RESUMEN
Transient Receptor Potential Melastatin 8 (TRPM8) from the melastatin TRP channel subfamily is a non-selective Ca2+-permeable ion channel with multimodal gating which can be activated by low temperatures and cooling compounds, such as menthol and icilin. Different conditions such as neuropathic pain, cancer, overactive bladder syndrome, migraine, and chronic cough have been linked to the TRPM8 mode of action. Despite the several potent natural and synthetic inhibitors of TRPM8 that have been identified, none of them have been approved for clinical use. The aim of this study was to discover novel blocking TRPM8 agents using automated patch clamp electrophysiology combined with a ligand-based virtual screening based on the SwissSimilarity platform. Among the compounds we have tested, nebivolol and carvedilol exhibited the greatest inhibitory effect, with an IC50 of 0.97 ± 0.15 µM and 9.1 ± 0.6 µM, respectively. This study therefore provides possible candidates for future drug repurposing and suggests promising lead compounds for further optimization as inhibitors of the TRPM8 ion channel.
RESUMEN
hERG1 potassium channels are widely expressed in human cancers of different origins, where they affect several key aspects of cellular behaviour. The present study was designed to evaluate the expression and clinical relevance of hERG1 protein in cancer tissues from patients suffering from neuroendocrine tumours (NETs) of ileal (iNETs) and pancreatic (pNETs) origin, with available clinicopathological history and follow-up. The study was carried out by immunohistochemistry with an anti-hERG1 monoclonal antibody. In a subset of samples, a different antibody directed against the hERG1/ß1 integrin complex was also used. The analysis showed for the first time that hERG1 is expressed in human NETs originating from either the ileum or the pancreas. hERG1 turned out to have a prognostic value in NETs, showing (i) a statistically significant positive impact on OS of patients affected by ileal NETs, regardless the TNM stage; (ii) a statistically significant positive impact on OS of patients affected by aggressive (TNM stage IV) disease, either ileal or pancreatic; (iii) a trend to a negative impact on OS of patients affected by less aggressive (TNM stage I-III) disease, either ileal or pancreatic. Moreover, in order to evaluate whether ERG1 was functionally expressed in a cellular model of pNET, the INS1E rat insulinoma cell line was used, and it emerged that blocking ERG1 with a specific inhibitor of the channel (E4031) turned out in a significant reduction in cell proliferation.
Asunto(s)
Canales de Potasio Éter-A-Go-Go , Tumores Neuroendocrinos , Animales , Anticuerpos Monoclonales/metabolismo , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Íleon/metabolismo , Integrina beta1/metabolismo , Páncreas/metabolismo , Pronóstico , RatasRESUMEN
In the present work, applying the whole-cell patch-clamp technique in voltage clamp mode, we have investigated the effects of different drugs, such as riluzole, Psora-4 and Tram-34, on the potassium currents in four human lymphoma cell lines. We focused on outward currents mediated by two potassium channels (Kv1.3 and KCa3.1), which are known to play a key physiological role in lymphoid cells. The currents were evoked by voltage ramps ranging from -120â¯mV to +40â¯mV and the conductance of the two potassium channels was measured between +20â¯mV and +40â¯mV, both in the absence and in the presence of the specific blockers Psora-4 (Kv1.3; 1⯵M) and Tram-34 (KCa3.1; 1⯵M). The effect of the latter was tested after KCa3.1 channels were activated by riluzole 10⯵M. Taken together, these data could be useful as an indication of the functional characteristics of the potassium channels in human lymphomas and represent a starting point for the study of potassium conductance in cellular models of these tumors.
RESUMEN
Many inflammation-associated diseases, including cancers, increase in women after menopause and with obesity. In contrast to anti-inflammatory actions of 17ß-estradiol, we find estrone, which dominates after menopause, is pro-inflammatory. In human mammary adipocytes, cytokine expression increases with obesity, menopause, and cancer. Adipocyte:cancer cell interaction stimulates estrone- and NFκB-dependent pro-inflammatory cytokine upregulation. Estrone- and 17ß-estradiol-driven transcriptomes differ. Estrone:ERα stimulates NFκB-mediated cytokine gene induction; 17ß-estradiol opposes this. In obese mice, estrone increases and 17ß-estradiol relieves inflammation. Estrone drives more rapid ER+ breast cancer growth in vivo. HSD17B14, which converts 17ß-estradiol to estrone, associates with poor ER+ breast cancer outcome. Estrone and HSD17B14 upregulate inflammation, ALDH1 activity, and tumorspheres, while 17ß-estradiol and HSD17B14 knockdown oppose these. Finally, a high intratumor estrone:17ß-estradiol ratio increases tumor-initiating stem cells and ER+ cancer growth in vivo. These findings help explain why postmenopausal ER+ breast cancer increases with obesity, and offer new strategies for prevention and therapy.
Asunto(s)
Neoplasias de la Mama/metabolismo , Estrógenos/metabolismo , Inflamación/metabolismo , Obesidad/metabolismo , Posmenopausia/metabolismo , Premenopausia/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Several studies suggest that an increase of glutathione (GSH) through activation of the transcriptional nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the dopaminergic neurons may be a promising neuroprotective strategy in Parkinson's disease (PD). Among Nrf2 activators, isothiocyanate sulforaphane (SFN), derived from precursor glucosinolate present in Brassica vegetables, has gained attention as a potential neuroprotective compound. Bioavailability studies also suggest the contribution of SFN metabolites, including erucin (ERN), to the neuroprotective effects of SFN. Therefore, we compared the in vitro neuroprotective effects of SFN and ERN at the same dose level (5 µM) and oxidative treatment with 6-hydroxydopamine (6-OHDA) in SH-SY5Y cells. The pretreatment of SH-SY5Y cells with SFN recorded a higher (p < 0.05) active nuclear Nrf2 protein (12.0 ± 0.4 vs 8.0 ± 0.2 fold increase), mRNA Nrf2 (2.0 ± 0.3 vs 1.4 ± 0.1 fold increase), total GSH (384.0 ± 9.0 vs 256.0 ± 8.0 µM) levels, and resistance to neuronal apoptosis elicited by 6-OHDA compared to ERN. By contrast, the simultaneous treatment of SH-SY5Y cells with either SFN or ERN and 6-OHDA recorded similar neuroprotective effects with both the isothiocyanates (Nrf2 protein 2.2 ± 0.2 vs 2.1 ± 0.1 and mRNA Nrf2 2.1 ± 0.3 vs 1.9 ± 0.2 fold increase; total GSH 384.0 ± 4.8 vs 352.0 ± 6.4 µM). Finally, in vitro finding was confirmed in a 6-OHDA-PD mouse model. The metabolic oxidation of ERN to SFN could account for their similar neuroprotective effects in vivo, raising the possibility of using vegetables containing a precursor of ERN for systemic antioxidant benefits in a similar manner to SFN.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Isotiocianatos/farmacología , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/prevención & control , Sulfuros/farmacología , Tiocianatos/farmacología , Animales , Brassica/química , Línea Celular Tumoral , Neuronas Dopaminérgicas/química , Glutatión/análisis , Humanos , Isotiocianatos/metabolismo , Isotiocianatos/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/análisis , Factor 2 Relacionado con NF-E2/genética , Neuroblastoma , Fármacos Neuroprotectores/uso terapéutico , Oxidación-Reducción , Oxidopamina/administración & dosificación , ARN Mensajero/análisis , Sulfuros/metabolismo , Sulfuros/uso terapéutico , Sulfóxidos , Tiocianatos/metabolismo , Tiocianatos/uso terapéuticoRESUMEN
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.
Asunto(s)
Cisplatino/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Canal de Potasio ERG1/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Animales , Apoptosis/efectos de los fármacos , Benzotiazoles/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacocinética , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Canal de Potasio ERG1/metabolismo , Células HCT116 , Células HT29 , Humanos , Concentración 50 Inhibidora , Ratones , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacología , Riluzol/farmacologíaRESUMEN
OBJECTIVES: Circulating endothelial progenitor cells (cEPCs) have been reported to be dysfunctional in diabetes mellitus (DM) patients, accounting for the vascular damage and the ensuing high risk for cardiovascular disease (CVD) characteristic of this disease. The aim of the present study was to evaluate the number of circulating cEPCs in type 1 DM (T1DM) patients, without clinical vascular damage, of different ages and with different disease duration. METHODS: An observational, clinical-based prospective study was performed on T1DM patients enrolled in two clinical centers. cEPCs were determined by flow cytometry, determining the number of CD34/CD133/VEGFR2-positive cells within peripheral blood mononuclear cells (PBMCs). RESULTS: The number of cEPCs was lower in adult T1DM patients, whilst higher in childhood/young patients, compared to controls of the same age range. When patients were grouped into two age groups (≥ or <20 years) (and categorized on the basis of the duration of the disease), the number of cEPCs in young (<20 years) patients was higher compared with older subjects, regardless of disease duration. A subset of patients with very high cEPCs was identified in the <20 years group. CONCLUSION: There is an association between the number of cEPCs and patients' age: childhood/young T1DM patients have significantly higher levels of cEPCs, respect to adult T1DM patients. Such difference is maintained also when the disease lasts for more than 10 years. The very high levels of cEPCs, identified in a subset of childhood/young patients, might protect vessels against endothelial dysfunction and damage. Such protection would be less operative in older subjects, endowed with lower cEPC numbers, in which complications are known to develop more easily.
RESUMEN
Sirtuins are NADâº-dependent deacetylases, lipoamidases, and ADP-ribosyltransferases that link cellular metabolism to multiple intracellular pathways that influence processes as diverse as cell survival, longevity, and cancer growth. Sirtuins influence the extent of neuronal death in stroke. However, different sirtuins appear to have opposite roles in neuronal protection. In Caenorhabditis elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the protective effect of sir-2.3 knock-out is enhanced by block of glycolysis and eliminated by a null mutation in daf-16/FOXO transcription factor, supporting the involvement of the insulin/IGF pathway. However, data in Caenorhabditis elegans cell culture suggest that the effects of sir-2.3 knock-out act downstream of the DAF-2/IGF-1 receptor. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under ischemic conditions in dietary deprivation (DD), but to a lesser extent than in wild type, suggesting more robust activation of a ROS scavenging system in this mutant in the absence of food. This work suggests a deleterious role of SIRT4 during ischemic processes in mammals that must be further investigated and reveals a novel pathway that can be targeted for the design of therapies aimed at protecting neurons from death in ischemic conditions.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Factores de Transcripción Forkhead/genética , Hidrolasas/genética , Isquemia/genética , Proteínas Mitocondriales/genética , Células Receptoras Sensoriales/metabolismo , Sirtuinas/genética , Animales , Azidas/toxicidad , Caenorhabditis elegans/genética , Muerte Celular/efectos de los fármacos , Técnicas de Inactivación de Genes , Glucólisis/efectos de los fármacos , Humanos , Insulina/genética , Isquemia/patología , Mitocondrias/genética , Mitocondrias/patología , Necrosis/genética , Necrosis/patología , Especies Reactivas de Oxígeno/metabolismo , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
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.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/metabolismo , Integrina beta1/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Canales de Potasio Éter-A-Go-Go/química , Canales de Potasio Éter-A-Go-Go/genética , Transferencia Resonante de Energía de Fluorescencia , Células HCT116 , Células HEK293 , Humanos , Immunoblotting , Integrina beta1/química , Integrina beta1/genética , Ratones Desnudos , Ratones SCID , Microscopía Confocal , Neoplasias/genética , Neoplasias/patología , Unión Proteica , Conformación Proteica , Trasplante HeterólogoRESUMEN
Epidermal growth factor receptor inhibitors (EGFR-TKIs) represent a class of compounds widely used in anticancer therapy. An increasing number of studies reports on combination therapies in which the block of the EGFR-TK activity is associated with inhibition of its downstream pathways, as PI3K-Akt. Sulforaphane targets the PI3K-Akt pathway whose dysregulation is implicated in many functions of cancer cells. According to these considerations, a series of multitarget molecules have been designed by combining key structural features derived from an EGFR-TKI, PD168393, and the isothiocyanate sulforaphane. Among the obtained molecules 1-6, compound 6 emerges as a promising lead compound able to exert antiproliferative and proapoptotic effects in A431 epithelial cancer cell line by covalently binding to EGFR-TK, and reducing the phosphorylation of Akt without affecting the total Akt levels.
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Antineoplásicos/química , Receptores ErbB/antagonistas & inhibidores , Isotiocianatos/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Quinazolinas/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Fosfatidilinositol 3-Quinasas , SulfóxidosRESUMEN
We defined the stem cell profile of K562 line, demonstrating the expression of the Embryonic Transcription Factors Oct3/4, Sox2, Klf4 and Nanog. This profile was associated with a high vulnerability to the physiological oxidizable substrate pyruvate. remarkably, this substrate was shown to be innocuous, even at the highest doses, to normal differentiated cells. This vulnerability is based on a complex metabolic trim centered on the cellular redox state expressed by the NADP/NADPH ratio geared by the mitochondrial respiratory chain. Flow cytometry revealed that the inhibition of this chain by antimycin A produced cell accumulation in the S phase of cell cycle and apoptosis. This block negatively interferes with the aerobic synthesis of purines, without affecting the anaerobic synthesis of pyrimidines. This imbalance was reproduced by using two antifolate agents, LY309887 and raltitrexed (TDX), inhibitors of purine or pyrimidine synthesis, respectively. All this revealed the apparent paradox that low doses of TDX stimulated, instead of inhibiting, leukemia cell growth. This paradox might have significant impact on therapy with regard to the effects of TDX during the intervals of administration, when the drug concentrations become so low as to promote maintenance of dormant cancer cells in hypoxic tissue niches.
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Antineoplásicos/farmacología , Fase G1/efectos de los fármacos , Leucemia/patología , Redes y Vías Metabólicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Fase S/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Antagonistas del Ácido Fólico/farmacología , Humanos , Células K562 , Factor 4 Similar a Kruppel , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Piruvatos/farmacología , Células Tumorales CultivadasRESUMEN
OBJECTIVES: The role of the lymphatic system in the connection between spondyloarthritis (SpA) and Crohn's disease (CD) remains yet to be elucidated. The aim of the present study was to investigate the circulating levels of lymphatic endothelial progenitor cells (LEPCs) and vascular endothelial growth factor-C (VEGF-C) and their possible correlation with clinical parameters in SpA, SpA associated with CD (SC), and CD. METHODS: Peripheral blood samples from SpA (n=36), SC (n=20) and CD (n=28) patients and 20 age- and sex-matched healthy controls were collected and used for quantification of circulating LEPCs and VEGF-C. LEPCs were identified by fluorescence-activated cell sorting using FITC-CD34, APC-CD133 and PE-VEGFR-3 antibodies. Serum levels of VEGF-C were measured by enzyme-linked immunosorbent assay. The possible correlations between disease duration (< or >10 years; < or >20 years) and clinical activity (BASDAI for SpA or CDAI for CD) and LEPC counts and VEGF-C levels were analysed. RESULTS: Circulating LEPC levels were significantly increased in SpA (p=0.0006) and SC (p=0.0058) patients compared with controls. In CD patients, LEPC counts negatively correlated with disease duration, with lower levels in longstanding disease (>20 years, p=0.018), but were not different from controls. No significant difference in VEGF-C levels was found in SpA, SC and CD compared with controls. Both LEPC and VEGF-C levels were independent of BASDAI and CDAI. CONCLUSIONS: On the basis of our observations, an active mobilisation of lymphatic endothelial cell precursors was observed only for spondylitis involvement.
Asunto(s)
Enfermedad de Crohn/diagnóstico , Células Progenitoras Endoteliales/patología , Endotelio Linfático/patología , Espondiloartritis/diagnóstico , Factor C de Crecimiento Endotelial Vascular/sangre , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Estudios de Casos y Controles , Recuento de Células , Separación Celular/métodos , Enfermedad de Crohn/sangre , Enfermedad de Crohn/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Estado de Salud , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Índice de Severidad de la Enfermedad , Espondiloartritis/sangre , Espondiloartritis/patología , Encuestas y Cuestionarios , Factores de TiempoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Cardiotoxinas , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Indoles/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinonas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/metabolismo , Femenino , Cobayas , Células HEK293 , Células HL-60 , Humanos , Indoles/química , Indoles/uso terapéutico , Leucemia de Células B/tratamiento farmacológico , Leucemia de Células B/metabolismo , Leucemia de Células B/patología , Masculino , Ratones , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Pirimidinas/uso terapéutico , Pirimidinonas/química , Pirimidinonas/uso terapéutico , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Histone deacetylase inhibitors (HDACi) are agents capable of inducing growth arrest and apoptosis in different tumour cell types. Previously, we reported a series of novel HDACi obtained by hybridizing SAHA or oxamflatin with 1,4-benzodiazepines. Some of these hybrids proved effective against haematological and solid cancer cells and, above all, compound (S)-8 has emerged for its activities in various biological systems. Here, we describe the effectiveness of (S)-8 against highly metastatic human A375 melanoma cells by using normal PIG1 melanocytes as control. (S)-8 prompted: acetylation of histones H3/H4 and α-tubulin; G0 /G1 and G2 /M cell cycle arrest by rising p21 and hypophos-phorylated RB levels; apoptosis involving the cleavage of PARP and caspase 9, BAD protein augmentation and cytochrome c release; decrease in cell motility, invasiveness and pro-angiogenic potential as shown by results of wound-healing assay, down-regulation of MMP-2 and VEGF-A/VEGF-R2, besides TIMP-1/TIMP-2 up-regulation; and also intracellular accumulation of melanin and neutral lipids. The pan-caspase inhibitor Z-VAD-fmk, but not the antioxidant N-acetyl-cysteine, contrasted these events. Mechanistically, (S)-8 allows the disruption of cytoplasmic HDAC6-protein phosphatase 1 (PP1) complex in A375 cells thus releasing the active PP1 that dephosphorylates AKT and blocks its downstream pro-survival signalling. This view is consistent with results obtained by: inhibiting PP1 with Calyculin A; using PPP1R2-transfected cells with impaired PP1 activity; monitoring drug-induced HDAC6-PP1 complex re-shuffling; and, abrogating HDAC6 expression with specific siRNA. Altogether, (S)-8 proved very effective against melanoma A375 cells, but not normal melanocytes, and safe to normal mice thus offering attractive clinical prospects for treating this aggressive malignancy.
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Apoptosis/efectos de los fármacos , Benzodiazepinonas/farmacología , Dibenzazepinas/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Melanoma/patología , Proteína Fosfatasa 1/metabolismo , Animales , Benzodiazepinonas/química , Caspasas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dibenzazepinas/química , Femenino , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas/química , Humanos , Masculino , Melanocitos/efectos de los fármacos , Melanocitos/patología , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , EstereoisomerismoRESUMEN
We report here that the established anticancer ruthenium(iii) complex NAMI-A induces potent and selective cytotoxic effects in a few leukaemia cell lines. These results sound very surprising after 20 years of intense studies on NAMI-A, commonly considered as a "non-cytotoxic" antimetastatic agent. In addition, evidence is given for selective inhibition of KCa 3.1 channels. The implications of these findings are discussed.
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Antineoplásicos/farmacología , Dimetilsulfóxido/análogos & derivados , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Compuestos Organometálicos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Humanos , Leucemia , Compuestos de RutenioRESUMEN
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.
Asunto(s)
Adenocarcinoma/metabolismo , Canales de Potasio Éter-A-Go-Go/metabolismo , Neoplasias Gástricas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Canal de Potasio ERG1 , Ensayo de Inmunoadsorción Enzimática , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/patología , TransfecciónRESUMEN
Despite the accumulating knowledge of alterations in pancreatic cancer molecular pathways, no substantial improvements in the clinical prognosis have been made and this malignancy continues to be a leading cause of cancer death in the Western World. The orphan nuclear receptor COUP-TFII is a regulator of a wide range of biological processes and it may exert a pro-oncogenic role in cancer cells; interestingly, indirect evidences suggest that the receptor could be involved in pancreatic cancer. The aim of this study was to evaluate the expression of COUP-TFII in human pancreatic tumors and to unveil its role in the regulation of pancreatic tumor growth. We evaluated COUP-TFII expression by immunohistochemistry on primary samples. We analyzed the effect of the nuclear receptor silencing in human pancreatic cancer cells by means of shRNA expressing cell lines. We finally confirmed the in vitro results by in vivo experiments on nude mice. COUP-TFII is expressed in 69% of tested primary samples and correlates with the N1 and M1 status and clinical stage; Kaplan-Meier and Cox regression analysis show that it may be an independent prognostic factor of worst outcome. In vitro silencing of COUP-TFII reduces the cell growth and invasiveness and it strongly inhibits angiogenesis, an effect mediated by the regulation of VEGF-C. In nude mice, COUP-TFII silencing reduces tumor growth by 40%. Our results suggest that COUP-TFII might be an important regulator of the behavior of pancreatic adenocarcinoma, thus representing a possible new target for pancreatic cancer therapy.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/patología , Factor de Transcripción COUP II/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/metabolismo , Anciano , Animales , Factor de Transcripción COUP II/biosíntesis , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones Desnudos , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/metabolismo , Pronóstico , Factor C de Crecimiento Endotelial Vascular/biosíntesis , Factor C de Crecimiento Endotelial Vascular/genética , Neoplasias PancreáticasRESUMEN
We have previously shown that peculiar metabolic features of cell adaptation and survival in hypoxia imply growth restriction points that are typical of embryonic stem cells and disappear with differentiation. Here we provide evidence that such restrictions can be exploited as specific antiblastic targets by physiological factors such as pyruvate, tetrahydrofolate, and glutamine. These metabolites act as powerful cytotoxic agents on cancer stem cells (CSCs) when supplied at doses that perturb the biochemical network, sustaining the resumption of aerobic growth after the hypoxic dormant state. Experiments were performed in vivo and in vitro using CSCs obtained from various anaplastic tumors: human melanoma, leukemia, and rat hepatoma cells. Pretreatment of melanoma CSCs with pyruvate significantly reduces their self-renewal in vitro and tumorigenicity in vivo. The metabolic network underlying the cytotoxic effect of the physiological factors was thoroughly defined, principally using AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem stage. This network, based on a tight integration of aerobic glycolysis, cellular redox state, and folate metabolism, is centered on the cellular NADP/NADPH ratio that controls the redox pathway of folate utilization in purine synthesis. On the whole, this study indicates that pyruvate, FH 4, and glutamine display anticancer activity, because CSCs are committed to survive and maintain their stemness in hypoxia. When CSC need to differentiate and proliferate, they shift from anaerobic to aerobic status, and the few mitochondria available makes them susceptible to the injury of the above physiological factors. This vulnerability might be exploited for novel therapeutic treatments.
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas Experimentales/patología , Células Madre Neoplásicas/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral/efectos de los fármacos , Ciclo del Ácido Cítrico , Glutamina/metabolismo , Glutamina/farmacología , Humanos , Leucemia/patología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/metabolismo , Melanoma/patología , Redes y Vías Metabólicas , Mitosis , NADP/metabolismo , Células Madre Neoplásicas/patología , Oxidación-Reducción , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacología , Ratas , Ratas Wistar , Tetrahidrofolatos/metabolismo , Tetrahidrofolatos/farmacologíaAsunto(s)
Antígenos CD/metabolismo , Epidermis/metabolismo , Regulación de la Expresión Génica , Inmunoglobulinas/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Células de Langerhans/metabolismo , Glicoproteínas de Membrana/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Colorantes/química , Citometría de Flujo , Humanos , Microscopía Fluorescente , Fenotipo , Antígeno CD83RESUMEN
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.