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1.
Braz. J. Pharm. Sci. (Online) ; 59: e21025, 2023. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1439501

RESUMO

Abstract The present study investigated the effects of valerian methanolic extract and valerenic acid on the expression of LL-37 gene and protein in A549 and MRC5 line cells. After preparing Valerian seeds, sowing them in March 2020, and harvesting the rhizome in October 2020, the extract was prepared from the valerian rhizome by maceration method. Valerian acid content was determined using high performance liquid chromatography (HPLC). Two cell lines (A549 and MRC-5) were used to study the effects of valerian extract, and the MTT test was used to evaluate cell viability. The expression of LL-37 mRNA and protein was assessed by Real-Time PCR and western blot, respectively. In vivo safety assessments and histopathological analysis were also conducted. Data was analyzed by Graphpad Prism 8 software. Valerian methanolic extract and valerenic acid upregulated the LL-37 mRNA and protein expression in both treated cell lines. Valerenic acid showed a greater effect on upregulating LL-37 expression than valerian methanolic extract. A549 cells were more sensitive to valerian methanolic extract compared to MRC5 cells, and its cell viability was reduced. Furthermore, liver and kidney-related safety assessments showed that valerian methanolic extract had no toxic effects. In general, it was concluded that the methanolic extract of valerian as well as the resulting valerenic acid as the most important component of the extract has the ability to upregulate LL-37expression. Therefore, methanolic extract of valerian and valerenic acid can be considered for improving the immune system.


Assuntos
Valeriana/efeitos adversos , Extratos Vegetais/efeitos adversos , Catelicidinas/efeitos adversos , Western Blotting/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Peptídeos Catiônicos Antimicrobianos/agonistas , Células A549/classificação , Genes/genética , Fígado/anormalidades
2.
Braz. J. Pharm. Sci. (Online) ; 58: e19801, 2022. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1394060

RESUMO

Abstract In the recent past, drug delivery through nanoparticles is considered an effective tool to treat various diseases. Biopolymeric nanoparticles such as protein based nanoparticles have vital role as drug carrier as it is non-antigenic, and easily biodegradable. Curcumin, plant polyphenolic anticancerous compound was loaded into the casein nanoparticles by coacervation method. Particle size and surface charge of spherical casein nanoparticles as observed to be 201.4 nm and -86.9 mV. The loading efficiency of curcumin loaded casein nanoparticles was found to 85.05 %. In vitro drug release was performed at different pH (7.4 and 3.0), and the cumulative release was observed to be 24.8 and 20.13% and at different temperatures (25°C and 37°C), the cumulative release was observed to be 24.8 and 28.60 % respectively in 48 h. Curcumin release from casein nanoparticles was shown to be in a steady, and prolonged rate. The nanoparticles were observed to have an effective antimocrobial activity than curcumin in free form. The drug loaded casein nanoparticles were found to be potent particles to protect cells from hydrogen peroxide and UV light damage. The cytotoxic activity of nanoparticles on MCF7 and A549 cells were assayed and was observed to have an IC50 value of 609 and 825.2µg/ml. Cell death was observed to be through apoptosis, accompanied by DNA fragmentation.


Assuntos
Humanos , Caseínas , Curcumina , Nanopartículas , Antineoplásicos/farmacologia , Técnicas In Vitro , Apoptose , Concentração Inibidora 50 , Curcumina/farmacocinética , Liberação Controlada de Fármacos , Células A549 , Antineoplásicos/farmacocinética
3.
Braz. J. Pharm. Sci. (Online) ; 58: e19194, 2022. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1383971

RESUMO

Abstract Increasing biological activity and phytochemical investigations on Eryngium species showed its potential as pharmaceutical approach. Eryngium kotschyi Boiss. is one of the species of Eryngium genus and is endemic to Turkey. It is known that this plant is traditionally used in the South-western part of Turkey for the treatment of various diseases. This study focuses on cytotoxic activities of methanol extract and ethyl acetate, n-butanol and water sub-extracts from E. kotschyi in A549, COLO 205 and MDA-MB-231 cell lines by Sulforhodamin B assay and qualitative and quantitative determination of phytochemical constituents in active extract by LC-MS/MS. From the result of the study, it was seen that E. kotschyi ethyl acetate (EKE) sub-extract showed the strongest cytotoxic effect with the low IC50 values (50.00; 31.96 and 22.26 µg/mL in A549; COLO 205 and MDA-MB-231 cells at 48 h, respectively). Preliminary examination of the mass spectrums revealed the presence of 15 phytochemical compounds in active sub-extract and 7 of them was quantified. According to quantitative analyses the main compounds of EKE sub-extract were rosmarinic acid (485.603 µg/mgextract), chlorogenic acid (62.355 µg/mgextract) and caffeic acid (59.266 µg/mgextract). Moreover, this preliminary study on inhibitory activity of EKE sub-extract suggests further toxicologic investigations and detailed investigation on cytotoxic effect of various combinations of determined compounds


Assuntos
Turquia/etnologia , Células/metabolismo , Eryngium/anatomia & histologia , Compostos Fitoquímicos/efeitos adversos , Preparações Farmacêuticas/administração & dosagem , Linhagem Celular/classificação , Células A549/metabolismo , Acetatos/administração & dosagem
4.
Braz. J. Pharm. Sci. (Online) ; 58: e19542, 2022. graf
Artigo em Inglês | LILACS | ID: biblio-1384004

RESUMO

Abstract The main aim of the study is to quantify the cytotoxic property of the Fucoidan extracted from the Turbinaria conoides using the MTT assay with the standard fucose. Fucoidan was extracted using the soaked water method and it was determined using the HPLC procedure the obtained Test sample Fucoidan extracted from the Turbinaria conoides and standard fucose was subjected to the cytotoxicity assay against the MCF7 Human breast cancer cell line, A549 lung cancer cell line, and L929 normal mouse fibroblast cell line. From the results it was found that the Test sample showed good IC50 value for MCF7 cell line then A549 with an increasing concentration 24 hours incubation at 37°C The IC50 for MCF7 was 115.21 µg/ml and A549 396.46µg/ml and the Fucoidan extract was checked for its cytotoxicity against the normal mouse fibroblast cell line L929, Fucoidan was found non-lethal to the L929 mouse fibroblast normal cell line. Standard fucose also gave a significant result towards MCF7 and against the L929. This indicates that the Fucoidan extracted from Tubinaria conoides shows better anticancer potential in it. Hence its application can be further extended in the pharmacological fields.


Assuntos
Técnicas In Vitro/instrumentação , Citotoxinas/efeitos adversos , Células MCF-7 , Células A549 , Neoplasias da Mama/patologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Concentração Inibidora 50 , Fibroblastos/classificação , Fucose/análogos & derivados , Neoplasias Pulmonares/patologia
5.
Biol. Res ; 54: 7-7, 2021. tab, graf, ilus
Artigo em Inglês | LILACS | ID: biblio-1505800

RESUMO

BACKGROUND: Leaves of the natural plant lotus are used in traditional Chinese medicine and tea production. They are rich in flavonoids. METHODS: In this study, lotus leaf flavonoids (LLF) were applied to human lung cancer A549 cells and human small cell lung cancer cells H446 in vitro to verify the effect of LLF on apoptosis in these cells through the ROS/p38 MAPK pathway. RESULTS: LLF had no toxic effect on normal cells at concentrations up to 500 µg/mL, but could significantly inhibit the proliferation of A549 cells and H446 cells. Flow cytometry showed that LLF could induce growth in A549 cells. We also found that LLF could increase ROS and MDA levels, and decrease SOD activity in A549 cells. Furthermore, qRT-PCR and western blot analyses showed that LLF could upregulate the expression of p38 MAPK (p-p38 MAPK), caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9 and Bax and downregulate the expression of Cu/Zn SOD, CAT, Nrf2, NQO1, HO-1, and Bcl-2 in A549 cells. Results of HPLC showed that LLF mainly contain five active substances: kaemp-feritrin, hyperoside, astragalin, phloridzin, and quercetin. The apoptosis-inducing effect of LLF on A549 cells came from these naturally active compounds. CONCLUSIONS: We have shown in this study that LLF is a bioactive substance that can induce apoptosis in A549 cells in vitro, and merits further research and development.


Assuntos
Humanos , Flavonoides/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Lotus/química , Neoplasias Pulmonares/patologia , Transdução de Sinais/efeitos dos fármacos , Folhas de Planta/química , Proliferação de Células , Compostos Fitoquímicos/farmacologia , Células A549 , Neoplasias Pulmonares/tratamento farmacológico
6.
Braz. arch. biol. technol ; 63: e20190364, 2020. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1132221

RESUMO

Abstract Chronic inflammation is a common indication of several diseases, e.g. asthma, chronic obstructive pulmonary disease (COPD), atherosclerosis, etc. Benzimidazole derivatives are preferable compounds to design new analgesic and anti-inflammatory substances due to their unique biological features. We aimed to investigate the effect of a newly synthesized benzimidazole derivative, ORT-83, on A549 human lung adenocarcinoma cell line. ORT-83 was synthesized, and a non-cytotoxic concentration of ORT-83 on A549 cells was detected with MTT assay. To analyze the anti-inflammatory effect of ORT-83, an inflammatory cell culture model was established by stimulating A549 cell line with IL1-β (10 ng/ml). After 2 hours of treatment with IL1-β to induce inflammation, A549 cells were exposed to ORT-83 (0.78 µg/ml) for 24 hours. Thereafter gene expression analyses were performed with qRT-PCR. We found that ORT-83 significantly suppressed the gene expression levels of the proinflammatory cytokines; IL-6, NFkB, and TNF-α. However, the increased levels of IL-10 (2.8 folds) by IL-1β induction did not change after ORT-83 and/or dexamethasone (Dex: positive control) treatments. While Dex; a COX-2 inhibitor, reduced the COX-2 expression level in inflammatory cells from 10.03 folds to 0.71 folds, ORT-83 reduced its level to 4.37 folds. iNOS expression levels did not change in any experimental groups. In conclusion, we showed that ORT-83 exerted its anti-inflammatory effects by repressing the gene expression of proinflammatory cytokines in the inflammation-induced A549 cell line. Although ORT-83 had a weaker COX-2 inhibitory effect compared to Dex, it was shown to be still a strong anti-inflammatory compound.


Assuntos
Humanos , Benzimidazóis/farmacologia , Drogas em Investigação , Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Testes de Toxicidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células A549
7.
J. venom. anim. toxins incl. trop. dis ; 25: e147418, 2019. graf, ilus
Artigo em Inglês | LILACS | ID: biblio-984697

RESUMO

Background: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. Methods: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. Results: The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 µg), goat (HU50 = 13.2 ± 0.3 µg), rabbit (HU50 = 34.7 ± 0.5 µg), and human (HU50 = 25.6 ± 0.6 µg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 µg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.(AU)


Assuntos
Humanos , Animais , Anêmonas-do-Mar , Venenos de Cnidários/isolamento & purificação , Neoplasias Pulmonares/terapia , Venenos/toxicidade , Espectrometria de Massas/métodos , Células A549
8.
Braz. J. Pharm. Sci. (Online) ; 55: e18172, 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1039039

RESUMO

Hesperidin, a natural compound, suppresses the epithelial-to-mesenchymal transition through the TGF-ß1/Smad signaling pathway. However, studies on the detailed effects and mechanisms of hesperidin are rare. The present study showed that, for A549 alveolar epithelial cells, the anti-proliferative effects of hesperidin occurred in a dose-dependent manner, with an IC50= 216.8 µM at 48 h. TGF-ß1 was used to activate the Smad signaling pathway and induce the epithelial to mesenchymal transition in cells. Treatment with hesperidin or SB431542 was used for antagonism of Smad pathway activation. Hesperidin inhibited the increase in ɑ-SMA and Col1ɑ-1 and the decrease in E-cadherin in a dose-dependent manner from concentration of 20 µM to 60 µM, as assessed by both ELISA and Western blotting assays; however, there was no significant effect on cellular morphological alterations. Moreover, the Western blotting assay showed that, in the cytoplasm, hesperidin and SB431542 had no significant effect on the protein expression of Smad 2, 3, 4, or 7 as well as 2/3. However, 60 µM hesperidin and SB431542 significantly decreased p-Smad2/3 protein expression. From the above results, it is concluded that hesperidin can partly inhibit the epithelial to mesenchymal transition in human alveolar epithelial cells; the effect accounts for the blockage of the phosphorylation of Smad2/3 in the cytoplasm rather than a change in Smad protein production in the cytoplasm


Assuntos
Transição Epitelial-Mesenquimal/genética , Hesperidina/análise , Hesperidina/efeitos adversos , Ensaio de Imunoadsorção Enzimática/instrumentação , Western Blotting/instrumentação , Fibrose Pulmonar Idiopática/fisiopatologia , Células A549
9.
Biol. Res ; 52: 7, 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1011410

RESUMO

BACKGROUND: Currently, the prognosis of patients with non-small cell lung cancer (NSCLC) remains dismal; hence, it is critical to identify effective anti-NSCLC agents with limited side effects. This study aimed to evaluate the therapeutic potential of flavonoid compound vitexin in human NSCLC cells and the underlying mechanisms. RESULTS: The experimental results indicated that vitexin reduced the viability of A549 cells in a dose-dependent manner with nearly no toxicity against normal human bronchial epithelial 16HBE cells. Vitexin also dose-dependently increased A549 cell apoptosis, accompanied by the decreased Bcl-2/Bax ratio and the increased expression of cleaved caspase-3. Moreover, the in vivo anticancer activity of vitexin was further determined in nude mice bearing A549 cells. In addition, vitexin induced the release of cytochrome c from the mitochondria to the cytosol and the loss of mitochondrial membrane potential. Vitexin also significantly reduced the levels of p-PI3K, p-Akt and p-mTOR, and the pro-apoptotic effect of vitexin on A549 cells was partly blocked by SC79, an Akt activator. CONCLUSIONS: Accordingly, we believed that vitexin could be used as a potential therapeutic agent for the treatment of NSCLC in the future.


Assuntos
Humanos , Animais , Camundongos , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Apigenina/farmacologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Serina-Treonina Quinases TOR/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Antineoplásicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células A549 , Neoplasias Pulmonares/metabolismo , Camundongos Nus , Mitocôndrias/efeitos dos fármacos
10.
Braz. j. med. biol. res ; 51(2): e6950, 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-889028

RESUMO

Alveolar epithelia play an essential role in maintaining the integrity and homeostasis of lungs, in which alveolar epithelial type II cells (AECII) are a cell type with stem cell potential for epithelial injury repair and regeneration. However, mechanisms behind the physiological and pathological roles of alveolar epithelia in human lungs remain largely unknown, partially owing to the difficulty of isolation and culture of primary human AECII cells. In the present study, we aimed to characterize alveolar epithelia generated from A549 lung adenocarcinoma cells that were cultured in an air-liquid interface (ALI) state. Morphological analysis demonstrated that A549 cells could reconstitute epithelial layers in ALI cultures as evaluated by histochemistry staining and electronic microscopy. Immunofluorescent staining further revealed an expression of alveolar epithelial type I cell (AECI) markers aquaporin-5 protein (AQP-5), and AECII cell marker surfactant protein C (SPC) in subpopulations of ALI cultured cells. Importantly, molecular analysis further revealed the expression of AQP-5, SPC, thyroid transcription factor-1, zonula occludens-1 and Mucin 5B in A549 ALI cultures as determined by both immunoblotting and quantitative RT-PCR assay. These results suggest that the ALI culture of A549 cells can partially mimic the property of alveolar epithelia, which may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.


Assuntos
Humanos , Meios de Cultivo Condicionados , Técnicas de Cultura de Células/métodos , Células Epiteliais Alveolares/fisiologia , Células A549/fisiologia , Valores de Referência , Fatores de Tempo , Microscopia Eletrônica de Varredura , Immunoblotting , Contagem de Células , Reprodutibilidade dos Testes , Análise de Variância , Proteína C Associada a Surfactante Pulmonar/análise , Aquaporina 5/análise , Mucina-5B/análise , Reação em Cadeia da Polimerase em Tempo Real , Proteína da Zônula de Oclusão-1/análise , Fator Nuclear 1 de Tireoide/análise
11.
São Paulo; s.n; s.n; 2017. 179 p. tab, graf, ilus.
Tese em Português | LILACS | ID: biblio-847737

RESUMO

O câncer de pulmão é a principal causa de morte relacionada ao câncer no mundo. Mutações em KRAS são altamente prevalentes no câncer e têm sido diretamente associadas ao processo tumorigênico. Apesar disso, até hoje todas as terapias visando inibir KRAS diretamente falharam e a caracterização de alvos indiretos, importantes para a oncogênese mediada por KRAS, é fundamental para o desenvolvimento de novas terapias contra o câncer de pulmão. Nós mostramos previamente que as quinases Aurora A (AURKA) e B (AURKB) são alvos a jusante de KRAS, importantes para o crescimento, viabilidade e oncogenicidade de linhagens celulares derivadas de tumores pulmonares mediados por KRAS. Aqui, nós aprofundamos os nossos estudos para melhor caracterizar AURKA e AURKB como potenciais alvos terapêuticos no câncer de pulmão. Os objetivos deste trabalho foram (1) investigar o mecanismo de perda de viabilidade induzido pela inibição de AURKA e/ou AURKB; (2) avaliar como a inibição de AURKA e/ou AURKB afeta propriedades oncogênicas relacionadas à agressividade tumoral; e (3) como a inibição destas quinases afeta o crescimento tumoral in vivo. Para tanto, nós utilizamos dois modelos celulares: (1) células A549 e H358, que apresentam mutações em KRAS, geneticamente modificadas para a expressão estável e induzível de shRNAs contra AURKA ou AURKB, e (2) células tumorais H1703, que não apresentam mutações em KRAS, geneticamente modificadas para a expressão induzível de KRASG12V, tratadas ou não com inibidores farmacológicos das quinases Aurora. A inibição farmacológica ou por interferência de RNA de AURKA e/ou AURKB em células H358 e A549 reduziu a proliferação celular, sendo esta inibição acompanhada de anomalias mitóticas, além de aneuploidia e poliploidia. A inibição destas quinases também induziu morte celular in vitro, tanto em mitose, quanto em interfase. Mais interessantemente, a inibição farmacológica dual de AURKA e AURKB induziu morte celular in vitro em células H1703, somente na presença de KRASG12V, indicando que a inibição das quinases Aurora afeta preferencialmente células portadoras de mutações em KRAS. Além disso, a inibição de AURKA e/ou AURKB reduziu propriedades malignas celulares relacionadas à agressividade tumoral, como migração, invasão e adesão. Finalmente, a inibição de AURKA por RNA de interferência em células A549 também reduziu a formação de tumores in vivo. Entretanto, como a inibição destas quinases levou a anomalias mitóticas e à instabilidade genética, nós resolvemos investigar se a inibição de TPX2, um substrato e ativador de AURKA, poderia ser uma abordagem alternativa para inibir esta via em câncer de pulmão induzido por KRAS. Primeiramente, nós observamos nos nossos modelos celulares que KRAS regula positivamente a expressão de TPX2. Além disso, a inibição de TPX2 em células pulmonares portadoras de KRAS oncogênica reduziu a viabilidade e proliferação celulares e induziu morte celular. Mais interessantemente, esses efeitos ocorreram preferencialmente em células que expressam KRAS oncogênica. Em conclusão, nossos resultados apoiam a hipótese de que a ativação de AURKA/TPX2 e AURKB por KRAS são eventos importantes no câncer de pulmão e sugerem a inibição destas vias, possivelmente em combinação com outras terapias citotóxicas, como uma nova abordagem terapêutica para o câncer de pulmão induzido por KRAS


Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS mutations are widespread in lung cancer and have been causally linked to tumorigenesis. Nonetheless, therapies targeting KRAS directly have so far failed and characterization of indirect KRAS targets, which play important roles in KRAS-mediated oncogenesis, is crucial for the development of new therapies for lung cancer. We have previously shown that mitotic kinases Aurora A (AURKA) and B (AURKB) are downstream targets of oncogenic KRAS, important for the growth, viability, and oncogenicity of KRAS-transformed lung cancer cell lines. Here, we studied these kinases more in depth in order to better characterize them as potential therapeutical targets for KRAS-induced lung cancer. The aims of this study were (1) to investigate the mechanism leading to loss of viability upon AURKA and/or AURKB targeting; (2) to evaluate how AURKA and/or AURKB inhibition affects malignant properties associated with tumor aggressiveness; and (3) to determine whether AURKA and/or AURKB inhibition reduces KRAS-induced tumor growth in vivo. For that purpose, we used two cell-based models: (1) KRAS mutant A549 and H358 cells with stable and inducible shRNA-mediated knockdown of AURKA or AURKB, and (2) KRAS wildtype H1703 tumor cell lines, genetically engineered to inducibly express oncogenic KRASG12V treated or not with Aurora kinase pharmacological inhibitors. Targeting AURKA and/or AURKB pharmacologically or by RNA interference in H358 and A549 cells led to decreased cell proliferation, which was accompanied by mitotic abnormalities, leading to aneuploidy and hyperploidy. Aurora kinase targeting also induced cell death in vitro, both during mitosis and interphase. More importantly, AURKA and AURKB inhibition with a dual pharmacological inhibitor in H1703 cells induced cell death in vitro, but only in the presence of KRASG12V, indicating that Aurora kinase targeting affects preferentially lung cells harboring oncogenic KRAS. Furthermore, AURKA and/or AURKB targeting reduced malignant properties associated with tumor aggressiveness, such as cell migration, invasion and adhesion. Finally, AURKA targeting by RNA interference in A549 cells also reduced growth of xenograft tumors in vivo. Nonetheless, since Aurora targeting was associated with mitotic abnormalities and genetic instability, we decided to investigate if targeting TPX2, a substrate and an activator of AURKA, could constitute an alternative approach to targeting this pathway in KRAS-induced lung cancer. First, using our cell-based models, we determined that KRAS positively regulates TPX2 expression. In addition, TPX2 inhibition by RNA interference in KRAS-positive lung cells reduced cell viability and proliferation and induced cell death. Finally, these effects occurred preferentially in cells harboring oncogenic KRAS. In conclusion, our results support the hypothesis that activation of AURKA/TPX2 and AURKB by KRAS are important events in lung cancer and suggest inhibition of these pathways, possibly in combination with other cytotoxic therapies, as a new approach for KRAS-induced lung cancer therapy


Assuntos
Oncogenes/genética , Aurora Quinase A/análise , Aurora Quinase B/análise , Testes de Carcinogenicidade , Sobrevivência Celular , /métodos , Células A549 , Neoplasias Pulmonares/complicações
12.
Braz. j. med. biol. res ; 49(7): e5263, 2016. graf
Artigo em Inglês | LILACS | ID: lil-785060

RESUMO

B7 homolog 1 (B7-H1) is the most potent immunoinhibitory molecule in the B7 family. In this study, we examined the effects of tumor-associated B7-H1 on T-cell proliferation in lung cancer. The expression of B7-H1 in human adenocarcinoma A549 and mouse Lewis lung carcinoma (LLC) cells were examined by flow cytometry. To assess the in vitro effect of tumor-associated B7-H1 on T-cell proliferation, we isolated T cells from peripheral blood mononuclear cells (PBMCs) of healthy individuals, labeled them with carboxyfluorescein succinimidyl ester, and co-cultured them with A549 cells in the absence or presence of anti-B7-H1 antibody. For in vivo analysis, LLC cells were subcutaneously injected into mice treated or not with anti-B7-H1 antibody. T-cell proliferation in both in vitro and in vivo assays was analyzed by flow cytometry. In vitro, co-culturing T cells with A549 cells significantly inhibited the proliferation of the former compared with the proliferation of T cells alone (P<0.01), and the addition of B7-H1 blocking antibody dramatically reversed the inhibition of T-cell proliferation by A549 cells. Similarly, in mice bearing LLC-derived xenograft tumors, in vivo administration of anti-B7-H1 antibody significantly increased the total number of spleen and tumor T cells compared to levels in control mice that did not receive anti-B7-H1 antibody. Functionally, in vivo administration of anti-B7-H1 antibody markedly reduced tumor growth. Tumor-associated B7-H1 may facilitate immune evasion by inhibiting T-cell proliferation. Targeting of this mechanism offers a promising therapy for cancer immunotherapy.


Assuntos
Humanos , Animais , Camundongos , Adenocarcinoma/patologia , Antígeno B7-H1/análise , Proliferação de Células , Neoplasias Pulmonares/patologia , Linfócitos T/patologia , Células A549 , Anticorpos Antineoplásicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Células Cultivadas , Citometria de Fluxo , Imunoterapia/métodos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Neoplasias Esplênicas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Biol. Res ; 48: 1-9, 2015. ilus, graf
Artigo em Inglês | LILACS | ID: biblio-950816

RESUMO

BACKGROUND: Glycoproteins play a critical role in the cellular activities of eukaryotes. Sialic acid is typically the outermost monosaccharide of glycolipids and glycoproteins, and is necessary for normal development. RESULTS: A strategy based on avidin-biotin affinity was established to enrich sialylated glycoproteins from HeLa cervical carcinoma, SW1990 pancreatic adenocarcinoma, and A549 lung adenocarcinoma cells. Using HPLC-MS/MS, western blot, real-time PCR, and enzyme-linked immunosorbent assay, gp96 was identified in all three cell lines. No significant difference in the protein expression of gp96 was detected at the whole cell level, but the amount of bioti-nylated gp96 in SW1990 cells was 30-40 % lower than that in A549 and HeLa cells, and the amount of sialylated gp96 in SW1990 cells was 30 % lower than that in A549 and HeLa cells. Immunoblotting results showed that the expression of sialyltransferase proteins in the total cell lysates from HeLa and A549 cells were higher than that in SW1990 cells. CONCLUSIONS: We established a new method for investigating the expression and sialylation of glycoproteins using metabolic labeling, click chemistry, and avidin-biotin affinity. We successfully used this method to purify sialylated glycoproteins from cancer cell lines. Our results showed that the levels of gp96 sialylation varied across different cancer cell lines, and this may be because of differences in sialyltransferase expression.


Assuntos
Humanos , Ácidos Siálicos/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicosiltransferases/metabolismo , Proteínas de Neoplasias/metabolismo , Ensaio de Imunoadsorção Enzimática , Células HeLa , Espectrometria de Massas em Tandem , Reação em Cadeia da Polimerase em Tempo Real , Células A549
14.
Biol. Res ; 48: 1-7, 2015. ilus, graf, tab
Artigo em Inglês | LILACS | ID: biblio-950804

RESUMO

BACKGROUND: We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells). RESULTS: MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells. CONCLUSION: Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.


Assuntos
Humanos , Benzopiranos/farmacologia , Extratos Vegetais/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Fabaceae/química , Antineoplásicos Fitogênicos/farmacologia , Benzopiranos/isolamento & purificação , Células HL-60 , Células Hep G2 , Células A549
15.
Acta bioquím. clín. latinoam ; 47(2): 343-351, abr.-jun. 2013. graf, tab
Artigo em Espanhol | LILACS | ID: lil-694557

RESUMO

Las estatinas son inhibidores competitivos de la 3-hidroxi-3-metilglutaril-coenzima A (HMG-CoA) reductasa ampliamente usados en los tratamientos contra las hipercolesterolemias. Los monoterpenos son componentes no nutritivos de la dieta presentes en aceites esenciales de varias plantas que han demostrado tener múltiples efectos en la vía del mevalonato. Se estudia el efecto y mecanismo de acción de monoterpenos presentes en aceites esenciales, así como la combinación de éstos entre sí y con simvastatina sobre la síntesis de colesterol, el metabolismo lipídico y la proliferación celular in vitro en células hepáticas Hep G2 y no hepáticas A549, e in vivo en ratones atímicos huéspedes y no huéspedes de tumores derivado de células A549 implantados en ellos. Se abre así una gran expectativa sobre la potencialidad de la administración conjunta de distintos monoterpenos y de extractos naturales de aceites esenciales en el mejoramiento de las terapias antihipercolesterolemiantes y/o el tratamiento del cáncer, como así también en el potencial sinergismo con estatinas como una alternativa para disminuir las dosis efectivas y los efectos indeseados y/o tóxicos.


Statins are competitive inhibitors of HMG-CoA reductase used in hypercholesterolemic patients. Monoterpenes are non-nutritive dietary components found in the essential oils of many plants with pharmacologic effects on mevalonate metabolism. The study is centered on the effects and action mechanisms of the monoterpene components of essential oils and the combination of monoterpenes between them and combined with simvastatin on cholesterogenesis, lipid metabolism and cellular proliferation in vitro using two established cell lines, Hep G2 (derived from a human hepatoblastoma), A549 (derived from a human lung adenocarcinoma) and in vivo in no host and host nude mice carrying implanted tumors derived from A549. This opens up great expectations about the potential of co-administration of different natural isoprenoids and essential oils in improving anti-cholesterolemic therapies and/or cancer treatment as well as in the potential synergism with statins as an alternative to lower effective doses, decreasing the likelihood of undesired and/or toxic effects.


As estatinas são inibidores competitivos da 3-hidroxi-3-metilglutaril - coenzima A (HMG-CoA) reductase amplamente utilizados nos tratamentos contra as hipercolesterolemias. Os monoterpenos são componentes não nutritivos da dieta encontrados em óleos essenciais de várias plantas que demonstraram ter múltiplos efeitos na via do mevalonato. Estudamos o efeito e o mecanismo de ação de monoterpenos encontrados em óleos essenciais, bem como a combinação deles entre si e com sinvastatina sobre a síntese de colesterol, o metabolismo lipídico e a proliferação celular in vitro em células hepáticas Hep G2 e não hepáticas A549 e in vivo em camundongos atímicos hospedeiros ou não hospedeiros de tumores derivados de células A549 implantadas neles. Isto abre grandes expectativas sobre o potencial da co-administração de diferentes monoterpenos e de extratos naturais de óleos essenciais na melhoria das terapias anti-hipercolesterolemiantes e/ou tratamento do câncer, assim como no potencial sinergismo com estatinas como uma alternativa para reduzir as doses efetivas e os efeitos indesejáveis e/ou tóxicos.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Monoterpenos/metabolismo , Células A549 , Anticolesterolemiantes , Células Hep G2 , Hepatócitos
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