Transwell Model Research Articles

Abstract 284: Crosstalk with postmenopausal obese mammary adipocytes impacts breast cancer cell metabolism and promotes tumorigenesis

Abstract Introduction: Obesity is associated with an increased risk and a poor prognosis of breast cancer (BC) in postmenopausal (PM) women. The mechanism(s) underlying obesity-related PM BC are not clearly understood. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the breast tumor microenvironment (TME) promotes the acquisition of an invasive BC cell phenotype and markedly accelerates tumor proliferation and progression. Methods: We asked whether cancer-associated mammary adipocytes (CA-MAd) differ from normal mammary adipocytes (N-MAd) in their ability to promote tumorigenesis of dormant BC cells and if so, what the underlying mechanism(s) might be. Using a 2D transwell model, BC cells (ER- and ER+), were cocultured with obese PM CA-MAd isolated from breast cancer patients or obese mammary N-MAds from PM disease-free women and analyzed for migration, proliferation, phenotypic, signaling and metabolic alterations. Results: Compared to monoculture (MC) conditions, BC cells cocultured (CC) with CA-MAd displayed significantly increased migration and proliferation. Quantitative phase imaging indicated that CA-MAd secretome-treated BC cells displayed increased motility, speed, distance covered and cell size/area compared to N-MAd. Consistent with these results, mesenchymal markers such as fibronectin, vimentin and SNAIL were upregulated whereas the epithelial marker E-cadherin was downregulated in CC vs. MC conditions suggesting that Ads in the TME promote EMT in BC cells. Interestingly, mature Ads CC with BC cells underwent delipidation and dedifferentiation as indicated by reduced Oil Red staining, decreased lipid content and reduced expression of Ad maturation markers such as FABP4, ADIPOQ and PLIN1. Consistently, we determined that CC BC cells exhibited increased lipid uptake and BODIPY staining and upregulated pAMPK, pACC and CPT1A signaling when compared to MC BC cells. Short term treatment of BC cells with both N-MAd and CA-MAd secretomes resulted in increased oxygen consumption rate (OCR), basal respiration, ATP-linked respiration and proton-linked respiration compared to control and as determined by the Seahorse extracellular flux assay. CC with MAds significantly downregulated OCR and all respiration parameters in BC cells suggesting decreased glucose uptake and glycolytic flux. Conclusions: Compared to MC, CC with obese M-Ads promoted functional, phenotypic and metabolic changes in BC cells. Taken together, our results indicate that crosstalk between BC cells and obese MAds promotes delipidation of MAds and a concomitant lipid uptake in BC cells and a switch to lipid metabolism signaling pathways that may, in turn, drive a metabolic shift in BC cells and promote tumor growth. [Supported by NIH RO1CA185530, the Breast Cancer Research Foundation, the Karp Family Foundation and the Nile Albright Research Foundation] Citation Format: Roopali Roy, Rama Aldakhlallah, Stephen Cobbs, Emily Man, Caleb Hallinan, Kwonmoo Lee, Margaret Lotz-Bousvaros, Susan Pories, Marsha A. Moses. Crosstalk with postmenopausal obese mammary adipocytes impacts breast cancer cell metabolism and promotes tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 284.

Circular RNA circCHSY1 silencing inhibits the malignant progression of esophageal squamous cell carcinoma

BackgroundCircRNAs play a crucial role in the regulation of various cancers. This study aims to investigate the involvement of circCHSY1 in the development of esophageal squamous cell carcinoma (ESCC).MethodsRNA levels were quantified using qRT-PCR, and protein levels were measured by western blot. The stability of circCHSY1 was analyzed using RNase R. The functional effect of circCHSY1 on cell behavior was evaluated by CCK-8, EdU, flow cytometry, transwell, tube formation, and xenograft tumor model assays. The associations among circCHSY1, miR-1229-3p, and Tectonic-1 (TCTN1) were certified by bioinformatics analysis, dual-luciferase reporter assay, and RNA pull-down assay.ResultsCircCHSY1 was up-regulated in both ESCC tissues and cell lines in comparison with the control groups. Knockdown of circCHSY1 inhibited the proliferation, migration, invasion, and tube formation and promoted apoptosis of ESCC cells. Mechanistically, circCHSY1 targeted miR-1229-3p, which was downregulated in ESCC tissues and cells. Inhibition of miR-1229-3p attenuated the effects mediated by circCHSY1 suppression. Besides, miR-1229-3p bound to TCTN1, and TCTN1 overexpression restored miR-1229-3p-induced effects in ESCC cells. Animal experiments revealed that circCHSY1 silencing suppressed tumor tumorigenesis in vivo.ConclusionCircCHSY1 contributed to ESCC cell malignancy, and the underlying mechanism involved the circCHSY1/miR-1229-3p/TCTN1 axis, providing potential therapeutic targets for ESCC.

Phenolic Characterization of a Purple Maize (Zea mays cv. "Moragro") by HPLC-QTOF-MS and Study of Its Bioaccessibility Using a Simulated In Vitro Digestion/Caco-2 Culture Model.

The present work aimed to characterize the phenolic and antioxidant content of the Argentinian purple maize "Moragro" cultivar. Additionally, the INFOGEST simulated in vitro digestion model was used to establish the effect of digestion on bioactive compounds. Finally, digestion samples were used to treat Caco-2 cells in the transwell model to better understand their bioavailability. Twenty-six phenolic compounds were found in purple maize cv. "Moragro", 15 nonanthocyanins and 11 anthocyanins. Several compounds were identified in maize for the first time, such as pyrogallol, citric acid, gallic acid, kaempferol 3-(6″-ferulylglucoside), and kaempferol 3-glucuronide. Anthocyanins accounted for 24.9% of total polyphenols, with the predominant anthocyanin being cyanidin-3-(6″ malonylglucoside). Catechin-(4,8)-cyanidin-3,5-diglucoside and catechin-(4,8)-cyanidin-3-malonylglucoside-5-glucoside were detected as characteristics of this American maize variety. Total polyphenol content (TPC; by the Folin-Ciocalteu method), HPLC-DAD/MSMS, and antioxidant activity [by DPPH and ferric-reducing antioxidant power (FRAP)] were evaluated throughout in vitro digestion. TPC, DPPH, and FRAP results were 2.71 mg gallic acid equivalents (GAE)/g, 24 μmol Trolox equiv/g, and 22 μmol Trolox eq/g, respectively. The in vitro digestion process did not cause significant differences in TPC. However, the antioxidant activity was significantly decreased. Moreover, the bioavailability of anthocyanins was studied, showing that a small fraction of polyphenols in their intact form was conserved at the end of digestion. Finally, a protective effect of digested maize polyphenols was observed in the Caco-2 cell viability. The results suggest that "Moragro" purple maize is a good source of bioavailable anthocyanins in the diet and an interesting source of this group of compounds for the food industry.

Tumor integrin targeted theranostic iron oxide nanoparticles for delivery of caffeic acid phenethyl ester: preparation, characterization, and anti-myeloma activities.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells preferentially in the bone marrow. Currently, emerging chemotherapy drugs with improved biosafety profiles, such as immunomodulatory agents and protease inhibitors, have been used in clinics to treat MM in both initial therapy or maintenance therapy post autologous hematopoietic stem cell transplantation (ASCT). We previously discovered that caffeic acid phenethyl ester (CAPE), a water-insoluble natural compound, inhibited the growth of MM cells by inducing oxidative stress. As part of our continuous effort to pursue a less toxic yet more effective therapeutic approach for MM, the objective of this study is to investigate the potential of CAPE for in vivo applications by using magnetic resonance imaging (MRI)-capable superparamagnetic iron oxide nanoparticles (IONP) as carriers. Cyclo (Arg-Gly-Asp-D-Phe-Cys) (RGD) is conjugated to IONP (RGD-IONP/CAPE) to target the overexpressed αvβ3 integrin on MMcells for receptor-mediated internalization and intracellular delivery of CAPE. A stable loading of CAPE on IONP can be achieved with a loading efficiency of 48.7% ± 3.3% (wt%). The drug-release studies indicate RGD-IONP/CAPE is stable at physiological (pH 7.4) and basic pH (pH 9.5) and subject to release of CAPE at acidic pH (pH 5.5) mimicking the tumor and lysosomal condition. RGD-IONP/CAPE causes cytotoxicity specific to human MM RPMI8226, U266, and NCI-H929 cells, but not to normal peripheral blood mononuclear cells (PBMCs), with IC50s of 7.97 ± 1.39, 16.75 ± 1.62, and 24.38 ± 1.71μM after 72-h treatment, respectively. Apoptosis assays indicate RGD-IONP/CAPE induces apoptosis of RPMI8226 cells through a caspase-9 mediated intrinsic pathway, the same as applying CAPE alone. The apoptogenic effect of RGD-IONP/CAPE was also confirmed on the RPMI8226 cells co-cultured with human bone marrow stromal cells HS-5 in a Transwell model to mimic the MM microenvironment in the bone marrow. In conclusion, we demonstrate that water-insoluble CAPE can be loaded to RGD-IONP to greatly improve the biocompatibility and significantly inhibit the growth of MMcells in vitro through the induction of apoptosis. This study paves the way for investigating the MRI-trackable delivery of CAPE for MM treatment in animal models in the future.

NSUN2 promotes lung adenocarcinoma progression through stabilizing PIK3R2 mRNA in an m5C-dependent manner.

It is well known that 5-methylcytosine (m5C) is involved in variety of crucial biological processes in cancers. However, its biological roles in lung adenocarcinoma (LAUD) remain to be determined. The LUAD samples were used to assess the clinical value of NOP2/Sun RNA Methyltransferase 2 (NSUN2). Dot blot was used to determine global m5C levels. ChIP and dual-luciferase assays were performed to investigate the MYC-associated zinc finger protein (MAZ)-binding sites in NSUN2 promoter. RNA-seq was used to explore the downstream molecular mechanisms of NSUN2. Dual luciferase reporter assay, m5C-RIP-qPCR, and mRNA stability assay were conducted to explore the effect of NSUN2-depletion on target genes. Cell viability, transwell, and xenograft mouse model were designed to demonstrate the characteristic of NSUN2 in promoting LUAD progression. The m5C methyltransferase NSUN2 was highly expressed and caused elevated m5C methylation in LUAD samples. Mechanistically,MAZ positively regulated the transcription of NSUN2 and was related to poor survival of LUAD patients. Silencing NSUN2 decreased the global m5C levels, suppressed proliferation, migration and invasion, and inhibited activation of PI3K-AKT signaling in A549 and SPAC-1 cells. Phosphoinositide-3-Kinase Regulatory Subunit 2 (PIK3R2) was upregulated by NSUN2-mediated m5C methylation by enhancing its mRNA stabilization and activated the phosphorylation of the PI3K-AKT signaling. The present study explored the underlying mechanism and biological function of NSUN2-meditated m5C RNA methylation in LUAD. NSUN2 was discovered to facilitate the malignancy progression of LUAD through regulating m5C modifications to stabilize PIK3R2 activating the PI3K-AKT signaling, suggesting that NSUN2 could be a novel biomarker and promising therapeutic target for LUAD patients.

DNA polymerase iota promotes EMT and metastasis of esophageal squamous cell carcinoma by interacting with USP7 to stabilize HIF-1α

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancer types, with a low 5-year survival rate of ~20%. Our prior research has suggested that DNA Polymerase iota (Pol ι), a member of Y-family DNA polymerase, plays a crucial role in the invasion and metastasis of ESCC. However, the underlying mechanism is not well understood. In this study, we utilized ChIP-PCR and luciferase reporter assays to investigate the binding of HIF-1α to the promoter of the Pol ι gene. Transwell, wound healing, and mouse models were employed to assess the impact of Pol ι and HIF-1α on the motility of ESCC cells. Co-immunoprecipitation and Western blot were carried out to explore the interaction between Pol ι and HIF-1α, while qRT-PCR and Western blot were conducted to confirm the regulation of Pol ι and HIF-1α on their downstream targets. Our results demonstrate that HIF-1α activates the transcription of the Pol ι gene in ESCC cells under hypoxic conditions. Furthermore, the knockdown of Pol ι impeded HIF-1α-induced invasion and metastasis. Additionally, we found that Pol ι regulates the expression of genes involved in epithelial-mesenchymal transition (EMT) and initiates EMT through the stabilization of HIF-1α. Mechanistically, Pol ι maintains the protein stability of HIF-1α by recruiting USP7 to mediate the deubiquitination of HIF-1α, with the residues 446–578 of Pol being crucial for the interaction between Pol ι and USP7. Collectively, our findings unveil a novel feedforward molecular axis of HIF-1α- Pol ι -USP7 in ESCC that contributes to ESCC metastasis. Hence, our results present an attractive target for intervention in ESCC.

PM2.5 Exposure Inhibits Transepithelial Anion Short-circuit Current by Downregulating P2Y2 Receptor/CFTR Pathway.

Fine particulate matter (PM2.5) can damage airway epithelial barriers. The anion transport system plays a crucial role in airway epithelial barriers. However, the detrimental effect and mechanism of PM2.5 on the anion transport system are still unclear. In this study, airway epithelial cells and ovalbumin (OVA)-induced asthmatic mice were used. In transwell model, the adenosine triphosphate (ATP)-induced transepithelial anion short-circuit current (Isc) and airway surface liquid (ASL) significantly decreased after PM2.5 exposure. In addition, PM2.5 exposure decreased the expression levels of P2Y2R, CFTR and cytoplasmic free-calcium, but ATP can increase the expressions of these proteins. PM2.5 exposure increased the levels of Th2-related cytokines of bronchoalveolar lavage fluid, lung inflammation, collagen deposition and hyperplasisa of goblet cells. Interestingly, the administration of ATP showed an inhibitory effect on lung inflammation induced by PM2.5. Together, our study reveals that PM2.5 impairs the ATP-induced transepithelial anion Isc through downregulating P2Y2R/CFTR pathway, and this process may participate in aggravating airway hyperresponsiveness and airway inflammation. These findings may provide important insights on PM2.5-mediated airway epithelial injury.

Developing an adult stem cell derived microphysiological intestinal system for predicting oral prodrug bioconversion and permeability in humans.

Microphysiological systems (MPS) incorporating human intestinal organoids have shown the potential to faithfully model intestinal biology with the promise to accelerate development of oral prodrugs. We hypothesized that an MPS model incorporating flow, shear stress, and vasculature could provide more reliable measures of prodrug bioconversion and permeability. Following construction of jejunal and duodenal organoid MPS derived from 3 donors, we determined the area under the concentration-time (AUC) curve for the active drug in the vascular channel and characterized the enzymology of prodrug bioconversion. Fosamprenavir underwent phosphatase mediated hydrolysis to amprenavir while dabigatran etexilate (DABE) exhibited proper CES2- and, as anticipated, not CES1-mediated de-esterification, followed by permeation of amprenavir to the vascular channel. When experiments were conducted in the presence of bio-converting enzyme inhibitors (orthovanadate for alkaline phosphatase; bis(p-nitrophenyl)phosphate for carboxylesterase), the AUC of the active drug decreased accordingly in the vascular channel. In addition to functional analysis, the MPS was characterized through imaging and proteomic analysis. Imaging revealed proper expression and localization of epithelial, endothelial, tight junction and catalytic enzyme markers. Global proteomic analysis was used to analyze the MPS model and 3 comparator sources: an organoid-based transwell model (which was also evaluated for function), Matrigel embedded organoids and finally jejunal and duodenal cadaver tissues collected from 3 donors. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) of global proteomic data demonstrated that all organoid-based models exhibited strong similarity and were distinct from tissues. Intestinal organoids in the MPS model exhibited strong similarity to human tissue for key epithelial markers via HCA. Quantitative proteomic analysis showed higher expression of key prodrug converting and drug metabolizing enzymes in MPS-derived organoids compared to tissues, organoids in Matrigel, and organoids on transwells. When comparing organoids from MPS and transwells, expression of intestinal alkaline phosphatase (ALPI), carboxylesterase (CES)2, cytochrome P450 3A4 (CYP3A4) and sucrase isomaltase (SI) was 2.97-, 1.2-, 11.3-, and 27.7-fold higher for duodenum and 7.7-, 4.6-, 18.1-, and 112.2-fold higher for jejunum organoids in MPS, respectively. The MPS approach can provide a more physiological system than enzymes, organoids, and organoids on transwells for pharmacokinetic analysis of prodrugs that account for 10% of all commercial medicines.

Development of an In Vitro Model to Study Mechanisms of Ultrasound-Targeted Microbubble Cavitation–Mediated Blood–Brain Barrier Opening

Ultrasound-targeted microbubble cavitation (UTMC)-mediated blood-brain barrier (BBB) opening is being explored as a method to increase drug delivery to the brain. This strategy has progressed to clinical trials for various neurological disorders, but the underlying cellular mechanisms are incompletely understood. In the study described here, a contact co-culture transwell model of the BBB was developed that can be used to determine the signaling cascade leading to increased BBB permeability. This BBB model consists of bEnd.3 cells and C8-D1A astrocytes seeded on opposite sides of a transwell membrane. Pulsed ultrasound (US) is applied to lipid microbubbles (MBs), and the change in barrier permeability is measured via transendothelial electrical resistance and dextran flux. Live cell calcium imaging (Fluo-4 AM) is performed during UTMC treatment. This model exhibits important features of the BBB, including endothelial tight junctions, and is more restrictive than the endothelial cell (EC) monolayer alone. When US is applied to MBs in contact with the ECs, BBB permeability increases in this model by two mechanisms: UTMC induces pore formation in the EC membrane (sonoporation), leading to increased transcellular permeability, and UTMC causes formation of reversible inter-endothelial gaps, which increases paracellular permeability. Additionally, this study determines that calcium influx into ECs mediates the increase in BBB permeability after UTMC in this model. Both transcellular and paracellular permeability can be used to increase drug delivery to the brain. Future studies can use this model to determine how UTMC-induced calcium-mediated signaling increases BBB permeability.

An Improved In Vitro Blood-Brain Barrier Model for the Evaluation of Drug Permeability Using Transwell with Shear Stress.

The development of drugs targeting the central nervous system (CNS) is challenging because of the presence of the Blood-Brain barrier (BBB). Developing physiologically relevant in vitro BBB models for evaluating drug permeability and predicting the activity of drug candidates is crucial. The transwell model is one of the most widely used in vitro BBB models. However, this model has limitations in mimicking in vivo conditions, particularly in the absence of shear stress. This study aimed to overcome the limitations of the transwell model using immortalized human endothelial cells (hCMEC/D3) by developing a novel dish design for an orbital shaker, providing shear stress. During optimization, we assessed cell layer integrity using trans-endothelial electrical resistance measurements and the % diffusion of lucifer yellow. The efflux transporter activity and mRNA expression of junctional proteins (claudin-5, occludin, and VE-cadherin) in the newly optimized model were verified. Additionally, the permeability of 14 compounds was evaluated and compared with published in vivo data. The cell-layer integrity was substantially increased using the newly designed annular shaking-dish model. The results demonstrate that our model provided robust conditions for evaluating the permeability of CNS drug candidates, potentially improving the reliability of in vitro BBB models in drug development.

Hsa_circ_0013561 promotes epithelial-mesenchymal transition and tumor progression by regulating ANXA2 via miR-23b-3p in ovarian cancer

Our preliminary experiment discovered that hsa_circ_0013561 was aberrantly expressed in OC. However, the underlying mechanism is unclear. The expression of hsa_circ_0013561 in OC cells and tissues was detected by RT-qPCR and fluorescence in situ hybridization. The effects of hsa_circ_0013561 on the proliferation and metastasis of OC were explored by functional experiments such as cell counting kit-8, transwell, and tumor xenograft models. To mechanistically understand the regulatory role of hsa_circ_0013561, bioinformatics analysis, Western blot, luciferase reporter assay, and a series of rescue experiments were applied. We found that the hsa_circ_0013561 expression was elevated in OC cells and tissues, and was correlated with metastasis formation. Downregulation of hsa_circ_0013561 suppressed the proliferation and migration of OC cells both in vitro and in vivo. Regarding the interactions of hsa_circ_0013561, the luciferase reporter assay verified that miR-23b-3p and Annexin A2 (ANXA2) were its downstream targets. MiR-23b-3p inhibition or ANXA2 overexpression reversed OC cell proliferation, migration, and epithelial-mesenchymal transition (EMT) post-hsa_circ_0013561 silencing. Moreover, ANXA2 overexpression also reversed OC cell migration, proliferation, and EMT after miR-23b-3p upregulation. Our data suggest that hsa_circ_0013561 increases the expression of ANXA2 by regulating miR-23b-3p competitively, resulting in EMT and metastasis of OC. Thus, hsa_circ_0013561 may serve as a novel oncogenic biomarker for OC progression.

Evaluation of a Microphysiological Human Placental Barrier Model for Studying Placental Drug Transfer

Understanding drug transport across the placental barrier is important for assessing the potential fetal drug toxicity and birth defect risks. Current in vivo and in vitro models have structural and functional limitations in evaluating placental drug transfer and toxicity. Microphysiological systems (MPSs) offer more accurate and relevant physiological models of human tissues and organs on a miniature scale for drug development and toxicology testing. MPSs for the placental barrier have been recently explored to study placental drug transfer. We utilized a multilayered hydrogel membrane-based microphysiological model composed of human placental epithelial and endothelial cells to replicate the key structure and function of the human placental barrier. A macroscale human placental barrier model was created using a transwell to compare the results with the microphysiological model. Placental barrier models were characterized by assessing monolayer formation, intercellular junctions, barrier permeability, and their structural integrity. Three small molecule drugs (glyburide, rifaximin, and caffeine) that are prescribed or taken during pregnancy were studied for their placental transfer. The results showed that all three drugs crossed the placental barrier, with transfer rates in the order: glyburide (molecular weight, MW = 494Da) < rifaximin (MW = 785.9Da) < caffeine (MW = 194.19Da). Using non-compartmental analysis, we estimated human pharmacokinetic characteristics based on in vitro data from both MPS and transwell models. While further research is needed, our findings suggest that MPS holds potential as an in vitro tool for studying placental drug transfer and predicting fetal exposure, offering insights into pharmacokinetics.

DDEL-07. BARRIER TO PROGRESS: DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) INDUCES A TIGHTENED BLOOD-BRAIN BARRIER PHENOTYPE

Abstract DIPG is an incurable paediatric brainstem tumour. Treatments have been significantly hindered by the necessity for drugs to penetrate the blood-brain barrier (BBB). We, and others, have shown that the BBB remains intact in DIPG, and it is thought that the BBB in the brainstem may be even less permeable in the presence of DIPG tumor cells. We performed in vivo studies in orthotopic DIPG mouse models that showed higher efficacy of mTOR inhibitor, temsirolimus, in cortex-injected DIPG compared to brainstem-injected DIPG. Analysis showed significantly increased survival, higher temsirolimus levels and decreased proliferating tumour cells in the cortex. Subsequently, the effect of DIPG on brain vasculature was interrogated by single-cell RNA sequencing. Microvascular endothelial cells were isolated from the brainstem and cortex, where DIPG cells or Matrigel were implanted. Transcriptomic analysis of DIPG-injected brainstem uncovered significant downregulation of inflammatory pathways, especially P38MAPK, downregulation of antigen presentation, and increased transporter Mfsd2a, suggesting a tightened BBB. Apoptotic signalling pathways were significantly upregulated, particularly MCL-1. Further analysis into cortex-injected DIPG revealed downregulation of interferon β pathways, suggesting a leakier phenotype. To overcome the tightened barrier in the brainstem, MCL-1 inhibitor S63845, was investigated as a BBB modulator. S63845 significantly reduced transendothelial electrical resistance, increased tracer dye leakage in an in vitro BBB transwell model with DIPG and decreased protein expression of claudin-5. In vivo, S63845 increased permeability to 3kDa Texas-red dextran after a single dose in DIPG mouse models. Overall, this study revealed that DIPG directly affects endothelial cell pathways associated with BBB permeability, resulting in a tightened BBB and reduced therapeutic efficacy. S63845 represents a promising BBB modulator that can be easily administered and may facilitate the delivery of therapeutics through the constricted BBB. This discovery has the potential to enhance the activity of anti-DIPG therapeutics that were previously considered impenetrable.

The novel HS-mimetic, Tet-29, regulates immune cell trafficking across barriers of the CNS during inflammation

BackgroundDisruption of the extracellular matrix at the blood–brain barrier (BBB) underpins neuroinflammation in multiple sclerosis (MS). The degradation of extracellular matrix components, such as heparan sulfate (HS) proteoglycans, can be prevented by treatment with HS-mimetics through their ability to inhibit the enzyme heparanase. The heparanase-inhibiting ability of our small dendrimer HS-mimetics has been investigated in various cancers but their efficacy in neuroinflammatory models has not been evaluated. This study investigates the use of a novel HS-mimetic, Tet-29, in an animal model of MS.MethodsNeuroinflammation was induced in mice by experimental autoimmune encephalomyelitis, a murine model of MS. In addition, the BBB and choroid plexus were modelled in vitro using transmigration assays, and migration of immune cells in vivo and in vitro was quantified by flow cytometry.ResultsWe found that Tet-29 significantly reduced lymphocyte accumulation in the central nervous system which, in turn, decreased disease severity in experimental autoimmune encephalomyelitis. The disease-modifying effect of Tet-29 was associated with a rescue of BBB integrity, as well as inhibition of activated lymphocyte migration across the BBB and choroid plexus in transwell models. In contrast, Tet-29 did not significantly impair in vivo or in vitro steady state-trafficking under homeostatic conditions.ConclusionsTogether these results suggest that Tet-29 modulates, rather than abolishes, trafficking across central nervous system barriers.

Effect of Verapamil, a P-glycoprotein-1 and Cytochrome P450 3A4 Inhibitor, on Pharmacokinetics and Metabolic Stability of Ripretinib: A Drug-Drug Interaction Study in Rats.

Ripretinib was developed to target a whole range of KIT proto-oncogene mutations and platelet-derived growth factor receptor A (PDGFR-A) kinases found in certain cancers and myeloproliferative neoplasms, particularly gastrointestinal stromal tumours (GISTs). This study investigated the effect of verapamil, a potential inhibitor of P-glycoprotein-1 (P-gp1) and cytochrome P450 3A4 (CYP3A4), on the pharmacokinetics of ripretinib in rats when administered orally together. This study also assessed the metabolic stability and in vitro cellular absorption of ripretinib in the presence of verapamil. A novel sensitive time-saving liquid chromatography tandem mass spectometry (LC-MS/MS) technique for determining ripretinib in rat plasma was developed and validated. A Zorbax SB C18 column was used for the separation and analysis of ripretinib with a mobile phase consisting of 50:50 (%v/v) acetonitrile and 10mM ammonium formate buffer at a flow rate of 0.4 mL/min. Imatinib was used as an internal standard (IS) in the method. The pharmacokinetic characteristics of ripretinib were evaluated in Wistar rats by successfully administering an oral dosage of 5 mg/kg body weight of ripretinib in the presence of verapamil (10 mg/kg body weight). Subsequently, rat liver microsomes were used to assess the effect of verapamil on ripretinib metabolic stability, and absorption was tested using a Caco-2 cell transwell model. Ripretinib and IS were identified using multiple reaction monitoring (MRM) modes by mass spectrometry and showed ion transitions of 510.09→94.06m/z and 494.26→ 394.16m/z, respectively. The high-performance liquid chromatography (HPLC) method successfully eluted ripretinib and IS at retention times of 0.91 and 0.68 min, respectively, and the method was validated for all parameters and met the criteria for acceptance. Co-administration of verapamil increased the maximum concentration (Cmax) of ripretinib from 437±84ng/mL to 492±50ng/mL (12%), and the area under the concentration-time curve from 0 to the last sampling time t (AUC0-t) increased by approximately 40.6%. Verapamil significantly reduced the basolateral-to-apical transfer of ripretinib through Caco-2 cells. Findings also showed that verapamil increased the metabolic stability of ripretinib. The study results indicate that the co-administration of ripretinib with CYP3A4 and/or P-gp1 inhibitors is associated with significant drug-drug interactions that affect the pharmacokinetics of ripretinib. Further research in human subjects is suggested to confirm dosage adjustment and therapeutic drug monitoring of ripretinib when administered along with P-gp1/CYP3A4 inhibitors ensuring patient safety and optimizing the therapeutic benefits of ripretinib.

Influence of Surface Ligand Density and Particle Size on the Penetration of the Blood-Brain Barrier by Porous Silicon Nanoparticles.

Overcoming the blood-brain barrier (BBB) remains a significant challenge with regard to drug delivery to the brain. By incorporating targeting ligands, and by carefully adjusting particle sizes, nanocarriers can be customized to improve drug delivery. Among these targeting ligands, transferrin stands out due to the high expression level of its receptor (i.e., transferrin receptor) on the BBB. Porous silicon nanoparticles (pSiNPs) are a promising drug nanocarrier to the brain due to their biodegradability, biocompatibility, and exceptional drug-loading capacity. However, an in-depth understanding of the optimal nanoparticle size and transferrin surface density, in order to maximize BBB penetration, is still lacking. To address this gap, a diverse library of pSiNPs was synthesized using bifunctional poly(ethylene glycol) linkers with methoxy or/and carboxyl terminal groups. These variations allowed us to explore different transferrin surface densities in addition to particle sizes. The effects of these parameters on the cellular association, uptake, and transcytosis in immortalized human brain microvascular endothelial cells (hCMEC/D3) were investigated using multiple in vitro systems of increasing degrees of complexity. These systems included the following: a 2D cell culture, a static Transwell model, and a dynamic BBB-on-a-chip model. Our results revealed the significant impact of both the ligand surface density and size of pSiNPs on their ability to penetrate the BBB, wherein intermediate-level transferrin densities and smaller pSiNPs exhibited the highest BBB transportation efficiency in vitro. Moreover, notable discrepancies emerged between the tested in vitro assays, further emphasizing the necessity of using more physiologically relevant assays, such as a microfluidic BBB-on-a-chip model, for nanocarrier testing and evaluation.

CircSETD2 inhibits YAP1 by interaction with HuR during breast cancer progression

ABSTRACT CircRNAs have been proven to play a pivotal role in cancer progression. The present study aims to explore the roles and related mechanisms of circSETD2 in breast cancer proliferation, migration and invasion. The expression of circSETD2 in BC was assessed by the GEO database and qRT‒PCR. The biological function and underlying molecular mechanism of circSETD2 in BC were explored using in vitro and in vivo experiments, including CCK8, transwell, RIP, western blot, and xenograft mouse models. The expression of circSETD2 was downregulated in BC tumors, in accordance with the GEO database. Overexpression of circSETD2 significantly suppressed cell growth, cell migration and invasion. Mechanistically, circSETD2 reduced the stabilization of YAP1 by competitively binding with HuR, resulting in inactivation of downstream targets such as CTGF, myc and Slug. Our work suggests that the novel signaling axis circSETD2/HuR/YAP1 plays an important role in BC progression. The molecular mechanism underlying this signaling axis may provide a potential therapeutic target for BC treatment.

Cytotoxic and Apoptotic Effects of Pinostilbene and Bortezomib Combination Treatment on Human Multiple Myeloma Cells.

Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow characterized by bone lesions, hypercalcemia, anemia, and renal failure. Bortezomib (BTZ), a common treatment for MM, is a proteasome inhibitor that induces apoptosis in MM cells. However, high doses of BTZ can be very toxic, signifying a need for a synergistic drug combination to improve treatment efficacy. Resveratrol (RES), a phenolic compound found in grapes, has been shown to inhibit MM cell growth. We sought to identify a synergistic combination of BTZ with a RES derivative and analyze the effects on reducing viability and inducing apoptosis in human MM cells. BTZ as well as RES and its derivatives pinostilbene (PIN) and piceatannol (PIC) decreased MM cell viability in a dose- and time-dependent manner and increased expression of cleaved proapoptotic proteins poly(ADP-ribose) polymerase 1 (PARP1) and caspase-3 in a dose-dependent manner. The combination of 5 nM BTZ and 5 μM PIN was identified to have synergistic cytotoxic effects in MM RPMI 8226 cells. MM RPMI 8226 cells treated with this combination for 24 h showed increased cleaved PARP1 and caspase-3 expression and higher percentages of apoptotic cells versus cells treated with the individual compounds alone. The treatment also showed increased apoptosis induction in MM RPMI 8226 cells co-cultured with human bone marrow stromal HS-5 cells in a Transwell model used to mimic the bone marrow microenvironment. Expression of oxidative stress defense proteins (catalase, thioredoxin, and superoxide dismutase) in RPMI 8226 cells were reduced after 24 h treatment, and cytotoxic effects of the treatment were ameliorated by antioxidant N-acetylcysteine (NAC), suggesting the treatment impacts antioxidant levels in RPMI 8226 cells. Our results suggest that this combination of BTZ and PIN decreases MM cell viability synergistically by inducing apoptosis and oxidative stress in MM cells.

Consumption of honey ameliorates lipopolysaccharide-induced intestinal barrier dysfunction via upregulation of tight junction proteins.

The leaky gut barrier is an important factor leading to various inflammatory gastrointestinal disorders. The nutritional value of honey and variety of its health benefits have long been recognized. This study was undertaken to assess the role of Indian mustard honey in preventing lipopolysaccharide (LPS)-induced intestinal barrier dysfunction using a combination of in vitro and in vivo experimental model systems. LPS was used to induce intestinal barrier damage in a trans-well model of Caco-2 cells (1µg/ml) and in Swiss albino mice (5mg/kg body weight). Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to analyse sugar and phenolic components in honey samples. The Caco-2 cell monolayer integrity was evaluated by transepithelial electrical resistance (TEER) and paracellular permeability assays. The histopathology of intestinal tissue was analysed by haematoxylin and eosin dual staining. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify the transcription of genes. The protein expression was analysed by immunofluorescence, western blot and ELISA-based techniques. The in vitro data showed that honey prevented LPS-induced intestinal barrier dysfunction dose dependently as was measured by TEER and paracellular flux of FITC-dextran dye. Further, the in vivo data showed a prophylactic effect of orally administered honey as it prevented the loss of intestinal barrier integrity and villus structure. The cellular localization and expression of tight junction (TJ) proteins were upregulated along with downregulation of pro-inflammatory cytokines in response to the administration of honey with LPS. The findings of this study suggest a propitious role of honey in the maintenance of TJ protein integrity, thereby preventing LPS-induced intestinal barrier disintegration.

Biomimetic Human Lung Alveolar Interstitium Chip with Extended Longevity.

Determining the mechanistic causes of lung diseases, developing new treatments thereof, and assessing toxicity whether from chemical exposures or engineered nanomaterials would benefit significantly from a preclinical human lung alveolar interstitium model of physiological relevance. The existing preclinical models have limitations because they fail to replicate the key anatomical and physiological characteristics of human alveoli. Thus, a human lung alveolar interstitium chip was developed to imitate key alveolar microenvironmental factors including an electrospun nanofibrous membrane as the analogue of the basement membrane for co-culture of epithelial cells with fibroblasts embedded in 3D collagenous gels, physiologically relevant interstitial matrix stiffness, interstitial fluid flow, and 3D breathing-like mechanical stretch. The biomimetic chip substantially improved the epithelial barrier function compared to transwell models. Moreover, the chip having a gel made of a collagen I-fibrin blend as the interstitial matrix sustained the interstitium integrity and further enhanced the epithelial barrier, resulting in a longevity that extended beyond eight weeks. The assessment of multiwalled carbon nanotube toxicity on the chip was in line with the animal study.