Cell-free Model Research Articles

Suitability of Artificial Membranes in Lipolysis-Permeation Assays of Oral Lipid-Based Formulations

PurposeTo evaluate the performance of artificial membranes in in vitro lipolysis-permeation assays useful for absorption studies of drugs loaded in lipid-based formulations (LBFs).MethodsPolycarbonate as well as PVDF filters were treated with hexadecane, or lecithin in n-dodecane solution (LiDo) to form artificial membranes. They were thereafter used as absorption membranes separating two compartments mimicking the luminal and serosal side of the intestine in vitro. Membranes were subjected to dispersions of an LBF that had been digested by porcine pancreatin and spiked with the membrane integrity marker Lucifer Yellow (LY). Three fenofibrate-loaded LBFs were used to explore the in vivo relevance of the assay.ResultsOf the explored artificial membranes, only LiDo applied to PVDF was compatible with lipolysis by porcine pancreatin. Formulation ranking based on mass transfer in the LiDo model exposed was the same as drug release in single-compartment lipolysis. Ranking based on observed apparent permeability coefficients of fenofibrate with different LBFs were the same as those obtained in a cell-based model.ConclusionsThe LiDo membrane was able to withstand lipolysis for a sufficient assay period. However, the assay with porcine pancreatin as digestive agent did not predict the in vivo ranking of the assayed formulations better than existing methods. Comparison with a Caco-2 based assay method nonetheless indicates that the in vitro in vivo relationship of this cell-free model could be improved with alternative digestive agents.

Epigallocatechin-3-gallate (EGCG) Alters Histone Acetylation and Methylation and Impacts Chromatin Architecture Profile in Human Endothelial Cells.

Epigallocatechin gallate (EGCG), the main green tea polyphenol, exerts a wide variety of biological actions. Epigenetically, the catechin has been classified as a DNMTs inhibitor, however, its impact on histone modifications and chromatin structure is still poorly understood. The purpose of this study was to find the impact of EGCG on the histone posttranslational modifications machinery and chromatin remodeling in human endothelial cells of both microvascular (HMEC-1) and vein (HUVECs) origin. We analyzed the methylation and acetylation status of histones (Western blotting), as well as assessed the activity (fluorometric assay kit) and gene expression (qPCR) of the enzymes playing a prominent role in shaping the human epigenome. The performed analyses showed that EGCG increases histone acetylation (H3K9/14ac, H3ac), and methylation of both active (H3K4me3) and repressive (H3K9me3) chromatin marks. We also found that the catechin acts as an HDAC inhibitor in cellular and cell-free models. Additionally, we observed that EGCG affects chromatin architecture by reducing the expression of heterochromatin binding proteins: HP1α, HP1γ. Our results indicate that EGCG promotes chromatin relaxation in human endothelial cells and presents a broad epigenetic potential affecting expression and activity of epigenome modulators including HDAC5 and 7, p300, CREBP, LSD1 or KMT2A.

Pomegranate Juice Enhances Iron Dialysability and Assimilation in In-Vitro Cell Free and Cell-Based Models.

Despite concerted programmatic efforts iron deficiency anemia (IDA) continues to be a global health problem. Per Ayurveda, an Indian Traditional Medical System, natural plant materials such as pomegranate juice (PJ) is indicated as a food supplement to manage IDA. We hypothesized that pomegranate could play a role in improving absorption and assimilation of iron. A cell free in vitro model simulating the gastric and intestinal digestive processes coupled with cell based (Caco-2 and HepG2) models were used to assess iron (FeSO4 form) dialysability in the presence of PJ. Iron assimilation into cells was measured in terms of the cellular ferritin content. PJ (containing ~13mg/100ml natural ascorbic acid equivalent) increased the dialysability of iron by >3 fold when compared to control in the cell free model. An equivalent concentration of ascorbic acid alone increased it only by 1.6-fold. PJ increased the iron uptake in Caco2 cells by ~6-fold and ferritin content by 30% when compared to the ascorbic acid control. Similarly, PJ enhanced the iron uptake in HepG2 cells by ~3 fold and iron assimilation by about 50%. This study establishes a scientific evidence for Ayurveda's claim of using pomegranate in the management of IDA by facilitating iron absorption and assimilation. It provides a simple solution for addressing the global problem of IDA. Synergistic action of multiple phytochemicals, over and above ascorbic acid, in PJ may be responsible for improving iron bioavailability.

Phytochemical characteristics of aerial part of Cissus quadrangularis (L) and its in-vitro inhibitory activity against leukemic cells and antioxidant properties.

Background and ObjectivesCissus quadrangularis Linn, is a rich bioresource for folk and traditional medicines from ancient times till date. The present study aimed to investigate the free radical scavenging and anticancer efficacy in vitro of the ethanolic and methanolic extract from the aerial parts of Cissus quadrangularis (L).Material and MethodsIn vitro cell-free antioxidant analyses were performed for the ethanolic extract of Cissus quadrangularis (L). (EECQ) and methanolic extract of Cissus quadrangularis (L). (MECQ) using different free radical scavenging assays includes DPPH, nitric oxide, superoxide, metal chelation, and hydrogen peroxide radical scavenging assays. In vitro leukemic cytotoxic assessment by MTT assay was performed both EECQ and MECQ extract against HL-60 cell lines.ResultsStrong antioxidant effects were recorded in EECQ and MECQ in all the cell-free models. The ethanolic extract exhibited a significant dose-dependent free radical activity in comparison with methanolic extracts. The EECQ and MECQ possess pronounced anticancer efficacy against leukemic cells HL-60 with an IC50 value of 36 μg/mL and 40 μg/mL respectively.ConclusionPresent data indicates the presence of marked antioxidant and anticancer behaviors in the extracts of aerial portions of Cissus quadrangularis (L). extracts. Thus, Cissus quadrangularis (L). poses as a promising safe chemopreventive plant to combat cancer.

Synaptotagmin-1 and Doc2b Exhibit Distinct Membrane-Remodeling Mechanisms

Synaptotagmin-1 (Syt1) is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared with that of Doc2b, which contains a conserved C2AB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both C2AB fragments cause a reduction in the membrane-bending modulus, as measured by a method based on atomic force microscopy. This lowering of the energy required for membrane deformation may contribute to Ca2+-induced fusion.

Downlink Interference Management in Cell-Free VLC Network

Visible light communication (VLC) network is a candidate complement to its radio frequency counterpart with the benefits of wide free spectrum and low energy consumption. Due to the dense distribution of access points (APs) and users in VLC network, interference management is crucial to ensure the quality of service. Meanwhile, VLC link relates not only to the transmission power and the orientations of APs, but also to the orientations of users. Hence, the coverage of APs can be irregular and aligned with the motion of users. Cell-free network model, which is composed of irregular cells, is suitable for the VLC network. Therefore, in consideration of the directionality of VLC links, we investigate the joint optimization of AP grouping, AP-user association and AP power allocation for interference management (IM) in the cell-free VLC network, which is formulated as a network utility maximization (NUM) problem. The NUM problem is a mixed non-convex and combinatorial problem, and a graph-based IM algorithm is proposed to solve it. Specifically, a heuristic AP grouping algorithm is proposed based on an interference graph, which is defined according to the locations of APs and users as well as their orientations, to preselect several candidate AP groupings from the huge number of feasible AP groupings. Given each select AP grouping, the AP-user association and AP power allocation is optimized via the successive convex approximation method. Then, the proposed IM algorithm will choose the AP grouping from those candidates, as well as the corresponding AP-user association and AP power allocation, that attain the highest network utility. Simulations demonstrate that the proposed IM algorithm could achieve a network utility close to the maximum one obtained by searching through the entire solution space, with comparatively low complexity, and outperforms the conventional IM schemes.

Qualitative Fingerprint Analysis and Multidirectional Assessment of Different Crude Extracts and Essential Oil from Wild Artemisia santonicum L.

Artemisia species are used as folk medicines in several countries. This work was aimed to shed more light on the effect of methanol, water, ethyl acetate extracts, and essential oil (EO) of A. santonicum on selected enzymes (cholinesterase, tyrosinase α-amylase, and α-glucosidase) as well of their antioxidant and pharmacological effects. The chemical profile of the essential oil was determined using gas chromatography coupled to mass spectrometry (GC-MS) analysis, while the extracts were chemically characterized by high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Forty-nine constituents were identified and camphor (36.6%), 1,8-cineole (10.2%), α-thujone (10.1%), borneol (4.5%), and β-thujone (3.6%) were the major components. Overall, 45, 74, and 67 components were identified from the ethyl acetate, methanol, and water extracts, respectively. The EO and extracts showed significant antioxidant properties, in a cell-free model; particularly, methanol and water extracts revealed promising sources of antioxidant compounds. Additionally, we evaluated protective effects of EO and extracts in isolated rat colon tissue challenged with lipopolysaccharide (LPS), as an ex vivo model of colon inflammation, and human colon cancer HCT116 cell line. Particularly, we observed that, among all tested samples, A. santonicum ethyl acetate displayed the best pharmacological profile, being able to blunt LPS-induced levels of all tested biomarkers of inflammation and oxidative stress, including colon nitrites, lactate dehydrogenase, prostaglandin E2, and serotonin. Additionally, this extract was also able to reduce HCT116 cell viability, thus suggesting potential antiproliferative effects against colon cancer cells. Based on our results, A. santonicum has great potential for developing novel functional agents including pharmaceuticals, cosmeceuticals, and nutraceuticals.

Phytochemistry and Antioxidant Activity of Aerial Parts of Phagnalon sordidum L.

One new natural monoterpene, 5-O-β-d-glucopyranosyl-2-hydroxy-p-cymene (1: ), and 11 known compounds were isolated through a biologically oriented approach from the aerial parts of Phagnalon sordidum L. The most active extract and fractions were selected using 3 complementary antioxidant activity assays. Results and the different methods were compared by relative antioxidant capacity index. In addition, the most active extract of P. sordidum was subjected to liquid chromatography coupled with electrospray ionization hybrid linear ion trap quadrupole Orbitrap mass spectrometry to quantify secondary metabolites. Antioxidant activities of ethyl acetate extract, and purified 3,4-dihydroxyacetophenone (3: ) and nebrodenside A (7: ) were demonstrated by in vitro cell free model assays, and their protective effect against H2O2-induced oxidative stress in a HepG2 (human hepatocellular carcinoma) cell line was established.

Abstract 3900: The Safety and efficacy profile of a PD-L1 directed, Engineered Toxin Body, as a novel targeted direct-cell kill approach for the treatment of PD-L1 expressing cancers

Abstract Engineered Toxin Bodies (ETBs) are comprised of a deimmunized Shiga-like toxin subunit A (SLTA) genetically fused to an antibody-like targeting domain. The antibody targeting domain allows for specific targeting of cancer cells while the SLTA component promotes self-internalization of ETBs, an activity that allows for the delivery of an enzymatic and permanent ribosomal destruction against targeted cells even in the context of non-or-poorly internalizing receptor targets. Molecular Templates has developed PD-L1-targeting ETBs as an approach to directly target tumor cells and overcome resistance mechanisms against PD-1 and PD-L1 antibodies. The cytotoxicity delivered by PD-L1-specific ETBs is engineered to be independent of a requirement for tumor infiltrating lymphocytes (TILs), high tumor mutational burden, or modulatory effects of the tumor microenvironment. Further, the activity is not dependent on blockade of the PD-1/PD-L1 checkpoint axis. Thus, PD-L1 targeting ETBs represent a distinct class of therapeutics with direct cell-kill mechanism of action and ability for activity in patients who have progressed on current standard of care or checkpoint therapy. In this study, we highlight the efficacy and safety profile of MT-6020, a human and cynomolgus cross-reactive, PD-L1 targeted, ETB. MT-6020 retains potent catalytic activity and mediates enzymatic destruction of ribosomes at comparable levels to wild-type SLTA in a cell free model. In addition, MT-6020 binds to human NSCLC, Melanoma, and TNBC tumor cell lines with nM affinity and mediates cellular cytotoxicity via ribosomal destruction at low nM to sub-nM potency. MT-6020 binds to cell lines expressing non-human primate (NHP)-PD-L1 and elicits cytotoxic responses comparable to those observed on human tumor target cells. MT-6020 demonstrated pharmacodynamic and pharmacokinetic effects and displayed a favorable tolerability profile in a repeat dose NHP study at doses that are above the presumed therapeutically active concentration. Further our lead PD-L1 ETB, MT-6035, is built upon the MT-6020 scaffold and can deliver a viral peptide for cell surface presentation to and targeting by a specific antiviral CTL population (antigen seeding technology (AST)) for a second and complementary mechanism for tumor cell destruction. MT-6020 and MT-6035 represent a novel approach to targeting and destroying tumors expressing PD-L1 that is unlikely to be inhibited by resistance mechanisms to current checkpoint inhibitors, is well tolerated in relevant toxicity models, and has the capacity for activity in indications where standard of care has failed. Molecular Templates is poised to initiate clinical development of the PD-L1 targeted-ETB (AST), MT-6035, in 2H - 2019. Citation Format: Hilario J. Ramos, Asis K. Sarkar, Sara Le Mar, Brigitte Brieschke, Joseph D. Dekker, Veronica R. Partridge, Pablo A. Maceda, Michaela M. Sousares, Garrett L. Robinson, Aimee Iberg, Shaoyou Chu, Jensing Liu, Jack P. Higgins, Erin K. Willert. The Safety and efficacy profile of a PD-L1 directed, Engineered Toxin Body, as a novel targeted direct-cell kill approach for the treatment of PD-L1 expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3900.

Dihydromyricetin and Salvianolic acid B inhibit alpha-synuclein aggregation and enhance chaperone-mediated autophagy

BackgroundProgressive accumulation of α-synuclein is a key step in the pathological development of Parkinson’s disease. Impaired protein degradation and increased levels of α-synuclein may trigger a pathological aggregation in vitro and in vivo. The chaperone-mediated autophagy (CMA) pathway is involved in the intracellular degradation processes of α-synuclein. Dysfunction of the CMA pathway impairs α-synuclein degradation and causes cytotoxicity.ResultsIn the present study, we investigated the effects on the CMA pathway and α-synuclein aggregation using bioactive ingredients (Dihydromyricetin (DHM) and Salvianolic acid B (Sal B)) extracted from natural medicinal plants. In both cell-free and cellular models of α-synuclein aggregation, after administration of DHM and Sal B, we observed significant inhibition of α-synuclein accumulation and aggregation. Cells were co-transfected with a C-terminal modified α-synuclein (SynT) and synphilin-1, and then treated with DHM (10 μM) and Sal B (50 μM) 16 hours after transfection; levels of α-synuclein aggregation decreased significantly (68% for DHM and 75% for Sal B). Concomitantly, we detected increased levels of LAMP-1 (a marker of lysosomal homeostasis) and LAMP-2A (a key marker of CMA). Immunofluorescence analyses showed increased colocalization between LAMP-1 and LAMP-2A with α-synuclein inclusions after treatment with DHM and Sal B. We also found increased levels of LAMP-1 and LAMP-2A both in vitro and in vivo, along with decreased levels of α-synuclein. Moreover, DHM and Sal B treatments exhibited anti-inflammatory activities, preventing astroglia- and microglia-mediated neuroinflammation in BAC-α-syn-GFP transgenic mice.ConclusionsOur data indicate that DHM and Sal B are effective in modulating α-synuclein accumulation and aggregate formation and augmenting activation of CMA, holding potential for the treatment of Parkinson’s disease.

Secretomes from metastatic breast cancer cells, enriched for a prognostically unfavorable LCN2 axis, induce anti-inflammatory MSC actions and a tumor-supportive premetastatic lung.

ABSTRACTCancer metastasis is responsible for the clear majority of cancer-related deaths. Survival and expansion of cancer cells at secondary sites requires that these premetastatic microenvironments be primed by primary tumor cells and their secreted factors. Efforts to date have been limited by immune-deficient in vivo models and/or the need for finely-tuned analysis time points that reduce contributions from early-disseminating cancer cells. In this regard, we developed a tumor cell-free syngeneic breast cancer model for characterizing tumor cell secretome-mediated reprogramming of premetastatic tissues. We demonstrate that secretomes from metastatic breast cancer cells differentially regulate the lung and brain, promoting a tumor-supportive lung microenvironment with both elevated CD73 expression and decreased TNFα expression. Using in vitro models of CD73-positive mesenchymal stem cells (MSCs) and macrophages/monocytes, we tested whether MSCs can mediate anti-inflammatory effects of metastatic breast cancer cells. Notably, conditioned media from metastatic Py230 cells reprogrammed the secretomes of MSCs toward an anti-inflammatory state. Mining transcriptome data from Py8119 and Py230 cells revealed a lipocalin 2 (LCN2) axis that is selectively expressed in the metastatic Py230 cells, predicts poor breast cancer patient survival and is elevated in circulating serum of mice chronically treated with conditioned media from Py230 cells. Taken together, these results establish the utility of an immune-competent tumor cell-free model for characterizing the mechanisms of breast cancer cell priming of the premetastatic niche, demonstrate that MSCs can mediate the anti-inflammatory effects of metastatic breast cancer cells and substantiate LCN2 as a promising therapeutic target for blocking breast cancer progression.

Identification of amentoflavone as a potent highly selective PARP-1 inhibitor and its potentiation on carboplatin in human non-small cell lung cancer

Nuclear protein poly (ADP-ribose) polymerase-1 (PARP-1) is a key enzyme in the repair of DNA and is a promising target in the development of chemosensitizers. This study first investigated the inhibitory effects of amentoflavone (AMF) and its derivatives on PARP-1 and the potentiation of AMF on carboplatin (CBP) in non-small cell lung cancer (NSCLC). This study aims to evaluate the inhibitory effect of AMF against PARP-1 and its potentiation on CBP in lung cancer both in vitro and in vivo. The inhibitory effect of AMF on PARP-1 was investigated using molecular docking and cell-free model of PARP-1 assay. Its potentiation on CBP in lung cancer was also evaluated. Fluorescence resonance energy transfer assay was used to detect the inhibitory effects of AMF and its analogues on PARP-1. Molecular docking was employed to predict the binding mode of AMF and PARP-1. MTT assay, isobologram analysis, Hoechst staining, and Annexin V-PI double staining were used to confirm the potentiation of AMF on CBP in vitro. siRNA (PARP-1)-A549 cells were used to reveal the action target of AMF. Western blot analysis, immunohistochemistry, and Tunnel assay were employed to evaluate the potentiation of AMF on CBP in A549 xenograft mice. AMF and its analogues exerted excellent inhibitory effects on PARP-1 with IC50 values ranging from 0.198 μM to 0.409 μM. Docking experiment showed that AMF can stably bind to PARP-1 with a comparable binding energy to olaparib. AMF can decrease the expression of PAR induced by H2O2in vitro. AMF synergistically increased the CBP anti-proliferative effect in A549. However, its potentiation nearly disappeared when the cells were transfected with siRNAs against PARP-1. Oral administration of AMF (100 mg/kg), combined with CBP, remarkably inhibited A549 tumor growth and ki67 expression, and increased apoptosis compared with CBP-alone group. All results suggest that AMF can be a potential PARP-1 inhibitor and a candidate adjuvant agent to boost the anticancer effect of CBP in NSCLC.

Uncovering the fine print of the CreERT2-LoxP system while generating a conditional knockout mouse model of Ssrp1 gene.

FAcilitates Chromatin Transcription (FACT) is a complex of SSRP1 and SPT16 that is involved in chromatin remodeling during transcription, replication, and DNA repair. FACT has been mostly studied in cell-free or single cell model systems because general FACT knockout (KO) is embryonically lethal (E3.5). FACT levels are limited to the early stages of development and stem cell niches of adult tissues. FACT is upregulated in poorly differentiated aggressive tumors. Importantly, FACT inhibition (RNAi) is lethal for tumors but not normal cells, making FACT a lucrative target for anticancer therapy. To develop a better understanding of FACT function in the context of the mammalian organism under normal physiological conditions and in disease, we aimed to generate a conditional FACT KO mouse model. Because SPT16 stability is dependent on the SSRP1-SPT16 association and the presence of SSRP1 mRNA, we targeted the Ssrp1 gene using a CreERT2- LoxP approach to generate the FACT KO model. Here, we highlight the limitations of the CreERT2-LoxP (Rosa26) system that we encountered during the generation of this model. In vitro studies showed an inefficient excision rate of ectopically expressed CreERT2 (retroviral CreERT2) in fibroblasts with homozygous floxed Ssrp1. In vitro and in vivo studies showed that the excision efficiency could only be increased with germline expression of two alleles of Rosa26CreERT2. The expression of one germline Rosa26CreERT2 allele led to the incomplete excision of Ssrp1. The limited efficiency of the CreERT2-LoxP system may be sufficient for studies involving the deletion of genes that interfere with cell growth or viability due to the positive selection of the phenotype. However, it may not be sufficient for studies that involve the deletion of genes supporting growth, or those crucial for development. Although CreERT2-LoxP is broadly used, it has limitations that have not been widely discussed. This paper aims to encourage such discussions.

P-152 - N-acyl derivatives of 4,6-di-tert-butyl-2-aminophenol protect neutrophils from halogenating stress

Hypochlorous acid is an important microbicidal agent mainly formed by neutrophil myeloperoxidase (MPO), however HOCl hyperproduction leads to halogenating stress associated with different diseases. It is necessary to search for substances that can selectively prevent excessive HOCl generation. We synthesized a number of N-acyl derivatives of 4,6-di-tert-butyl-2-aminophenol which exhibit antiradical activity in cell free model systems. The aim of the work is to study the effect of (N-(4,6-di-tert-butyl-2-aminophenol)R, where R=-COCH3 (1), -COC2H5 (2), -COC3H7 (3) on ROS formation, secretory degranulation and viability of neutrophils exposed to HOCl. It has been shown that all compounds in micromolar concentrations reduce neutrophil ROS formation by 75% or more, inhibit MPO secretion and reduce HOCl production by MPO. It has been found that compounds 1–3 effectively intercept HOCl (up to 78%), but do not interact with H2O2. In concentrations of up to 1 μmol/l, compound 1 increases cell survival in the absence of exogenous HOCl, while compounds 2 and 3 do not influence it. Compound 1 in higher concentrations prevents destruction of neutrophils caused by addition of 300 μmol/l HOCl. Thus, compounds 1–3 can be considered as potential cytoprotectors under halogenating stress.

Acrolein-mediated neuronal cell death and alpha-synuclein aggregation: Implications for Parkinson's disease

Growing evidence suggests that oxidative stress plays a critical role in neuronal destruction characteristic of Parkinson's disease (PD). However, the molecular mechanisms of oxidative stress-mediated dopaminergic cell death are far from clear. In the current investigation, we tested the hypothesis that acrolein, an oxidative stress and lipid peroxidation (LPO) product, is a key factor in the pathogenesis of PD. Using a combination of in vitro, in vivo, and cell free models, coupled with anatomical, functional, and behavioral examination, we found that acrolein was elevated in 6-OHDA-injected rats, and behavioral deficits associated with 6-OHDA could be mitigated by the application of the acrolein scavenger hydralazine, and mimicked by injection of acrolein in healthy rats. Furthermore, hydralazine alleviated neuronal cell death elicited by 6-OHDA and another PD-related toxin, rotenone, in vitro. We also show that acrolein can promote the aggregation of alpha-synuclein, suggesting that alpha-synuclein self-assembly, a key pathological phenomenon in human PD, could play a role in neurotoxic effects of acrolein in PD models. These studies suggest that acrolein is involved in the pathogenesis of PD, and the administration of anti-acrolein scavengers such as hydralazine could represent a novel strategy to alleviate tissue damage and motor deficits associated with this disease.

INVERSE RELATIONSHIP BETWEEN THE ANTIOXIDANT ACTIVITY OF STRUCTURALLY RELATED SYNTHETIC MONOPHENOLS AND THEIR TOXICITY IN TUMOR CELLS

The aim of the study was to study investigate the relationship between the antioxidant activity of new synthetic structurally related water-soluble monophenols and their effect on the tumor cell viability in vitro. Material and methods. Five original hydrophilic sulfur- and selenium-containing monophenols with varying length of the hydrocarbon chain of the para -alkylthiosulfonate substituent, the amount of tert -butyl ortho -substituents and the «S-S» fragment structure are synthesized: sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)ethyl thiosulfonate (TS-12), sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)propyl sulfonate (S-13), sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)propyl seleniumsulfonate (SeS-13), sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)propyl thiosulfonate (TS-13), sodium 3-(3',5'-di- tert -butyl-4’-hydroxyphenyl)propyl thiosulfonate (TS-17). The antioxidant activity of the compounds was determined in a cell-free model test system by the ability to inhibit the luminol-dependent chemiluminescence of free radical-generating azo compound AAPH. Antitumor cytotoxicity was evaluated by their effect on the viability of human histiocytic lymphoma cell line U937 (monocyte/macrophage-like cells) and human breast adenocarcinoma cell line MCF-7 in the MTT test. Results and discussion. All tested phenolic compounds exerted antioxidant activity in the cell-free model system (inhibition of azo initiator radical AAPH • ) and cytotoxicity against U937 and MCF-7 cells, the effect depended on the dose and structure of the molecule. There was a direct relationship between the structure of monophenols and their ability to inhibit the viability of tumor cells of different lines, regardless of the origin of the latter, myeloid (U937) or epithelial (MCF-7), and growth type (respectively anchorage-independent and attached), as well as the concentration range of compounds (respectively from 100 to 500 μM and from 2 to 150 μM). At the same time, the relationship between the antioxidant activity of monophenols and their cytotoxicity was inverse (when SeS-13 was excluded from analysis), which may be related both to the ability of the tumor to self-defense against reactivate oxygen metabolites and to the indirect pro-oxidant effect of phenolic compounds.

Antioxidant and Antisenescence Effects of Bergamot Juice.

Aging is one of the main risk factor for the onset of cardiovascular diseases; one of the possible explanations could be linked to the age-associated overproduction of free radicals. This increase of oxidative stress can be overcome with a high intake of food antioxidants. In this context, a number of studies have been addressed to assess the antiaging potential of natural antioxidant compounds. Recently, it has been shown that the juice of bergamot (Citrus bergamia Risso et Poiteau), a fruit mostly produced in the Ionian coastal areas of Southern Italy (Calabria), is a valuable source of health-promoting constituents with, among other, antioxidant properties. In order to investigate the potential antiaging effects of this Mediterranean natural antioxidant source, bergamot juices of three different cultivars (“fantastico,” “femminello,” and “castagnaro”) were herein characterized by the mean of high-performance liquid chromatography-photodiode array-electrospray ionization-tandem mass spectrometry. Then, juices were investigated for the evaluation of total polyphenolic and flavonoid contents, cell-free model antioxidant activities, and in vitro antiaging properties on two different cellular models of induced myocardial senescence. The best performing juice was also assessed in vivo. The phytochemical profiles confirmed that juices were rich in flavonoids, both flavone and flavanone glycosides. In addition, two limonoid glycosides were also identified in all cultivars. Each cultivar showed different phenolic and flavonoid contents. In tube results showed the juice robust antioxidant activities that correlate with their phenolic and flavonoid contents. Moreover, for the first time, the ability of juice to counteract the chemical-induced senescence was here demonstrated in both cellular models. Lastly, the in vivo data obtained from mouse hearts evidenced an increase in transcription of genes involved in antiaging and antioxidant responses. The overall results suggest that bergamot juice exerts antioxidant and antisenescence effects, making it useful for nutraceutical purposes.

Antioxidant activity of hydroxytyrosyl esters studied in liposome models

The properties and the antioxidant activity of a series of hydroxytyrosyl esters having different carbon chain lengths (C4, C8, C12 and C18) have been measured in phosphatidylcholine model membrane (liposomes) using specific probes for the bilayer and liposome lumen microenvironment, i.e., 1,6-diphenyl-1,3,5-hexatriene (DPH) and 2′,7′-dichlorodihydrofluorescein (H2DCF), respectively.Antioxidants self-assembly and their interaction with liposomes has been evaluated by light scattering, fluorescence, turbidimetry, gel filtration chromatography and microfiltration measurements, allowing the determination of critical aggregation concentration, bound fraction, capacity of crossing the lipid bilayer.The distribution of hydroxytyrosyl long chain esters has been proved to depend quite specifically on their lipophilic chain length, and this turns to have deep effects on their antioxidant behaviour. Shedding new light on the cut off effect and antioxidant behaviour of phenolipids, this study also put forward the relevance of cell-free liposome-based cellular models, like giant liposomes, for further characterization of analogous systems.

Assessment of Passive Intestinal Permeability Using an Artificial Membrane Insert System

Despite reasonable predictive power of current cell-based and cell-free absorption models for the assessment of intestinal drug permeability, high costs and lengthy preparation steps hamper their use. The use of a simple artificial membrane (without any lipids present) as intestinal barrier substitute would overcome these hurdles. In the present study, a set of 14 poorly water-soluble drugs, dissolved in 2 different media (fasted state simulated/human intestinal fluids [FaSSIF/FaHIF]), were applied to the donor compartment of an artificial membrane insert system (AMI-system) containing a regenerated cellulose membrane. Furthermore, to investigate the predictive capacity of the AMI-system as substitute for the well-established Caco-2 system to assess intestinal permeability, the same set of 14 drugs dissolved in FaHIF were applied to the donor compartment of a Caco-2 system. For 14 drugs, covering a broad range of physicochemical parameters, a reasonable correlation between both absorption systems was observed, characterized by a Pearson correlation coefficient r of 0.95 (FaHIF). Using the AMI-system, an excellent predictive capacity of FaSSIF as surrogate medium for FaHIF was demonstrated (r = 0.96). Based on the acquired data, the AMI-system appears to be a time- and cost-effective tool for the early-stage estimation of passive intestinal permeability for poorly water-soluble drugs.

Aβ42-mediated proteasome inhibition and associated tau pathology in hippocampus are governed by a lysosomal response involving cathepsin B: Evidence for protective crosstalk between protein clearance pathways.

Impaired protein clearance likely increases the risk of protein accumulation disorders including Alzheimer’s disease (AD). Protein degradation through the proteasome pathway decreases with age and in AD brains, and the Aβ42 peptide has been shown to impair proteasome function in cultured cells and in a cell-free model. Here, Aβ42 was studied in brain tissue to measure changes in protein clearance pathways and related secondary pathology. Oligomerized Aβ42 (0.5–1.5 μM) reduced proteasome activity by 62% in hippocampal slice cultures over a 4-6-day period, corresponding with increased tau phosphorylation and reduced synaptophysin levels. Interestingly, the decrease in proteasome activity was associated with a delayed inverse effect, >2-fold increase, regarding lysosomal cathepsin B (CatB) activity. The CatB enhancement did not correspond with the Aβ42-mediated phospho-tau alterations since the latter occurred prior to the CatB response. Hippocampal slices treated with the proteasome inhibitor lactacystin also exhibited an inverse effect on CatB activity with respect to diminished proteasome function. Lactacystin caused earlier CatB enhancement than Aβ42, and no correspondence was evident between up-regulated CatB levels and the delayed synaptic pathology indicated by the loss of pre- and postsynaptic markers. Contrasting the inverse effects on the proteasomal and lysosomal pathways by Aβ42 and lactacystin, such were not found when CatB activity was up-regulated two-fold with Z-Phe-Ala-diazomethylketone (PADK). Instead of an inverse decline, proteasome function was increased marginally in PADK-treated hippocampal slices. Unexpectedly, the proteasomal augmentation was significantly pronounced in Aβ42-compromised slices, while absent in lactacystin-treated tissue, resulting in >2-fold improvement for nearly complete recovery of proteasome function by the CatB-enhancing compound. The PADK treatment also reduced Aβ42-mediated tau phosphorylation and synaptic marker declines, corresponding with the positive modulation of both proteasome activity and the lysosomal CatB enzyme. These findings indicate that proteasomal stress contributes to AD-type pathogenesis and that governing such pathology occurs through crosstalk between the two protein clearance pathways.