Reactivity of human fetal and adult immortalized hepatocytes to potentially toxic bilirubin and bile acid species

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaCholestasis is the reduction or stoppage of bile flow. When bile flow is interrupted, the bile compounds, namely bile acids and bilirubin, accumulate into the hepatocyte causing cellular injury and cell death. The hepatocyte function under cholestasis must be studied using liver cell lines closely resembling human primary hepatocytes. However, most of the cell lines used is derived from hepatic tumors which have altered gene expression. In this project, we used the novel non-neoplasic cell line, HHL-5, which retain primary adult hepatocyte phenotype. In addition, we used the fetal hepatocyte cell line WRL-68, which was shown to present similar morphological properties and antigenic profile of human fetal hepatocytes in situ. Using these two cell lines, we evaluated whether fetal and adult hepatocytes respond differently to conditions that mimic cholestasis with associated jaundice, never evaluated in vitro before. The hepatocytes were exposed to 100 μM glycochenodeoxycholic acid(GCDCA), 100 μM conjugated bilirubin (CB), 100 μM unconjugated bilirubin (UCB), 100 μM GCDCA + 100 μM CB + 100 μM UCB or vehicle alone, in the presence of 100 μM human serum albumin (HSA), at various time points. After, we assessed cellular toxicity analyzing cytolysis by lactate dehydrogenase (LDH) release and apoptosis by caspase-3 activity and nuclear fragmentation. There was a significant increase of all these parameters in hepatocytes stimulated with the GCDCA+CB+UCB mixture. LDH release significantly increased after 48 h incubation for GCDCA+CB+UCB treatment (~40%, P<0.01) in the adult cell line. This increase was also statistically significant when compared to GCDCA, CB or UCB incubation alone (P<0.05). Relatively to WRL-68 cells, the release of LDH also increased significantly (~22%) after 48 h incubation with GCDCA+CB+UCB when compared to control (P<0.05), GCDCA (P<0.05) or UCB (P<0.01) incubations. Regarding caspase-3 activity, the increase was evident for GCDCA+CB+UCB treatment. In the adult cell line, it increased ~1.5-fold after 6 h (P<0.01 vs. control, P<0.01 vs. GCDCA, P<0.05 vs. UCB), peaking at 12 h (~3.5-fold, P<0.05 vs. control, P<0.05 vs. GCDCA) and remaining elevated after 24 h (P<0.01 vs. control, P<0.01 vs. GCDCA, P<0.05 vs. CB, P<0.01 vs. UCB). In the fetal cell line, the peak of caspase-3 activity with the same treatment occurred earlier and in a higher magnitude. Indeed, caspase-3 activity increased 4-fold after GCDCA+CB+UCB treatment at 6 h incubation (P<0.05) and this activation was sustained until 12 h incubation(P<0.01). Concerning nuclear fragmentation, in both cell lines, hepatocytes incubated with GCDCA+CB+UCB exhibited profound changes in nuclear morphology, consistent with apoptosis, in a more marked way than when incubated with GCDCA, CB or UCB alone. The results showed similar data to those previously obtained for caspase-3 activity

Biomedical properties of cystoseira species: insights into nutra- and pharmaceutical applications

Tese de doutoramento, Ciências Biológicas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016Brown algae contain interesting nutritional profiles, usually with low levels of lipids but relatively high amounts of polyunsaturated fatty acids (PUFA) and minerals. Moreover, brown algae have secondary metabolites that display several biological activities. However, studies on the chemical composition, bioactive compounds and respective biological activities of brown algae are scarce. Here it is shown that Cystoseira species have potential biotechnological applications. Among the species evaluated, C. tamariscifolia and C. baccata were those that in general had the highest ash, protein and lipid contents, while the highest levels of moisture and total carbohydrates were detected in C. nodicaulis and C. compressa. Cystoseira species had also high amounts of K, Ca and Fe, and a favorable Na/K ratio. C. tamariscifolia, C. compressa and C. nodicaulis stood out for their high polyunsaturated/saturated fatty acids (PUFA/SFA) and low n-6/n- 3 PUFA ratios as well as appropriate unsaturation, atherogenicity and thrombogenicity indices, suggesting a high nutritional value. C. tamariscifolia hexane extract had the highest antioxidant and anti-proliferative activities against a panel of tumoral cells. This extract was particularly selective for hepatocarcinoma cells (HepG2) when compared to non-tumoral cells. HepG2 cells presented pro-apoptotic features and disaggregation on 3D multicellular tumor spheroids after incubation with the extract. Demethoxy cystoketal chromane was isolated and identified as an anti-proliferative compound, selective towards HepG2 cells. Furthermore, isololiolide was isolated for the first time also from C. tamariscifolia hexane extract. The latter compound exhibited significant cytotoxic activity against three human tumoral cell lines, namely HepG2 cells, whereas no cytotoxicity was found in non-malignant human fibroblasts. Isololiolide disrupted the HepG2 normal cell cycle and induced apoptosis. Moreover, it altered the expression of proteins that are important in the apoptotic cascade, increasing PARP cleavage and p53 protein expression, and decreasing procaspase-3 and Bcl-2 expression levels. Taken together, the results here presented highlight the potential of Cystoseira macroalgae as sources of products for nutra- and pharmaceutical applications

Polyunsaturated fatty acids of marine macroalgae: potential for nutritional and pharmaceutical applications

As mammals are unable to synthesize essential polyunsaturated fatty acids (PUFA), these compounds need to be taken in through diet. Nowadays, obtaining essential PUFA in diet is becoming increasingly difficult; therefore this work investigated the suitability of using macroalgae as novel dietary sources of PUFA. Hence, 17 macroalgal species from three different phyla (Chlorophyta, Phaeophyta and Rhodophyta) were analyzed and their fatty acid methyl esters (FAME) profile was assessed. Each phylum presented a characteristic fatty acid signature as evidenced by clustering of PUFA profiles of algae belonging to the same phylum in a Principal Components Analysis. The major PUFA detected in all phyla were C-18 and C-20, namely linoleic, arachidonic and eicosapentaenoic acids. The obtained data showed that rhodophytes and phaeophytes have higher concentrations of PUFA, particularly from the n-3 series, thereby being a better source of these compounds. Moreover, rhodophytes and phaeophytes presented. healthier. Sigma(n-6/)Sigma(n-3) and PUFA/saturated fatty acid ratios than chlorophytes. Ulva was an exception within the Chlorophyta, as it presented high concentrations of n-3 PUFA, alpha-linolenic acid in particular. In conclusion, macroalgae can be considered as a potential source for large-scale production of essential PUFA with wide applications in the nutraceutical and pharmacological industries.SEABIOMED [PTDC/MAR/103957/2008]; Foundation for Science and Technology (FCT); Portuguese National Budget; FCT [SFRH/BPD/65116/2009, SFRH/BD/81425/2011

Maritime halophyte species from southern Portugal as sources of bioactive molecules

Extracts of five halophytes from southern Portugal (Arthrocnemum macrostachyum, Mesembryanthemum edule, Juncus acutus, Plantago coronopus and Halimione portulacoides), were studied for antioxidant, anti-inflammatory and in vitro antitumor properties. The most active extracts towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were the methanol extracts of M. edule (IC50 = 0.1 mg/mL) and J. acutus (IC50 = 0.4 mg/mL), and the ether extracts of J. acutus (IC50 = 0.2 mg/mL) and A. macrostachyum (IC50 = 0.3 mg/mL). The highest radical scavenging activity (RSA) against the 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical was obtained in the ether extract of J. acutus (IC50 = 0.4 mg/mL) and H. portulacoides (IC50 = 0.9 mg/mL). The maximum total phenolic content (TPC) was found in the methanol extract of M. edule (147 mg gallic acid equivalents (GAE)/g) and in the ether extract of J. acutus (94 mg GAE/g). Significant decreases in nitric oxide (NO) production were observed after incubation of macrophages with lipopolysaccharide (LPS) and the chloroform extract of H. portulacoides (IC50 = 109 mu g/mL) and the hexane extract of P. coronopus (IC50 = 98.0 mu g/mL). High in vitro cytotoxic activity and selectivity was obtained with the ether extract of J. acutus. Juncunol was identified as the active compound and for the first time was shown to display selective in vitro cytotoxicity towards various human cancer cells.SEABIOMED project [PTDC/MAR/103957/2008]; XtremeBio project - Foundation for Science and Technology (FCT); Portuguese National Budget; Danish Research Council for Independent Research, Nature and Universe [10-085264]info:eu-repo/semantics/publishedVersio

Isololiolide, a carotenoid metabolite isolated from the brown alga Cystoseira tamariscifolia, is cytotoxic and able to induce apoptosis in hepatocarcinoma cells through caspase-3 activation, decreased Bcl-2 levels, increased p53 expression and PARP cleavage

Background: Brown macroalgae have attracted attention because they display a wide range of biological activities, including antitumoral properties. In this study we isolated isololiolide from Cystoseira tamariscifolia for the first time.Purpose: To examine the therapeutical potential of isololiolide against tumor cell lines.Methods/Study design: The structure of the compound was established and confirmed by 1D and 2D NMR as well as HRMS spectral analysis. The in vitro cytotoxicity was analyzed by colorimetric 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay in tumoral as well as in non-tumoral cell lines. Cell cycle arrest and induction of apoptosis were assessed by flow cytometry. Alteration of expression levels in proteins important in the apoptotic cascade was analyzed by western blotting.Results: Isololiolidewas isolated for the first time from the brown macroalga C. tamariscifolia. Isololiolide exhibited significant cytotoxic activity against three human tumoral cell lines, namely hepatocarcinoma HepG2 cells, whereas no cytotoxicity was found in non-malignant MRC-5 and HFF-1 human fibroblasts. Isololiolide completely disrupted the HepG2 normal cell cycle and induced significant apoptosis. Moreover, western blot analysis showed that isololiolide altered the expression of proteins that are important in the apoptotic cascade, increasing PARP cleavage and p53 expression while decreasing procaspase-3 and Bcl-2 levels.Conclusion: Isololiolide isolated from C. tamariscifolia is able to exert a selective cytotoxic activity on hepatocarcinoma HepG2 cells as well as induce apoptosis through the modulation of apoptosis-related proteins. (C) 2016 Elsevier GmbH. All rights reserved

Human hair proteins as natural reactive oxygen species scavengers for in vitro applications

Human hair proteins are recognized for their intrinsically high cysteine content. They can be solubilized while preserving their highly reductive thiol groups for free radical scavenging applications. The presence of aromatic and nucleophilic amino acids such as methionine, serine, phenylalanine and threonine further contribute to the antioxidative potential of this material. Herein, utilizing the DPPH (2,2-diphenyl-1-picrylhydrazyl) and acellular 2',7'- dichlorodihydrofluorescein diacetate (H2DCFDA) assays, keratins are demonstrated to possess the highest radical scavenging activity among the studied hair proteins. Consequently, protection against hydrogen peroxide-induced oxidative stress in Human Dermal Fibroblasts (HDF) cultured in human hair keratin supplemented media is demonstrated. Quenching of reactive oxygen species(ROS) in the HDF is observed using the CellROX Green dye and the expression levels of antioxidant (HMOX1, SOD2, GPX1) and tumor suppressor (TP53) genes is analyzed using qPCR. Collectively, this study presents further evidence and demonstrates the in vitro application potential of hair proteins, especially keratins, as an antioxidizing supplement.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Submitted/Accepted versionThis research was supported by the Agency for Science, Technology and Research (A*STAR) under its Wound Care Innovation for the Tropics (IAF-PP H17/01/a0/0L9) and the Ministry of Education Tier 1 funding (RT10/20)

Composite hydrogels in three-dimensional in vitro models

3-dimensional (3D) in vitro models were developed in order to mimic the complexity of real organ/tissue in a dish. They offer new possibilities to model biological processes in more physiologically relevant ways which can be applied to a myriad of applications including drug development, toxicity screening and regenerative medicine. Hydrogels are the most relevant tissue-like matrices to support the development of 3D in vitro models since they are in many ways akin to the native extracellular matrix (ECM). For the purpose of further improving matrix relevance or to impart specific functionalities, composite hydrogels have attracted increasing attention. These could incorporate drugs to control cell fates, additional ECM elements to improve mechanical properties, biomolecules to improve biological activities or any combinations of the above. In this Review, recent developments in using composite hydrogels laden with cells as biomimetic tissue- or organ-like constructs, and as matrices for multi-cell type organoid cultures are highlighted. The latest composite hydrogel systems that contain nanomaterials, biological factors, and combinations of biopolymers (e.g., proteins and polysaccharide), such as Interpenetrating Networks (IPNs) and Soft Network Composites (SNCs) are also presented. While promising, challenges remain. These will be discussed in light of future perspectives toward encompassing diverse composite hydrogel platforms for an improved organ environment in vitro.Agency for Science, Technology and Research (A*STAR)Published versionThis research was supported by the Agency for Science, Technology and Research (A*STAR) under its Acne and Sebaceous Gland Program & Wound Care Innovation for the Tropics IAF-PP (H17/01/a0/008 and H17/01/a0/0L9)

Anti-inflammatory potential of simvastatin loaded nanoliposomes in 2D and 3D foam cell models

Atherosclerosis is a multifactorial disease triggered and sustained by risk factors such as high cholesterol, high blood pressure and unhealthy lifestyle. Inflammation plays a pivotal role in atherosclerosis pathogenesis. In this study, we developed a simvastatin (STAT) loaded nanoliposomal formulation (LIPOSTAT) which can deliver the drug into atherosclerotic plaque when administered intravenously. This formulation is easily prepared, stable, and biocompatible with minimal burst release for effective drug delivery. 2D and 3D in vitro models were examined towards anti-inflammatory effects of STAT, both free and in combination with liposomes. LIPOSTAT induced greater cholesterol efflux in the 2D foam cells and significantly reduced inflammation in both 2D and 3D models. LIPOSTAT alleviated inflammation by reducing the secretion of early and late phase pro-inflammatory cytokines, monocyte adherence marker, and lipid accumulation cytokines. Additionally, the 3D foam cell spheroid model is a convenient and practical approach in testing various anti-atherosclerotic drugs without the need for human tissue.Accepted versionThis study was supported by the NTU–Northwestern Institute for Nanomedicine (04INS000156C150)