RESUMO
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.
Assuntos
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
KV11.1 (hERG1) channels are often overexpressed in human cancers. In leukemias, KV11.1 regulates pro-survival signals that promote resistance to chemotherapy, raising the possibility that inhibitors of KV11.1 could be therapeutically beneficial. However, because of the role of KV11.1 in cardiac repolarization, blocking these channels may cause cardiac arrhythmias. We show that CD-160130, a novel pyrimido-indole compound, blocks KV11.1 channels with a higher efficacy for the KV11.1 isoform B, in which the IC50 (1.8 µM) was approximately 10-fold lower than observed in KV11.1 isoform A. At this concentration, CD-160130 also had minor effects on Kir2.1, KV 1.3, Kv1.5, and KCa3.1. In vitro, CD-160130 induced leukemia cell apoptosis, and could overcome bone marrow mesenchymal stromal cell (MSC)-induced chemoresistance. This effect was caused by interference with the survival signaling pathways triggered by MSCs. In vivo, CD-160130 produced an antileukemic activity, stronger than that caused by cytarabine. Consistent with its atypical target specificity, CD-160130 did not bind to the main binding site of the arrhythmogenic KV11.1 blockers (the Phe656 pore residue). Importantly, in guinea pigs CD-160130 produced neither alteration of the cardiac action potential shape in dissociated cardiomyocytes nor any lengthening of the QT interval in vivo. Moreover, CD-160130 had no myelotoxicity on human bone marrow-derived cells. Therefore, CD-160130 is a promising first-in-class compound to attempt oncologic therapy without cardiotoxicity, based on targeting KV11.1. Because leukemia and cardiac cells tend to express different ratios of the A and B KV11.1 isoforms, the pharmacological properties of CD-160130 may depend, at least in part, on isoform specificity.
Assuntos
Antineoplásicos/farmacologia , Cardiotoxinas , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Indóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinonas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Cobaias , Células HEK293 , Células HL-60 , Humanos , Indóis/química , Indóis/uso terapêutico , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/metabolismo , Leucemia de Células B/patologia , Masculino , Camundongos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Pirimidinas/uso terapêutico , Pirimidinonas/química , Pirimidinonas/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
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.
Assuntos
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
Brain tumors, including the majority gliomas, are the leading cause of cancer-related death in children. World Health Organization has divided pediatric brain tumors into different grades and, based upon cDNA microarray data identifying gene expression profiles (GEPs), it has become evident in the last decade that the various grades involve different types of genetic alterations. However, it is not known whether ion channel and transporter genes, intimately involved in brain functioning, are associated with such GEPs. We determined the GEPs in an available cohort of 10 pediatric brain tumors initially by comparing the data obtained from four primary tumor samples and corresponding short-term cultures. The correspondence between the two types of samples was statistically significant. We then performed bioinformatic analyses on those samples (a total of nine) which corresponded to tumors of glial origin, either tissues or cell cultures, depending on the best "RNA integrity number." We used R software to evaluate the genes which were differentially expressed (DE) in gliomas compared with normal brain. Applying a p-value below 0.01 and fold change ≥4, led to identification of 2284 DE genes. Through a Functional Annotation Analysis (FAA) using the NIH-DAVID software, the DE genes turned out to be associated mainly with: immune/inflammatory response, cell proliferation and survival, cell adhesion and motility, neuronal phenotype, and ion transport. We have shown that GEPs of pediatric brain tumors can be studied using either primary tumor samples or short-term cultures with similar results. From FAA, we concluded that, among DE genes, pediatric gliomas show a strong deregulation of genes related to ion channels and transporters.
RESUMO
BACKGROUND AIMS: Retroviral transduction of anti-CD19 chimeric antigen receptors significantly enhances the cytotoxicity of natural killer (NK) cells against B-cell malignancies. We aimed to validate a more practical, affordable and safe method for this purpose. METHODS: We tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after electroporation with the corresponding mRNA using a clinical-grade electroporator. The cytotoxic capacity of the transfected NK cells was tested in vitro and in a mouse model of leukemia. RESULTS: Median anti-CD19-BB-ζ expression 24 h after electroporation was 40.3% in freshly purified (n =18) and 61.3% in expanded (n = 31) NK cells; median cell viability was 90%. NK cells expressing anti-CD19-BB-ζ secreted interferon (IFN)-γ in response to CD19-positive target cells and had increased cytotoxicity. Receptor expression was detectable 6 h after electroporation, reaching maximum levels at 24-48 h; specific anti-CD19 cytotoxicity was observed at 96 h. Levels of expression and cytotoxicities were comparable with those achieved by retroviral transduction. A large-scale protocol was developed and applied to expanded NK cells (median NK cell number 2.5 × 10(8), n = 12). Median receptor expression after 24 h was 82.0%; NK cells transfected under these conditions exerted considerable cytotoxicity in xenograft models of B-cell leukemia. CONCLUSIONS: The method described here represents a practical way to augment the cytotoxicity of NK cells against B-cell malignancies. It has the potential to be extended to other targets beyond CD19 and should facilitate the clinical use of redirected NK cells for cancer therapy.
Assuntos
Antígenos CD19 , Terapia Baseada em Transplante de Células e Tecidos , Células Matadoras Naturais/citologia , Linfoma não Hodgkin , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animais , Antígenos CD19/genética , Antígenos CD19/imunologia , Antígenos CD19/uso terapêutico , Complexo CD3/genética , Complexo CD3/uso terapêutico , Linhagem Celular Tumoral , Citotoxicidade Imunológica/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Linfoma não Hodgkin/terapia , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , RNA Mensageiro/genética , RNA Mensageiro/uso terapêutico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/uso terapêuticoRESUMO
A model is proposed to study the process of hypoxia-induced angiogenesis in cancer cells. The model accounts for the role played by the vascular endothelial growth factor (VEGF)-A in regulating the oxygen intake. VEGF-A is dynamically controlled by the HIF-1α concentration. If not degraded, HIF-1α can bind to the subunit termed HIF-1ß and so experience translocation to the nucleus, to exert its proper transcriptional activity. The delicate balance between these opposing tendencies translates into the emergence of distinct macroscopic behaviors in terms of the associated molecular concentrations that we here trace back to normoxia, hypoxia and death regimes. These aspects are firstly analyzed with reference to the ideal mean-field scenario. Stochastic fluctuations are also briefly discussed and shown to seed a cooperative interaction among cellular units, competing for the same oxygen reservoir.
Assuntos
Modelos Biológicos , Neoplasias/patologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Comunicação Celular , Morte Celular , Hipóxia Celular , Linhagem Celular Tumoral , Simulação por Computador , Humanos , Processos Estocásticos , Fatores de Tempo , Ensaio Tumoral de Célula-TroncoRESUMO
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.
Assuntos
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
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.