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Soft tissue sarcomas (STSs) are a heterogeneous group of rare tumors. Although constituting only 1% of all human malignancies, STSs represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. Over 100 histologic subtypes have been characterized to date (occurring predominantly in the trunk, extremity, and retroperitoneum), and many more are being discovered due to molecular profiling. STS mortality remains high, despite adjuvant chemotherapy. New prognostic stratification markers are needed to help identify patients at risk of recurrence and possibly apply more intensive or novel treatments. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the most relevant cellular, molecular and metabolic biomarkers for STS, and highlight advances in STS-related biomarker research.
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Macrolide antibiotics, such as Clarithromycin (Cla), have been proven to exert anti-tumour activity in several preclinical models of different types of cancer. Cla can exert its anti-tumour effects through different mechanisms, e.g. by blocking the autophagic flux, inducing apoptosis or inhibiting tumour-induced angiogenesis. The clinical benefit of Cla in treating various tumours in combination with conventional treatment was confirmed in extensive clinical studies in patients suffering from non-small cell lung cancer, breast cancer, multiple myeloma and other haematological malignancies. Data regarding the anti-cancer effect of Cla on Colorectal Cancer (CRC) are still lacking. This article shares data on the in vivo efficacy of Cla in two xenograft models of CRC. Our results show that Cla treatment reduces tumour growth and increases the overall survival in CRC mouse xenograft models. Moreover, the Western blot analysis of autophagic and apoptotic markers suggests that the anti-tumour effects of Cla are related to a modulation of both cellular processes. The data suggest that it will worth consider Cla as treatment option for CRC patients.
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The definition of the gene expression profile of genes encoding Ion Channels and Transporters (ICT-GEP) represents a novel and attracting aspect in cancer. We determined the ICT-GEP of Follicular Lymphoma (FL), and compared it with that of the more aggressive Diffuse Large B Cell Lymphoma (DLBCL). cDNA microarray data were collected both from patients enrolled for this study, and from public datasets. In FL the ICT-GEP indicated the overexpression of both the K+ channel encoding gene KCNN4, and SLC2A1, which encodes the Glut1 glucose transporter. SLC2A1 turned out to represent the hub of a functional network, connecting channels and transporters in FL. Relapsed FL patients were characterised by 38 differentially expressed ICT genes, among which ATP9A, SLC2A1 and KCNN4 were under-expressed, indicating a down-regulation of both excitability and glycolysis. A completely different profile of K+ channel encoding genes emerged in DLBCL accompanied by the over-expression of the fatty acid transporter-encoding gene SLC27A1 as well as of the metabolism regulator NCoR1. This indicates a change in excitability and a shift towards an oxidative metabolism in DLBCL. Overall, the ICT-GEP may contribute to identifying novel lymphoma biomarkers related to excitability and metabolic pathways, with particular relevance for drug resistant, relapsed FL.
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Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Canais Iônicos/genética , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Proteínas de Membrana Transportadoras/genética , Idoso , Estudos de Coortes , Bases de Dados Genéticas , Feminino , Redes Reguladoras de Genes , Humanos , Canais Iônicos/metabolismo , Linfoma Folicular/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Pessoa de Meia-IdadeRESUMO
In recent years, a few successful attempts were made to repurpose the clinically approved antiarthritic gold drug, Auranofin (AF), as an anticancer agent. The present study shows that the iodido(triethylphosphine)gold(I) complex, (Et 3 PAuI hereafter)-an AF analogue where the thiosugar ligand is simply replaced by one iodide ligand-manifests a solution chemistry resembling that of AF and exerts similar cytotoxic and proapoptotic effects on A2780 human ovarian cancer cells in vitro. However, when evaluated in a preclinical orthotopic model of ovarian cancer, Et 3 PAuI produces a far superior anticancer action than AF inducing a nearly complete tumor remission. The highly promising in vivo performances here documented for Et 3 PAuI warrant its further evaluation as a drug candidate for ovarian cancer treatment.
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Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-à-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging. The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the VH domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the "native" anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo. In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.
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The purpose of this research has been deciphering the Warburg paradox, the biochemical enigma unsolved since 1923. We solved it by demonstrating that its specific character, i.e. the forced aerobic lactate exportation, represents a crucial metabolic device to counteract the cytotoxic effect produced by an excess of pyruvate at the connection of glycolysis with the Krebs cycle. This solution was verified by exposing cancer cells of different histogenesis to pyruvate concentrations higher than the physiological ones, after showing that these concentrations are totally innocuous when injected into mice. The mechanism of the pyruvate cytotoxicity relies on the saturation of the respiratory chain, leading to a negative shift of the cytosolic NADP/NADPH ratio and the consequent restriction of the purine synthesis and the related cell apoptosis. The reducing equivalents generated by glycolysis and by cytosolic metabolism compete each other for their disposal trough the respiratory chain; this makes it that the cytotoxicity of pyruvate is inversely related to the mitochondrial number and efficiency of various cell types. Thus, the cytotoxicity is high in anaplastic cancer stem cells, whose mitochondria are extremely few and immature (cristae-poor); on the contrary, no inhibition is brought about in adult differentiated cells, physiologically rich of mature mitochondria. All this generates the pyruvate anticancer selectivity, together with the lack of a general toxicity, making pyruvate represent an ideal candidate for a radical non toxical anticancer treatment.
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Antineoplásicos/farmacologia , Glicólise/efeitos dos fármacos , Animais , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Hipóxia Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Platinum-based drugs such as Cisplatin are commonly employed for cancer treatment. Despite an initial therapeutic response, Cisplatin treatment often results in the development of chemoresistance. To identify novel approaches to overcome Cisplatin resistance, we tested Cisplatin in combination with K+ channel modulators on colorectal cancer (CRC) cells. METHODS: The functional expression of Ca2+-activated (KCa3.1, also known as KCNN4) and voltage-dependent (Kv11.1, also known as KCNH2 or hERG1) K+ channels was determined in two CRC cell lines (HCT-116 and HCT-8) by molecular and electrophysiological techniques. Cisplatin and several K+ channel modulators were tested in vitro for their action on K+ currents, cell vitality, apoptosis, cell cycle, proliferation, intracellular signalling and Platinum uptake. These effects were also analysed in a mouse model mimicking Cisplatin resistance. RESULTS: Cisplatin-resistant CRC cells expressed higher levels of KCa3.1 and Kv11.1 channels, compared with Cisplatin-sensitive CRC cells. In resistant cells, KCa3.1 activators (SKA-31) and Kv11.1 inhibitors (E4031) had a synergistic action with Cisplatin in triggering apoptosis and inhibiting proliferation. The effect was maximal when KCa3.1 activation and Kv11.1 inhibition were combined. In fact, similar results were produced by Riluzole, which is able to both activate KCa3.1 and inhibit Kv11.1. Cisplatin uptake into resistant cells depended on KCa3.1 channel activity, as it was potentiated by KCa3.1 activators. Kv11.1 blockade led to increased KCa3.1 expression and thereby stimulated Cisplatin uptake. Finally, the combined administration of a KCa3.1 activator and a Kv11.1 inhibitor also overcame Cisplatin resistance in vivo. CONCLUSIONS: As Riluzole, an activator of KCa3.1 and inhibitor of Kv11.1 channels, is in clinical use, our results suggest that this compound may be useful in the clinic to improve Cisplatin efficacy and overcome Cisplatin resistance in CRC.
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Cisplatino/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Canal de Potássio ERG1/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzotiazóis/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacocinética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Canal de Potássio ERG1/metabolismo , Células HCT116 , Células HT29 , Humanos , Concentração Inibidora 50 , Camundongos , Bloqueadores dos Canais de Potássio/farmacologia , Pirazóis/farmacologia , Riluzol/farmacologiaRESUMO
Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-go-go-related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the ß1 integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the ß1 integrin-hERG1 interaction. Although open hERG1 channels did not interact as strongly with ß1 integrins as did closed channels, current flow through hERG1 channels was necessary to activate the integrin-dependent phosphorylation of Tyr397 in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K+ flow, whereas metastasis of breast cancer cells was reduced when the hERG1/ß1 integrin interaction was disrupted. We conclude that the interaction of ß1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression.
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Canais de Potássio Éter-A-Go-Go/metabolismo , Integrina beta1/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Progressão da Doença , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/genética , Transferência Ressonante de Energia de Fluorescência , Células HCT116 , Células HEK293 , Humanos , Immunoblotting , Integrina beta1/química , Integrina beta1/genética , Camundongos Nus , Camundongos SCID , Microscopia Confocal , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Conformação Proteica , Transplante HeterólogoRESUMO
The trinuclear platinum cluster [Pt3(µ-PBut2)3(CO)3]CF3SO3 (I) was designed featuring the presence of a nearly equilateral platinum triangle bridged by three di-tert-butylphosphide ligands; in addition, each platinum center bears a terminal carbonyl ligand. This triplatinum cluster was initially developed in view of applications in the field of cluster-containing innovative materials. Yet, due to the large success of platinum complexes in cancer treatment, we also decided to explore its cytotoxic and anticancer properties. Accordingly, the solubility profile of this compound in several solvents was preliminarily investigated, revealing a conspicuous solubility in DMSO and DMSO/buffer mixtures; this makes the biological testing of I amenable. UV-Vis measurements showed that the triplatinum cluster is stable for several hours under a variety of conditions, within aqueous environments. No measurable reactivity was observed for I toward two typical model proteins, i.e. lysozyme and cytochrome c. On the contrary, a significant reactivity was evidenced when reacting I with small sulfur-containing ligands. In particular, a pronounced reactivity with reduced glutathione and cysteine emerged from ESI-MS experiments, proving complete formation of I-GSH and I-Cys derivatives, with the loss of a single carbonyl ligand. Starting from these encouraging results, the cytotoxic potential of I was assayed in vitro against a panel of representative cancer cell lines, and potent cytotoxic properties were disclosed. Of particular interest is the finding that the triplatinum species manifests potent antiproliferative properties toward Triple Negative Breast Cancer Cells, often refractory to most anticancer drugs. Owing to the reported encouraging results, a more extensive biological and pharmacological evaluation of this Pt cluster is now warranted to better elucidate its mode of action.
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Antineoplásicos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Compostos Organoplatínicos , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HL-60 , Humanos , Células MCF-7 , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/química , Compostos Organoplatínicos/farmacologiaRESUMO
Cancer must be viewed as a 'tissue', constituted of both transformed cells and a heterogeneous microenvironment, the 'tumour microenvironment' (TME). The TME undergoes a complex remodelling during the course of multistep tumourigenesis, hence strongly contributing to tumour progression. Ion channels and transporters (ICTs), being expressed on both tumour cells and in the different cellular components of the TME, are in a strategic position to sense and mediate signals arising from the TME. Often, this transmission is mediated by integrin adhesion receptors, which are the main cellular receptors capable of mediating cell-to-cell and cell-to-matrix bidirectional signalling. Integrins can often operate in conjunction with ICT because they can behave as functional partners of ICT proteins. The role of integrin receptors in the crosstalk between tumour cells and the TME is particularly relevant in the context of pancreatic cancer (PC), characterized by an overwhelming TME which actively contributes to therapy resistance. We discuss the possibility that this occurs through integrins and ICTs, which could be exploited as targets to overcome chemoresistance in PC.
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Adesão Celular/fisiologia , Integrinas/metabolismo , Canais Iônicos/metabolismo , Modelos Biológicos , Neoplasias Pancreáticas/fisiopatologia , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Humanos , Neoplasias Pancreáticas/metabolismoRESUMO
PURPOSE: hERG1 channels are aberrantly expressed in several types of human cancers, where they affect different aspects of cancer cell behavior. A thorough analysis of the functional role and clinical significance of hERG1 channels in gastric cancer is still lacking. EXPERIMENTAL DESIGN: hERG1 expression was tested in a wide (508 samples) Italian cohort of surgically resected patients with gastric cancer, by immunohistochemistry and real-time quantitative PCR. The functional link between hERG1 and the VEGF-A was studied in different gastric cancer cell lines. The effects of hERG1 and VEGF-A inhibition were evaluated in vivo in xenograft mouse models. RESULTS: hERG1 was positive in 69% of the patients and positivity correlated with Lauren's intestinal type, fundus localization of the tumor, G1-G2 grading, I and II tumor-node-metastasis stage, and VEGF-A expression. hERG1 activity modulated VEGF-A secretion, through an AKT-dependent regulation of the transcriptional activity of the hypoxia inducible factor. Treatment of immunodeficient mice xenografted with human gastric cancer cells, with a combination of hERG1 blockers and anti-VEGF-A antibodies, impaired tumor growth more than single-drug treatments. CONCLUSION: Our results show that hERG1 (i) is aberrantly expressed in human gastric cancer since its early stages; (ii) drives an intracellular pathway leading to VEGF-A secretion; (iii) can be exploited to identify a gastric cancer patients' group where a combined treatment with antiangiogenic drugs and noncardiotoxic hERG1 inhibitors could be proposed.
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Adenocarcinoma/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Neoplasias Gástricas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Canal de Potássio ERG1 , Ensaio de Imunoadsorção Enzimática , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/patologia , TransfecçãoRESUMO
Titanium dioxide (TiO2) has been widely used in many nanotechnology areas including nanomedicine, where it could be proposed for the photodynamic and sonodynamic cancer therapies. However, TiO2 nanoformulations have been shown to be toxic for living cells. In this article, we report the development of a new delivery system, based on nontoxic TiO2 nanoparticles, further conjugated with a monoclonal antibody against a novel and easily accessible tumor marker, e.g., the Kv 11.1 potassium channel. We synthesized, by simple solvothermal method, dicarboxylic acid-terminated PEG TiO2 nanocrystals (PEG-TiO2 NPs). Anti-Kv 11.1 monoclonal antibodies (Kv 11.1-Mab) were further linked to the terminal carboxylic acid groups. Proper conjugation was confirmed by X-ray photoelectron spectroscopy analysis. Kv 11.1-Mab-PEG-TiO2 NPs efficiently recognized the specific Kv 11.1 antigen, both in vitro and in pancreatic ductal adenocarcinoma (PDAC) cells, which express the Kv 11.1 channel onto the plasma membrane. Both PEG TiO2 and Kv 11.1-Mab-PEG-TiO2 NPs were not cytotoxic, but only Kv 11.1-Mab-PEG-TiO2 NPs were efficiently internalized into PDAC cells. Data gathered from this study may have further applications for the chemical design of nanostructures to be applied for therapeutic purposes in pancreatic cancer.
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Angiogenesis is a potential target for cancer therapy. We identified a novel signaling pathway that sustains angiogenesis and progression in colorectal cancer (CRC). This pathway is triggered by ß1 integrin-mediated adhesion and leads to VEGF-A secretion. The effect is modulated by the human ether-à-go-go related gene 1 (hERG1) K(+) channel. hERG1 recruits and activates PI3K and Akt. This in turn increases the Hypoxia Inducible Factor (HIF)-dependent transcription of VEGF-A and other tumour progression genes. This signaling pathway has novel features in that the integrin- and hERG1-dependent activation of HIF (i) is triggered in normoxia, especially after CRC cells have experienced a hypoxic stage, (ii) involves NF-kB and (iii) is counteracted by an active p53. Blocking hERG1 switches this pathway off also in vivo, by inhibiting cell growth, angiogenesis and metastatic spread. This suggests that non-cardiotoxic anti-hERG1 drugs might be a fruitful therapeutic strategy to prevent the failure of anti-VEGF therapy.
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Neoplasias Colorretais/patologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Integrina beta1/metabolismo , Neovascularização Patológica/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/genética , Células HCT116 , Células HT29 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Nus , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transplante Heterólogo , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: There is a need to identify new markers to assess recurrence risk in early-stage colorectal cancer (CRC) patients. We explored the prognostic impact of ether-a-gò-gò-related gene 1 channels and some hypoxia markers, in patients with nonmetastatic (stage I, II, and III) CRC. METHODS: The expression of hERG1, vascular endothelial growth factor A (VEGF-A), glucose transporter 1, carbonic anhydrase IX (CA-IX), epidermal growth factor receptor (EGF-R), and p53 was tested by immunohistochemistry in 135 patients. The median follow-up was 35 months. Clinicopathologic parameters and overall survival were evaluated. RESULTS: hERG1 displayed a statistically significant association with Glut-1, VEGF-A, CA-IX, and EGF-R; p53 with VEGF-A and CA-IX; Glut-1 with the age of the patients; and EGF-R with TNM and mucin content. TNM and CA-IX were prognostic factors at the univariate analysis; TNM, hERG1, and Glut-1, at the multivariate analysis. Risk scores calculated from the final multivariate model allowed to stratify patients into four different risk groups: A) stage I-II, Glut-1 positivity, any hERG1; B) stage I-II, Glut-1 and hERG1 negativity; C) stage I-II, Glut-1 negativity, hERG1 positivity; D) stage III, any Glut-1 and any hERG1. CONCLUSIONS: hERG1 positivity with Glut-1 negativity identifies a patient group with poor prognosis within stage I-II CRC. The possibility that these patients might benefit from adjuvant therapy, independently from the TNM stage, is discussed. IMPACT: More robust prognostic and predictive markers, supplementing standard clinical and pathologic staging, are needed for node-negative patients.
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Bone marrow mesenchymal cells (MSCs) can protect leukemic cells from chemotherapy, thus increasing their survival rate. We studied the potential molecular mechanisms underlying this effect in acute lymphoblastic leukemia (ALL) cells. Coculture of ALL cells with MSCs induced on the lymphoblast plasma membrane the expression of a signaling complex formed by hERG1 (human ether-à-go-go-related gene 1) channels, the ß(1)-integrin subunit, and the chemokine receptor CXC chemokine receptor-4. The assembly of such a protein complex activated both the extracellular signal-related kinase 1/2 (ERK1/2) and the phosphoinositide 3-kinase (PI3K)/Akt prosurvival signaling pathways. At the same time, ALL cells became markedly resistant to chemotherapy-induced apoptosis. hERG1 channel function appeared to be important for both the initiation of prosurvival signals and the development of drug resistance, because specific channel blockers decreased the protective effect of MSCs. NOD/SCID mice engrafted with ALL cells and treated with channel blockers showed reduced leukemic infiltration and had higher survival rates. Moreover, hERG1 blockade enhanced the therapeutic effect produced by corticosteroids. Our findings provide a rationale for clinical testing of hERG1 blockers in the context of antileukemic therapy for patients with ALL.
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Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Animais , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Técnicas de Cocultura , Doxorrubicina/farmacologia , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Humanos , Integrina beta1/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Complexos Multiproteicos/metabolismo , Piperidinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prednisona/farmacologia , Piridinas/farmacologia , Interferência de RNA , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immunotherapy of tumors and of melanoma in particular has a long history, and recently this therapeutic approach found a reliable scientific rationale. This biological therapy aims to teach the patient's immune system to recognize the antigens expressed on tumor cells and destroy them, leaving normal cells intact. The success of this therapy highly depends on the selection of target antigens that are essential for tumors growth and progression. The overexpression of GM(3) ganglioside 1 and especially the expression of its metabolite GM(3) lactone 2 characterize murine and human melanomas, playing an important role in tumor progression and making such self-antigens potential targets for the immunotherapy of these neoplasms. Although more immunogenic than its precursor, GM(3) lactone 2 is unsuitable to be used in immunotherapy as a melanoma-associated antigen (MAA) because it is unstable under physiological conditions. We designed and synthesized the hydrolytically stable mimetic 3, which is remarkably simpler than the native lactone 2; after conjugation of 3 to the protein carrier keyhole-limpet hemocyanin (KLH), the obtained glycoprotein 5 was used as the immunogen in vivo to successfully elicit specific antimelanoma antibodies. In fact, no appreciable binding to GM(1) was observed. Capitalizing on the stability and on the reduced structural complexity of mimetic 3, the immunostimulant 5 we report represents a new promising synthetic glycoconjugate for the immunotherapy of melanoma.
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Anticorpos/imunologia , Gangliosídeo G(M3)/análogos & derivados , Melanoma/imunologia , Mimetismo Molecular/imunologia , Animais , Anticorpos/química , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Configuração de Carboidratos , Simulação por Computador , Gangliosídeo G(M3)/química , Gangliosídeo G(M3)/genética , Gangliosídeo G(M3)/imunologia , Hemocianinas/química , Hemocianinas/imunologia , Humanos , Imunoterapia , Melanoma/genética , Melanoma/terapia , Camundongos , Mimetismo Molecular/genéticaRESUMO
A common feature of tumor cells is the aberrant expression of ion channels on their plasma membrane. The molecular mechanisms regulating ion channel expression in cancer cells are still poorly known. K(+) channels that belong to the human ether-a-go-go-related gene 1 (herg1) family are frequently misexpressed in cancer cells compared to their healthy counterparts. We describe here a posttranslational mechanism for the regulation of hERG1 channel surface expression in cancer cells. This mechanism is based on the activity of hERG1 isoforms containing the USO exon. These isoforms (i) are frequently overexpressed in human cancers, (ii) are retained in the endoplasmic reticulum, and (iii) form heterotetramers with different proteins of the hERG family. (iv) The USO-containing heterotetramers are retained intracellularly and undergo ubiquitin-dependent degradation. This process results in decreased hERG1 current (I(hERG1)) density. We detailed such a mechanism in heterologous systems and confirmed its functioning in tumor cells that endogenously express hERG1 proteins. The silencing of USO-containing hERG1 isoforms induces a higher I(hERG1) density in tumors, an effect that apparently regulates neurite outgrowth in neuroblastoma cells and apoptosis in leukemia cells.
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Canais de Potássio Éter-A-Go-Go/metabolismo , Ativação do Canal Iônico , Processamento de Proteína Pós-Traducional , Processamento Alternativo/genética , Apoptose , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Clonagem Molecular , DNA Complementar/genética , Canais de Potássio Éter-A-Go-Go/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Saúde , Humanos , Espaço Intracelular/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Neuritos/metabolismo , Isoformas de Proteínas/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
Voltage-dependent K+ channels are the main determinants in controlling cellular excitability within the central nervous system. Among voltage-dependent K+ channels, the ERG subfamily is deeply involved in the control of cellular excitability, both in mammals and in invertebrates. ERG channels are encoded by different genes: the erg1 gene, which can generate two alternative transcripts (erg1a and erg1b), erg2 and erg3. The aim of the present study was to determine the expression pattern and cellular localization of ERG proteins (ERG1, ERG2, and ERG3) in the mouse CNS, differentiating, for the first time, the ERG1A and ERG1B isoforms. To this purpose, novel specific antibodies were raised against the various channel proteins and their specificity and immunoreactivity tested. It emerged that: 1) all the erg genes were indeed translated in neuronal tissue; 2) ERG proteins distribution in the mouse CNS often overlapped, and only in specific areas each ERG protein showed a distinct pattern of expression; and 3) ERG proteins were generally expressed in neuronal soma, but dendritic and/or white matter labeling could be detected in specific areas. The finding that ERG proteins often have an overlapping expression suggests that neuronal ERG currents could be determined, at least in part, by heterotetrameric ERG channels. This suggestion is demonstrated to occur for ERG1A/ERG1B by showing that the two isoforms coassemble in mouse brain.
Assuntos
Encéfalo/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Neurônios/metabolismo , Medula Espinal/metabolismo , Animais , Encéfalo/citologia , Canal de Potássio ERG1 , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas , Medula Espinal/citologia , Distribuição TecidualRESUMO
Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adhesion. Additionally, integrins trigger multiple signaling pathways that are involved in cell migration, proliferation, survival, and differentiation. We previously demonstrated that the activation of integrins containing the beta(1) subunit leads to a selective increase in potassium currents carried by the human ether-a-go-go-related gene (hERG) channels in neuroblastoma and leukemia cells; this current activation modulates adhesion-dependent differentiation in these cells. We hypothesized that the cross-talk between integrins and hERG channels could be traced back to the assembly of a macromolecular signaling complex comprising the two proteins. We tested this hypothesis in both SH-SY5Y neuroblastoma cells and in human embryonic kidney 293 cells stably transfected with hERG1 and, therefore, expressing only the full-length hERG1 protein on the plasma membrane. The beta(1) integrin and hERG1 coprecipitate in these cells and colocalize in both intracellular and surface membrane compartments. The two proteins also coprecipitate with caveolin-1, suggesting the localization of the complex in lipid rafts/caveolae. hERG1-transfected cells undergo an activation of hERG currents after beta(1) integrin-mediated adhesion to fibronectin; concomitant with this activation, the focal adhesion kinase associates with the hERG1 protein and becomes tyrosine phosphorylated. Using hERG1-specific inhibitors, we show that the tyrosine phosphorylation of focal adhesion kinase is strictly dependent on hERG channel activity. Similarly, the activity of the small GTPase Rac1 turned out to be dependent on hERG currents. On the whole, these data indicate that the hERG1 protein associates with beta(1) integrins and modulates adhesion receptor signaling.
Assuntos
Adesão Celular , Canais de Potássio Éter-A-Go-Go/metabolismo , Cadeias beta de Integrinas/metabolismo , Transdução de Sinais , Técnicas de Cultura de Células , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Canal de Potássio ERG1 , Fibronectinas/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Cadeias beta de Integrinas/análise , Membranas Intracelulares/metabolismo , Rim/citologia , Rim/embriologia , Modelos Biológicos , Neuroblastoma/patologia , Técnicas de Patch-Clamp , Fosforilação , Testes de Precipitina , Transfecção , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The acquisition of the capacity to invade surrounding tissues confers a more malignant phenotype to tumor cells and is necessary for the establishment of metastases. The understanding of the molecular mechanisms underlying cell invasion in human solid tumors such as colorectal cancers could provide not only more sensitive prognostic analyses but also novel molecular targets for cancer therapy. We report in this article that K(+) ion channels belonging to the HERG family are important determinants for the acquisition of an invasive phenotype in colorectal cancers. The herg1 gene and HERG1 protein are expressed in many colon cancer cell lines, and the activity of HERG channels modulates colon cancer cell invasiveness. Moreover, the amount of HERG1 protein expressed on the plasma membrane is directly related to the invasive phenotype of colon cancer cells. Finally, both the herg1 gene and HERG1 protein were expressed in a high percentage of primary human colorectal cancers, with the highest incidence occurring in metastatic cancers, whereas no expression could be detected either in normal colonic mucosa or in adenomas.