore T cell-mediated cancer killing. Together, these data provide compelling in vitro and in vivo evidence showing novel mechanisms underlying the IL-33-macrophage-MMP-9 axis-mediated immune tolerance against cancer cells. Targeting each of these signaling components, including IL-33 and MMP-9 provides a new therapeutic paradigm for improving anticancer efficacy by immune therapy.

Insight into the structural requirement of aryl sulphonamide based gelatinases (MMP-2 and MMP-9) inhibitors - Part I: 2D-QSAR, 3D-QSAR topomer CoMFA and Naïve Bayes studies - First report of 3D-QSAR Topomer CoMFA analysis for MMP-9 inhibitors and jointly inhibitors of gelatinases together.

Gelatinases [gelatinase A - matrix metalloproteinase-2 (MMP-2), gelatinase B - matrix metalloproteinase-9 (MMP-9)] play key roles in many disease conditions including cancer. Despite some research work on gelatinases inhibitors both jointly and individually had been reported, challenges still exist in achieving potency as well as selectivity. Here in part I of a series of work, we have reported the structural requirement of some arylsulfonamides. In particular, regression-based 2D-QSARs, topomer CoMFA (comparative molecular field analysis) and Bayesian classification models were constructed to refine structural features for attaining better gelatinase inhibitory activity. The 2D-QSAR models exhibited good statistical significance. The descriptors nsssN, SHBint6, SHBint7, PubchemFP629 were directly correlated with the MMP-2 binding affinities whereas nsssN, SHBint10 and AATS2i were directly proportional to MMP-9 binding affinities. The topomer CoMFA results indicated that the steric and electrostatic fields play key roles in gelatinase inhibition. The established Naïve Bayes prediction models were evaluated by fivefold cross validation and an external test set. Furthermore, important molecular descriptors related to MMP-2 and MMP-9 binding affinities and some active/inactive fragments were identified. Thus, these observations may be helpful for further work of aryl sulphonamide based gelatinase inhibitors in future.

Carnosine Protects against Cerebral Ischemic Injury by Inhibiting Matrix-Metalloproteinases.

Stroke is one of the leading causes of death and disability worldwide. However, treatment options for ischemic stroke remain limited. Matrix-metalloproteinases (MMPs) contribute to brain damage during ischemic strokes by disrupting the blood-brain barrier (BBB) and causing brain edemas. Carnosine, an endogenous dipeptide, was found by us and others to be protective against ischemic brain injury. In this study, we investigated whether carnosine influences MMP activity. Brain MMP levels and activity were measured by gelatin zymography after permanent occlusion of the middle cerebral artery (pMCAO) in rats and in vitro enzyme assays. Carnosine significantly reduced infarct volume and edema. Gelatin zymography and in vitro enzyme assays showed that carnosine inhibited brain MMPs. We showed that carnosine inhibited both MMP-2 and MMP-9 activity by chelating zinc. Carnosine also reduced the ischemia-mediated degradation of the tight junction proteins that comprise the BBB. In summary, our findings show that carnosine inhibits MMP activity by chelating zinc, an essential MMP co-factor, resulting in the reduction of edema and brain injury. We believe that our findings shed new light on the neuroprotective mechanism of carnosine against ischemic brain damage.

Biphenyl substituted lysine derivatives as recognition elements for the matrix metalloproteinases MMP-2 and MMP-9.

Matrix metalloproteinases (MMPs) are an important factor in cancer progression and metastasis, especially gelatinases MMP-2 and MMP-9. A simple methodology for their detection and monitoring is highly desirable. Molecular probes have been very widely and successfully applied to study the activity of MMPs in cellular processes in vitro. We thus synthesized a small compound library of MMP-2 and MMP-9 binding probes based on drug molecules and endowed with free amine groups for the functionalization of transducer surfaces. In this study, we combined experimental results obtained by a kinetic fluorogenic peptide substrate cleavage assay with molecular modeling studies in order to assess the ability of the probe to bind to their target enzymes. The synthesized biphenyl substituted lysine derivatives showed IC50-values in the low nanomolar concentration range against MMP-2 (ligands 3a-d: 3 nM to 8 µM, ligands 4a-d: 45 nM to 350 µM) and low micromolar range against MMP-9 (ligands 3a-d: 350 nM to 60 µM, ligands 4a-d: 5 µM to 600 µM), with a selectivity up to more than 160-fold for MMP-2. The experimental results correlated well with molecular modelling with FleXAID and X-score functions. We showed that in our compound series, the side chain remained far away from the S1' cavity and the ligand for all the docked minima. Ligands 4a-d with their free amine group on the side chain may thus be bound to transducer surfaces for the fabrication of sensors, while retaining their activity against their target enzymes.

Expression of soluble recombinant human matrix metalloproteinase 9 and generation of its monoclonal antibody.

We have successfully produced a recombinant human matrix metalloproteinase 9 (hMMP9) antigen with high yield and purity and used it to generate a hybridoma cell-culture-based monoclonal anti-hMMP9 antibody. We selected the most effective antibody for binding antigens and successfully identified its nucleotide sequence. The entire antigen and antibody developmental procedures described herein can be a practical approach for producing large amounts of monoclonal antibodies against hMMP9 and other antigens of interest. Additionally, the nucleotide sequence information of the anti-hMMP9 monoclonal antibody revealed herein will be useful for the generation of recombinant antibodies or antibody fragments against hMMP9.

Structural characterisation of inhibitory and non-inhibitory MMP-9-TIMP-1 complexes and implications for regulatory mechanisms of MMP-9.

MMP-9 plays a number of important physiological functions but is also responsible for many pathological processes, including cancer invasion, metastasis, and angiogenesis. It is, therefore, crucial to understand its enzymatic activity, including activation and inhibition mechanisms. This enzyme may also be partially involved in the "cytokine storm" that is characteristic of COVID-19 disease (SARS-CoV-2), as well as in the molecular mechanisms responsible for lung fibrosis. Due to the variety of processing pathways involving MMP-9 in biological systems and its uniqueness due to the O-glycosylated domain (OGD) and fibronectin-like (FBN) domain, specific interactions with its natural TIMP-1 inhibitor should be carefully studied, because they differ significantly from other homologous systems. In particular, earlier experimental studies have indicated that the newly characterised circular form of a proMMP-9 homotrimer exhibits stronger binding properties to TIMP-1 compared to its monomeric form. However, molecular structures of the complexes and the binding mechanisms remain unknown. The purpose of this study is to fill in the gaps in knowledge. Molecular modelling methods are applied to build the inhibitory and non-inhibitory MMP-9-TIMP-1 complexes, which allows for a detailed description of these structures and should allow for a better understanding of the regulatory processes in which MMP-9 is involved.

Sustained delivery of MMP-9 siRNA via thermosensitive hydrogel accelerates diabetic wound healing.

Excessive expression of matrix metalloproteinase 9 (MMP-9) impedes healing of diabetic chronic wounds, thus wound dressing that could effectively inhibit the expression of MMP-9 offers significant clinical translation for diabetic wound healing. Herein, a hybrid hydrogel dressing was developed for localized and sustained delivery of MMP-9 siRNA (siMMP-9). siMMP-9 was complexed with Gly-TETA (GT), the GT/siMMP9 complex was then loaded into a thermosensitive hydrogel based on Pluronic F-127 (PF) and methylcellulose (MC). In vitro, this hybrid hydrogel dressing exhibited negligible cytotoxicity, prolonged the release of GT/siMMP-9 for up to 7 days, and significantly reduced MMP-9 expression. In vivo assessment in diabetic rats demonstrated that hydrogel provided localized and sustained delivery via the thermosensitive controlled release of entrapped GT/siMMP-9 into wound tissues for 7 days, resulting in dramatic MMP-9 silencing which significantly improved diabetic wound closure. This hybrid hydrogel dressing exhibited excellent biocompatibility, with no observed systemic toxicity in rats. Taken together, the hybrid hydrogel dressing may constitute an effective and biocompatible means of enhancing diabetic wound healing through effective silencing of the MMP-9 gene, and this hydrogel delivery system also offers a platform for in vivo delivery of siRNA for the treatment of other diseases.

Celastrol inhibit the proliferation, invasion and migration of human cervical HeLa cancer cells through down-regulation of MMP-2 and MMP-9.

The present study evaluated the anticancer potential of celastrol through down-regulation of matrix metalloproteinase-2 (MMP-2) and MMP-9. HeLa cells were incubated with different concentrations of celastrol (1, 10 and 100 µM) for 48h. Doxorubicin was used as a reference drug. Cancer cell migration, apoptosis, cell viability and mitochondrial fragmentation were evaluated following celastrol treatment. In addition, the expression level of MMP-2, MMP-9 and caspase-3 was evaluated following celastrol treatment. HeLa cell viability was 94.1 ± 7, 53.4 ± 4 and 36.3 ± 2% at 1-100 µM of celastrol, respectively. Apoptotic cell numbers were increased, and inhibition of larger wounds in cancer cells was observed following celastrol treatment. Celastrol-treated cells showed condensed nuclei and clumped mitochondria. Reduced expression of MMP-2 and MMP-9 and increased expression of caspase-3 were observed following celastrol treatment. Based on the experimental results, we are concluding that the celastrol was effective against HeLa cervical cancer cells.

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies.

A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.

Crocin and Metformin suppress metastatic breast cancer progression via VEGF and MMP9 downregulations: in vitro and in vivo studies.

Metastatic breast cancer remains a serious health concern and numerous investigations recommended medicinal plants as a complementary therapy. Crocin is one of the known anticancer bio-component. Recently, the inhibitory effect of metformin has been studied on the various aspects of cancer. However, no study reported their combination effects on metastatic breast cancer. In the present study, we have assessed their anti-metastatic effects on in vitro and in vivo breast cancer models. Using MTT assay, scratch, and adhesion tests, we have evaluated the cytotoxic, anti-invasive and anti-adhesion effects of crocin and metformin on 4T1 cell line, respectively. Their protective effects and MMP9 as well as VEGF protein expression levels (Western blotting) investigated in the 4T1 murine breast cancer model. Our results showed that both crocin and metformin reduced cell viability, delayed scratch healing and inhibited the cell adhesion, in vitro. While crocin alone restored the mice's weight reduction, crocin, metformin, and their combination significantly reduced the tumor volume size and enhanced animal survival rate in murine breast cancer model, responses that were associated with VEGF and MMP9 down-regulation. These findings suggest that a combination of crocin and metformin could serve as a novel therapeutic approach to enhance the effectiveness of metastatic breast cancer therapy.