RESUMO
Fusarium wilt of bananas (FWB) is seriously affecting the sustainable development of the banana industry and is caused by the devastating soil-borne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Biological control is a promising strategy for controlling Fusarium wilt in bananas. We previously identified Streptomyces hygroscopicus subsp. hygroscopicus 5-4 with strong antifungal activity against the FWB. The most possible antimicrobial mechanism of strain 5-4 was explored using the metabolomics approach, light microscopy imaging, and transmission electron microscopy (TEM). The membrane integrity and ultrastructure of Foc TR4 was damaged after extract treatment, which was supported by the degradation of mycelium, soluble protein content, extracellular reducing sugar content, NADH oxidase activity, malondialdehyde content, mitochondrial membrane potential, and mitochondrial respiratory chain complex enzyme activity. The extracts of strain 5-4 cultivated at different times were characterized by a liquid chromatography-mass spectrometer (LC-MS). 647 known metabolites were detected in the extracts of strains 5-4. Hygromycin B, gluten exorphin B4, torvoside G, (z)-8-tetradecenal, piperitoside, sarmentosin, pubescenol, and other compounds were the main differential metabolites on fermentation culture for 7 days. Compared with strain 5-4 extracts, hygromycin B inhibited the mycelial growth of Foc TR4, and the EC50 concentration was 7.4 µg/mL. These results showed that strain 5-4 could destroy the cell membrane of Foc TR4 to inhibit the mycelial growth, and hygromycin B may be the key antimicrobial active metabolite. Streptomyces hygroscopicus subsp. hygroscopicus 5-4 might be a promising candidate strain to control the FWB and provide a scientific basis for the practical application of hygromycin B as a biological control agent.
RESUMO
Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is one of the most disastrous fungal diseases. Biological control is a promising strategy for controlling Fusarium wilt of banana. To explore endophytic actinomycetes as biocontrol resources against Foc TR4, antagonistic strains were isolated from different tissues of medicinal plants. Here, a total of 144 actinomycetes were isolated and belonged to Nonomuraea, Kitasatospora, and Streptomyces. Forty-three isolates exhibited antifungal activities against Foc TR4. The strain labeled with 5-4 isolated from roots of Piper austrosinense had a broad-spectrum antifungal activity by the production of chitinase and ß-1,3-glucanase and was identified as Streptomyces hygroscopicus subsp. hygroscopicus 5-4. Furthermore, disease index of banana wilt was significantly reduced by application of strain 5-4 in comparison with application of Foc TR4 alone. Exogenous application of strain 5-4 increased the expression levels of defense genes such as (PAL), peroxidase (POD), pathogenesis-related protein 1 (PR-1), hydrolytic enzymes (ß-1,3-glucanase), lysin motif receptor kinase 1 (LYK-1), and mitogen-activated protein kinase 1 (MPK-1). The antifungal mechanism assay demonstrated that extracts of strain 5-4 inhibited spore gemination and hyphal growth of Foc TR4, and caused abnormally swollen, deformity, and rupture of Foc TR4 hypha. Thus, S. hygroscopicus subsp. hygroscopicus 5-4 could be used as a potential biological agent for controlling Fusarium wilt of banana.
Assuntos
Fusarium , Musa , Streptomyces , Antifúngicos/farmacologia , Fusarium/genética , Perfilação da Expressão Gênica , Musa/microbiologia , Doenças das Plantas/microbiologia , Streptomyces/genéticaRESUMO
Banana residues are an important energy resource after fruit harvesting. The optionally dumping and burning causes severely environmental problems. Traditional compost efficiency was limited by lignocellulosic composition of banana residues. Inoculation with cellulase-producing microbes provides an efficient strategy for improving degradation of lignocellulosic materials. In our study, a newly isolated cellulolytic bacterium Acetobacter orientalis XJC-C with a salt and high temperature resistance was identified from a marine soft coral. By contrast, the strain can biodegrade different lignocellulosic agricultural residues, especially banana straw. The highest cellulolytic and ligninolytic enzyme activities were detected during composting at 40 days. Compared with the negative and positive control groups, the lignin degradation rate reached 76.24% in the A. orientalis XJC-C group, increased by 47.08% and 21.85%, respectively. Moreover, the strain improved significantly the metabolic activity and functional diversity of bacterial community. Hence, A. orientalis XJC-C will be a promising candidate for degrading lignocellulosic agricultural residues.
Assuntos
Compostagem , Musa , Acetobacter , Biomassa , Lignina , SoloRESUMO
Fatty acids in avocado fruit are crucial components influencing taste as well as fruit quality and nutritional value. Changes to fatty acid contents and concentrations in avocado fruit are important because of the associated effects on sensory properties. Hence, plant physiologists and molecular biologists interested in elucidating the influence of transcription factors on fatty acid accumulation in avocado fruit. In this study, APETALA2/ethylene-responsive factor (AP2/ERF) family members in avocado (Persea americana Mill.) were systematically and comprehensively analyze to identify potential PaAP2/ERF genes related to fatty acid accumulation. The results of bioinformatics analysis and the expression profiles of the AP2/ERF members suggested that 10 highly expressed PaAP2/ERF genes may encode transcription factors with functions related to the fatty acid accumulation in the avocado mesocarp. Furthermore, PaWRI1 and PaWRI2, two AP2/ERF transcription factor genes in avocado, were functionally characterized regarding their effects on fatty acid accumulation. The transcriptome and biochemical analyses of PaWRI1-2-overexpressing transgenic tomato plants revealed the up-regulated expression of 17 unigenes related to fatty acid synthesis and triacylglycerol assembly as well as increased fatty acid contents relative to the corresponding levels in the wild-type plants. In contrast, the overexpression of PaWRI2 in transgenic tomato plants up-regulated the expression of only six unigenes associated with fatty acid synthesis and triacylglycerol assembly and negligibly affected fatty acid accumulation when compared with wild-type plants. This systematic analysis provides a foundation for future studies regarding AP2/ERF functions associated with fatty acid accumulation.
Assuntos
Ácidos Graxos/metabolismo , Persea , Proteínas de Plantas/metabolismo , Solanum lycopersicum/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Persea/genética , Persea/metabolismo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Fatores de Transcrição/genéticaRESUMO
Banana is a key staple food and fruit in countries all over the world. However, the development of the global banana industry is seriously threatened by Fusarium wilt disease, which is caused by Fusarium oxysporum f. sp. cubense (Foc). In particular, Foc tropical race 4 (Foc TR4) could infect more than 80% of global banana and plantain crops. Until now, there were no commercial chemicals or resistant cultivars available to control the disease. Biological control using actinomycetes is considered a promising strategy. In this study, 88 actinomycetes were isolated from a banana orchard without symptoms of Fusarium wilt disease for more than 10 years. An actinobacterial strain labeled as JBS5-6 has exhibited strong antifungal activities against Foc TR4 and other selected 10 phytopathogenic fungi. Based on phenotypic and biochemical traits as well as complete genome analysis, strain JBS5-6 was assigned to Streptomyces violaceusniger. Extracts of the strain inhibited the mycelial growth and spore germination of Foc TR4 by destroying membrane integrity and the ultrastructure of cells. The complete genome of strain JBS5-6 was sequenced and revealed a number of key function gene clusters that contribute to the biosynthesis of active secondary metabolites. Sixteen chemical compounds were further identified by gas chromatography-mass spectrometry (GC-MS). 5-hydroxymethyl-2-furancarboxaldehyde was one of the dominant components in strain JBS5-6 extracts. Moreover, fermentation broth of strain JBS5-6 significantly reduced the disease index of banana seedlings by inhibiting the infection of Foc TR4 in a pot experiment. Hence, strain JBS5-6 is a potential biocontrol agent for the management of disease and the exploitation of biofertilizer.
RESUMO
Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive diseases, severely limiting the development of banana industry. Especially, Foc tropical race 4 (Foc TR4) can infect and destroy almost all banana cultivars. Until now, there is still a lack of an effective method for controlling fusarium wilt. A biocontrol strategy using Actinobacteria is considered as a promising method for management of disease and pest. In this study, 229 Actinobacteria were isolated from rhizosphere soil samples of a primitive ecological mountain. An actinobacterium strain marked with YYS-7 exhibited a high antifungal activity against Foc TR4. Combining the physiological and biochemical characteristics as well as alignment of the 16S rRNA sequence, the strain YYS-7 was assigned to Streptomyces sp. The crude extracts of Streptomyces sp. YYS-7 obviously inhibited the mycelial growth of Foc TR4. The cell integrity and ultrastructure were seriously destroyed. In addition, Streptomyces sp. YYS-7 and crude extracts also showed a broad-spectrum antifungal activity against the selected seven phytopathogenic fungi. A gas chromatography-mass spectrometry (GC-MS) was used to predict the antifungal metabolites. A total of eleven different compounds were identified, including phenolic compounds, hydrocarbons, esters and acids. In the pot experiment, the crude extracts can significantly improve the banana plant's resistance to Foc TR4. Hence, Streptomyces sp. YYS-7 will be a potential biocontrol agent for the biofertilizer exploitation and the discovery of new bioactive substances.
RESUMO
A new isolated cellulolytic bacterium from a soft coral was named as Fictibacillus sp YS-26 based on the morphologic and molecular characteristics. It can degrade different lignocellulosic agricultural residues by producing cellulolytic enzymes, α-amylase, protease, pectinase and xylanase. Especially, Fictibacillus sp. YS-26 exhibited the highest cellulolytic activities in the soybean meal medium. By contrast, the fermentation broth of Fictibacillus sp. YS-26 significantly enhanced utilization efficiency of carboxylic acids and polymers by soil microorganisms as well as the microbial metabolism function and community diversity in rhizosphere soil of banana plantlets. The fermentation broth also improved soil characters and increased the growth of banana plantlets. We found that soil total nitrogen and electrical conductivity had a positive relationship with the increase of microbial diversity. Hence, Fictibacillus sp. YS-26 will be a promising candidate for biodegradating lignocellulosic biomass and improving the soil microbial diversity.
Assuntos
Microbiota , Rizosfera , Carbono , Lignina , Solo , Microbiologia do SoloRESUMO
Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc) is a disastrous soil-borne fungal disease. Foc tropical race 4 (Foc TR4) can infect almost all banana cultivars. Until now, there is a shortage of safety and effective control methods and commercial banana cultivars with a resistance against Foc TR4. Biocontrol using environmentally friendly microbes is a promising strategy for the management of Foc TR4. Here, a strain 5-10, newly isolated from a medicinal plant (Curculigo capitulata), exhibited a high antifungal activity against Foc TR4. Combing the morphological characteristics and molecular identification, strain 5-10 was classified as a Streptomyces genus. The sequenced genome revealed that more than 39 gene clusters were involved in the biosynthesis of secondary metabolites. Some multidrug resistance gene clusters were also identified such as mdtD, vatB, and vgaE. To improve the anti-Foc TR4 activity of the strain 5-10 extracts, an optimization method of fermentation broth was established. Antifungal activity increased by 72.13% under the fermentation system containing 2.86 g/L of NaCl and 11.57% of inoculation amount. After being treated with the strain 5-10 extracts, the Foc TR4 hyphae shrinked, deformed, and ruptured. The membrane integrity and cell ultrastructure incurred irreversible damage. Streptomyces sp. 5-10 extracts play a fungicidal role in Foc TR4. Hence, Streptomyces sp. 5-10 will be a potential biocontrol agent to manage fungal diseases by exploring the microbial fertilizer.
RESUMO
Avocado (Persea americana Mill.) is an economically important crop because of its high nutritional value. However, the absence of a sequenced avocado reference genome has hindered investigations of secondary metabolism. For next-generation high-throughput transcriptome sequencing, we obtained 365,615,152 and 348,623,402 clean reads as well as 109.13 and 104.10 Gb of sequencing data for avocado mesocarp and seed, respectively, during five developmental stages. High-quality reads were assembled into 100,837 unigenes with an average length of 847.40 bp (N50 = 1725 bp). Additionally, 16,903 differentially expressed genes (DEGs) were detected, 17 of which were related to carotenoid biosynthesis. The expression levels of most of these 17 DEGs were higher in the mesocarp than in the seed during five developmental stages. In this study, the avocado mesocarp and seed transcriptome were also sequenced using single-molecule long-read sequencing to acquired 25.79 and 17.67 Gb clean data, respectively. We identified 233,014 and 238,219 consensus isoforms in avocado mesocarp and seed, respectively. Furthermore, 104 and 59 isoforms were found to correspond to the putative 11 carotenoid biosynthetic-related genes in the avocado mesocarp and seed, respectively. The isoform numbers of 10 out of the putative 11 genes involved in the carotenoid biosynthetic pathway were higher in the mesocarp than those in the seed. Besides, alpha- and beta-carotene contents in the avocado mesocarp and seed during five developmental stages were also measured, and they were higher in the mesocarp than in the seed, which validated the results of transcriptome profiling. Gene expression changes and the associated variations in gene dosage could influence carotenoid biosynthesis. These results will help to further elucidate carotenoid biosynthesis in avocado.
Assuntos
Carotenoides/metabolismo , Regulação da Expressão Gênica de Plantas , Persea/genética , Persea/metabolismo , Sementes/genética , Sementes/metabolismo , Transcriptoma , Vias Biossintéticas , Biologia Computacional/métodos , Dosagem de Genes , Perfilação da Expressão Gênica , Ontologia Genética , Metaboloma , Metabolômica/métodos , Anotação de Sequência Molecular , Desenvolvimento Vegetal/genéticaRESUMO
Avocado (Persea americana) is a tropical fruit that has drawn great interest its oil for foods and cosmetic industries; however, avocado oil processing by-product is a potential source of edible protein. Herein, edible protein was prepared from defatted avocado meal, and it's physicochemical, functional and emulsion properties were investigated. The avocado protein showed U-shaped exhibiting strong effect of pH, and a minimum solubility being observed at pH 4.5, confirming the isoelectric point of avocado protein. Nutritionally, the avocado protein contains all the essential amino acids. Avocado protein provided higher water and oil absorption capacities, higher radical scavenging capacity but lower in-vitro digestibility compared with soy protein. Furthermore, the avocado protein as emulsifier afforded a stability oil-in-water emulsion system, resulting in a greater emulsifying stability than that of soy protein. The present results highlight the potential source of edible protein from avocado oil processing by-products for functional food ingredients.
Assuntos
Emulsões , Persea/química , Óleos de Plantas/química , Proteínas de Plantas/química , Digestão , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Plantas/metabolismo , Solubilidade , Propriedades de SuperfícieRESUMO
Lots of bananas were wasted before commercialization. It is necessary to search potential industrial applications of banana. In the present study, starches from seven banana cultivars (labeled as A-G) were isolated and then characterized. These starches presented different and irregular shapes, such as sphere, long spheroid and polygonal granules. The distribution of size and analyses of average molecular weight showed more small granules in samples B, D, F and G than other samples. The amylose content varied from 22.59% to 38.40%. The crystal types of these starches were a mixture of B-type and C-type, and the relative crystallinity varied greatly. The differential scanning calorimetry (DSC) results showed that the onset temperature of gelatinization increased as follows: Aâ¯<â¯Bâ¯<â¯Eâ¯<â¯Câ¯≈â¯Dâ¯<â¯F. The maximum viscosity of banana starch decreased as follows: Gâ¯>â¯Câ¯>â¯Dâ¯>â¯Fâ¯>â¯Eâ¯=â¯Bâ¯>â¯A. The in vitro digestibility test showed that the content of resistant starch was very high in banana starches. These results would be useful to the application of those starches in food and nonfood industries.
Assuntos
Fenômenos Químicos , Digestão , Musa/química , Amido/química , Amido/metabolismo , Pomadas , TemperaturaRESUMO
Focal segmental glomerulosclerosis (FSGS) comprises a group of uncommon disorders that present with marked proteinuria, nephrotic syndrome, progressive renal failure and characteristic glomerular lesions on histopathology. The current standard of care for patients with FSGS include immunosuppressive drugs such as glucocorticoids followed by calcineurin inhibitors, if needed for intolerance or inadequate response to glucocorticoids. Renin-angiotensin-aldosterone (RAAS) blockers are also used to control proteinuria, an important signature of FSGS. Existing treatments, however, achieved only limited success. Despite best care, treatment failure is common and FSGS is causal in a significant proportion of end stage renal disease. Thus, an unmet need exists for novel disease modifying treatments for FSGS. We employed two widely-used murine models of FSGS to test the hypothesis that systemic inhibition of chemokine receptor CCR2 would have therapeutic benefit. Here we report that administration CCX872, a potent and selective small molecule antagonist of CCR2, achieved rapid and sustained attenuation of renal damage as determined by urine albumin excretion and improved histopathological outcome. Therapeutic benefit was present when CCX872 was used as a single therapy, and moreover, the combination of CCX872 and RAAS blockade was statistically more effective than RAAS blockade alone. In addition, the combination of CCR2 and RAAS blockade was equally as effective as endothelin receptor inhibition. We conclude that specific inhibition of CCR2 is effective in the Adriamycin-induced and 5/6 nephrectomy murine models of FSGS, and thus holds promise as a mechanistically distinct therapeutic addition to the treatment of human FSGS.
Assuntos
Albuminúria , Glomerulosclerose Segmentar e Focal , Glomérulos Renais , Receptores CCR2/antagonistas & inibidores , Sistema Renina-Angiotensina/efeitos dos fármacos , Albuminúria/tratamento farmacológico , Albuminúria/patologia , Albuminúria/urina , Animais , Linhagem Celular , Modelos Animais de Doenças , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/urina , Humanos , Glomérulos Renais/lesões , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR2/metabolismoRESUMO
BACKGROUND: Keratinocyte growth factor (KGF) acts at the KGF receptor (KGFR) to produce a rapid stimulation of breast cancer cell proliferation and motility which is mediated via the Erk signaling pathway. Enhancement of KGF/KGFR signal transduction may be an early step in the metastatic progression of breast cancer. Receptor modeling of KGFR was used to identify selective KGFR tyrosine kinase (TK) inhibitor molecules that have the potential to bind selectively to the KGFR. The present study evaluated the biological activity of 57 of these KGFR TK inhibitor compounds on breast cancer cells. MATERIALS AND METHODS: These compounds were tested for their ability to inhibit KGF-mediated breast cancer cell proliferation in MCF-7 breast cancer cells. Furthermore, the effects of the most effective proliferation inhibitors were examined on Erk signaling and on the relative density of cell membrane KGFR. RESULTS: It was observed that 27 of the 57 compounds tested produced a 20% or greater reduction in KGF-mediated proliferation; while five compounds produced greater than 50% inhibition. In addition, the most potent inhibitors also reduced Erk signaling and cell membrane density of the KGFR. CONCLUSION: The compounds examined appear to be selective KGFR inhibitors which inhibit KGF-mediated activity and reduce the expression of KGFR on cancer cells. These results may lead to the development of a novel class of anticancer agents for the prevention of metastatic cancer progression.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Fator 7 de Crescimento de Fibroblastos/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Neoplasias da Mama/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fator 7 de Crescimento de Fibroblastos/farmacologia , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinolonas/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
BACKGROUND: We have reported the development of a novel fusion protein (FP) consisting of an amino-terminal fragment of urokinase linked to the amino terminus of the enzyme L-methioninase (L-M). The present study compared the effect of this novel FP on the proliferation of human ovarian, skin, breast endometrial and pancreatic cancer cell lines. METHODS: The FP, L-M and a mutated FP, with reduced L-M activity, were produced by recombinant methods. The effect of treatment with FP, L-M and mutated FP on the proliferation of the cancer cells was measured in vitro using an MTS assay. RESULTS: The inhibitory effect of the FP was found to be significantly greater than that of L-M alone or the mutated FP. In addition, the FP produced a greater inhibitory effect on an ovarian cancer cell line than on comparable normal, non-cancerous cells. Further, the FP produced a dose-dependent inhibition of the proliferation of pancreatic cancer cell lines. CONCLUSION: These results suggest that this FP is a potent and selective inhibitor of the proliferation of various cancer cell lines and has potential as a therapeutic agent for the treatment of various methionine-dependent cancers.
Assuntos
Antineoplásicos/farmacologia , Liases de Carbono-Enxofre/genética , Proliferação de Células/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Humanos , Mutação , Proteínas Recombinantes de Fusão/genéticaRESUMO
BACKGROUND: Keratinocyte growth factor (KGF increases the proliferation and motility of many epithelial cells and is known to be up-regulated in pancreatic cancer. The present study examined the hypothesis that KGF may initiate or enhance the progression of pancreatic cancer by increasing the proliferation and motility of pancreatic cancer cells. MATERIALS AND METHODS: HPAF-II pancreatic cancer cell migration and proliferation was evaluated using a culture wounding assay 24 and 48 hours following KGF treatment. KGF receptor (KGFR) localization in these cells was established by immunohistochemistry. RESULTS: KGF treatment significantly increased the proliferation and motility of the HPAF-II cells. In addition, KGF enhanced the motile morphology of these cancer cells. CONCLUSION: The results of this study indicate that KGF has a rapid influence on the proliferation and motility of HPAF-II cells and suggest that KGF may be involved in the progression of pancreatic cancer.
Assuntos
Movimento Celular , Proliferação de Células , Fator 7 de Crescimento de Fibroblastos/farmacologia , Neoplasias Pancreáticas/patologia , Adesão Celular , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pancreáticas/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Keratinocyte growth factor (KGF) produces a rapid increase in the proliferation and motility of estrogen receptor (ER)-positive breast cancer cells which is abolished by estrogen deprivation and/or anti-estrogen treatment. The present study examined the hypothesis that ER-alpha is involved in the KGF proliferation in MCF-7 cancer cells using small interfering RNA (siRNA) to selectively inhibit ER-alpha expression. MATERIALS AND METHODS: At 48 hours following ER-alpha siRNA transfection, the MCF-7 cells were treated with KGF (50 ng/ml) or vehicle for 24 hours. Cell proliferation was measured using a MTT assay. ER-alpha protein levels were quantified by Western blotting. RESULTS: ER-alpha siRNA transfection significantly reduced ER-alpha expression and MCF-7 cell proliferation. KGF-mediated enhancement of cell proliferation and motile cell morphology were reduced or absent in the siRNA transfected MCF-7 cells. CONCLUSION: ER-alpha expression is associated with KGF-induced proliferation of breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/antagonistas & inibidores , Fator 7 de Crescimento de Fibroblastos/farmacologia , RNA Interferente Pequeno/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/genética , Fator 7 de Crescimento de Fibroblastos/antagonistas & inibidores , Humanos , TransfecçãoRESUMO
BACKGROUND: Keratinocyte growth factor (KGF) has been shown to induce breast cancer metastasis in animal models. cDNA microarrays have revealed that KGF increased Wilms tumor 1 (WT1) and focal adhesion kinase (FAK) expression in breast cancer cells. The role of WT1 and FAK in KGF signaling was investigated. MATERIALS AND METHODS: A cell culture wounding model was used to study the effects of WT1 and FAK down-regulation on KGF-induced proliferation and motility in breast cancer cells. RESULTS: WT1 down-regulation inhibited KGF-mediated proliferation and motility of breast cancer cells, while FAK down-regulation inhibited proliferation, but had no significant effect on cell motility. WT1 down-regulation, but not FAK down-regulation, led to Erk1,2 inactivation. CONCLUSION: KGF-mediated signaling employs WT1 and FAK to regulate breast cancer cell proliferation and motility and may represent therapeutic targets for the prevention of breast cancer progression.
Assuntos
Neoplasias da Mama/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação para Baixo , Ativação Enzimática , Fator 7 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 7 de Crescimento de Fibroblastos/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/biossíntese , Proteína Adaptadora GRB2/biossíntese , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Nucleares/biossíntese , Fosforilação , Fatores de Processamento de RNA , Proteínas Recombinantes/farmacologia , Transdução de SinaisRESUMO
BACKGROUND: Previously, we reported that a novel fusion protein consisting of an amino-terminal fragment of urokinase linked to the amino terminus of the enzyme L-methioninase inhibited MCF-7 breast cancer cells in vitro to a greater extent than treatment with L-methioninase. MATERIALS AND METHODS: The fusion protein, L-methioninase and a mutated fusion protein without L-methioninase activity were produced by recombinant methods. The effects of fusion protein, L-methioninase, and mutated fusion protein treatment on the proliferation and motility of SK-LU-i lung and PC-3 prostate and cancer cells were measured in vitro using a culture wounding assay. RESULTS: The fusion protein produced a dose-dependent inhibition of the proliferation and motility of both cancer cell lines. In addition, the fusion protein was found to be significantly more effective than L-methioninase alone or mutated fusion protein. CONCLUSION: Our results suggest that this fusion protein has potential as a selective therapeutic agent for the treatment of various methionine-dependent cancers.
Assuntos
Liases de Carbono-Enxofre/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Liases de Carbono-Enxofre/genética , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Sistemas de Liberação de Medicamentos , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Neoplasias da Próstata/metabolismo , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes de Fusão/genéticaRESUMO
BACKGROUND: The mammary glands of adult female animals are remarkably sensitive to keratinocyte growth factor (KGF). KGF acts at the KGF receptor (KGFR) to produce a rapid and profound stimulation of breast cancer cell proliferation and motility. Further, KGF-induced motility in breast cancer cells is mediated via the Erk1/2 signaling pathway. Thus, enhancement of KGF/KGFR signal transduction may be an early step in the metastatic progression of breast cancer. Receptor modeling of KGFR was used to identify selective KGFR tyrosine kinase inhibitor (TKI) molecules with high receptor affinity. The present study describes the synthesis and biological activity of three of the KGFR TKI compounds. MATERIALS AND METHODS: Computer modeling of the KGFR was used to create a virtual library of compounds that have the potential to bind with high affinity to the KGFR. Three of these compounds were synthesized and tested in this study. The compounds were tested for their ability to inhibit KGF-mediated breast cancer cell proliferation and motility using a culture wounding assay. In addition, the effect of the most potent KGFR TKI compound on the relative density of cell membrane KGFR was measured using immunocytochemistry. RESULTS: It was observed that the KGFR TKIs decreased KGF-mediated activity as predicted by computer modeling. In addition, the most potent inhibitor also reduced the density of the KGFR on the membrane of the cancer cells. CONCLUSION: The novel inhibitors identified in this project are selective KGFR inhibitors which appear to reduce the expression of KGFR on cancer cells. These results may lead to the development of a novel class of anticancer agents for the chemoprevention of metastatic cancer development and provide a new approach in the treatment of breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Desenho de Fármacos , Humanos , Imuno-Histoquímica , Quinolonas/síntese química , Quinolonas/farmacologiaRESUMO
In a previous study we compared the influence of several growth factors on breast cancer cells in culture and observed that epidermal growth factor (EGF) enhanced the invasiveness of estrogen receptor-positive breast cancer cells. The objective of the present study was to determine the influence of three unique antiestrogens on EGF-mediated movement of human breast cancer cells. The rate of movement of MCF-7 breast cancer cells was measured using time-lapse videomicroscopy (TLVM). The MCF-7 cells were pretreated with antiestrogen (either tamoxifen, ICI-182-780 (ICI) or 1,1-dichloro-cis-2,3-diarylcyclopropane (AII)) at 10(-6) mol/l for 4 days, and then treated with EGF (10(-10) mol/l) immediately prior to TLVM. EGF enhanced the motility of the MCF-7 cells at 30-90 min post-administration. However, EGF-mediated motility of the MCF-7 cells was inhibited by antiestrogen pretreatment, with TAM and ICI producing complete inhibition of EGF-induced motility. In conclusion, this study demonstrates that EGF enhances the rate of movement of MCF-7 breast cancer cells and that antiestrogen pretreatment inhibits EGF-mediated motility.