RESUMO
Breast cancer is the most prevalent cancer in women. Metabolic abnormalities, particularly increased lipid synthesis and uptake, impact the onset and progression of the disease. However, the influence of lipid metabolism in breast cancer varies according to the disease stage and patient's hormone status. In postmenopausal patients, obesity is associated with a higher risk and poor prognosis of luminal tumors, while in premenopausal individuals, it is correlated to BRCA mutated tumors. In fact, the tumor's lipid profile may be used to distinguish between HER2+, luminal and BRCA-mutated tumors. Moreover, drug resistance was associated with increased fatty acid synthesis and alterations in membrane composition, impacting its fluidity and spatial subdomains such as lipid rafts. Here, we discuss the subtype-specific lipid metabolism alterations found in breast cancer and the potentiality of its modulation in a clinical setting.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/metabolismo , Lipídeos , Obesidade/complicações , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Transdução de SinaisRESUMO
A water-soluble sulfated heterorhamnan (Gb1) was isolated from the green seaweed Gayralia brasiliensis and purified by ultrafiltration, yielding a homogeneous polysaccharide (Gb1r). Both fractions contained rhamnose, xylose, galacturonic and glucuronic acids, galactose, and glucose. Chemical and spectroscopic methods allowed the determination of Gb1 and Gb1r chemical structure. Their backbones were constituted by 3-, 2-, and 2,3-linked rhamnosyl units (1:0.49:0.13 and 1:0.58:0.17, respectively), which are unsulfated (13.5 and 14.6%), disulfated (16.6 and 17.8%) or monosulfated at C-2 (8 and 8.6%) and C-4 (24.5 and 23.4%). Gb1 was oversulfated giving rise to Gb1-OS, which presented ~2.5-fold higher content of disulfated rhamnosyl units than Gb1, as determined by methylation analyses and NMR spectroscopy. Gb1 and Gb1-OS potently reduced the viability of U87MG human glioblastoma cells. Gb1 caused cell cycle arrest in the G1 phase, increased annexin V-stained cells, and no DNA fragmentation, while Gb1-OS increased the percentage of cells in the S and G2 phases and the levels of fragmented DNA and cells double-stained with annexin V/propidium iodide, suggesting an apoptosis mechanism. The results suggest that the different effects of Gb1 and Gb1-OS were related to differences in the sulfate content and position of these groups along the polysaccharide chains.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Mananas/farmacologia , Alga Marinha , Sulfatos/farmacologia , Antineoplásicos/isolamento & purificação , Neoplasias Encefálicas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glioma/patologia , Humanos , Mananas/isolamento & purificação , Estrutura Molecular , Alga Marinha/química , Relação Estrutura-Atividade , Sulfatos/isolamento & purificaçãoRESUMO
In this study we described the synthesis of a hybrid polysaccharide harboring moieties of ulvan and kappa-carrabiose. Alkylamines (1,3-diaminopropane and 1,6-diaminohexane) were selectively inserted into ß-D-GlcAp and α-L-IdoAp units in the ulvan structure via an amide bond formation producing ulvan-amide derivatives F-DAP (N% = 1.77; Mw = 208 kg mol-1) and F-DAH (N% = 1.77; Mw = 202 kg mol-1), which were reacted with kappa-carrabiose via reductive amination to produce hybrid ulvan-kappa-carrabiose polysaccharides F-DAP-Kb (N% = 1.56; Mw = 206 kg mol-1) and F-DAH-Kb (N% = 1.16; Mw = 200 kg mol-1). All the ulvan derivatives were characterized by 1H and 13C NMR spectroscopy and did not show cytotoxicity against human dermal fibroblasts (HDFa) at the concentrations of 25, 100, and 500 µg mL-1, neither anticoagulant properties at the range of 10-150 µg mL-1. Therefore, the ulvan-amide derivatives and the hybrid ulvan-kappa-carrabiose polysaccharides showed good biocompatibility in vitro, presenting as worthy candidates for tailoring scaffolds for biomedical applications.
Assuntos
Anticoagulantes/farmacologia , Carragenina/farmacologia , Polissacarídeos/farmacologia , Anticoagulantes/síntese química , Anticoagulantes/toxicidade , Carragenina/síntese química , Carragenina/toxicidade , Fibroblastos/efeitos dos fármacos , Humanos , Tempo de Tromboplastina Parcial , Polissacarídeos/síntese química , Polissacarídeos/toxicidadeRESUMO
Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.
Assuntos
Proteínas Ligadas por GPI/metabolismo , Genes Supressores de Tumor , Neurogênese/genética , Teratocarcinoma/metabolismo , Animais , Sítios de Ligação , Citometria de Fluxo , Proteínas Ligadas por GPI/genética , Regulação Neoplásica da Expressão Gênica/genética , Camundongos , Células PC12 , Regiões Promotoras Genéticas , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Teratocarcinoma/genética , Tubulina (Proteína)/metabolismo , Regulação para CimaRESUMO
Kraft lignin, so far useful for energy generation, has been gathering considerable attention as an alternative material to replace fossil-based resources mainly due to its high phenolic content. However, the wide molecular weight distribution and chemical composition heterogeneity led to the development of fractionation methods. Herein, to narrow such characteristics we used eucalypt kraft lignin fractionated at pH's 9, 7, 5 and 3 by sequential acid precipitation. These lignin fractions were first characterised by simultaneous pyrolysis and trimethylsilylation (SPyT) with N-Methyl-N-(trimethylsilyl) trifluoroacetamide with posterior tests of antioxidant, antibacterial, and antitumour activities. We observed higher ratio of syringyl/guaiacyl groups and increase in antioxidant activity in those fractions with lower molecular weight (precipitated at lower pH's). Fractions precipitated at pH's 9 and 7 have shown an outstanding antibacterial activity against five bacteria. Moreover, fractions 7 and 5 presented at cytotoxicity tests higher ability to inhibit the growth of U87MG and T98G glioma cells, while only a slight inhibition of adult human fibroblasts (non-tumour cells) was detected.
Assuntos
Antibacterianos/química , Antineoplásicos/química , Antioxidantes/química , Lignina/química , Madeira/química , Ácidos/química , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fracionamento Químico/métodos , Fibroblastos/efeitos dos fármacos , Humanos , Lignina/farmacologiaRESUMO
Exotic fruits and their co-products may be valuable sources of antioxidant dietary fibres (DF) which are useful for food industry and human health. In this study, we aimed to characterize DF obtained from guavira fruit pomace and investigate its antioxidant potential employing TEAC assay as well as a cell model. The DF were chemically characterized as containing arabinan, highly-methoxylated homogalacturonan and arabinogalactan. The DF-containing fraction (CPW) presented ABTS free radical scavenger activity. MTT and DCFH-DA assay were performed to assess, respectively, changes in cell viability and the potential intracellular antioxidant activity against H2O2-induced oxidative stress in murine NIH 3T3 fibroblast. CPW exhibited no effects on cell viability, moreover, when administered 48 h prior the induction of H2O2 toxic effects, it protected the cells, significantly increasing the cell viability compared to control. This protection may be related to the observed reduction of reactive oxygen species levels. Thus, the pre-treatment of cells with guavira DF for 48 h remarkably induced a cytoprotection against pro-oxidant conditions, and may be a valuable functional compound recovered from an unexploited agroindutrial waste.
Assuntos
Antioxidantes/análise , Antioxidantes/farmacologia , Fibras na Dieta/análise , Frutas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Galactanos/química , Peróxido de Hidrogênio/toxicidade , Camundongos , Células NIH 3T3 , Estresse Oxidativo , Pectinas/química , Polissacarídeos/químicaRESUMO
Neuroblastoma is a common childhood cancer with high mortality. We evaluated the capacity of the flavonoid, isoliquiritigenin (4,2',4'-trihydroxychalcone; ISL) to inhibit cellular proliferation and migration in the human neuroblastoma cell line SH-SY5Y. Incubation of cultured SH-SY5Y cells with 20-100⯵M ISL decreased cell confluency (15-70%) after 24â¯h incubation, while 10-100⯵M ISL (24â¯h) depleted intracellular ATP stores (15-90% vs vehicle-treated control) after 24â¯h incubation. ISL-mediated cell toxicity did not involve intracellular caspase 3/7 activation, externalization of phosphatidylserine on the cell membrane or stimulation of TNF and IL-1ß release, all indicating that the flavonoid did not induce apoptosis. Pre-treatment of cells with necrostatin-1, a necroptosis inhibitor, significantly restored ATP levels (ATP levels increased 12-42%) in ISL-treated neuroblastoma cells indicative of enhanced viability. By contrast, RIP1 phosphorylation status remained unchanged in cells treated with ISL although the intracellular ratio of phosphorylated/total parental RIP1 increased after ISL treatment on SH-SY5Y cells indicating that ISL decreased levels of native RIP1. In addition, ISL treatment inhibited SH-SY5Y cell migration/proliferation in a scratch assay and arrested cell cycle transition by significantly decreasing the number of cells in G0/G1 phase and increasing populations by ~10% in S (primarily) and G2/M (lesser extent) phases. The intracellular ratio of phosphorylated/total ERK 1/2 and p38 remained unchanged after ISL treatment (up to 40⯵M); ERK activation was only determined at ISL dose well above the experimental IC50 value as judged by ELISA analyses and this did not correlate with ISL cytotoxicity at lower dose <40⯵M; Western blot assay confirmed the detection of phosphorylated (p-)ERK1/2 and (p-)p38 in ISL treated cells. Together the results suggest that ISL exerts anti-proliferative and cytotoxic activity on SHSY5Y cells through the loss of ATP, induction of cell cycle arrest, and cell death largely via a necroptotic mechanism in the absence of apoptotic activity.
Assuntos
Proliferação de Células/efeitos dos fármacos , Chalconas/farmacologia , Flavonoides/farmacologia , Trifosfato de Adenosina/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Interleucina-1beta/análise , Interleucina-1beta/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosforilação/efeitos dos fármacos , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A new series of amphiphilic η6-areneruthenium(II) compounds containing phenylazo ligands (group I: compounds 1a, 1b, 2a and 2b) and phenyloxadiazole ligands (group II: compounds 3a, 3b, 4a and 4b) were synthesized and characterized for their anti-glioblastoma activity. The effects of the amphiphilic η6-areneruthenium(II) complexes on the viability of three human glioblastoma cell lines, U251, U87MG and T98G, were evaluated. The azo-derivative ruthenium complexes (group I) showed high cytotoxicity to all cell lines, whilst most oxadiazole-derivative complexes (group II) were less cytotoxic, except for compound 4a. The cationic complexes 2a, 2b and 4b were more cytotoxic than the neutral complexes. Compounds 2a and 2b caused a significant reduction in the percentage of cells in the G0/G1 phase, with concomitant increases in the G2/M phase and fragmented DNA in the T98G cell line. The η6-areneruthenium(II) compounds were also tested in cell lines that overexpress the multidrug ABC transporters P-gp, MRP1 and ABCG2. Compounds 2b and 4a were substrates for the P-gp protein, with resistance indexes of 8.6 and 1.9, respectively. Compound 2b was also a substrate for ABCG2 and MRP1 proteins, with lower resistance indexes (1.8 and 1.6, respectively). The contribution of multidrug ABC transporters to the cytotoxicity of compound 2b in T98G cells was evidenced, since verapamil (a characteristic inhibitor of MRP1) increased the cytotoxicity of compound 2b at concentrations up to 20⯵molâ¯L-1, whilst GF120918 and Ko143 (specific inhibitors of P-gp and ABCG2, respectively) had no significant effect. In addition, we showed that compound 2b interacts with glutathione (GSH), which could explain its cellular efflux by MRP1. Our results showed that the amphiphilic η6-areneruthenium(II) complexes are promising anti-glioblastoma compounds, especially compound 2b, which was cytotoxic for all three cell lines, although it is transported by the three main multidrug ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos , Glioblastoma/tratamento farmacológico , Rutênio/farmacologia , Compostos Azo , Transporte Biológico , Linhagem Celular Tumoral , Glioblastoma/patologia , Humanos , Ligantes , Compostos Organometálicos/farmacologia , OxidiazóisRESUMO
Malpighia emarginata is a tropical fruit plant, found naturally in the Caribbean islands and South America that produces an edible fruit known as acerola or Barbados Cherry. Its polysaccharides were obtained by aqueous extraction, subjected to a freezing and thawing process and ultrafiltration. A homogeneous fraction (ACWS-01E) was analyzed by sugar composition, HPSEC, methylation and NMR spectroscopy analyses. The results showed an arabinan-rich pectic polysaccharide, with 6.1×104g/mol and formed mainly by a high methyl esterified (DM=86%) homogalacturonan and branched arabinan. This latter is anchored in type I rhamnogalacturonan regions. The main chain of arabinan consisted of (1â5)-linked α-Araf, branched only at O-3. The potential ACWS-01E intracellular antioxidant activity against H2O2-induced oxidative stress in murine fibroblast cell line (3T3) was determined by DCFH-DA assay. The treatment with ACWS-01E significantly reduced H2O2-induced cytotoxic effect and the levels of reactive oxygen species (ROS). These findings suggested that ACWS-01E protected and improved NIH 3T3 cell viability from H2O2-induced toxicity by decreasing intracellular levels of ROS.
Assuntos
Antioxidantes/química , Peróxido de Hidrogênio/antagonistas & inibidores , Malpighiaceae/química , Pectinas/química , Polissacarídeos/química , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Sequência de Carboidratos , Sobrevivência Celular/efeitos dos fármacos , Frutas/química , Peróxido de Hidrogênio/farmacologia , Extração Líquido-Líquido/métodos , Camundongos , Células NIH 3T3 , Pectinas/isolamento & purificação , Pectinas/farmacologia , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Água/químicaRESUMO
We synthesized 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP), characterized it by electrochemistry, spectroelectrochemistry, and electron paramagnetic resonance techniques, and evaluated its cytotoxicity to human cancer cell lines. The results revealed that HHTP has accessible higher-oxidation states, especially the tris-semiquinone monoradical. This species is stable and is formed after being stored for months. HHTP exhibited cytotoxic effects on 5 human cancer cell lines, including glioma and lung cancer cells. The cytotoxic effect was evaluated based on the decrease in cell viability, increases in the percentage of cells with fragmented DNA, and elevated numbers of annexin V-PI-positive cells after HHTP treatment.
Assuntos
Fenantrenos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Fenantrenos/síntese química , Fenantrenos/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The oxidation of the free nucleoside 2'-deoxyguanosine (dGuo) by singlet molecular oxygen ((1)O2) has been studied over the three last decades due to the major role of DNA oxidation products in process such as ageing, mutation and carcinogenesis. In the present work we investigated the dGuo oxidation by (1)O2 in the presence of the important low molecular antioxidant, glutathione, in its reduced (GSH) and oxidized (GSSG) forms. There were applied different conditions of concentration, pH, time of incubation, and the use of a [(18)O]-labeled thermolabile endoperoxide naphthalene derivative as a source of [(18)O]-labeled (1)O2. Data was obtained through high performance liquid chromatography (HPLC) and HPLC coupled to micrOTOF Q-II analysis of the main oxidation products: the diastereomers of spiroiminodihydantoin-2'-deoxyribonucleosides (dSp) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). An intriguing result was that 8-oxodGuo levels increased by 100 fold when dGuo was oxidized by (1)O2 in the presence of GSH and by 2 fold in the presence of GSSG, while dSp levels dropped to zero for both conditions. All data from dGuo, 8-oxodGuo and dSp quantification together with the analysis of residual GSH/GSSG content in each sample strongly suggest that glutathione modifies the mechanism of dGuo oxidation by (1)O2 by disfavoring the pathway of dSp formation.
Assuntos
Desoxiguanosina/metabolismo , Glutationa/metabolismo , Oxigênio Singlete/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Glutationa/química , Dissulfeto de Glutationa/química , Dissulfeto de Glutationa/metabolismo , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Técnicas In Vitro , Modelos Químicos , Oxirredução , Oxigênio Singlete/química , Compostos de Espiro/química , Compostos de Espiro/metabolismoRESUMO
Hepatocellular carcinoma is the third most common cause of cancer-related deaths worldwide. Furthermore, the existing pharmacological-based treatments are insufficiently effective and generate many side effects. Hispidulin (6-methoxy-5,7,4'-trihydroxyflavone) is a flavonoid found in various medicinal herbs that present antineoplastic properties. Here we evaluated how modulation of reactive oxygen species (ROS) and alterations of antioxidant defenses could be associated to the antiproliferative effects of hispidulin in HepG2 cells. In addition, we studied the inhibitory activity of hispidulin on the efflux of drugs mediated by ABC transporters involved in multidrug resistance. In order to understand the increase of intracellular ROS promoted by hispidulin, we investigated the mRNA expression levels and activities of antioxidant enzymes, and the GSH/GSSG ratio. We showed that hispidulin significantly down-regulated the transcription levels of catalase, leading to reduction of enzyme activity and decrease of the GSH content. We also observed that, in the presence of N-acetylcysteine or exogenous catalase, the proliferation was lowered back to the control levels. These data clearly indicate a strong involvement of intracellular ROS levels for triggering the antiproliferative effects. We also demonstrated that the inhibition produced by hispidulin on drug efflux was specific for ABCG2, since no effects were observed with ABCB1 and ABCC1. Furthermore, HepG2 cells were more sensitive to hispidulin-mediated cell death than immortalized L929 fibroblasts, suggesting a differential toxicity of this compound between tumor and non-tumor cell lines. Our results suggest that hispidulin constitutes a promising candidate to sensitize chemoresistant cancer cells overexpressing ABCG2.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antioxidantes/farmacologia , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Flavonas/farmacologia , Neoplasias Hepáticas/patologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Catalase/biossíntese , Catalase/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Células L , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Mitoxantrona/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Plantas Medicinais/metabolismo , RNA Mensageiro/biossíntese , Espécies Reativas de Oxigênio/metabolismoRESUMO
Sulfated heterorhamnans produced by Gayralia oxysperma were utilized for the preparation of two homogeneous and highly sulfated Smith-degraded products (M(w) of 109 and 251 kDa), which were constituted principally by 3-linked α-L-rhamnosyl units 2- or 4-sulfate and 2-linked α-L-rhamnosyl units 4- or 3,4-sulfate, in different percentages. The homogeneous products and the crude extracts containing the sulfated heterorhamnans showed cytotoxic effect against U87MG cells. These sulfated polysaccharides induced an increase in the number of cells in G1 phase with concomitant increase of the mRNA levels of p53 and p21. The presence of 2-linked disulfated rhamnose residues together with the molecular weight could be important factors to be correlated with the inhibitory effect on human glioblastoma cells.
Assuntos
Clorófitas/química , Desoxiaçúcares/farmacologia , Mananas/farmacologia , Sulfatos/química , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxiaçúcares/química , Desoxiaçúcares/isolamento & purificação , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mananas/química , Mananas/isolamento & purificação , Estrutura Molecular , Polimerização , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Flavones have received considerable attention because of their antiproliferative properties and selective effects on cancer cells, making them good candidates for use in cancer therapy. In contrast to other flavones, little is known about the effects of the flavone core structure (2-phenyl-4H-1-benzopyran-4one) on cancer cells. Here, we report that flavone induces cell death in human hepatoma HepG2 cells. Furthermore, annexin-V+/PI- and SubG1 populations of HepG2 cells increased after flavone treatment. Exposure of HepG2 to flavone did not result in either cytochrome c release into the cytosol or changes in the mitochondrial membrane potential. Treatment of HepG2 cells with flavone for 24 h reduced the accumulation of intracellular ROS, which correlated with upregulation of Gred, CuZnSOD and MnSOD mRNA levels. Taken together, our results provided useful insights into the mechanism of cell death caused by flavones, in order to evaluate their future application in hepatocarcinoma therapy.
Assuntos
Morte Celular/efeitos dos fármacos , Flavonas/farmacologia , Citocromos c/metabolismo , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
The invasive phenotype of many tumors is associated with an imbalance between the matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), and the membrane-anchored reversion-inducing cysteine-rich protein with Kazal motifs (RECK). RECK inhibits MMP-2, MMP-9, and MT1-MMP, and has been linked to patient survival and better prognosis in several types of tumors. However, despite the wide implication of these MMPs in melanoma establishment and progression, the role of RECK in this type of tumor is still unknown. Here, we analyzed the expression of RECK, TIMP1, TIMP2, TIMP3, MT1MMP, MMP2, and MMP9 in two publicly available melanoma microarray datasets and in a panel of human melanoma cell lines. We found that RECK is downregulated in malignant melanoma, accompanied by upregulation of MT1MMP and TIMP2. In both datasets, we observed that the group of samples displaying higher RECK levels show lower median expression levels of MT1MMP and TIMP2 and higher levels of TIMP3. When tested in a sample-wise manner, these correlations were statistically significant. Inverse correlations between RECK, MT1MMP, and TIMP2 were verified in a panel of human melanoma cell lines and in a further reduced model that includes a pair of matched primary tumor-derived and metastasis-derived cell lines. Taken together, our data indicate a consistent correlation between RECK, MT1MMP, and TIMP2 across different models of clinical samples and cell lines and suggest evidence of the potential use of this subset of genes as a gene signature for diagnosing melanoma.
Assuntos
Proteínas Ligadas por GPI/biossíntese , Metaloproteinase 14 da Matriz/biossíntese , Melanoma/genética , Melanoma/metabolismo , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Linhagem Celular Tumoral , Regulação para Baixo , Proteínas Ligadas por GPI/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , Melanoma/enzimologia , Fenótipo , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Inibidor Tecidual de Metaloproteinase-3/genéticaRESUMO
Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis.
Assuntos
Melaninas/biossíntese , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Cloreto de Amônio/farmacologia , Animais , Apoptose , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Pontos de Checagem da Fase G1 do Ciclo Celular , Expressão Gênica , Genes Supressores de Tumor , Melanoma Experimental/genética , Camundongos , Proteínas de Neoplasias/metabolismo , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tirosina/farmacologiaAssuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/farmacologia , Cromonas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Triptaminas/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/síntese química , Cromonas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Estrutura Molecular , Proteínas de Neoplasias/metabolismo , Relação Estrutura-AtividadeRESUMO
ABCG2 plays a major role in anticancer-drug efflux and related tumor multidrug resistance. Potent and selective ABCG2 inhibitors with low cytotoxicity were investigated among a series of 44 chalcones and analogues (1,3-diarylpropenones), by evaluating their inhibitory effect on the transport of mitoxantrone, a known ABCG2 substrate. Six compounds producing complete inhibition with IC(50) values below 0.5 µM and high selectivity for ABCG2 were identified. The number and position of methoxy substituents appeared to be critical for both inhibition and cytotoxicity. The best compounds, with potent inhibition and low toxicity, contained an N-methyl-1-indolyl (compound 38) or a 6'-hydroxyl-2',4'-dimethoxy-1-phenyl (compound 27) moiety (A-ring) and two methoxy groups at positions 2 and 6 of the 3-phenyl moiety (B-ring). Methoxy substitution contributed to inhibition at positions 3 and 5, but had a negative effect at position 4. Finally, methoxy groups at positions 3, 4, and 5 of the B-ring markedly increased cytotoxicity and, therefore, should be avoided.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/síntese química , Chalconas/síntese química , Proteínas de Neoplasias/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Chalconas/química , Chalconas/farmacologia , Células HEK293 , Humanos , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Modelos Moleculares , Proteínas de Neoplasias/genética , Relação Quantitativa Estrutura-Atividade , TransfecçãoRESUMO
The ABCG2 multidrug transporter is known to confer cancer cell multidrug resistance by causing the efflux of anticancer drugs; therefore, selective inhibitors have the potential to improve chemotherapeutic treatments. Here, various methoxy derivatives of resveratrol are shown to be potent inhibitors of mitoxantrone efflux by ABCG2: among a series of 11 derivatives, compound 9 (3,5,3',4'-tetramethoxy trans-stilbene) had an IC(50) of 0.16 µM and showed a maximal inhibition of 75%, as measured by flow cytometry. It was not transported, as shown by HPLC fractionation and mass spectrometry titration and the lack of any cross-resistance in cell survival experiments. Compound 9 had a very low intrinsic cytotoxicity and was able to chemosensitize the growth of resistant ABCG2-transfected HEK293 cells at submicromolar concentrations. Drug-efflux inhibition was specific for ABCG2 since very low effects were observed with ABCB1 and ABCC1. The action mechanism of compound 9 was different from that of GF120918, which produced a complete and partly competitive but not ABCG2-specific inhibition, since ABCB1 was even more strongly inhibited. The two inhibitors also displayed different effects on the ABCG2 vanadate-sensitive ATPase activity, suggesting that they either bound to distinct sites or induced different conformational changes. Mitoxantrone efflux was fully inhibited by combining low concentrations of compound 9 with either GF120918 or a transport substrate such as prazosin or nilotinib. We conclude that methoxy derivatives of stilbene are good candidates for investigating future in vivo modulation of ABCG2 drug-efflux activity.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/metabolismo , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mitoxantrona/metabolismo , Proteínas de Neoplasias/metabolismo , Estilbenos/química , Estilbenos/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , ResveratrolRESUMO
A series of 13 disubstituted chromones was synthesized. Two types of substituents, on each side of the scaffold, contributed to both the potency of ABCG2 inhibition and the cytotoxicity. The best compound, 5-(4-bromobenzyloxy)-2-(2-(5-methoxyindolyl)ethyl-1-carbonyl)-4H-chromen-4-one (6g), displayed high-affinity inhibition and low cytotoxicity, giving a markedly high therapeutic index. The chromone derivative specifically inhibited ABCG2 versus other multidrug ABC transporters and was not transported. It constitutes a highly promising candidate for in vivo chemosensitization of ABCG2-expressing tumors.