RESUMEN
Stabilization of p53 in erythroid precursors in response to nucleosomal stress underlies the hypoplastic anemia in myelodysplastic syndromes (MDS) with chromosome 5q deletion [del(5q)]. We investigated whether cenersen, a clinically active 20-mer antisense oligonucleotide complementary to TP53 exon10, could suppress p53 expression and restore erythropoiesis in del(5q) MDS. Cenersen treatment of ribosomal protein S-14-deficient erythroblasts significantly reduced cellular p53 and p53-up-regulated modulator of apoptosis expression compared with controls, accompanied by a significant reduction in apoptosis and increased cell proliferation. In a two-stage erythroid differentiation assay, cenersen significantly suppressed nuclear p53 in bone marrow CD34+ cells isolated from patients with del(5q) MDS, whereas erythroid burst recovery increased proportionally to the magnitude of p53 suppression without evidence of del(5q) clonal suppression (r = -0.6; P = 0.005). To explore the effect of p53 suppression on erythropoiesis in vivo, dexamethasone, a glucocorticoid receptor-dependent p53 antagonist, was added to lenalidomide treatment in eight lower-risk, transfusion-dependent, del(5q) MDS patients with acquired drug resistance. Transfusion independence was restored in five patients accompanied by expansion of erythroid precursors and decreased cellular p53 expression. We conclude that targeted suppression of p53 could support effective erythropoiesis in lenalidomide-resistant del(5q) MDS.
Asunto(s)
Eritropoyesis/efectos de los fármacos , Síndromes Mielodisplásicos/metabolismo , Oligonucleótidos/farmacología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Secuencia de Bases , Dexametasona , Resistencia a Medicamentos/fisiología , Células Precursoras Eritroides/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Lenalidomida , Datos de Secuencia Molecular , Síndromes Mielodisplásicos/genética , Oligonucleótidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Estadísticas no Paramétricas , Talidomida/análogos & derivados , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. We observed that SMAD7, a negative regulator of transforming growth factor-beta (TGF-ß) receptor-I kinase, is markedly reduced in MDS and leads to ineffective hematopoiesis by overactivation of TGF-ß signaling. To determine the cause of SMAD7 reduction in MDS, we analyzed the 3'UTR of the gene and determined that it contains a highly conserved putative binding site for microRNA-21. We observed significantly elevated levels of miR-21 in MDS marrow samples when compared with age-matched controls. miR-21 was shown to directly bind to the 3'UTR of SMAD7 and reduce its expression in hematopoietic cells. Next, we tested the role of miR-21 in regulating TGF-ß signaling in a TGF-ß-overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with a chemically modified miR-21 inhibitor led to significant increases in hematocrit and led to an increase in SMAD7 expression in vivo. Inhibition of miR-21 also led to an increase in erythroid colony formation from primary MDS bone marrow progenitors, demonstrating its ability in stimulating hematopoiesis in vitro. Taken together, these studies demonstrate the role of miR-21 in regulating overactivated TGF-ß signaling in MDS.
Asunto(s)
Hematopoyesis/genética , MicroARNs/genética , Síndromes Mielodisplásicos/genética , Transducción de Señal/genética , Factor de Crecimiento Transformador beta1/genética , Regiones no Traducidas 3'/genética , Anciano , Anciano de 80 o más Años , Animales , Sitios de Unión/genética , Células de la Médula Ósea/metabolismo , Línea Celular , Células Cultivadas , Femenino , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células K562 , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Persona de Mediana Edad , Mutación , Síndromes Mielodisplásicos/metabolismo , Proteína smad7/genéticaRESUMEN
BACKGROUND: Circulating tumor cells (CTCs) are an important diagnostic tool for understanding the metastatic process and the development of cancer. METHODS: This review covers the background, relevance, and potential limitations of CTCs as a measurement of cancer progression and how information derived from CTCs may affect treatment efficacy. It also highlights the difficulties of characterizing these rare cells due to the limited cell surface molecules unique to CTCs and each particular type of cancer. RESULTS: The analysis of cancer in real time, through the measure of the number of CTCs in a " liquid" biopsy specimen, gives us the ability to monitor the therapeutic efficacy of treatments and possibly the metastatic potential of a tumor. CONCLUSIONS: Through novel and innovative techniques yielding encouraging results, including microfluidic techniques, isolating and molecularly analyzing CTCs are becoming a reality. CTCs hold promise for understanding how tumors work and potentially aiding in their demise.
Asunto(s)
Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Biomarcadores de Tumor , Separación Celular , Transición Epitelial-Mesenquimal/fisiología , Humanos , Técnicas Analíticas Microfluídicas , Técnicas de Amplificación de Ácido NucleicoRESUMEN
We examined whether lenalidomide exposure up-regulates miRNAs and mRNAs, previously shown to play a role in the disease phenotype of del(5q) myelodysplastic syndrome, in pre-treatment CD34(+) marrow cells. We hypothesized that increased expression would predict for clinical response. Changes in miR-143, miR-145, miR-146a, miR-146b, miR-378, miR-584, SPARC and RPS14 were examined in del(5q) (n=10) and non-del(5q) (n=18) myelodysplastic syndrome patient samples. Significantly increased expression of miR-143 (1.8-fold and 1.5-fold in del(5q) and non-del(5q), respectively), and miR-145 (1.9-fold and 1.6-fold in del(5q) and non-del(5q), respectively) was observed. In the del(5q) myelodysplastic syndrome cohort, transfusion independence correlated with a 1.3-fold or more increase in miR-145 expression and response over 12 months correlated with a 1.5-fold or more increase. Knockdown of miR-143 and miR-145 in cord blood CD34(+) cells resulted in increased erythroid progenitor activity. Lenalidomide selectively abrogated progenitor activity in cells depleted of miR-143 and miR-145 supporting a key role for miR-143/145 in the sensitivity to lenalidomide of del(5q) myelodysplastic syndrome patients.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 5 , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , MicroARNs/genética , Síndromes Mielodisplásicos/genética , Talidomida/análogos & derivados , Animales , Antígenos CD34/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Lenalidomida , Ratones , Síndromes Mielodisplásicos/tratamiento farmacológico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Talidomida/farmacología , Talidomida/uso terapéutico , Resultado del TratamientoRESUMEN
BACKGROUND: Leptomeningeal disease (LMD) occurs as a late complication of several human cancers and has no rationally designed treatment options. A major barrier to developing effective therapies for LMD is the lack of cell-based or preclinical models that recapitulate human disease. Here, we describe the development of in vitro and in vivo cultures of patient-derived cerebrospinal fluid circulating tumor cells (PD-CSF-CTCs) from patients with melanoma as a preclinical model to identify exploitable vulnerabilities in melanoma LMD. METHODS: CSF-CTCs were collected from melanoma patients with melanoma-derived LMD and cultured ex vivo using human meningeal cell-conditioned media. Using immunoassays and RNA-sequencing analyses of PD-CSF-CTCs, molecular signaling pathways were examined and new therapeutic targets were tested for efficacy in PD-CSF-CTCs preclinical models. RESULTS: PD-CSF-CTCs were successfully established both in vitro and in vivo. Global RNA analyses of PD-CSF-CTCs revealed several therapeutically tractable targets. These studies complimented our prior proteomic studies highlighting IGF1 signaling as a potential target in LMD. As a proof of concept, combining treatment of ceritinib and trametinib in vitro and in vivo demonstrated synergistic antitumor activity in PD-CSF-CTCs and BRAF inhibitor-resistant melanoma cells. CONCLUSIONS: This study demonstrates that CSF-CTCs can be grown in vitro and in vivo from some melanoma patients with LMD and used as preclinical models. These models retained melanoma expression patterns and had signaling pathways that are therapeutically targetable. These novel models/reagents may be useful in developing rationally designed treatments for LMD.
Asunto(s)
Melanoma , Neoplasias Meníngeas , Células Neoplásicas Circulantes , Medios de Cultivo Condicionados , Humanos , Melanoma/patología , Neoplasias Meníngeas/patología , Proteómica , Proteínas Proto-Oncogénicas B-raf/genética , ARNRESUMEN
The myelodysplastic syndromes (MDS) display both haematological and biological heterogeneity with variable leukaemia potential. MicroRNAs play an important role in tumour suppression and the regulation of self-renewal and differentiation of haematopoietic progenitors. Using a microarray platform, we evaluated microRNA expression from 44 patients with MDS and 17 normal controls. We identified a thirteen microRNA signature with statistically significant differential expression between normal and MDS specimens (P < 0·01), including down-regulation of members of the leukaemia-associated MIRLET7 family. A unique signature consisting of 10 microRNAs was closely associated with International Prognostic Scoring System (IPSS) risk category permitting discrimination between lower (Low/Intermediate-1) and higher risk (Intermediate-2/High) disease (P < 0·01). Selective overexpression of MIR181 family members was detected in higher risk MDS, indicating pathogenetic overlap with acute myeloid leukaemia. Survival analysis of an independent cohort of 22 IPSS lower risk MDS patients revealed a median survival of 3·5 years in patients with high expression of MIR181 family compared to 9·3 years in patients with low MIR181 expression (P = 0·002). Our pilot study suggested that analysis of microRNA expression profile offers diagnostic utility, and provide pathogenetic and prognostic discrimination in MDS.
Asunto(s)
MicroARNs/genética , Síndromes Mielodisplásicos/genética , Anciano , Anciano de 80 o más Años , Progresión de la Enfermedad , Métodos Epidemiológicos , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Hematopoyesis/genética , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/diagnóstico , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
A risk mitigation strategy was implemented to determine if a higher prophylactic voriconazole dosage in patients with CYP2C19 rapid metabolizer neutropenic acute myeloid leukemia (AML) reduces the incidence of subtherapeutic trough concentrations. Patients with AML (n = 263) were preemptively genotyped for CYP2C19*2, *3, and *17 alleles as part of a single-center prospective, interventional, quality improvement study. CYP2C19 rapid metabolizers (CYP2C19*1/*17) were recommended to receive interventional voriconazole 300 mg twice daily, ultrarapid metabolizers (CYP2C19*17/*17) were recommended to avoid voriconazole, and all others received the standard prophylactic dosage of 200 mg twice daily. In this real-world setting, 202 patients (76.8%) were prescribed prophylactic voriconazole, and of these patients 176 (87.1%) received CYP2C19-guided prophylactic dosing. Voriconazole trough concentrations were obtained for 41 of the 58 (70.7%) CYP2C19 rapid metabolizers prescribed prophylactic voriconazole. Interventional voriconazole resulted in higher plasma trough concentrations (median 2.7 µg/mL) compared with the standard prophylactic dosage (median 0.6 µg/mL; P = 0.001). Subtherapeutic concentrations were avoided in 83.8% of CYP2C19 rapid metabolizers receiving interventional dosage compared to 46.2% receiving standard dosage (P = 0.02). CYP2C19 genotyping to preemptively guide prophylactic voriconazole dosing is feasible and may be a potential strategy for reducing the risk of subtherapeutic trough concentrations that potentiate breakthrough fungal infections.
Asunto(s)
Antifúngicos/administración & dosificación , Citocromo P-450 CYP2C19/genética , Leucemia Mieloide Aguda/complicaciones , Micosis/prevención & control , Voriconazol/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Antifúngicos/farmacocinética , Relación Dosis-Respuesta a Droga , Femenino , Genotipo , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Neutropenia/etiología , Estudios Prospectivos , Gestión de Riesgos , Voriconazol/farmacocinética , Adulto JovenRESUMEN
In a subset of patients with non-del(5q) myelodysplastic syndrome (MDS), lenalidomide promotes erythroid lineage competence and effective erythropoiesis. To determine the mechanism by which lenalidomide promotes erythropoiesis, we investigated its action on erythropoietin receptor (EpoR) cellular dynamics. Lenalidomide upregulated expression and stability of JAK2-associated EpoR in UT7 erythroid cells and primary CD71+ erythroid progenitors. The effects of lenalidomide on receptor turnover were Type I cytokine receptor specific, as evidenced by coregulation of the IL3-Rα receptor but not c-Kit. To elucidate this mechanism, we investigated the effects of lenalidomide on the E3 ubiquitin ligase RNF41. Lenalidomide promoted EpoR/RNF41 association and inhibited RNF41 auto-ubiquitination, accompanied by a reduction in EpoR ubiquitination. To confirm that RNF41 is the principal target responsible for EpoR stabilization, HEK293T cells were transfected with EpoR and/or RNF41 gene expression vectors. Steady-state EpoR expression was reduced in EpoR/RNF41 cells, whereas EpoR upregulation by lenalidomide was abrogated, indicating that cellular RNF41 is a critical determinant of drug-induced receptor modulation. Notably, shRNA suppression of CRBN gene expression failed to alter EpoR upregulation, indicating that drug-induced receptor modulation is independent of cereblon. Immunohistochemical staining showed that RNF41 expression decreased in primary erythroid cells of lenalidomide-responding patients, suggesting that cellular RNF41 expression merits investigation as a biomarker for lenalidomide response. Our findings indicate that lenalidomide has E3 ubiquitin ligase inhibitory effects that extend to RNF41 and that inhibition of RNF41 auto-ubiquitination promotes membrane accumulation of signaling competent JAK2/EpoR complexes that augment Epo responsiveness. Cancer Res; 76(12); 3531-40. ©2016 AACR.
Asunto(s)
Receptores de Eritropoyetina/efectos de los fármacos , Talidomida/análogos & derivados , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Células Cultivadas , Humanos , Janus Quinasa 2/fisiología , Lenalidomida , Péptido Hidrolasas/fisiología , Receptores de Eritropoyetina/análisis , Talidomida/farmacología , Ubiquitina-Proteína Ligasas/fisiología , UbiquitinaciónRESUMEN
Uveal melanoma is a rare subtype of melanoma, accounting for only 3-5% of all melanoma cases in the USA. Although fewer than 4% of uveal melanoma patients present with metastasis at diagnosis, approximately half will develop metastasis, more than 90% of which disseminate to the liver. Infrequently, a number of malignancies can lead to leptomeningeal metastases, a devastating and terminal complication. In this case report, we present an exceedingly rare case of a patient with uveal melanoma who developed leptomeningeal carcinomatosis as the sole site of metastasis. After conventional methods to diagnose leptomeningeal carcinomatosis fell short, a diagnosis was confirmed on the basis of identification and genomic analysis of melanoma circulating tumor cells in the cerebrospinal fluid.
Asunto(s)
Melanoma/complicaciones , Carcinomatosis Meníngea/etiología , Neoplasias Cutáneas/complicaciones , Neoplasias de la Úvea/complicaciones , Femenino , Humanos , Melanoma/patología , Persona de Mediana Edad , Neoplasias Cutáneas/patología , Neoplasias de la Úvea/patologíaRESUMEN
The application of in vitro and in vivo ATP bioluminescence systems as an integrated approach for preclinical research and development of new chemotherapeutic drugs is described. This approach includes both (a) the in vitro tumor response assay (TRA) system that utilizes new technologies for cell culture and ATP measurement of clinical specimens and (b) the use of human tumor cell lines transfected with Photinus pyralis luciferase (luc) gene for both in vitro and in vivo studies. Dried reagent microplates for TRA culture and counting procedures are described for a two-stage TRA method, which can be used to evaluate drug sensitivity and resistance of cells from clinical specimens after initial drug exposure in vitro. The use of dried reagent counting plates for screening and testing of agents against tumor cell lines is described, as well as an alternative method for screening and testing chemotherapeutic drugs in vitro with luc-transfected human tumor cell lines. The potential application of luc-transfected reporter cell lines for in vivo studies of drug activity with photon imaging for analysis is discussed.
Asunto(s)
Adenosina Trifosfato/metabolismo , Antineoplásicos/farmacología , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Luciferasas/metabolismo , Mediciones Luminiscentes , Células Tumorales Cultivadas/efectos de los fármacos , Animales , Anticuerpos Monoclonales/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Diagnóstico por Imagen , Ensayos de Selección de Medicamentos Antitumorales/estadística & datos numéricos , Femenino , Humanos , Luciferasas/genética , Ratones , Ratones Desnudos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fotones , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Células Tumorales Cultivadas/trasplanteRESUMEN
A chemically synthesized 34-amino-acid peptide and a select analog have been studied to determine their activities against the growth of prostate and breast cancer tumors. It was of interest to determine if the peptide has anti-prostate cancer activity. Previously, the peptide was shown to inhibit the growth of breast cancer tumor cells. The peptide inhibited the growth of both breast and prostate tumors. A novel experimental design for the peptide was in a study in which a time-release pellet was used to give daily peptide doses to mice that were subjected to a breast cancer tumor. The peptide was effective in inhibiting the growth of tumors in the mice. The 2 C-->2 A analog peptide, in which the two cysteines were replaced by alanines, was also active in inhibition of the growth of prostate and breast cancer cell lines and in an in vivo assay against breast cancer. A scrambled amino acid sequence of the peptide was used as a control in these tumor studies, and it had virtually no anti-cancer activity.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , alfa-Fetoproteínas/farmacología , Secuencia de Aminoácidos , Animales , Antineoplásicos/administración & dosificación , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Implantes de Medicamentos , Femenino , Inhibidores de Crecimiento/administración & dosificación , Inhibidores de Crecimiento/farmacología , Humanos , Masculino , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Fragmentos de Péptidos/administración & dosificación , Neoplasias de la Próstata/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , alfa-Fetoproteínas/administración & dosificaciónRESUMEN
Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (pâ=â0.005, raft number; pâ=â0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.
Asunto(s)
Células Precursoras Eritroides/efectos de los fármacos , Factores Inmunológicos/farmacología , Microdominios de Membrana/metabolismo , Receptores de Eritropoyetina/metabolismo , Talidomida/análogos & derivados , Actinas/metabolismo , Anciano , Anciano de 80 o más Años , Amidas/farmacología , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Células Precursoras Eritroides/fisiología , Femenino , Humanos , Lenalidomida , Masculino , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/patología , Multimerización de Proteína/efectos de los fármacos , Piridinas/farmacología , Transducción de Señal , Talidomida/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units.
Asunto(s)
Microdominios de Membrana/metabolismo , Receptores de Eritropoyetina/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Humanos , Microdominios de Membrana/efectos de los fármacos , Fosfoproteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/antagonistas & inhibidoresRESUMEN
Transmembrane drug export mediated by the ATP-binding cassette (ABC) transporter P-glycoprotein contributes to clinical resistance to antineoplastics. In this study, we identified the substituted quinoline HG-829 as a novel, noncompetitive, and potent P-glycoprotein inhibitor that overcomes in vitro and in vivo drug resistance. We found that nontoxic concentrations of HG-829 restored sensitivity to P-glycoprotein oncolytic substrates. In ABCB1-overexpressing cell lines, HG-829 significantly enhanced cytotoxicity to daunorubicin, paclitaxel, vinblastine, vincristine, and etoposide. Coadministration of HG-829 fully restored in vivo antitumor activity of daunorubicin in mice without added toxicity. Functional assays showed that HG-829 is not a Pgp substrate or competitive inhibitor of Pgp-mediated drug efflux but rather acts as a noncompetitive modulator of P-glycoprotein transport function. Taken together, our findings indicate that HG-829 is a potent, long-acting, and noncompetitive modulator of P-glycoprotein export function that may offer therapeutic promise for multidrug-resistant malignancies.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Quinolinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Interacciones Farmacológicas , Resistencia a Antineoplásicos , Femenino , Células HEK293 , Humanos , Células K562 , Ratones , Ratones SCIDAsunto(s)
Enfermedad de las Cadenas Pesadas/complicaciones , Enfermedad de las Cadenas Pesadas/diagnóstico , Leucemia Linfocítica Granular Grande/complicaciones , Leucemia Linfocítica Granular Grande/diagnóstico , Esplenomegalia/diagnóstico , Anciano , Femenino , Humanos , Imagen Multimodal , Tomografía de Emisión de Positrones , Bazo/patología , Tomografía Computarizada por Rayos XRESUMEN
The DExD/H box RNA helicase retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) are key intracellular receptors that recognize virus infection to produce type I IFN. A third helicase gene, Lgp2, is homologous to Rig-I and Mda5 but lacks a caspase activation and recruitment domain. We generated Lgp2-deficient mice and report that the loss of this gene greatly sensitizes cells to cytosolic polyinosinic/polycytidylic acid-mediated induction of type I IFN. However, negative feedback inhibition of IFN-beta transcription was found to be normal in the absence of LGP2, indicating that LGP2 is not the primary negative regulator of type I IFN production. Our data further indicate that Lgp2-/- mice exhibited resistance to lethal vesicular stomatitis virus infection, a virus whose replicative RNA intermediates are recognized specifically by RIG-I rather than by MDA5 to trigger the production of type I IFN. However, mice lacking LGP2 were observed to exhibit a defect in type I IFN production in response to infection by the encephalomyocarditis virus, the replication of which activates MDA5-dependent innate immune responses. Collectively, our data indicate a disparate regulatory role for LGP2 in the triggering of innate immune signaling pathways following RNA virus infection.
Asunto(s)
Infecciones por Cardiovirus/enzimología , Infecciones por Cardiovirus/prevención & control , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , Infecciones por Rhabdoviridae/enzimología , Infecciones por Rhabdoviridae/prevención & control , Animales , Infecciones por Cardiovirus/genética , Infecciones por Cardiovirus/inmunología , Células Cultivadas , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/fisiología , Virus de la Encefalomiocarditis/inmunología , Femenino , Inmunidad Innata/genética , Helicasa Inducida por Interferón IFIH1 , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular , Infecciones por Rhabdoviridae/genética , Infecciones por Rhabdoviridae/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
Studies were performed to compare green fluorescent protein (GFP)-transfected and fi re fl y luciferase (Luc)-transfected MCF-7 human breast tumour cells both in vitro and in vivo. For in vitro studies, cells were serially diluted in 96-well microplates and analysed using a NightOwl LB 981 Molecular Light Imager and a Victor multilabel reader. For in vivo studies, nude mice were injected either intraperitoneally, intravenously or subcutaneously with transfected cells and then imaged using the NightOwl Imager after intraperitoneal injection of d-luciferin for Luc tumours, or excitation at 470 nm for GFP tumours. In vitro imaging studies revealed that both GFP and Luc transfectants were quantifiable. However, the Luc-transfected cells were detectable at a significantly lower concentration compared to GFP transfectants. In vivo studies demonstrated that GFP-transfected tumours were detectable as subcutaneous and intraperitoneal tumours but not as deep tissue lesions, whereas Luc-transfected tumours were detectable as subcutaneous and intraperitoneal tumours and as deep tissue lesions resulting from intraperitoneal or intravenous inoculation. These findings demonstrate that GFP-transfected cells may be useful for imaging studies of superficial tumours where both excitation and emission wavelengths are able to penetrate tissues, whereas luciferase-transfected cells appear superior for imaging studies of primary and metastatic tumours in distant sites and deep tissues.
Asunto(s)
Diagnóstico por Imagen/métodos , Luciferasas , Proteínas Luminiscentes , Neoplasias Experimentales/patología , Animales , Línea Celular Tumoral , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Luciferasas/genética , Proteínas Luminiscentes/genética , Ratones , Ratones Desnudos , Neoplasias Experimentales/diagnóstico , Transfección , Trasplante HeterólogoRESUMEN
Human DU-145 prostate and MCF-7 breast tumor cell lines were stably transfected with plasmid pcDNA3.1-Luc expressing firefly luciferase. Studies were performed with the transfected cell lines to evaluate luminescent imaging for measuring the efficacy of anti-cancer agents. In vitro experiments demonstrated a dose response of both cell lines to topotecan (Hycamtin) with an IC50 of 0.013 microM for MCF-7 Luc cells and 0.002 microM for DU-145 Luc cells. In vivo imaging experiments were performed using athymic nude mice inoculated i.p. with 5 x 10(6) MCF-7 cells or s.c. with 5 x 10(6) DU-145 cells and then treated with topotecan at 2.5 mg/kg body weight. Tumor progression and regression were monitored for 27 days. Animals inoculated s.c. with DU-145 Luc cells and then treated with topotecan demonstrated significant tumor growth and regression as measured with calipers and luminescent imaging. High correlation was observed between caliper and imaging results. The correlation coefficient was 0.75 for the control untreated group and 0.93 for the topotecan-treated group. Similarly, tumor progression and regression were measurable using luminescent imaging for untreated and topotecan-treated mice inoculated i.p. with MCF-7 Luc cells. These data indicate that luminescent imaging is a useful tool for evaluating anti-cancer drugs in vivo and may prove to be particularly useful for the development of novel agents. Luminescent imaging could also be used to locate and harvest residual tumors in drug-treated animals in order to study mechanisms of drug resistance.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Luciferasas , Neoplasias de la Próstata/tratamiento farmacológico , Topotecan/uso terapéutico , Animales , Femenino , Humanos , Masculino , Ratones , Resultado del Tratamiento , Células Tumorales CultivadasRESUMEN
Hembras adultas de cachama C. macropomum en la etapa de desove, fueron inyectadas intraperitonealmente con 5 mg/Kg de peso corporal de extracto hipofisiario (EH) de cachama obtenido de ejemplares adultos capturados en el príodo de predesove sólo o en combinación con un análogo de la hormona liberadora de la hormona luteinizante ([desGLI10D-Ala6] LHRH etilamida) (LHRH-A) (50 *g/kg de peso corporal), administrada 12 horas después de la administración de EH. La administración de EH produjo el desove en 2 de los 5 animales tratados. Una inyección de EH seguida 12 horas más tarde de LHRH-A causó un 60%de desove. La calidad de los huevos fué mejor en los animales inyectados con EH que en los tratados con la combinación de EH+LHRH-A