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- Elucidating the mechanisms Involved in the cytotoxicity induced by marine cyanobacteria strains against the RKO colon carcinoma cell linePublication . Freitas, S.; Urbatzka, R.; Osório, H.; Costa, M.; Barros, Piedade; Vasconcelos, V.; Martins, RosárioCyanobacteria are known to synthesize secondary metabolites that may have potential as drugs for the treatment of human diseases such as cancer. Previous studies on marine cyanobacteria isolated from the Portuguese coast revealed strains of the picoplanktonic genera Cyanobium and Synechocystis as potential sources of anticancer compounds. The ethyl acetate fraction of the strains Cyanobium sp. LEGE06113 and the Synechocystis salina LEGE06155 was found to reduce cell viability of cancer cell lines. This work aimed to elucidate the mechanisms involved in the cytotoxicity of this strains in the colon adenocarcinoma cell line RKO by employing real-time PCR (RT-PCR) for genes involved in cell cycle and apoptosis, by flow cytometry for cell cycle and by two-dimensional gel electrophoresis for protein expression. RT-PCR results revealed differences in mRNA expression of genes CCNB1 (cell cycle) and BCL-2 (apoptosis). Flow cytometry results revealed a decrease in the G0/G1 and S phase and increased its number in the G2/M phase, which is in accordance with the lower expression of CCNB1. The proteomic results demonstrated different protein patterns comparing treatment groups with control. Proteins differentially regulated in exposed RKO cells were involved in cell cycle regulation, apoptosis, cell structure, protein regulation and cell metabolism. Although several of these proteins were identified in cells exposed to both cyanobacterial extracts, the data provides an indication that the cytotoxicity is induced by different toxic mechanisms in LEGE06113 and LEGE06155 fractions.
- Evaluating the effect of the Ionic liquid [C16Pyr] [Amp] in hormone-resistant tumors using an in vivo zebrafish assayPublication . Barros, Piedade; Martins, Rosário; Cruz, Agostinho; Silva, ReginaThe NASYTHOR project, aimes to study the potential of novel natural and synthetic compounds as anti-cancer drugs on hormone-resistant tumors. One of the objectives is the evaluation of the in vivo efficacy of the anticancer activity of the compounds using Danio rerio, zebrafish embryo as a model. In this work we describe the general methodology, advantages and disadvantages. Among a large range of advantages in the use of zebrafish in cancer research, the high level of genetic and physiologic homology with humans, including brain, digestive tract, musculature and vasculature can be highlighted. Also, the immature immune system of the embryos favors xenotransplantation of human cancer cells, and makes these animals a promising experimental model to tumorigenesis, angiogenesis, invasion and metastasis. The ionic liquid cetylpyridinium ampicillin [C16Pyr] [Amp] induces cytotoxicity in hormone-resistant breast and prostate cancer cell lines. To study the possible use of [C16Pyr] [Amp] as an anticancer, the toxicity towards zebrafish embryos should be evaluated first by an acute toxic assay that should be carried out following the OECD 236 Guideline.
- Citotoxicity of marine cyanobacteria from the portuguese coast agains breast cancer cellsPublication . Costa, Margarida; Leão, Pedro; Ribeiro, Maria João; Costa-Rodrigues, João; Garcia, Mónica; Barros, Piedade; Fernandes, Helena; Vasconcelos, Vítor; Martins, RosárioThe marine environment possesses an unmeasured chemical and biological diversity, being considered an extraordinary resource for the discovery of new therapeutic drugs. Between others, marine cyanobacteria have emerged as a prominent source of bioactive secondary metabolites with a promising anticancer potential, as the described and intensively studied dolastatins.
- Cytotoxicity of picocyanobacteria strains of the genera Cyanobium on osteosarcoma cellsPublication . Martins, Rosário; Costa, Margarida; Garcia, Mónica; Barros, Piedade; Costa-Rodrigues, João; Vasconcelos, Vítor; Fernandes, MariaMarine cyanobacteria have been recognized as an important source of bioactive compounds. The cytotoxicity on cancer cell lines has been extensively explored and several cyanobacteria metabolites are already described as potential anticancer compounds or are considered useful templates for the design of new anticancer drugs. The majority of compounds have been isolated from filamentous or colonial cyanobacteria that growth in high densities along shores. In contrast, picoplanktonic forms have rarely been explored since, for these strains, there is a need for culture for biomass production. From our LEGE cyanobacteria culture collection we selected a panel of seven strains of the picocyanobacteria genera Cyanobium in order to explore it’s potential as anticancer agents. Strains were cultured under laboratory conditions. Freeze-dried biomass was extracted using methanol and dichloromethane to a crude extract and then fractionated using hexane, ethyl acetate and methanol. The cytotoxicity of crude extracts and fractions was evaluated in the osteosarcoma cell line MG63 by the reduction of the bromide 3-(4,5-dimetil-tiazol-2-il)-2,5-difenil-tetrazolio (MTT) and confirmed by the lactate dehydrogenase (LDH) assay. From the results, four of the seven Cyanobium strains were found to induce a significant decrease in cell viability. The highest percentage of inhibition of tumor cells growth was observed within the ethyl acetate, which is therefore, promising in terms of isolation of bioactive compounds
- Harmful cyanobacterial toxic blooms in waste stabilization pondsPublication . Barros, Piedade; Martins, RosarioA coccoid picocyanobacterium Synechocystis sp. (0.6-2 µm of cell diameter) was found to be dominant during summer period in the experimental wastewater stabilisation pond of Marrakesh. The taxonomy of this isolated strain was confirmed by electron microscope study. The general patterns of ultrastructure and the mode of cell division resemble Chroococcales. The cyanobacterium strain was axenic and cultured on both inorganic Z8 and BG13 media. Mammalian toxicity was confirmed by mice bioassay. The major sympton of poisoning was severe diarrhoea. Histopathological study shows a slight hepatotoxicosis associated with a pronounced change in the intestinal mucosa which shows swelling and destruction of villi epithelium and shedding of enterocytes into the lumen. Although slow, these kinds of poisoning are comparable to those induced by okadiac acid intraperitoneal mice injection (diarrhetic shellfish poisoning “DSP” toxins). By using the enzyme-linked immunosorbent assay (ELISA), the amount of hepatotoxins “microcystins” was determined. The result shows that Synechocystis can produce a small amount of total microcystine [an average of 15 µg-1 dry weight corresponding to 20 ng(109cell)-1]. These findings lead us to consider Synechocystis as both a potent neurotoxin and hepatotoxin producer. Because of the confirmed cyanobacterium toxicity, an eventual ecological implication should be considered. However, a toxic chronic test experiment on Daphnia was simultaneously carried out. Juvenile D. magna (less than 24 hours old), were fed three concentrations (104 , 106 , 108 cells / ml) of Synechocystis. A group of organisms fed with Chlorella vulgaris (3. 105 cells/ml) and another group without food, were studied as control treatments. Only animals cultured with 104 cells/ml of cyanobacterium survived at 80% until the end of the test (21 days). Reproduction and normal growth occurred in control treatments fed with Chlorella vulgaris and the group fed with the lowest concentration of Synechocystis. One-way ANOVA statistical analyses show significant differences in Daphnia survival and growth, between treatments with and without Synechocystis and between treatments with and without food. In terms of this study, there is evidence that toxic picocyanobacteria blooms occurring in wastewater stabilization ponds of Marrakesh, could have harmful repercussions on zooplanktonic, bacteria and other algae communities. Consequently, this will constitute a possible hindrance for sewage self-purification process and system treatment performance. In addition, the reuse of such treated wastewater effluent for irrigation will constitute an additional, potent, health hazard for animals and human’s. Keywords Chlorella; daphnia; microcystins; picocyanobacteria; synechocystis; wastewater.
- Cytotoxic activity of marine cyanobacteria against cancer cell linesPublication . Ribeiro, Maria João; Costa, Margarida; Leão, Pedro; Barros, Piedade; Vasconcelos, Vítor; Martins, RosárioMarine cyanobacteria possess an extensive capacity to produce compounds that were found to induce biological activities against cell lines and organisms. Many of the marine cyanobacteria compounds already described were found to induce toxicity against cancer cells.
- Insights into the potential of picoplanktonic marine cyanobacteria strains for cancer therapies – Cytotoxic mechanisms against the RKO colon cancer cell linePublication . Freitas, Sara; Martins, Rosario; Campos, Alexandre; Azevedo, Joana; Osório, Hugo; Costa, Margarida; Barros, Piedade; Vasconcelos, Vitor; Urbatzka, RalphIn this work, we analysed the potential of picoplanktonic marine cyanobacteria strains as a source of anticancer compounds by elucidating the cytotoxic mechanisms of an ethyl acetate fraction of Cyanobium sp. (LEGE06113) and the Synechocystis salina (LEGE06155) on the RKO colon adenocarcinoma cell line.
- Embryotoxicity assessment of a Microcystis extract with Daphnia in vitro egg testPublication . Barros, Piedade; Martins, RosárioMicrocystis aeruginosa is a colonial unicellular cyanobacteria, usually found in eutrophic and hipereutrophic waters. Microcystins are the main toxins produced. As chronic toxicity test for daphnid is time consuming, the developmental stages of Daphnia eggs in vitro has been used in several works as an alternative to the 21 days reproduction test.
- Marine cyanobacteria compounds with anticancer properties: a review on the implication of apoptosisPublication . Costa, Margarida; Costa-Rodrigues, Joao; Fernandes, Maria Helena; Barros, Piedade; Vasconcelos, Vítor; Martins, RosarioMarine cyanobacteria have been considered a rich source of secondary metabolites with potential biotechnological applications, namely in the pharmacological field. Chemically diverse compounds were found to induce cytoxicity, anti-inflammatory and antibacterial activities. The potential of marine cyanobacteria as anticancer agents has however been the most explored and, besides cytotoxicity in tumor cell lines, several compounds have emerged as templates for the development of new anticancer drugs. The mechanisms implicated in the cytotoxicity of marine cyanobacteria compounds in tumor cell lines are still largely overlooked but several studies point to an implication in apoptosis. This association has been related to several apoptotic indicators such as cell cycle arrest, mitochondrial dysfunctions and oxidative damage, alterations in caspase cascade, alterations in specific proteins levels and alterations in the membrane sodium dynamics. In the present paper a compilation of the described marine cyanobacterial compounds with potential anticancer properties is presented and a review on the implication of apoptosis as the mechanism of cell death is discussed.
- Cytotoxicity of marine cyanobacteria extracts on osteosarcoma cellsPublication . Costa-Rodrigues, João; Costa, M. M.; Costa, S.; Garcia, M.; Fernandes, M. H.; Vasconcelos, V.; Barros, Piedade; Martins, RosárioMarine cyanobacteria have been identified as a rich source of secondary metabolites with potential pharmacological applications. Anti-inflammatory, antibacterial and anticancer activities are some examples of properties described for cyanobacteria compounds, being the cytoxicity against cancer cell lines one of the most documented. The Laboratory of Ecotoxicology Genomics and Evolution (LEGE) — CIIMAR, Porto, Portugal, possesses a large collection of cyanobacteria strains isolated from the Portuguese coast. In order to investigate the interest of genera such as Cyanobium, Synechocystis, Synechococcus, Leptolyngbya and Pseudoanabaena which are genera that have been largely overlooked in terms of bioactivity, we have been screening their ability to induce cytotoxicity on human cancer cell lines. Assays have been conducted with a crude extract obtained by a dichloromethane and methanol extraction of freeze dried biomass and three fractions obtained using Si column chromatography with a gradient from 100% hexane, to 100% ethyl acetate to 100% methanol. The cytotoxicity of cyanobacteria crude extract and fractions has been evaluated by the MTT assay at 24, 48 and 72 h. Here we present the results concerning the cytotoxicity of 24 cyanobacteria strains on the osteosarcoma cell line MG63. The results show both inhibitory and stimulatory effects on cell growth within the same cyanobacteria strain. However, five cyanobacteria strains were found to induce a decrease in cell viability that reached the 80% within the ethyl acetate fraction, which makes this fraction interesting for the isolation and characterization of new bioactive compounds.