Expression Levels Of MMP-9 Research Articles

MiR-1180 Targets FXYD5 to Regulate Pancreatic Cancer Cells Migration and Invasion.

Pancreatic cancer is a fatal malignancy typically diagnosed in older males and has an aggressive progression. The function of the miR-1180/FXYD5 axis in pancreatic cancer malignant behaviors was investigated. 20 pairs of pancreatic cancer and adjacent normal tissue samples were harvested from pancreatic cancer patients, and qRT-PCR, IHC, and western blot assays were performed, respectively, to detect the mRNA expression and protein levels of miR-1180 or FXYD5. Transwell and scratch assays were conducted to detect the migratory and invasive ability of pancreatic cancer cells; a Dual-luciferase reporter assay was employed to validate miR-1180 targeting FXYD5. miR-1180 targeting FXYD5 to regulate the migratory and invasive ability of pancreatic cancer cells was validated in mouse xenograft tumor models. FXYD5 expression was increased in pancreatic cancer tissue samples than in adjacent normal tissue samples (P < 0.01), and FXYD5 expression exhibited a positive correlation with the migratory and invasive ability of pancreatic cancer cells. miR-1180 targeted FXYD5 and negatively regulated FXYD5. Restoring miR-1180 expression could inhibit the migratory and invasive ability of pancreatic cancer cells (P < 0.01), and this effect could potentially be alleviated by FXYD5 overexpression. The miR-1180/FXYD5 axis positively regulated E-cadherin and negatively regulated MMP2 and MMP9 expression levels. In vivo findings demonstrated that miR-1180 overexpression inhibited tumor growth and lung metastasis (P < 0.05), while FXYD5 overexpression promoted tumor growth and lung metastasis (P < 0.05). In conclusion, the miR-1180 /FXYD5 axis could be involved in pancreatic cancer metastasis through the regulation of EMT and extracellular matrix degradation.

Potential effects of parietin on apoptosis and cell cycle related genes in SH-SY5Y neuroblastoma cells

Background: Ingredients obtained from natural products have been used in cancer treatments for years. High diversity and non-toxicity compared to chemotherapeutic agents are the main reasons for their preference. Lichens having potential for treatment of cancer consist of fungus and 1-2 species of algae. Under the name of lichen substances, many of them have also been synthesized as specific substances. The secondary metabolites in lichens are generally insoluble in water, and have many biological activities such as antiviral, antitumor, antibacterial, and antioxidant; they store in the fungal cell or on the surface of the hyphae and can only be extracted with organic solvents.&#x0D; Parietin extracted from lichen species such as xanthoria parietina is an anthraquinone pigment and a secondary metabolite. &#x0D; In our study, the effects of parietin on cytotoxicity, gene expression, migration, invasion, and colony formation in neuroblastoma cells treated with parietin were investigated. The SH-SY5Y neuroblastoma (NB), which is not treated with parietin, was administered as a control group.&#x0D; Methods: The IC50 value of the parietin was determined using XTT assay. The total RNA extractions were performed from the cells using the Tri-Reagent kit. The expressions of BCL-XL, BCL-2, MMP2, MMP9, P21, CYCLIN D1, CASAPASE-3, CASAPASE-9, BAX, P53, PUMA, and NOXA genes were investigated by Lightcycler 480 (Roche) using SYBR Green dye. Migration analysis of the control and the dose group cells were performed in accordance with the Wound-healing assay protocol. Invasion activities were determined using the “Invasion Chamber” (BD Biosciences) protocol. The colony assay was treated with crystal violet and the cells were observed under the light microscope.&#x0D; Results: The IC50 value of the parietin used for 48-hour treatment on the cells was determined as 35 µM. It was found that the expression levels of BCL-XL, BCL-2, MMP2, MMP9, P21, and CYCLIN D1 mRNA were downregulated, and it was also shown the expression levels of CASAPASE-3, CASAPASE-9, BAX, P53, PUMA, and NOXA to be upregulated. It was determined that parietin suppressed both cell invasion and migration, and colony formation in the neuroblastoma cells. &#x0D; Conclusions: Thus, it can be possible parietin to be used as an alternative, complementary, and supportive agent together with the other drugs in the treatment of neuroblastoma. However, more comprehensive studies supporting these significant effects of parietin will increase its potential in the application.

Triptolide inhibits proliferation and invasion of colorectal cancer cells by blocking Nrf2 expression.

Triptolide (TPL), the main active ingredient of Tripterygium wilfordii, has anti-inflammatory, immunomodulatory, and antitumor actions. It can also inhibit cell proliferation and metastasis while promoting apoptosis of several tumors, such as colorectal cancer (CRC). However, the mechanism of TPL against CRC is not clear. This study was designed to investigate the effects and molecular mechanisms of TPL on the proliferation and invasion ability of CRC cells. A human CRC cell line (HT29 cell line) cultured invitro was treated with different concentrations of TPL (0, 25, 50, and 100 nmol/L). The proliferation of cells was detected by MTT, the invasion ability of cells by Transwell, and the apoptosis level by flow cytometry. The protein expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), matrix metalloproteinase (MMP)-2, and MMP-9 were detected by western blotting. After transfection with sh-Nrf2, HT29 cells were divided into NC group, NC + TPL group and sh-Nrf2 + TPL group, and the above assays were repeated for each group. TPL significantly inhibited the proliferation and invasion ability of HT29 cells and promoted apoptosis (p < .05). Notably, its inhibitory or promotional effects were concentration-dependent, which were enhanced with increasing drug concentration (p < .05). After silencing Nrf2 expression, the proliferation, and invasion ability of HT29 cells were further significantly inhibited while cells apoptosis was further promoted (p < .05). Besides, the decreased Nrf2 expression reduced the protein expression levels of MMP-2 and MMP-9 (p < .05). TPL can effectively inhibit the proliferation and invasion while promoting apoptosis of HT29 cells. And its mechanism of action may be related to the inhibition of Nrf2 signaling expression.

POTENTIAL FOR PATHOGENETIC CORRECTION OF PROTEASE-ANTIPROTEASE SYSTEM IMBALANCE IN PATIENTS WITH COMORBIDITY OF STABLE ANGINA PECTORIS OF THE II-III FUNCTIONAL CLASS AND STAGE II-III CHRONIC OBSTRUCTIVE PULMONARY DISEASE

The aim of this study is to assess the clinical effectiveness of the therapy including exogenous L-arginine and statins under the comorbidity of stable angina pectoris of the II-III functional class and chronic obstructive pulmonary disease of the II-III stage.&#x0D; Materials and methods. The study included 50 patients with coronary heart disease and chronic obstructive pulmonary disease (mean age 57 years; male/female ratio 78/22%). In order to assess the clinical effectiveness of the therapy for combined cardiopulmonary pathology, the patients were randomized into 2 subgroups: subgroup 1 included 25 patients who took metabolitotropic medication under high-intensity statin therapy (the basic therapy included L-arginine and rosuvastatin as a hypolipidemic agent in a dose of 20 mg/day); subgroup 2 included 25 patients who received only basic therapy under moderately intensive statin therapy (daily doses of atorvastatin 20 mg, rosuvastatin 10 mg)). The groups were comparable in terms of gender distribution and demographic characteristics. Comparative analysis of smoking experience, initial smoking age and smoking history did not differ significantly between the groups. After 12±1 weeks, a control examination was carried out.&#x0D; Results. The analysis of the ratio of the MMP-9 activity level before and after the therapy showed that the level of MMP-9 expression decreased by 43.28% (p&lt;0.05) in the 1st subgroup and by 13.18% in the 2nd subgroup until the end of the therapy. TIMP activity after 12 weeks of the treatment was +33.11% (p&lt;0.05) and +11.27% for the 1st and 2nd subgroups respectively. The difference between subgroups 1 and 2 in the level of the CRP marker before and after the therapy was -45.65% (p&lt;0.05) and -9.09%, respectively. 76% of people from the 1st subgroup reached the target level of healthy people before the completion of the therapy that is significantly higher than the similar indicator in the 2nd subgroup (32%) at χ2=8.05, p&lt;0.01.&#x0D; Conclusions: The application of L-arginine alongside high-intensity statin therapy for patients presenting with stable angina pectoris of the II-III functional class and stage II-III chronic obstructive pulmonary disease (COPD) resulted in more notable positive changes in the modulation of protease-antiprotease status disorders. This was evidenced by a reduction in the heightened expression of MMP-9 and an increase in the level of TIMP during the course of treatment.

Inflammatory Factors Predicting Dry Eye Syndrome in a Model Using Osmotic Pressure

Purpose: To identify inflammatory factors that may serve as biomarkers for dry eye syndrome using a hyperosmotic dry eye syndrome model.Methods: Cultured human conjunctival epithelial cells were subjected to 400, 450, 500, and 550 mOsm/L NaCl, and cell viability was assessed in response to osmolarity. The relative expression of inflammatory factors was evaluated by real-time polymerase chain reaction of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, metalloproteinase (MMP)-2, and MMP-9 according to the NaCl concentration.Results: An increase in the concentration of NaCl led to a notable decrease in cell viability (&lt;i&gt;p&lt;/i&gt; &lt; 0.05). IL-8, TNF-α, and MMP-9 expression levels increased significantly in proportion to NaCl concentration (&lt;i&gt;p&lt;/i&gt; &lt; 0.05).Conclusions: IL-8, TNF-α, and MMP-9 may serve as effective biomarkers in dry eye studies.

An enhanced model for environmental dry eye: Exploring pathological features and underlying factors

This study aimed to develop an enhanced environmental dry eye (EDE) model that accurately reproduces the etiology of prolonged visual fatigue and investigates the underlying pathological features. A total of 40 adult SPF-grade Wistar rats were randomly assigned to control (n = 20) and model (n = 20) groups. Rats in the control group were maintained under normal conditions, while rats in the model group were exposed to a controlled frontal airflow of 2–4 m/s from a fan for 7.5 h daily while placed on a suspended cylindrical wire mesh frame. Various assessments were performed at different time points during the 14-day experiment, including blink frequency, tear secretion (phenol red thread test), tear film breakup time (BUT), fluorescein staining (FL), corneal epithelial status (light microscopy), ultrastructure of corneal epithelial cells (electron microscopy), and expression levels of inflammatory cytokines (IL-1β, TNF-α) in tears (enzyme-linked immunosorbent assay). Additionally, mRNA and protein expression levels of MMP-9, IL1β, IL6, TNF-α, IFN-γ, and caspase-3 in corneal tissues were quantified (real-time quantitative PCR and Western blotting). Compared to the control group, the model group rats exhibited significant decreases in blink frequency (P < 0.001), tear secretion (Schirmer I test) values (P < 0.001), and tear film breakup time levels (P < 0.001). There was also a significant increase in fluorescein staining scores (P < 0.001) in the model group. Histological examination revealed distinct differences of the corneal epithelium between groups. The corneal epithelium of the model group appeared thicker, with disorganized cell arrangement in the superficial and basal layers, partial defects or detachment of superficial epithelial cells, and a rough, uneven surface. Scanning electron microscopy observations showed a rough corneal epithelial surface with numerous cracks and scattered vesicular-like structures in the model group. Furthermore, the model group rats exhibited a significant increase in expression of IL-1β and TNF-α in tears (P < 0.001), and upregulated expression levels of MMP-9, TNF-α, IL-1β, caspase-3, IL-6, and IFN-γ at both the mRNA and protein levels in corneal tissues (P < 0.001). In conclusion, the modified “wire-meshing cylindrical board” model effectively overcomes the limitations of the traditional “jogging board " dry eye model and successfully simulates the etiology of prolonged visual fatigue. This innovative EDE model demonstrates a high degree of relevance to dry eye conditions resulting from prolonged visual tasks, with a high success rate of model induction. Moreover, it proves to be a simple, practical, and easily replicable model, making it highly suitable for further studies on prolonged visual fatigue and facilitating its widespread adoption in research and clinical applications.

Anti-breast cancer activity of biosynthesized selenium nanoparticles using Bacillus coagulans supernatant

BackgroundIn the present study, Selenium Nanoparticles (SeNPs) were prepared using Bacillus coagulans, which is a type of Lactic Acid Bacteria (LAB), and then they were applied to treat breast cancer cells. MethodsThe chemicophysical properties of the bioengineered SeNPs were investigated by Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FE-SEM), zeta potential, dynamic light scattering, Fourier Transform Infrared Spectroscopy (FT-IR), energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction analysis (XRD). The cytotoxic potential of SeNPs was evaluated by MTT assay against MCF-7 breast cancer cell line. The expression levels of apoptotic genes including BAX, BCL2, VEGF, ERBB2, CASP3, CASP9, CCNE1, CCND1, MMP2 and MMP9 were determined by real-time PCR. The rate of apoptosis and necrosis of the cancer cells as well as the results of the cell cycle were evaluated by flow cytometry method. ResultsThe synthesized SeNPs had an average particle size of about 24–40 nm and a zeta potential of –16.1 mV, indicating the high stability of SeNPs. EDX results showed presence of SeNPs because amount of selenium in SeNPs was 86.6 % by weight. The cytotoxicity results showed a concentration-dependent effect against MCF-7 cells. The half-maximal inhibitory concentration (IC50) values of B. coagulans supernatant and SeNPs against breast cancer cells were 389.7 µg/mL and 17.56 µg/mL, respectively. In addition, SeNPs synthesized by the green process exhibited enhanced apoptotic potential in MCF-7 cancer cells compared with bacterial supernatants. Cancer cells treated with IC50 concentration of SeNPs induced 32 % apoptosis compared to untreated cells (3 % apoptosis). The gene expression levels of BAX, CASP3, and CASP9 were upregulated, while the expression levels of BCL2, CCNE1, CCND1, MMP2, MMP9, VEGF, and ERBB2 were downregulated after SeNPs treatment of cells. The potential of SeNPs to induce cell apoptosis was demonstrated by the increase in the expression level of BAX gene and the decrease in the expression level of BCL2 after treatment of cancer cells with SeNPs. ConclusionThe obtained results indicated that SeNPs had strong potential to induce significant cell apoptosis and are cytotoxic against the MCF-7 cancer cell line.

Inhibitory effects and mechanisms of low-temperature plasma on hypertrophic scar

Recent advancements in comprehending the properties of low-temperature plasmas (LTPs) have spurred the creation of plasma medicine. Nonetheless, there exists limited scientific evidence concerning its mechanism of impeding proliferative scarring. This study aims to investigate the role of LTPs in hypertrophic scar (HS) formation. Establishing rabbit ear scar models, two groups were individually treated with salvianolic acid B (SAB) gel and low-temperature plasmas for six weeks, while the control group was not treated. The expression levels of cytokines, including TGF-β1, p-Smad3, and MMP-2, in rabbit serum were assessed using ELISA in this study. Additionally, hematoxylin-eosin staining and Masson’s trichrome staining were conducted on proliferative scar tissue to observe the arrangement of collagen fibers and determine the density of fibroblasts. Immunohistochemical analysis was also performed to obtain the percentage of type I collagen and α-SMA positive expression area. The findings indicated that the scars in both the SAB and LTP groups were narrower than those in the model group. The scar tissues treated with LTP or SAB showed a lower level of TGF-β1 and p-Smad3. In addition, α-SMA was significantly reduced in the LTP-treated group. Furthermore, the type I collagen expression was lower in the LTP group. These results suggest that LTP could have a comparable effect to SAB in hindering the development of HSs. It could potentially enhance skin scarring by impeding collagen deposition and fibroblast proliferation in HSs via the TGF-β/Smad signaling pathway. This investigation may provide a new perspective on HS treatment.

Novel nano-SiRNA delivery system loaded with anti-ESR1 siRNA and its application as a cyclosporine drug carrier in dry eye caused by diabetes

Dry eye is a relatively common clinical condition caused by abnormal fluid dynamics and tear quality, for which effective pharmacological treatment is lacking. In this study, a new nano-siRNA delivery system loaded with anti-ESR1 siRNA was designed and synthesized, and its effect in the treatment of dry eye was evaluated. The system significantly inhibited the NaCl-induced increase in inflammatory factors, thereby suppressing the inflammatory response. In addition, the system downregulated the expression level of MMP-9 and STRA6. This system could be developed as a drug for the treatment of diabetes-induced dry eye.

Dual silencing of tumor-intrinsic VISTA and CTLA-4 stimulates T-cell mediated immune responses and inhibits MCF7 breast cancer development

BackgroundAs inhibitory immune checkpoint molecules, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and V-domain Ig suppressor of T-cell activation (VISTA) can be expressed in tumoral cells and facilitate immune evasion of tumoral cells. Herein, we studied the significance of tumor-intrinsic CTLA-4 and VISTA silencing in tumor development and inflammatory factors expression in a co-culture system with MCF7 and T-cells. MethodsMCF7 cells were transfected with 60 pmol of CTLA-siRNA, VISTA-siRNA, and dual VISTA-/CTLA-4-siRNA. The MTT assay was performed to study the effect of CTLA-4 and VISTA knockdown on the viability of MCF7 cells. Colony formation and wound-healing assays were performed to investigate the effect of CTLA-4 and VISTA silencing on the clonogenicity and migration of MCF7 cells. Flow cytometry was used to study the significance of CTLA-4 and VISTA knockdown on the apoptosis and cell cycle of MCF7 cells. Also, a co-culture system with MCF7 and T-cells was developed to study the expression levels of IL-2, IFN-γ, TNF-α, TGF-β, and IL-10 following CTLA-4 and VISTA knockdown. The expression levels of caspase3, Bax, Bcl2, and MMP-9 were also investigated using quantitative real-time PCR. Finally, the TCGA Breast Cancer and GSE45827 datasets were analyzed to study the potential prognostic values of VISTA and CTLA-4, their expression difference in luminal A breast cancer and non-tumoral tissues, and their correlation in luminal A breast cancer tissues. ResultsCombined knockdown of tumor-intrinsic VISTA and CTLA-4 is superior in upregulating IL-2, IFN-γ, and TNF-α, downregulating TGF-β and IL-10 in T lymphocytes. Also, the combined silencing arrests the cell cycle at the sub-G1 phase, decreases migration, inhibits clonogenicity, and reduces cell viability of MCF7 cells. This combined treatment upregulates caspase 9 and BAX and downregulates MMP-9 in MCF7 cells. Our in-silico results have demonstrated a significant positive correlation between CTLA-4 and VISTA in luminal A breast cancer. ConclusionThe additive effect of the combined knockdown of tumor-intrinsic VISTA and CTLA-4 can substantially upregulate pro-inflammatory factors, downregulate anti-inflammatory factors, and inhibit tumor development in MCF7 cells. The significant positive correlation between VISTA and CTLA-4 in luminal A breast cancer might support the idea that a network of inhibitory immune checkpoint molecules regulates anti-tumoral immune responses; thus, combinational immune checkpoint molecules blockade can be suggested.

The mechanism of action of myricetin against lung adenocarcinoma based on bioinformatics, in silico and in vitro experiments.

Myricetin is a natural flavonoid with anti-cancer and anti-inflammatory effects, but its mechanism for treating lung adenocarcinoma (LUAD) remains unclearly. Therefore, bioinformatics, in silico and in vitro experiments were employed to elucidate this issue in this study. The core targets of myricetin against LUAD were screened by PharmaMapper (v2017), Assistant for Clinical Bioinformatics, STRING (v11.5) and Cytoscape (v3.8.1). Using Kaplan-Meier Plotter (v2022.04.20), UALCAN (v2021.12.13) and GEPIA (v2.0) databases, the correlation between core genes and the prognosis of LUAD patients were analyzed, and the expression levels of core genes were verified. In silico studies were used to analyze the binding energies and sites of myricetin with core genes. The effects of myricetin on H1975 cells were explored through thiazolyl blue (MTT), cell migration, colony formation and western blot assays. A total of 72 potential targets of myricetin against LUAD were identified through bioinformatics. Among the four core targets obtained by multiple networks and in silico assays, the up-regulated MMP9 (HR = 1.14 (1-1.29), logrank P = 0.046) and down-regulated PIK3R1 (HR = 0.58 (0.51-0.66), logrank P < 1E-16) were positively correlated with poor survival outcomes in LUAD patients. In vitro experiments demonstrated that myricetin inhibited the proliferation and migration of H1975 cells, promoting their apoptosis. Myricetin inhibits the proliferation of H1975 cells and induces cell apoptosis through its influence on the expression levels of MMP1, MMP3, MMP9, and PIK3R1 and regulating the multiple pathways these genes participate in. Both MMP9 and PIK3R1 are potential biomarkers for LUAD.

TGF-β1 Inhibits Osteoclast Differentiation and Abnormal Angiogenesis in Intervertebral Disc Degeneration: Evidence from RNA Sequencing and Animal Studies.

Mechanisms involved in developing intervertebral disc degeneration (IDD) are poorly understood, thus making developing effective therapies difficult. This study aimed to suggest a possible molecular mechanism, based on transcriptome sequencing-identified transforming growth factor (TGF-β), underlying the effects on bone homeostasis in IDD. A mouse model for IDD was established. Transcriptome sequencing of nucleus pulposus tissue from mice (n = 3) identified differentially expressed mRNAs and key genes impacting bone homeostasis. A protein-protein interaction network pinpointed core genes. GO and KEGG analysis revealed gene functions. Expression levels of TGF-β1, tartrate-resistant acid phosphatase (TRAP), and cathepsin K (CTSK) were measured. Micro-CT evaluated vertebral structures and vascular imaging. Western Blot measured expression levels of Vegf, Opn, MMP3, and MMP13. Safranin O-Fast Green and TRAP staining were performed on intervertebral discs and endplates. Transcriptomic analysis found 1790 differentially expressed mRNAs in IDD mice. Twenty-eight genes related to bone homeostasis in IDD were identified. TGF-β1 was confirmed as the core gene. GO and KEGG showed TGF-β1 regulates osteoclast markers like CTSK and TRAP through pathways including NF-κB and MAPK. Experimental validation revealed lower TGF-β1 expression in IDD mice than controls, and increased TRAP and CTSK expression. Micro-CT showed decreased bone mass and intervertebral disc space in IDD mice. Vascular imaging showed increased vascular volume in IDD cartilaginous endplates. Western blot displayed increased VEGF and OPN levels, but decreased MMP3 and MMP13 in IDD mice. Safranin O-fast green staining revealed severe IDD degeneration. However, TGF-β1 injection improved bone parameters in IDD mice. In vitro experiments confirmed TGF-β1 inhibits bone marrow macrophages differentiation into osteoclasts. From our data, we conclude that TGF-β1 repressed osteoclast differentiation and aberrant bone-associated angiogenesis in cartilage endplates (EPs) to alleviate IDD, which may be instrumental for the therapeutic targeting of IDD.

TLR2 mediates renal apoptosis in neonatal mice subjected experimentally to obstructive nephropathy.

Urinary tract obstruction during renal development leads to inflammation, tubular apoptosis, and interstitial fibrosis. Toll like receptors (TLRs) expressed on leukocytes, myofibroblasts and renal cells play a central role in acute inflammation. TLR2 is activated by endogenous danger signals in the kidney; its contribution to renal injury in early life is still a controversial topic. We analyzed TLR2 for a potential role in the neonatal mouse model of congenital obstructive nephropathy. Inborn obstructive nephropathies are a leading cause of end-stage kidney disease in children. Thus, newborn Tlr2-/- and wild type (WT) C57BL/6 mice were subjected to complete unilateral ureteral obstruction (UUO) or sham-operation on the 2nd day of life. The neonatal kidneys were harvested and analyzed at days 7 and 14 of life. Relative expression levels of TLR2, caspase-8, Bcl-2, Bax, GSDMD, GSDME, HMGB1, TNF, galectin-3, α-SMA, MMP-2, and TGF-β proteins were quantified semi-quantitatively by immunoblot analyses. Tubular apoptosis, proliferation, macrophage- and T-cell infiltration, tubular atrophy, and interstitial fibrosis were analyzed immunohistochemically. Neonatal Tlr2-/- mice kidneys exhibited less tubular and interstitial apoptosis as compared to those of WT C57BL/6 mice after UUO. UUO induced neonatally did trigger pyroptosis in kidneys, however to similar degrees in Tlr2-/- and WT mice. Also, tubular atrophy, interstitial fibrosis, tubular proliferation, as well as macrophage and T-cell infiltration were unremarkable. We conclude that while TLR2 mediates apoptosis in the kidneys of neonatal mice subjected to UUO, leukocyte recruitment, interstitial fibrosis, and consequent neonatal obstructive nephropathy might lack a TLR2 involvement.

Combined Therapy of Zanubrutinib and Zilovertamab in the Inhibition of Invasive Capability of Chronic Lymphocytic Leukemia Cells

Signaling pathways such as CXCR4/CXCL12 can regulate migration and trafficking of chronic lymphocytic leukemia (CLL) cells from the blood to lymphoid tissues, where they receive growth/survival signals from accessory cells within the lymphoid-tissue microenvironment. This requires extravasation of CLL cells that is contingent upon the leukemia-cells' ability to degrade the extracellular matrix via matrix metallopeptidases (MMP), such as MMP9, which is a type IV collagenase. We find that CLL cells that express high-levels of the receptor tyrosine kinase-like orphan receptor 1 (ROR1 Hi) express high-levels of MMP9 relative to that of CLL cells with low-level expression of ROR1 (ROR1 Low). However, short-term culture of ROR1 Hi CLL cells in serum-free medium causes attenuation in MMP9 expression, which can be counteracted by addition of exogenous Wnt5a, which is a non-canonical Wnt factor and ligand for ROR1 that is expressed at significantly higher levels in the plasma of patients with CLL than that of healthy adults. We found that stimulation of serum-starved CLL cells with Wnt5a enhance expression and release of MMP9 into the culture media. Such effects of Wnt5a could be inhibited by zilovertamab (also known as UC-961, or previously known as cirmtuzumab; 20 µg/ml), a humanized mAb highly-specific for ROR1 that can block CLL-cell ROR1-signaling (Choi et al., Cell Stem Cell, 22:951, 2018), indicating that MMP9 may be regulated in part by Wnt5a-induced ROR1-signaling. To test this hypothesis, we used the CLL-cell derived cell line, MEC1, which expresses endogenous Wnt5a, but lacks expression of ROR1, and MEC1-Wnt5a -/- cells in which we deleted functional genes encoding Wnt5a via CRISPR-Cas9. Stable transfection of MEC1 or MEC1-Wnt5a -/- cells with an expression vector encoding ROR1 generated MEC1-ROR1 and MEC1-Wnt5a -/--ROR1 that each expressed high-levels of surface ROR1, as assessed via flow cytometry. We found that MEC1-ROR1 cells had significantly higher expression levels of MMP9 and greater capacity to invade Boyden -Chamber Matrigel than MEC1 cells or MEC1-Wnt5a -/- or MEC1-Wnt5a -/--ROR1 cells. Noting the gene encoding MMP9 possesses a promoter that may be induced by activation of NF-kB, we examined for NF-kB-p65 and found MEC1-ROR1 cells had significantly higher activation of NF-kB-p65 than MEC1 cells or MEC1-Wnt5a -/- or MEC1-Wnt5a -/--ROR1 cells. Similarly, serum-starved ROR1 + CLL cells also expressed activated NF-kB-p65 when treated with Wnt5a, an effect that also could be inhibited by zilovertamab. Consistent with this model, we found that an inhibitor (CAS 545380-34-5, 20 nM) of NF-kB also could block Wnt5a induced expression of MMP9 in ROR1 + CLL cells. We examined whether such effects also could be observed for zanubrutinib, a covalent inhibitor of BTK that recently was approved for treatment of patients with CLL. We found that zanubrutinib (0.5 µM) could not inhibit Wnt5a-induced activation of NF-kB-p65 or expression of MMP9 in ROR1 + CLL cells. We also use the Boyden -Chamber Matrigel Invasion Assay to study the response to CXCL12 without or with Wnt5a of primary CLL cells in vitro. We found that CLL could invade the Boyden -Chamber Matrigel in response to CXCL12 and the extent of such invasion was enhanced by Wnt5a. We also found that zanubrutinib (0.5 µM) could inhibit the CXCL12 induced capacity of CLL cells to invade Matrigel, however, this drug was unable to block Wnt5a enhanced invasiveness of CLL cells. Zilovertamab could inhibit Wnt5a enhanced invasiveness of CLL cells. Moreover, the combined treatment of zanubrutinib and zilovertamab had additive activity in inhibiting Matrigel invasiveness by CLL cells, supporting potential evaluation of the combined use of zilovertamab and zanubrutinib in the treatment of patients with CLL.

Quantitative evaluation of warm acupuncture-moxibustion in improvement of cartilage damage in rabbits with early knee osteoarthritis based on MR T2 mapping.

To observe the application value of MR T2 mapping for evaluating the effect of warm acupuncture-moxibustion on articular cartilage degeneration, and to observe the relationship between T2 value and expression of matrix metalloproteinases (MMP)-1 and MMP-13 of chondrocytes in rabbits with early knee osteoarthritis (KOA). Thirty male New Zealand rabbits were randomly divided into blank control, KOA model and warm acupuncture-moxibustion groups, with 10 rabbits in each group. The early KOA model was established by right hind limb tubular plaster extension fixation method for 2 weeks. The rabbits of the warm acupuncture-moxibustion group received warm acupuncture-moxibustion stimulation at "Heding"(EX-LE2), "Neixiyan"(EX-LE4), "Waixiyan" (EX-LE5) and"Zusanli"(ST36) on the right hind limb for 15 min, once a day for 2 weeks. After intervention, MR T2 mapping of the right knee joint was performed in each group. The H.E. staining was used to evaluate the histopathological changes of cartilage, followed by giving a score according to the standards of Mankin scoring. The TUNEL method was used to analyze the apoptosis state of chondrocytes, and the positive expressions of MMP-1 and MMP-13 in the articular cartilage were detected by immunohistochemical staining. Compared with the blank control group, the Mankin score, chondrocyte apoptosis index, T2 value and the positive expressions of MMP-1 and MMP-13 in the cartilage tissue were significantly increased in the model group (P<0.01). Compared with the model group, the Mankin score, chondrocyte apoptosis index, T2 value and the positive expressions of MMP-1 and MMP-13 in the cartilage tissue were markedly decreased in the warm acupuncture-moxibustion group (P<0.01). The T2 value was positively correlated with the expression levels of MMP-1 and MMP-13 (P<0.01). H.E. staining showed disordered arrangement of chondrocytes and thinner cartilage layer in the model group, and a clear and relative ordered arrangement of chondrocyte in the warm acupuncture-moxibustion group. Warm acupuncture-moxibustion can reduce the T2 value of articular cartilage in early KOA rabbits, which is positively correlated with the decreased expression of MMP-1 and MMP-13 in the extracellular matrix of cartilage. The MR T2 mapping has certain value in evaluating the effect of warm acupuncture-moxibustion on KOA rabbits with early cartilage degeneration.

PF-04449913 Inhibits Proliferation and Metastasis of Colorectal Cancer Cells by Down-regulating MMP9 Expression through the ERK/p65 Pathway.

Colorectal cancer remains a life-threatening malignancy with increasing morbidity and mortality worldwide. Therefore, new and effective anti-colorectal cancer therapeutics are urgently needed. In this study, we have studied the anti-tumor properties and potential mechanisms of PF-04449913. Colorectal cancer cell viability was reduced by PF-04449913 in a dose-dependent manner. The migration and invasion ability of malignant colon cells were attenuated by the drug, as demonstrated by the Transwell test. Moreover, PF-04449913 repressed the phosphorylation levels of ERK and other proteins, and the expression levels of MMP9. The anti-tumor effects of the drug in vivo were demonstrated in BALB/c-nude mice models, and PF-04449913 inhibited the malignant phenotype of colorectal cancer cells, including reduction of tumor size and promotion of apoptosis. At the molecular level, PF-04449913 induced a significant decrease in ERK and p65 protein phosphorylation levels and inhibited MMP9 protein expression. Both in vivo and in vitro results showed PF-04449913 to demonstrate antitumor effects, which have been proposed to be mediated through blockade of the ERK/p65 signaling pathway, and subsequent repression of MMP9 expression. Our study provides a new perspective on the potential clinical application of PF-04449913 in the treatment of colorectal cancer.

Mechanistic Evaluation of Antiarthritic Effects of Citronellol in CFA-Induced Arthritic Rats.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by systemic inflammation, joint tissue damage, pain, and synovitis. It leads to deformity of joints, disability, and even premature death. Markers of inflammation are highly expressed in synovium fluid and serum of arthritic patients and play an important role in the pathophysiology of RA. These transcription factors promote the fabrication of type I interferons and inflammatory cytokines. In RA, degradation of synovial cartilage and bone results from stimulation of proinflammatory cytokines. Citronellol (Ct), a monoterpene alcohol, is found in citrus fruits and essential oils of many aromatic plants. It possesses numerous pharmacological properties such as antioxidant activity and potential antinociceptive and anti-inflammatory effects. Keeping in view the significant anti-inflammatory role of Ct, a trial of 28 days was conducted. Ct was administered orally at three different doses (25, 50, and 100) mg/kg in Freund's adjuvant-induced arthritic rats, and the results were compared with piroxicam, chosen as the standard drug. The antiarthritic activity of the compound was evaluated through measurements of arthritic scoring and plethysmometry before and after treatment. The blood biochemical and hematological parameters and histopathological analyses were performed. Additionally, qPCR was conducted to analyze the mRNA expression levels of TNF-α, IL-1β, NF-κB, MMP3, IL-6, and IL-4 in the blood. ELISA was performed to evaluate the levels of PGE2. The results demonstrated that Ct showed significant results at all doses, but the highest dose proved to be most significant in terms of decreasing arthritic scoring and paw edema, indicating the antiarthritic potential of Ct. Furthermore, the compound was found to downregulate all the proinflammatory cytokines (TNF-α, IL-1β, NF-κB, MMP3, and IL-6) and upregulate the anti-inflammatory cytokine (IL-4). The levels of PGE2 were also reduced which further supported the antiarthritic effects of Ct and validated it as a potential antiarthritic candidate.

Baicalein improves the chemoresistance of ovarian cancer through regulation of CirSLC7A6

PurposeThe present study aimed to investigate whether baicalein improves the sensitivity of resistant ovarian cancer cells to cisplatin.MethodsTranscriptomic sequencing and bioinformatics analysis were used to screen differentially expressed CirSLC7A6 in A2780 and A2780/CDDP cells. RT-qPCR was performed to examine the expression levels of CirSLC7A6, miR-2682-5p, and SLC7A6. Cell proliferation and apoptosis were examined using a Cell Counting Kit-8 assay and flow cytometry, and cell migration and invasion were analyzed using wound healing and Transwell assays. Cell suspensions were inoculated into the subcutaneous tissues of the bilateral interscapular region of nude mice. Saline, cisplatin, baicalein and cisplatin plus baicalein were intraperitoneally injected to observe the effects on tumor growth. Toxicity analyses in the liver and kidney were performed using H&E staining. RT-qPCR and immunohistochemistry were used to detect the expression of CirSLC7A6, miR-2682-5p, and SLC7A6 in tumor tissues, and western blot analysis was carried out to measure protein expression levels.ResultsCirSLC7A6 was markedly upregulated in A2780/CDDP cells compared with the A2780 cells. CirSLC7A6 knockdown notably increased the expression of miR-2682-5p and decreased SLC7A6 expression. The rates of inhibition and apoptosis in the group treated with a combination of cisplatin and baicalein were significantly higher than those of the cisplatin and baicalein groups of A2780/CDDP shCirSLC7A6 cells. In A2780/CDDP shCirSLC7A6 cells, migration and invasion were significantly higher in the cisplatin and baicalein groups, compared with the combined treatment group. In the A2780/CDDP shCirSLC7A6 cell xenograft, the tumor weight of the combined treatment group was significantly lower than that of the cisplatin and baicalein groups. In addition, the combination of cisplatin and baicalein did not induce higher levels of toxicity in the liver or kidney. Baicalein alone and in combination with cisplatin notably reduced the expression of CirSLC7A6 and SLC7A6, and increased the expression of miR-2682-5p in the A2780/CDDP shCirSLC7A6 cell xenograft. In A2780/CDDP shCirSLC7A6 cells, the expression levels of P-Akt, P-mTOR, P-Erk, Bcl-2 and MMP2 were lower in the combined treatment group than in the control group.ConclusionsTreatment with baicalein improved the sensitivity of ovarian cancer cells to cisplatin and inhibited cell proliferation, metastasis and tumor growth.

The role and mechanism of biological collagen membranes in repairing cartilage injury through the p38MAPK signaling pathway

ObjectiveTo explore the mechanism of the p38MAPK signaling pathway in repairing articular cartilage defects with biological collagen membranes.MethodsThirty-two healthy adult male rabbits were randomly divided into a control group (n = 8), model group (n = 8), treatment group (n = 8) and positive drug group (n = 8). The control group was fed normally, and the models of bilateral knee joint femoral cartilage defects were established in the other three groups. The knee cartilage defects in the model group were not treated, the biological collagen membrane was implanted in the treatment group, and glucosamine hydrochloride was intragastrically administered in the positive drug group. Twelve weeks after the operation, the repair of cartilage defects was evaluated by histological observation (HE staining and Masson staining), the degree of cartilage repair was quantitatively evaluated by the Mankin scoring system, the mRNA expression levels of p38MAPK, MMP1 and MMP13 were detected by real-time fluorescence quantitative PCR (qRT-PCR), and the protein expression levels of p38MAPK, p-p38MAPK, MMP1 and MMP13 were detected by Western blotting. The results after the construction of cartilage defects, histological staining showed that the articular cartilage wound was covered by a large capillary network, the cartilage tissue defect was serious, and a small amount of collagen fibers were formed around the wound, indicating the formation of a small amount of new bone tissue. In the treatment group and the positive drug group, the staining of cartilage matrix was uneven, the cytoplasmic staining was lighter, the chondrocytes became hypertrophic as a whole, the chondrocytes cloned and proliferated, some areas were nest-shaped, the cells were arranged disorderly, the density was uneven, and the nucleus was stained deeply. The Mankin score of the model group was significantly higher than that of the control group, while the Mankin scores of the treatment group and positive drug group were significantly lower than that of the model group. The results of qRT-PCR detection showed that compared with the control group, the expression level of the p38MAPK gene in the model group did not increase significantly, but the gene expression levels of MMP1 and MMP13 in the model group increased significantly, while the gene expression levels of MMP1 and MMP13 decreased significantly in the treatment group and positive drug group compared with the model group. The results of Western blot detection showed that compared with the control group, the expression level of p38MAPK protein in the model group was not significantly increased, but the phosphorylation level of p38MAPK protein and the protein expression levels of MMP1 and MMP13 were significantly increased in the model group, while the phosphorylation level of p38MAPK protein and the protein expression levels of MMP1 and MMP13 in the treatment group and positive drug group were significantly lower than those in the model group.ConclusionThe biological collagen membrane can regulate the expression of MMP1 and MMP13 and repair the activity of chondrocytes by reducing the phosphorylation level of p38MAPK and inhibiting the activation of the p38MAPK signaling pathway, thus improving the repair effect of articular cartilage defects in rabbits. The P38MAPK signaling pathway is expected to become an important molecular target for the clinical treatment of cartilage defects in the future.

The occurrence and development of vertebral osteoporosis regulated by IL-8.

Vertebral osteoporotic fracture is a common type of fracture, and the incidence is higher in the elderly. However, the relationship between vertebral osteoporotic fractures and interleukin-8 (IL-8) remains unclear. A total of 163 patients with osteoporotic vertebral fractures were recruited. Clinical and follow-up data were recorded, and the expression levels of IL1, MMP9, IL-8, and C-reactive protein in blood were measured. Pearson Chi-square test and Spearman correlation coefficient were used to analyze the relationship between vertebral osteoporotic fractures and related parameters. Univariate and multivariate logistic regression and univariate and multivariate Cox proportional hazards regression were used for further analysis. Pearson chi-square test, Spearman correlation coefficient and Logistic regression analysis showed that IL1 and IL-8 were significantly associated with vertebral osteoporotic fractures. Univariate Cox regression analysis showed that age and IL-8 expression level were significantly associated with maintenance time from recovery to recurrence of vertebral osteoporotic fractures. Multivariate Cox regression analysis showed that IL-8 expression level was significantly associated with maintenance time from recovery to recurrence of vertebral osteoporotic fractures. The higher the expression level of IL-8, the more likely it is to develop vertebral osteoporotic fracture, and the more likely it is to relapse in a short time.