Percorrer por autor "Lima, Raquel T."
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- Aminodi(hetero)arylamines in the thieno[3,2-b]pyridine series: synthesis, effects in human tumor cells growth, cell cycle analysis, apoptosis and evaluation of toxicity using non-tumor cellsPublication . Calhelha, Ricardo C.; Ferreira, Isabel C.F.R.; Peixoto, Daniela; Abreu, Rui M.V.; Vale-Silva, Luís A.; Pinto, Eugénia; Lima, Raquel T.; Alvelos, M. Inês; Vasconcelos, M. Helena; Queiroz, Maria João R.P.Three aminodi(hetero)arylamines were prepared via a palladium-catalyzed C-N Buchwald-Hartwig coupling of methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate with different bromonitrobenzenes, followed by reduction of the nitro groups of the coupling products to the corresponding amino compounds. The aminodi(hetero)arylamines thus obtained were evaluated for their growth inhibitory effect on four human tumor cell lines MCF-7 (breast adenocarcinoma), A375-C5 (melanoma), NCI-H460 (non-small cell lung cancer) and HepG2 (hepatocellular carcinoma). The toxicity to non-tumor cells was also evaluated using a porcine liver primary cell culture (PLP1), established by us. The aminodi(hetero)arylamine with the NH2 group in the ortho position and an OMe group in the para position to the NH of the di(hetero)arylamine, is the most promising compound giving the lowest GI50 values (1.30–1.63 μM) in all the tested human tumor cell lines, presenting no toxicity to PLP1 at those concentrations. The effect of this compound on the cell cycle and induction of apoptosis was analyzed in the NCI-H460 cell line. It was observed that it altered the cell cycle profile causing a decrease in the percentage of cells in the G0/G1 phase and an increase of the apoptosis levels.
- An aqueous extract of tuberaria lignosa inhibits cell growth, alters the cell cycle profile, and induces apoptosis of NCI-H460 tumor cellsPublication . Pereira, Joana M.; Lopes-Rodrigues, Vanessa; Xavier, Cristina; Lima, M. João; Lima, Raquel T.; Ferreira, Isabel C.F.R.; Vasconcelos, M. HelenaTuberaria lignosa (Sweet) Samp. is found in European regions, and has antioxidant properties due to its composition in ascorbic acid and phenolic compounds. Given its traditional use and antioxidant properties, the tumor cell growth inhibitory potential of aqueous extracts from T. lignosa (prepared by infusion and decoction) was investigated in three human tumor cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and HCT-15 (human colorectal adenocarcinoma). Both extracts inhibited the growth of these cell lines; the most potent one being the T. lignosa extract obtained by infusion in the NCI-H460 cells (GI50 of approximately 50 μg/mL). Further assays were carried out with this extract in NCI-H460 cells. At 100 μg/mL or 150 μg/mL it caused an increase in the percentage of cells in the G0/G1 phase and a decrease of cells in S phase of the cell cycle. Additionally, these concentrations caused an increase in the percentage of apoptotic cells. In agreement, a decrease in total poly (ADP-ribose) polymerase (PARP) and pro-caspase 3 levels was found. In conclusion, the T. lignosa extract obtained by infusion was more potent in NCI-H460 cells, altering the cell cycle progression and inducing apoptosis. This work highlights the importance of T. lignosa as a source of bioactive compounds with tumor cell growth inhibitory potential.
- Anti-hepatocellular carcinoma activity using human HepG2 cells and hepatotoxicity of 6-substituted methyl 3-aminothieno[3,2-b]pyridine-2- carboxylate derivatives: In vitro evaluation, cell cycle analysis and QSAR studiesPublication . Abreu, Rui M.V.; Ferreira, Isabel C.F.R.; Calhelha, Ricardo C.; Lima, Raquel T.; Vasconcelos, M. Helena; Adega, Filomena; Chaves, Raquel; Queiroz, Maria João R.P.Hepatocellular carcinoma (HCC) is a highly complex cancer, resistant to commonly used treatments and new therapeutic agents are urgently needed. A total of thirty-two thieno[3,2-b]pyridine derivatives of two series: methyl 3-amino- -(hetero)arylthieno[3,2-b]pyridine-2-carboxylates (1ae1t) and methyl 3-amino-6-[(hetero)arylethynyl]thieno[3,2-b]pyridine-2-carboxylates (2ae2n), previously prepared by some of us, were evaluated as new potential anti-HCC agents by studying their in vitro cell growth inhibition on human HepG2 cells and hepatotoxicity using a porcine liver primary cell culture (PLP1). The presence of amino groups linked to a benzene moiety emerges as the key element for the anti-HCC activity. The methyl 3-amino-6-[(3-aminophenyl)ethynyl]thieno[3,2-b]pyridine-2-carboxylate (2f) is the most potent compound presenting GI50 values on HepG2 cells of 1.2 mM compared to 2.9 mM of the positive control ellipticine, with no observed hepatotoxicity (PLP1 GI50 > 125 mM against 3.3 mM of ellipticine). Moreover this compound changes the cell cycle profile of the HepG2 cells, causing a decrease in the % of cells in the S phase and a cell cycle arrest in the G2/M phase. QSAR studies were also performed and the correlations obtained using molecular and 1D descriptors revealed the importance of the presence of amino groups and hydrogen bond donors for anti-HCC activity, and hydrogen bond acceptors for hepatotoxicity. The best correlations were obtained with 3D descriptors belonging to different subcategories for anti-HCC activity and hepatotoxicity, respectively. These results point to different molecular mechanisms of action of the compounds in anti-HCC activity and hepatotoxicity. This work presents some promising thieno[3,2-b]pyridine derivatives for potential use in the therapy of HCC. These compounds can also be used as scaffolds for further synthesis of more potent analogs.
- A cold methanolic extract of Ganoderma lucidum induces autophagy in a gastric cancer cell linePublication . Reis, Filipa S.; Lima, Raquel T.; Morales, Patricia; Ferreira, Isabel C.F.R.; Vasconcelos, M. HelenaBeneficial effects have been attributed to Ganoderma lucidum including antioxidant, antitumor and antibacterial properties [1-3]. Previous work from our group has identified a methanolic extract (prepared at 25 °C) of this mushroom as being a potent modulator of autophagy in a gastric cancer cell line (AGS) [4]. In the present work, the antitumor potential and autophagy modulatory activity of a methanolic extract of G. lucidum, obtained under cold extraction (-20°C), was further investigated. The chemical characterization of the extract was previously published by some of the present authors [1]. Tumour cell growth screening was carried out in four human tumor cell lines: AGS (gastric adenocarcinoma), MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and HCT-15 (colorectal adenocarcinoma). The effect of the extract was further studied in the most sensitive cells (AGS), particularly regarding effect in autophagy. The presence of autophagossomes was observed following transfection of cells with a mCherry-LC3 expression vector and the levels of some autophagic proteins was analysed by Western Blot. To confirm if the increase in LC3-II levels was a result from autophagy induction or from a decrease in autophagic flux, cells were treated with the extract and with lysossomal protease inhibitors (E-64d/pepstatin, to prevent formation of the autophagolysosome), and the expression levels of autophagic proteins was analysed. We verified that this extract provided a Gl50 of 66.59 iJQ/mL in the AGS cell line. In addition, treatment with this extract (G150 concentration) caused an increase in the expression of LC3-II. Additionally, the extract increased the formation of autophagossomes and when cells were treated with the extract together with E-64d/pepstatin a further increase in the LC3-IIIevels was verified, indicating that the extract caused an induction of autophagy.
- Cordyceps militaris (L.) link fruiting body reduces NCI-H460 cellular viability through a mechanism involving p53 and p21Publication . Bizarro, Ana; Ferreira, Isabel C.F.R.; Soković, Marina; Van Griensven, Leo J.L.D.; Sousa, Diana; Vasconcelos, M. Helena; Lima, Raquel T.Mushroom extracts are recognized by their numerous potential medicinal properties. Recently, a methanolic extract from Cordyceps militaris (L.) Link (an edible entomopathogenic fungus widely used in traditional Chinese medicine) has been shown to inhibit cell growth of several human tumour cell lines [1,2]. However, its mechanism of action remained unknown. The aim of the present work was to study the antitumour mechanism of action of the methanolic extract of C. militaris, in the NCI-H460 cell line which is representative of non-small cell lung cancer. Results showed that the extract reduced viable cell number (observed with the trypan blue exclusion assay) by: i) decreasing cellular proliferation (analysed with the BrdU incorporation assay), ii) inducing cell cycle arrest at GO/GI phase (analysed by flow cytometry following propidium iodide-PI labelling) and iii) increasing apoptosis (analysed by flow cytometry following Annexin V-FITC and PI labelling). In addition, results also showed that treatment with the extract increased the cellular levels of p53 and p21. Moreover, this study also showed evidences of DNA damage caused by this extract, since an increase in the levels ofp-H2A.X and 53BP1 were observed, together with an increase in the number of 53BP1 foci/cell. In conclusion, this extract reduced NCI-H460 cellular viability, possibly through a mechanism which involves DNA damage and p53.
- Cordyceps militaris (L.) Link fruiting body reduces the growth of a non-small cell lung cancer cell line by increasing cellular levels of p53 and p21Publication . Bizarro, Ana; Ferreira, Isabel C.F.R.; Soković, Marina; Van Griensven, Leo J.L.D.; Sousa, Diana; Vasconcelos, M. Helena; Lima, Raquel T.Cordyceps militaris (L.) Link, an edible entomopathogenic fungus widely used in traditional Chinese medicine, has numerous potential medicinal properties including antitumor activity. The methanolic extract of C. militaris fruiting body was recently shown to have tumor cell growth inhibitory activity in several human tumor cell lines. Nonetheless, the mechanism of action involved is still not known. This work aimed at further studying the effect of the methanolic extract of C. militaris regarding its antitumor mechanism of action, using the non-small cell lung cancer cell line (NCI-H460) as a model. Results showed that treatment with the extract decreased cellular proliferation, induced cell cycle arrest at G0/G1 and increased apoptosis. In addition, the extract increased the levels of p53 and p21. Moreover, an increase in p-H2A.X and 53BP1 levels, together with an increase in the number of 53BP1 foci/cell (all indicative of DNA damage), were also observed after treatment with the extract. This work suggests that this extract affected NCI-H460 cellular viability through a mechanism involving DNA damage and p53 activation. This further supports the potential of this extract as a source of bioactive compounds, which may be used in anticancer strategies.
- Effect of Cordyceps militaris methanolic extract in NCI-H460 tumor cellsPublication . Bizarro, Ana; Ferreira, Isabel C.F.R.; Soković, Marina; Van Griensven, Leo J.L.D.; Vasconcelos, M. Helena; Lima, Raquel T.Mushroom extracts have been studied extensively for their potential antitumor effect in tumor cell lines and in animal models [1]. Recently, some of our collaborators have described that the methanolic extract from the medicinal mushroom Cordyceps militaris (L.) Link presented tumor cell growth inhibitory activity. In particular, this extract inhibited the growth of the nonsmall cell lung cancer (NSCLC) cell line NCI-H460, presenting a GI50 of approximately 50 μg/ml [2]. The aim of this work was to further study the effect of C. militaris methanolic extract in NCIH460 cells, regarding its mechanism of action. NCI-H460 cells were treated with C. militaris methanolic extract (at 25 and 50 μg/ml) for 48 h. Viable cell number was then assessed with the Trypan blue exclusion assay. Cellular proliferation was analyzed with the BrdU incorporation assay and cell cycle profile with flow cytometry following propidium iodide (PI) labeling. Levels of apoptotic cell death were determined with flow cytometry following Annexin V-FITC /PI labeling. Treatment of cells with the C. militaris extract caused a dose-dependent decrease in viable cell number. Moreover, a clear and strong decrease in cellular proliferation was observed. In addition, alterations in cell cycle profile were found, with a strong decrease in the S and G2/M phases of the cell cycle together with an increase in G0/G1 phase. Furthermore, treatment with the extract also induced apoptosis in this cell line. In conclusion, C. militaris methanolic extract was shown to interfere with cell proliferation, cell cycle and to induce apoptosis of NCI-H460 cells. Further studies will aim at further understanding the mechanism of action of this extract.
- Flower extracts of Filipendula ulmaria (L.) Maxim inhibit cell growth of human tumour cell linesPublication . Lima, M. João; Sousa, Diana; Lima, Raquel T.; Carvalho, Ana Maria; Ferreira, Isabel C.F.R.; Vasconcelos, M. HelenaAccording to the World Health Organization, cancer is the leading cause of death worldwide and its mortality is expected to rise in the next few years. Despite all efforts, the current therapeutic arsenal is not sufficient to reduce these numbers. Therefore, it is imperative to identify new sources of anticancer drugs. Filipendula ulmaria (L.) Maxim is part of the ethnobothanical patrimony of the Iberian Peninsula. For centuries, it has been used as a medicinal species due to its rich antioxidant content, which includes flavonoids and ascorbic acid [l]. Nonetheless, little is known about its antiproliferative activity in cancer cells. Thus, the aims of this project were to: i) investigate if different flower extracts of F. ulmaria have cell growth inhibitory activity in human tumour cell lines and ii) study the mechanism of action of one of the most potent extracts. Four flower extracts obtained by different extraction methods (decoction, infusion, methanol and methanol:water 80:20, v!v) were screened for tumour cell growth inhibitory activity in three human tumour cell lines: NCI-H460 (non-small cell lung cancer), A375-C5 (melanoma) and MCF-7 (breast adenocarcinoma). One of the most potent extracts (obtained by decoction) was further studied in the NCI-H460 cell line (one of the most sensitive), by investigating its effect on viable cell number, programmed cell death, cellular proliferation and cell cycle profile. Results showed that all extracts have growth inhibitory activity in the studied cell lines, in particular the extract obtained by decoction (GI50 of 70.0 ± 8.6, 96.0 ± 12.4 and 63.3 ± 7.6 flg/mL in the NCI-H460, MCF-7 and A373-C5 cells, respectively). Further studies in the NCI-H460 cell line showed that this extract reduced viable cell number. Moreover, treatment with this extract resulted in a strong reduction of cellular proliferation, with a slight increase in the percentage of cells in the G l phase of the cell cycle. No significant alterations in programmed cell death were observed, although results showed a statistically significant increase in the cellular levels of p53 and p2l. Future work will confirm if this extract is non-toxic to human non-tumour cells.
- Flower extracts of Filipendula ulmaria (L.) Maxim inhibit the proliferation of human tumour cell linesPublication . Lima, M. João; Sousa, Diana; Lima, Raquel T.; Carvalho, Ana Maria; Ferreira, Isabel C.F.R.; Vasconcelos, M. HelenaEthnobotanical surveys document wild plants that have been commonly used worldwide to prepare homemade remedies. Filipendula ulmaria (L.) Maxim (meadowsweet) is good example ofa popular medicinal species that can be found throughout most Europe and Asia.
- Flower extracts of Filipendula ulmaria (L.) Maxim inhibit the proliferation of the NCI-H460 tumour cell linePublication . Lima, M. João; Sousa, Diana; Lima, Raquel T.; Carvalho, Ana Maria; Ferreira, Isabel C.F.R.; Vasconcelos, M. HelenaFilipendula ulmaria (L.) Maxim (meadowsweet) is a popular medicinal species that can be found throughout most Europe and Asia. The plant is known for its rich antioxidants content, having compounds such as flavonoids and ascorbic acid. Therefore, the aim of this work was to investigate if the flower extracts of F. ulmaria (L.) Maxim have cell growth inhibitory activity in three human tumour cell lines representative of non-small cell lung cancer (NCI-H460), melanoma (A375-C5) and breast adenocarcinoma (MCF-7). One of the most potent extracts was selected to be further studied in the NCI-H460 cells, by investigating its antiproliferative potential, the effect on cell cycle profile and on programmed cell death. The obtained results showed that all the extracts inhibited the growth of the mentioned cell lines, with the one obtained by decoction being the most potent. Its effect on the NCI-H460 cells was due to a reduction in cellular proliferation, but not to alterations in programmed cell death. Interestingly, cellular treatment with the extract caused a statistically significant increase in the cellular levels of p21. Data obtained highlight the potential interest of F. ulmaria as a source of bioactive compounds.