Browsing by Author "Gaspar, Helena"
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- Adverse outcome pathways induced by 3,4‑dimethylmethcathinone and 4‑methylmethcathinone in differentiated human SH‑SY5Y neuronal cellsPublication . Soares, Jorge; Costa, Vera Marisa; Gaspar, Helena; Santos, Susana; Bastos, Maria de LourdesCathinones (β-keto amphetamines), widely abused in recreational settings, have been shown similar or even worse toxicological profile than classical amphetamines. In the present study, the cytotoxicity of two β-keto amphetamines [3,4-dimethylmethcathinone (3,4-DMMC) and 4-methylmethcathinone (4-MMC)], was evaluated in differentiated dopaminergic SH-SY5Y cells in comparison to methamphetamine (METH). MTT reduction and NR uptake assays revealed that both cathinones and METH induced cytotoxicity in a concentration- and time-dependent manner. Pre-treatment with trolox (antioxidant) partially prevented the cytotoxicity induced by all tested drugs, while N-acetyl-l-cysteine (NAC; antioxidant and glutathione precursor) and GBR 12909 dopamine transporter inhibitor) partially prevented the cytotoxicity induced by cathinones, as evaluated by the MTT reduction assay. Unlike METH, cathinones induced oxidative stress evidenced by the increase on intracellular levels of reactive oxygen species (ROS), and also by the decrease of intracellular glutathione levels. Trolox prevented, partially but significantly, the ROS generation elicited by cathinones, while NAC inhibited it completely. All tested drugs induced mitochondrial dysfunction, since they led to mitochondrial membrane depolarization and to intracellular ATP depletion. Activation of caspase-3, indicative of apoptosis, was seen both for cathinones and METH, and confirmed by annexin V and propidium iodide positive staining. Autophagy was also activated by all drugs tested. Pre-incubation with bafilomycin A1, an inhibitor of the vacuolar H+- ATPase, only protected against the cytotoxicity induced by METH, which indicates dissimilar toxicological pathways for the tested drugs. In conclusion, the mitochondrial impairment and oxidative stress observed for the tested cathinones may be key factors for their neurotoxicity, but different outcome pathways seem to be involved in the adverse effects, when compared to METH.
- Anti-Hepatocellular Carcinoma (HepG2) Activities of Monoterpene Hydroxy Lactones Isolated from the Marine Microalga Tisochrysis LuteaPublication . Gangadhar, Katkam N.; Rodrigues, Maria João; Pereira, Hugo; Gaspar, Helena; Malcata, F. Xavier; Barreira, Luísa; Varela, JoãoTisochrysis lutea is a marine haptophyte rich in omega-3 polyunsaturated fatty acids (e.g., docosahexaenoic acid (DHA)) and carotenoids (e.g., fucoxanthin). Because of the nutraceutical applications of these compounds, this microalga is being used in aquaculture to feed oyster and shrimp larvae. In our earlier report, T. lutea organic crude extracts exhibited in vitro cytotoxic activity against human hepatocarcinoma (HepG2) cells. However, so far, the compound(s) accountable for the observed bioactivity have not been identified. Therefore, the aim of this study was to isolate and identify the chemical component(s) responsible for the bioactivity observed. Bioassay-guided fractionation through a combination of silica-gel column chromatography, followed by preparative thin layer chromatography (PTLC), led to the isolation of two diastereomers of a monoterpenoid lactone, namely, loliolide (1) and epi-loliolide (2), isolated for the first time in this species. The structural elucidation of both compounds was carried out by GC-MS and 1D (1 H and13 C APT) and 2D (COSY, HMBC, HSQC-ed, and NOESY) NMR analysis. Both compounds significantly reduced the viability of HepG2 cells and were considerably less toxic towards a non-tumoral murine stromal (S17) cell line, although epi-loliolide was found to be more active than loliolide. © 2020 by the authors.
- Antioxidant and neuroprotective potential of the brown seaweed Bifurcaria bifurcata in an in vitro Parkinson’s Disease ModelPublication . Silva, Joana; Alves, Celso; Freitas, Rafaela; Martins, Alice; Pinteus, Susete; Ribeiro, Joana; Gaspar, Helena; Alfonso, Amparo; Pedrosa, RuiBifurcaria bifurcata is a marine brown seaweed mainly found on the Atlantic coast. Herein, we report the antioxidant and neuroprotective activities of seven fractions (F1–F7) obtained by normal phase chromatography from the B. bifurcata dichloromethane extract, as well as of its two major isolated diterpenes. Total phenolic content of fractions was determined by the Folin–Ciocalteu method, while antioxidant activity was evaluated by the DPPH, ORAC, and FRAP assays. Neuroprotective effects were evaluated in a neurotoxic model induced by 6-hydroxydopamine (6-OHDA) in a human neuroblastoma cell line (SH-SY5Y), while the mechanisms associated to neuroprotection were investigated by the determination of mitochondrial membrane potential, H2O2 production, Caspase-3 activity, and by observation of DNA fragmentation. Fractions F4 and F5 exhibited the best neuroprotective and antioxidant activities, respectively. F4 fraction prevented changes in mitochondrial potential, and induced a reduction of H2O2 levels production and an increase in cell viability, suggesting that it may contain multi-target compounds acting on different pathways. Hence, this fraction was subjected to purification steps, affording the known diterpenes eleganolone and eleganonal. Both compounds exhibited antioxidant potential, being interesting candidates for further neuroprotective studies.
- Antiulcerogenic potential of the ethanolic extract of Ceiba speciosa (A. St.-Hil.) Ravenna evaluated by in vitro and in vivo studiesPublication . Dörr, Juliana Andréa; Majolo, Fernanda; Bortoluzzi, Luísa; Vargas, Evelin Zen de; Silva, Joana; Pasini, Manoela; Stoll, Stefani Natali; Rosa, Rafael Lopes da; Figueira, Mariana Moreira; Fronza, Marcio; Beys-da-Silva, Walter O.; Martins, Alice; Gaspar, Helena; Pedrosa, Rui; Laufer, StefanGastrointestinal diseases, such as peptic ulcers, are caused by a damage in the gastric mucosa provoked by several factors. This stomach injury is regulated by many inflammatory mediators and is commonly treated with proton-pump inhibitors, histamine H2 receptor blockers and antacids. However, various medicinal plants have demonstrated positive effects on gastric ulcer treatment, including plants of the Ceiba genus. The aim of this study was to evaluate the antiulcer and anti-inflammatory activities of the stem bark ethanolic extract of Ceiba speciosa (A. St.-Hil.) Ravenna. We performed a preliminary quantification of phenolic compounds by high-performance liquid chromatography-diode array detection (HPLC-DAD), followed by the prospection of other chemical groups through nuclear magnetic resonance (NMR) spectroscopy. A set of in vitro assays was used to evaluate the extract potential regarding its antioxidant activity (DPPH: 19.83 +- 0.34 ug/mL; TPC: 307.20 +- 6.20 mg GAE/g of extract), effects on cell viability and on the release of TNF-α in whole human blood. Additionally, in vivo assays were performed to evaluate the leukocyte accumulation and total protein quantification in carrageenan-induced air pouch, as well as the antiulcerogenic effect of the extract on an ethanol-induced ulcer in rats. The extract contains flavonoids and phenolic compounds, as well as sugars and quinic acid derivatives exhibiting potent antioxidant activity and low toxicity. The extract reduced the release of TNF-α in human blood and inhibited the activity of p38α (1.66 ug/mL), JAK3 (5.25 ug/mL), and JNK3 (8.34 ug/mL). Moreover, it reduced the leukocyte recruitment on the pouch exudate and the formation of edema, reverting the effects caused by carrageenan. The extract presented a significant prevention of ulcer formation and a higher reduction than the reference drug, Omeprazole. Therefore, C. speciosa extract has demonstrated relevant therapeutic potential for the treatment of gastric diseases, deserving the continuation of further studies to unveil the mechanisms of action of plant bioactive ingredients.
- Bromoditerpenes from the red seaweed Sphaerococcus coronopifolius as potential cytotoxic agents and proteasome inhibitors and related mechanisms of actionPublication . Alves, Celso; Silva, Joana; Pintéus, Susete; Guedes, Romina A.; Guedes, Rita C.; Alvariño, Rebeca; Freitas, Rafaela; Goettert, Márcia I.; Gaspar, Helena; Alfonso, Amparo; Alpoím, Maria C.; Botana, Luis M.; Pedrosa, RuiSeaweeds are a great source of compounds with cytotoxic properties with the potential to be used as anticancer agents. This study evaluated the cytotoxic and proteasome inhibitory activities of 12R-hydroxy-bromosphaerol, 12S-hydroxy-bromosphaerol, and bromosphaerol isolated from Sphaerococcus coronopifolius. The cytotoxicity was evaluated on malignant cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, and SK-MEL-28) using the MTT and LDH assays. The ability of compounds to stimulate the production of hydrogen peroxide (H2O2) and to induce mitochondrial dysfunction, the externalization of phosphatidylserine, Caspase-9 activity, and changes in nuclear morphology was also studied on MCF-7 cells. The ability to induce DNA damage was also studied on L929 fibroblasts. The proteasome inhibitory activity was estimated through molecular docking studies. The compounds exhibited IC50 values between 15.35 and 53.34 μM. 12Rhydroxy-bromosphaerol and 12S-hydroxy-bromosphaerol increased the H2O2 levels on MCF-7 cells, and bromosphaerol induced DNA damage on fibroblasts. All compounds promoted a depolarization of mitochondrial membrane potential, Caspase-9 activity, and nuclear condensation and fragmentation. The compounds have been shown to interact with the chymotrypsin-like catalytic site through molecular docking studies; however, only 12S-hydroxy-bromosphaerol evidenced interaction with ALA20 and SER169, key residues of the proteasome catalytic mechanism. Further studies should be outlined to deeply characterize and understand the potential of those bromoditerpenes for anticancer therapeutics.
- Cytotoxic mechanism of sphaerodactylomelol, an uncommon bromoditerpene isolated from sphaerococcus coronopifoliusPublication . Alves, Celso; Silva, Joana; Pinteus, Susete; Alonso, Eva; Alvarino, Rebeca; Duarte, Adriana; Marmitt, Diorge; Geottert, Márcia Inês; Gaspar, Helena; Alfonso, Amparo; Alpoim, Maria C.; Botana, Luis M; Pedrosa, RuiMarine natural products have exhibited uncommon chemical structures with relevant antitumor properties highlighting their potential to inspire the development of new anticancer agents. The goal of this work was to study the antitumor activities of the brominated diterpene sphaerodactylomelol, a rare example of the dactylomelane family. Cytotoxicity (10–100 M; 24 h) was evaluated on tumor cells (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, SK-ML-28) and the effects estimated by MTT assay. Hydrogen peroxide (H2O2) levels and apoptosis biomarkers (membrane translocation of phosphatidylserine, depolarization of mitochondrial membrane potential, Caspase-9 activity, and DNA condensation and/or fragmentation) were studied in the breast adenocarcinoma cellular model (MCF-7) and its genotoxicity on mouse fibroblasts (L929). Sphaerodactylomelol displayed an IC50 range between 33.04 and 89.41 M without selective activity for a specific tumor tissue. The cells’ viability decrease was accompanied by an increase on H2O2 production, a depolarization of mitochondrial membrane potential and an increase of Caspase-9 activity and DNA fragmentation. However, the DNA damage studies in L929 non-malignant cell line suggested that this compound is not genotoxic for normal fibroblasts. Overall, the results suggest that the cytotoxicity of sphaerodactylomelol seems to be mediated by an increase of H2O2 levels and downstream apoptosis.
- Disclosing the antitumour potential of the marine bromoditerpene sphaerococcenol a on distinct cancer cellular modelsPublication . Alves, Celso; Silva, Joana; Afonso, Marta B.; Guedes, Romina A.; Guedes, Rita C.; Alvariño, Rebeca; Pinteus, Susete; Gaspar, Helena; Goettert, Marcia I.; Alfonso, Amparo; Rodrigues, Cecília M. P.; Alpoim, Maria C.; Botana, Luis; Pedrosa, RuiNature has revealed to be a key source of innovative anticancer drugs. This study evaluated the antitumour potential of the marine bromoditerpene sphaerococcenol A on different cancer cellular models. Dose-response analyses (0.1–100 μM; 24 h) were accomplished in eight different tumour cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, SK-MEL-28). Deeper studies were conducted on MFC-7 cells, namely, determination of hydrogen peroxide (H2O2) levels and evaluation of apoptosis biomarkers (phosphatidylserine membrane translocation, mitochondrial dysfunction, Caspase-9 activity, and DNA changes). The ability of the compound to induce genotoxicity was verified in L929 fibroblasts. Sphaerococcenol A capacity to impact colorectal-cancer stem cells (CSCs) tumourspheres (HT29, HCT116, SW620) was evaluated by determining tumourspheres viability, number, and area, as well as the proteasome inhibitory activity. Sphaerococcenol A hepatoxicity was studied in AML12 hepatocytes. The compound exhibited cytotoxicity in all malignant cell lines (IC50 ranging from 4.5 to 16.6 μM). MCF-7 cells viability loss was accompanied by H2O2 generation, mitochondrial dysfunction, Caspase-9 activation and DNA nuclear morphology changes. Furthermore, the compound displayed the lowest IC50 on HT29-derived tumourspheres (0.70 μM), followed by HCT116 (1.77 μM) and SW620 (2.74 μM), impacting the HT29 tumoursphere formation by reducing their number and area. Finally, the compound displayed low cytotoxicity on AML12 hepatocytes without genotoxicity. Overall, sphaerococcenol A exhibits broad cytotoxic effects on different tumour cells, increasing H2O2 production and apoptosis. It also affects colorectal CSCs-enriched tumoursphere development. These data highlight the elevance to include sphaerococcenol A in further pharmacological studies aiming cancer treatments.
- Disclosing the potential of eleganolone for Parkinson’s disease therapeutics: neuroprotective and anti-inflammatory activitiesPublication . Silva, Joana; Alves, Celso; Pinteus, Susete; Susano, Patrícia; Simões, Marco; Guedes, Miguel; Martins, Alice; Rehfeldt, Stephanie; Gaspar, Helena; Goettert, Márcia Inês; Alfonso, Amparo; Pedrosa, RuiThe treatment of Parkinson´s disease (PD) has benefited from significant advances resulting from the increasing research efforts focused on new therapeutics. However, the current treatments for PD are mostly symptomatic, alleviating disease symptoms without reversing or retarding disease progression. Thus, it is critical to find new molecules that can result in more effective treatments. Within this framework, this study aims to evaluate the neuroprotective and anti-inflammatory effects of three compounds (eleganolone, eleganonal and fucosterol) isolated from the brown seaweed Bifurcaria bifurcata. In vitro neuroprotective effects were evaluated on a PD cellular model induced by the neurotoxin 6-hydroxydopamine (6-OHDA) on SH-SY5Y human cells, while lipopolysaccharide (LPS) -stimulated RAW 264.7 macrophages were used to evaluate the anti-inflammatory potential. Additionally, the underlying mechanisms of action were also investigated. Compounds were isolated by preparative chromatographic methods and their structural elucidation attained by NMR spectroscopy. Among the tested compounds, eleganolone (0.1–1 μM; 24 h) reverted the neurotoxicity induced by 6-OHDA in about 20%. The neuroprotective effects were mediated by mitochondrial protection, reduction of oxidative stress, inflammation and apoptosis, and inhibition of NF-kB pathway. The results suggest that eleganolone may provide advantages in the treatment of neurodegenerative conditions and, therefore, should be considered for future preclinical studies.
- Exploring marine resources against neurological disorders: the neuroprotective and anti‑inflammatory potential of the brown seaweed Bifurcaria bifurcataPublication . Silva, Joana; Alves, Celso; Susano, Patrícia; Martins, Alice; Pinteus, Susete; Gaspar, Helena; Alfonso, Amparo; Pedrosa, RuiOxidative stress is strongly involved in the pathogenesis of neurodegenerative diseases, like Parkinson´s disease (PD). Particularly, an excess of reactive oxygen species (ROS) released by the cells promotes an oxidative stress condition, which is a main cause of tissue injury leading to nervous system dysfunction. In this work, the antioxidant, neuroprotective and antiinflammatory activities of different fractions from the brown seaweed Bifurcaria bifurcata are presented and related with their chemical profile. The antioxidant capacity was evaluated by the Folin-Ciocalteu method, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Neuroprotective capacity was evaluated to prevent neurological cell death mediated by the neurotoxin 6-hydroxydopamine (6-OHDA) on SH-SY5Y cells, and their anti-inflammatory effects on RAW 264.7 macrophages. The ethyl acetate fractions (100 μg mL−1) exhibited significant antioxidant and neuroprotective activities in the in vitro models assayed. Furthermore, two of the most polar fractions obtained with methanol and water also evidenced a significant neuroprotective potential. Bifurcaria bifurcata fractions treatment decreased ROS production, mitochondrial dysfunction, and Caspase-3 activity. Regarding the anti-inflammatory potential, five fractions (100 μg mL−1) inhibited nitric oxide (NO) production and reduced the interleukin – 6 (IL-6) and tumor necrosis factor (TNF-α) levels. Mannitol, identified as the major component of the most bioactive fraction, protected SH-SY5Y cells against the 6-OHDA neurotoxicity mediating ROS generation mitigation, mitochondrial dysfunction, and DNA damage, together with the Caspase-3 activity inhibition. Results suggest that B. bifurcata is a relevant source of neuroprotective agents, with particular interest for preventive therapeutics.
- From marine origin to therapeutics: the antitumor potential of marine algae-derived compoundsPublication . Alves, Celso; Silva, Joana; Pinteus, Susete; Gaspar, Helena; Alpoim, Maria C.; Botana, Luís M.; Pedrosa, RuiMarine environment has demonstrated to be an interesting source of compounds with uncommon and unique chemical features on which themolecularmodeling and chemical synthesis of new drugs can be based with greater efficacy and specificity for the therapeutics. Cancer is a growing public health threat, and despite the advances in biomedical research and technology, there is an urgent need for the development of new anticancer drugs. In this field, it is estimated that more than 60% of commercially available anticancer drugs are natural biomimetic inspired. Among the marine organisms, algae have revealed to be one of the major sources of new compounds of marine origin, including those exhibiting antitumor and cytotoxic potential. These compounds demonstrated ability to mediate specific inhibitory activities on a number of key cellular processes, including apoptosis pathways, angiogenesis, migration and invasion, in both in vitro and in vivo models, revealing their potential to be used as anticancer drugs. This review will focus on the bioactive molecules from algae with antitumor potential, from their origin to their potential uses, with special emphasis to the alga Sphaerococcus coronopifolius as a producer of cytotoxic compounds.
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