RESUMO
Trichomonas vaginalis, the causative agent of trichomoniasis is generally known to inhabit the genitourinary tract. However, several case reports with supporting molecular and immunological identifications have documented its occurrence in the respiratory tract of neonates and adults. In addition, the reports have documented that its occurrence is associated with respiratory failures. The medical significance or consequence of this association is unclear. Thus, to establish the possible outcome from the interaction of T. vaginalis with lung cells, the cytopathic effects of the parasites were evaluated using monolayer cultures of the human lung alveolar basal carcinoma epithelial cell line A549. The possible effect of association of T. vaginalis with A549 epithelial cells was analyzed using phase-contrast, scanning electron microscopy and fluorescence microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), crystal-violet and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assays were conducted for cytotoxicity testing. The results demonstrate that T. vaginalis: (1) adheres to A549 epithelial cells, suggesting a density-dependent parasite-cell association; (2) adherence on A549 is through flagella, membrane and axostyle; (3) causes cell detachment and cytotoxicity (50-72.4%) to A549 and this effect is a function of parasite density; and (4) induces apoptosis in A549 about 20% after 6 h of incubation. These observations indicate that T. vaginalis causes cytopathic effects on A549 cell. To date, this is the first report showing a possible interaction of T. vaginalis with the lung cells using A549 monolayer cultures. Further studies are recommended to completely elucidate this association.
Assuntos
Adenocarcinoma Bronquioloalveolar/patologia , Carcinoma Basocelular/patologia , Neoplasias Pulmonares/patologia , Trichomonas vaginalis/patogenicidade , Adenocarcinoma Bronquioloalveolar/parasitologia , Apoptose , Carcinoma Basocelular/parasitologia , Adesão Celular , Linhagem Celular Tumoral , Colorimetria , Violeta Genciana , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Pulmonares/parasitologia , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Trichomonas vaginalis/ultraestruturaRESUMO
BACKGROUND: The genus Aglaia (Meliaceae) is an established source of many anticancer compounds. The study evaluated the leaf extracts of Aglaia loheri, a tree native to the Philippines, as potential source of anticancer compounds. METHODS: Using bioassay-guided fractionation, A. loheri leaf extract was subjected to various chromatographic techniques and step-wise application of MTT assay on human colorectal carcinoma cells, HCT116, to determine the cytotoxic fractions. The most cytotoxic HPLC isolate was structurally identified using 1D and 2D NMR and its apoptotic effect was assessed by JC-1 staining, caspase 3/7 assay and TUNEL assay. RESULTS: After stepwise chromatography fractionation, an HPLC isolate, structurally identified as aglaforbesin derivative (AFD), demonstrated potent cytotoxicity against HCT116. AFD exhibited strong toxicity (IC50 = 1.13 ±0.07 µg/mL) and high selectivity on HCT116 than normal human kidney cells (HK-2). AFD-induced toxicity to HCT116 is possibly through the stimulation of the apoptotic signaling pathway via caspase 3/7 activation and DNA fragmentation independent of mitochondrial membrane depolarization. CONCLUSION: AFD exhibited selective cytotoxicity and apoptotic activity to HCT116 and could be further developed as anticancer drug lead.
Assuntos
Aglaia/química , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Neoplasias Colorretais/patologia , Mitocôndrias/patologia , Extratos Vegetais/farmacologia , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Humanos , Mitocôndrias/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The bioactive chemicals in L. cuneata were investigated by repeated column chromatography and their effect on aldose reductase (AR), obtained from rat lenses, was examined. Results showed that the ethyl acetate and n-butanol fractions of L. cuneata exhibited potential inhibitory effect against AR with IC50 values of 0.57 and 0.49 µg/mL, respectively. Phytochemical analysis of these two fractions resulted in the isolation of five flavonoids namely, acacetin (1), afzelin (2), astragalin (3), kaempferol (4) and scutellarein 7-O-glucoside (5). The AR inhibitory effect of compounds 1-5 was explored; compounds 2, 3 and 5 showed potential AR-inhibitory effects with IC50 values of 2.20, 1.91 and 12.87 µM, respectively. Quantitative analysis of afzelin (2) and astragalin (3) in L. cuneata by high performance liquid chromatography with ultraviolet detection revealed its content to be 0.722-11.828 and 2.054-7.006 mg/g, respectively. Overall, this study showed that L. cuneata is rich in flavonoids with promising AR-inhibitory activities, which can be utilized for the development of natural therapies for treating and managing diabetic complications.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Flavonoides , Quempferóis , Lespedeza/química , Manosídeos , Proantocianidinas , Aldeído Redutase/metabolismo , Animais , Flavonoides/análise , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Quempferóis/análise , Quempferóis/isolamento & purificação , Quempferóis/farmacologia , Cristalino/enzimologia , Manosídeos/análise , Manosídeos/isolamento & purificação , Manosídeos/farmacologia , Extratos Vegetais/química , Proantocianidinas/análise , Proantocianidinas/isolamento & purificação , Proantocianidinas/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Chemically defined stem cell culture media are often costly, and the use of mitotically arrested mouse embryonic fibroblasts (MEFs) as feeder cells is a popular and cost-efficient way to maintain induced pluripotent stem cells (iPSCs). However, the commonly used mitotic inhibitor mitomycin-C (MMC) is known to cause cellular metabolic stress. Therefore, our aim was to determine whether such stress in feeder cells indirectly affects iPSC growth during coculture. We report that prolonged exposure to MMC causes metabolic stress in MEFs in the form of oxidative dysregulation. Through optimization of MMC exposure time, we show how to effectively arrest MEFs without inducing oxidative stress, thus promoting significantly better colony growth rates (p < 0.05), improved viability and longer periods between passages of iPSCs in coculture.
Assuntos
Técnicas de Cocultura/métodos , Células Alimentadoras , Células-Tronco Pluripotentes Induzidas , Mitomicina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacosRESUMO
The chemotherapy drug doxorubicin (DOX) is effective in treating many types of cancers. However, due to its pro-inflammatory and cardiotoxic side effects, other remedies have also been explored as alternative treatments. The plant Alangium longiflorum was reported to contain cytotoxic activity against cancer cells, but it is unclear whether this plant would also yield side effects similar to doxorubicin. Hence, this study investigated cytotoxic activity of A. longiflorum leaf extract against lung cancer cells and compared its pro-inflammatory and cardiotoxic side effects with those of DOX. METHODS: Cytotoxic activity of A. longiflorum in human lung (A549) and breast (MCF-7) cancer cells was initially assessed by MTT assay and then was compared with doxorubicin. Presence of secondary metabolites in the leaf extract was examined by phytochemical screening. The ability of the plant extract to induce apoptosis was determined by measuring caspase-3/7 activity and apoptosis-related gene expression. Pro-inflammatory response was assessed by quantifying NFκB transcriptional activity and nuclear translocation with dual luciferase reporter and immunofluorescence assays, respectively. Cardiotoxicity was measured using zebrafish as a model organism. RESULTS: A. longiflorum leaf extract displayed high cytotoxic activity against A549 versus MCF-7, which led this study to focus further on A549. Phytochemical screening showed that the extract contained terpenoids, alkaloids, phenols, cardiac glycosides, and tannins. The extract induced apoptosis through activation of caspase-3/7 and upregulation of pro-apoptotic genes without causing NFκB transcriptional activation and nuclear localization. The extract also did not significantly reduce heart function in zebrafish. CONCLUSION: Overall, our data suggested that extract from leaves of A. longiflorum can have the potential to serve as apoptotic agent towards lung cancer without inducing significant cardiotoxicity.
Assuntos
Alangiaceae/química , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Cardiotoxicidade/tratamento farmacológico , Neoplasias Pulmonares/patologia , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Animais , Antibióticos Antineoplásicos/farmacologia , Cardiotoxicidade/etiologia , Cardiotoxicidade/patologia , Proliferação de Células , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , NF-kappa B/genética , Folhas de Planta/química , Ativação Transcricional , Células Tumorais Cultivadas , Peixe-ZebraRESUMO
Aralia continentalis (AC) is a perennial herb that has long been used as a traditional medicine for many diseases. Continentalic acid (CA) and kaurenoic acid (KA) are major diterpenoids in AC, which are known to exert various pharmacological activities. This study focuses on the optimization of the extraction of CA and KA from dried AC roots by evaluating the influence of different extraction conditions on their yield. Five extraction variables were examined: sample weight, solvent concentration, extraction time, solid matrix and the number of repeated extractions. The analytical method used in this study was also validated in terms of linearity, limit of detection, limit of quantification, precision and accuracy. The CA and KA yields were measured by high-performance liquid chromatography analysis. The results show that CA and KA were the highest when unpulverized samples (3.75 g) were subjected to a single extraction for 5 h using 50% ethanol (300 mL) as the solvent. These conditions are proposed for the optimization of the extraction of CA and KA from AC.
Assuntos
Aralia/química , Cromatografia Líquida de Alta Pressão/métodos , Diterpenos/análise , Diterpenos/química , Diterpenos/isolamento & purificação , Limite de Detecção , Modelos Lineares , Extratos Vegetais/química , Raízes de Plantas/química , Reprodutibilidade dos Testes , Espectrofotometria UltravioletaRESUMO
BACKGROUND: Cancer is one of the leading causes of illness and death worldwide. Only palliative therapeutic options are available for many types of cancers, and most anticancer drugs in clinical use exhibit significant side effects. It is therefore important to develop new anticancer drugs that are more effective and less toxic. In this study, we evaluate the bioactivity of a Philippine endemic plant, "katmon" or Dillenia philippinensis, and its potential use in cancer therapy. METHODS: The cytotoxicity of the crude leaf extract, partitions, and isocratic column chromatography fractions of Dillenia philippinensis was determined in vitro by MTT assay against drug-sensitive cancer cell lines MCF7 (human breast adenocarcinoma) and HCT 116 (human colorectal carcinoma), as well as against moderately multidrug resistant (MDR) cancer cell line HCT-15 (human colorectal carcinoma) and its highly MDR subline HCT-15/Dox. The selectivity of the extract to cancer cells was determined by computing for the selectivity index (SI) with respect to normal mouse embryonic fibroblasts (NIH/3T3) cell line. To check for a possible mechanism for overcoming cancer multiple drug resistance, Calcein-AM assay was performed to assess the activity of the extract against P-glycoprotein-activated efflux pump. RESULTS: Dillenia philippinensis (DP1) fraction from the hexane partition exhibited cytotoxicity (IC50< 30 µg/ml) against MCF7, HCT 116, HCT-15, and HCT-15/Dox cells. DP1 also exhibited a moderate level of selectivity against cancer cells over normal cells as supported by the SI computed from the IC50 value obtained for the normal cell line. DP1 was able to inhibit P-glycoprotein (P-gp) activity in a dose-dependent manner, suggesting its possible role in targeting cancer cells with overexpressed P-gp. CONCLUSION: The present findings thus demonstrate the potential chemotherapeutic properties of D. philippinensis which can be promising for future drug development against cancer.
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Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Neoplasias do Colo/patologia , Dilleniaceae/química , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Células Tumorais CultivadasRESUMO
The dichloromethane extract from the leaves of Calotropis gigantea Linn. was strongly cytotoxic against non-small cell lung carcinoma (A549), colon carcinoma (HCT 116) and hepatocellular carcinoma (Hep G2), and non toxic to Chinese hamster ovary (AA8). The extract afforded uscharin (1), 3,5,8-trihydroxy-24-methylcholest-6,22-diene (2), a mixture of (24R)-3beta-hydroxy-24-ethylcholest-5-en-7-one (3a) and 6beta-hydroxy-24-ethylcholest-4,22-dien-3-one (3b), and another mixture of (24R)-24-ethylcholest-4-en-3-one (4a) and (24S)-24-ethylcholest-4,22-dien-3-one (4b). Cardenolide 1 exhibited extreme toxicity to A549, HCT 116 and Hep G2 with IC50 values of 0.003 microg/mL, 0.013 microg/mL, and 0.018 microg/mL, respectively, while sample 3 exhibited an IC50 of 1.35 microg/mL, 4.46 microg/mL, and 3.83 microg/mL, respectively.