RESUMEN
Autosomal recessive whole gene deletions of nephrocystin-1 (NPHP1) result in abnormal structure and function of the primary cilia. These deletions can result in a tubulointerstitial kidney disease known as nephronophthisis and retinal (Senior-Løken syndrome) and neurological (Joubert syndrome) diseases. Nephronophthisis is a common cause of end-stage kidney disease (ESKD) in children and up to 1% of adult onset ESKD. Single nucleotide variants (SNVs) and small insertions and deletions (Indels) have been less well characterised. We used a gene pathogenicity scoring system (GenePy) and a genotype-to-phenotype approach on individuals recruited to the UK Genomics England (GEL) 100,000 Genomes Project (100kGP) (n = 78,050). This approach identified all participants with NPHP1-related diseases reported by NHS Genomics Medical Centres and an additional eight participants. Extreme NPHP1 gene scores, often underpinned by clear recessive inheritance, were observed in patients from diverse recruitment categories, including cancer, suggesting the possibility of a more widespread disease than previously appreciated. In total, ten participants had homozygous CNV deletions with eight homozygous or compound heterozygous with SNVs. Our data also reveals strong in-silico evidence that approximately 44% of NPHP1 related disease may be due to SNVs with AlphaFold structural modelling evidence for a significant impact on protein structure. This study suggests historical under-reporting of SNVS in NPHP1 related diseases compared with CNVs.
Asunto(s)
Enfermedades Renales Quísticas , Fallo Renal Crónico , Humanos , Proteínas de la Membrana/genética , Proteínas del Citoesqueleto/genética , Enfermedades Renales Quísticas/genética , Fallo Renal Crónico/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Homocigoto , Fenotipo , Nucleótidos , Reino UnidoRESUMEN
Nephronophthisis is an autosomal recessive tubulointerstitial nephropathy, belonging to the ciliopathy disorders, characterized by fibrosis and/or cysts. It is the most common genetic cause of kidney failure in children and young adults. Clinically and genetically heterogeneous, it is caused by variants in ciliary genes, resulting in either an isolated kidney disease or syndromic forms in association with other manifestations of ciliopathy disorders. No curative treatment is currently available. Over the past 2 decades, advances in understanding disease mechanisms have identified several dysregulated signaling pathways, some shared with other cystic kidney diseases. Notably, molecules previously developed to target these pathways have shown promising beneficial effects in orthologous mouse models. In addition to these knowledge-based repurposing approaches, unbiased "in cellulo" phenotypic screens of "repurposing" libraries identified small molecules able to rescue the ciliogenesis defects observed in nephronophthisis conditions. Those compounds appeared to act on relevant pathways and, when tested, showed beneficial nephronophthisis-associated kidney and/or extrarenal defects in mice. In this review, we have summarized those studies that highlight the drug repurposing strategies in the context of a rare disorders, such as nephronophthisis-related ciliopathies, with broad genetic heterogeneity and systemic manifestations but with shared disease mechanisms.
Asunto(s)
Ciliopatías , Enfermedades Renales Quísticas , Enfermedades Renales Poliquísticas , Insuficiencia Renal , Animales , Ratones , Riñón/patología , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Quísticas/tratamiento farmacológico , Enfermedades Renales Quísticas/genética , Ciliopatías/tratamiento farmacológico , Ciliopatías/genética , Insuficiencia Renal/complicaciones , Fibrosis , Cilios/patologíaRESUMEN
A timely diagnosis is a key challenge for many rare diseases. As an expanding group of rare and severe monogenic disorders with a broad spectrum of clinical manifestations, ciliopathies, notably renal ciliopathies, suffer from important underdiagnosis issues. Our objective is to develop an approach for screening large-scale clinical data warehouses and detecting patients with similar clinical manifestations to those from diagnosed ciliopathy patients. We expect that the top-ranked similar patients will benefit from genetic testing for an early diagnosis. The dependence and relatedness between phenotypes were taken into account in our similarity model through medical concept embedding. The relevance of each phenotype to each patient was also considered by adjusted aggregation of phenotype similarity into patient similarity. A ranking model based on the best-subtype-average similarity was proposed to address the phenotypic overlapping and heterogeneity of ciliopathies. Our results showed that using less than one-tenth of learning sources, our language and center specific embedding provided comparable or better performances than other existing medical concept embeddings. Combined with the best-subtype-average ranking model, our patient-patient similarity-based screening approach was demonstrated effective in two large scale unbalanced datasets containing approximately 10,000 and 60,000 controls with kidney manifestations in the clinical data warehouse (about 2 and 0.4% of prevalence, respectively). Our approach will offer the opportunity to identify candidate patients who could go through genetic testing for ciliopathy. Earlier diagnosis, before irreversible end-stage kidney disease, will enable these patients to benefit from appropriate follow-up and novel treatments that could alleviate kidney dysfunction.
RESUMEN
Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using a medium-throughput drug-screen in NPHP1 knockdown cells, we identified 51 Food and Drug Administration-approved compounds by their ability to alleviate the cellular phenotypes associated with the loss of NPHP1; 11 compounds were further selected for their physicochemical properties. Among those compounds, prostaglandin E1 (PGE1) rescued ciliogenesis defects in immortalized patient NPHP1 urine-derived renal tubular cells, and improved ciliary and kidney phenotypes in our NPH zebrafish and Nphp1 knockout mouse models. Furthermore, Taprenepag, a nonprostanoid prostaglandin E2 receptor agonist, alleviated the severe retinopathy observed in Nphp1−/− mice. Finally, comparative transcriptomics allowed identification of key signaling pathways downstream PGE1, including cell cycle progression, extracellular matrix, adhesion, or actin cytoskeleton organization. In conclusion, using in vitro and in vivo models, we showed that prostaglandin E2 receptor agonists can ameliorate several of the pleotropic phenotypes caused by the absence of NPHP1; this opens their potential as a first therapeutic option for juvenile NPH-associated ciliopathies.
Asunto(s)
Ciliopatías , Enfermedades Renales Poliquísticas , Animales , Cilios/metabolismo , Ciliopatías/tratamiento farmacológico , Ciliopatías/genética , Ciliopatías/metabolismo , Femenino , Humanos , Enfermedades Renales Quísticas/congénito , Masculino , Ratones , Enfermedades Renales Poliquísticas/metabolismo , Prostaglandinas/metabolismo , Receptores de Prostaglandina E/metabolismo , Pez CebraRESUMEN
Epithelial ovarian cancer is the fourth cause of death among cancer-bearing women and frequently associated with carboplatin resistance, underlining the need for more efficient and targeted therapies. F14512 is an epipodophylotoxin-core linked to a spermine chain which enters cells via the polyamine transport system (PTS). Here, we investigate this novel concept of vectorization in ovarian cancer. We compared the effects of etoposide and F14512 on a panel of five carboplatin-sensitive or resistant ovarian cancer models. We assessed the incorporation of F17073, a spermine-linked fluorescent probe, in these cells and in 18 clinical samples. We then showed that F14512 exhibits a high anti-proliferative and pro-apoptotic activity, particularly in cells with high levels of F17073 incorporation. Consistently, F14512 significantly inhibited tumor growth compared to etoposide, in a cisplatin-resistant A2780R subcutaneous model, at a dose of 1.25 mg/kg. In addition, ex vivo analysis indicated that 15 out of 18 patients presented a higher F17073 incorporation into tumor cells compared to normal cells. Overall, our data suggest that F14512, a targeted drug with a potent anti-tumor efficacy, constitutes a potential new therapy for highly PTS-positive and platinum-resistant ovarian cancer-bearing patients.
Asunto(s)
Antineoplásicos/farmacología , Poliaminas Biogénicas/metabolismo , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Podofilotoxina/análogos & derivados , Inhibidores de Topoisomerasa II/farmacología , Animales , Apoptosis/efectos de los fármacos , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Femenino , Humanos , Ratones , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Podofilotoxina/farmacologíaRESUMEN
(-)-Cryptopleurine 1 is one of the most potent anti-proliferative member of the phenanthroquinolizidine class of alkaloids. We report here the synthesis of (-)-6-O-desmethylcryptopleurine (-)-2 and (-)-6-O-desmethyl-(15R)-hydroxycryptopleurine (-)-4 in their enantiomerically enriched form through a convergent synthetic route, where the chirality is introduced by the use of commercially available (R)-methyl piperidine-2-carboxylate hydrochloride 17. Anti-proliferative activities of these compounds were evaluated on a panel of four cancer cell lines, revealing that compounds (-)-2 and (-)-4 are potent cytotoxic compared to cryptopleurine.
Asunto(s)
Alcaloides/síntesis química , Alcaloides/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Evaluación Preclínica de Medicamentos/métodos , HumanosRESUMEN
BACKGROUND: No biological or molecular marker of primary melanoma tumor cells has been shown to predict clinical outcome in melanoma. OBJECTIVE: To determine whether CD10, CD133, nestin and CD20 may evaluate the prognosis of melanoma. METHODS: The differential expression of these molecules was assessed in pairs of cell lines. We evaluated, by both immunohistochemical staining and RT-qPCR, their expression in a cohort of 32 patients (68 samples) with a history of metastatic melanoma, divided into two groups according to their clinical outcome profile. RESULTS: CD10 over expression in cancer cell lines was associated with more aggressive behavior in vitro. A CD10-positive staining was more frequent in patients in the "rapidly progressive" group than those in the "long survivor" group (23/35 versus 2/18, p<10(-4)). CD10 expression was associated with a lower median overall survival (1.15 year - IQR: [0.50-2.58] versus 4.27 - IQR: [1.66-6.33]; p=10(-4)). The Odds Ratio of displaying a "rapidly progressive" melanoma when tumor cells expressed CD10 was 15 (95% confidence interval: [3-78]). After adjusting for confounding factors, CD10 expression in melanoma tumor cells remained associated with an increased risk of death and more rapid disease progression (p=6×10(-4); HR=3.71). CONCLUSION: CD10 may predict clinical outcome in melanoma patients.
Asunto(s)
Melanoma/genética , Melanoma/secundario , Neprilisina/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Antígeno AC133 , Anciano , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD20/genética , Antígenos CD20/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Masculino , Melanoma/metabolismo , Ratones , Ratones Desnudos , Persona de Mediana Edad , Trasplante de Neoplasias , Neprilisina/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , Péptidos/genética , Péptidos/metabolismo , Valor Predictivo de las Pruebas , Pronóstico , Neoplasias Cutáneas/metabolismo , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Metastatic melanoma is the most aggressive skin cancer. Recently, phenotypically distinct subpopulations of tumor cells were identified. Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties. In addition, ABCB5(+) cells are thought to participate to chemoresistance through a potential efflux function of ABCB5. Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified. Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells. Using a melanoma xenograft model (WM266-4), we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression. These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine. In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs. Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5. This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , 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/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Dacarbazina/farmacología , Dacarbazina/uso terapéutico , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Neoplasia Residual , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The polyamines transport system (PTS) is usually enhanced in cancer cells and can be exploited to deliver anticancer drugs. The spermine-conjugated epipodophyllotoxin derivative F14512 is a topoisomerase II poison that exploits the PTS to target preferentially tumor cells. F14512 has been characterized as a potent anticancer drug candidate and is currently in phase 1 clinical trials. Here we have analyzed the mechanisms of cell death induced by F14512, compared to the parent drug etoposide lacking the polyamine tail. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages. The cytotoxic action of F14512 is extremely rapid (within 3 h) and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased ß-galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent (methuosis-like) vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 compared to etoposide, and this difference may account for their distinct pharmacological profiles and the markedly superior activity of F14512 in vivo. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor activity.
Asunto(s)
Antineoplásicos/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Podofilotoxina/análogos & derivados , Poliaminas/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Ciclo Celular , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Etopósido/química , Etopósido/farmacología , Humanos , Estructura Molecular , Podofilotoxina/química , Podofilotoxina/farmacologíaRESUMEN
Triptolide, a natural product extracted from the Chinese plant Tripterygium wilfordii, possesses antitumor properties. Despite numerous reports showing the proapoptotic capacity and the inhibition of NF-kappaB-mediated transcription by triptolide, the identity of its cellular target is still unknown. To clarify its mechanism of action, we further investigated the effect of triptolide on RNA synthesis in the human non-small cell lung cancer cell line A549. Triptolide inhibited both total RNA and mRNA de novo synthesis, with the primary action being on the latter pool. We used 44K human pan-genomic DNA microarrays and identified the genes primarily affected by a short treatment with triptolide. Among the modulated genes, up to 98% are down-regulated, encompassing a large array of oncogenes including transcription factors and cell cycle regulators. We next observed that triptolide induced a rapid depletion of RPB1, the RNA polymerase II main subunit that is considered a hallmark of a transcription elongation blockage. However, we also show that triptolide does not directly interact with the RNA polymerase II complex nor does it damage DNA. We thus conclude that triptolide is an original pharmacologic inhibitor of RNA polymerase activity, affecting indirectly the transcription machinery, leading to a rapid depletion of short-lived mRNA, including transcription factors, cell cycle regulators such as CDC25A, and the oncogenes MYC and Src. Overall, the data shed light on the effect of triptolide on transcription, along with its novel potential applications in cancers, including acute myeloid leukemia, which is in part driven by the aforementioned oncogenic factors.
Asunto(s)
Diterpenos/química , Regulación hacia Abajo/efectos de los fármacos , Fenantrenos/química , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa I/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Diterpenos/farmacología , Compuestos Epoxi/química , Compuestos Epoxi/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenantrenos/farmacología , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Factores de Tiempo , Proteína p53 Supresora de Tumor , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
The synthesis of a series of conjugated spermine derivatives with benzoxadiazole, phenylxanthene or bodipy fluorophores is described. These fluorescent probes were used to identify the activity of the polyamine transport system (PTS). N(1)-Methylspermine NBD conjugate 5 proved to have the optimal fluorescence characteristics and was used to show a selectivity for PTS-proficient CHO versus PTS-deficient CHO-MG cells. It can therefore be used as a tool for the selection of cells sensitive to cytotoxic compounds vectored through the PTS.
Asunto(s)
Compuestos de Boro/química , Química Farmacéutica/métodos , Colorantes Fluorescentes/farmacología , Oxadiazoles/síntesis química , Rodaminas/química , Espermina/síntesis química , Animales , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Diseño de Fármacos , Colorantes Fluorescentes/química , Humanos , Oxadiazoles/farmacología , Poliaminas/química , Espermina/análogos & derivados , Espermina/química , Espermina/farmacologíaRESUMEN
The anaplastic lymphoma kinase (ALK) is a validated target for the therapy of different malignancies. Aberrant expression of constitutively active ALK chimeric proteins has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL) and has been detected in other cancers such as inflammatory myofibroblastic tumors, diffuse large B-cell lymphomas, certain non-small-cell lung cancers, rhabdomyosarcomas, neuroblastomas and glioblastomas. In the course of a screening program aimed at identifying kinase inhibitors with novel scaffolds, the two pyridoisoquinoline derivatives F91873 and F91874, were identified as multikinase inhibitors with activity against ALK in a biochemical screen. F91873 and F91874 also inhibited nucleophosmin-ALK and signal transducer and activator of transcription 3 phosphorylation in the ALCL cell line COST with the same potency. Both F91873 and F91874 behaved as ATP noncompetitive inhibitors and inhibited cell proliferation of the ALK(+) ALCL cell lines COST, PIO, and Karpas299 ALCL. This growth inhibition effect was associated with a G1-phase cell cycle arrest. Furthermore, administration of F91874 to severe combined immunodeficient mice bearing COST tumor xenografts resulted in a significant antitumor efficacy at 15 mg/kg/day, illustrating the potential utility of such compounds in the treatment of ALK-related pathologies.
Asunto(s)
Antineoplásicos/uso terapéutico , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinolizinas/uso terapéutico , Tiazoles/uso terapéutico , Quinasa de Linfoma Anaplásico , Animales , Antineoplásicos/síntesis química , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Femenino , Fase G1/efectos de los fármacos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Linfoma Anaplásico de Células Grandes/enzimología , Linfoma Anaplásico de Células Grandes/patología , Ratones , Ratones Endogámicos ICR , Ratones SCID , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , Inhibidores de Proteínas Quinasas/síntesis química , Estructura Terciaria de Proteína , Quinolizinas/síntesis química , Proteínas Tirosina Quinasas Receptoras , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Tiazoles/síntesis química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The polyamine transport system (PTS) is an energy-dependent machinery frequently overactivated in cancer cells with a high demand for polyamines. We have exploited the PTS to selectively deliver a polyamine-containing drug to cancer cells. F14512 combines an epipodophyllotoxin core-targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. The polyamine tail supports three complementary functions: (a) facilitate formulation of a water-soluble compound, (b) increase DNA binding to reinforce topoisomerase II inhibition, and (c) facilitate selective uptake by tumor cells via the PTS. F14512 is 73-fold more cytotoxic to Chinese hamster ovary cells compared with CHO-MG cells with a reduced PTS activity. A decreased sensitivity of L1210 leukemia cells to F14512 was observed in the presence of putrescine, spermidine, and spermine. In parallel, the spermine moiety considerably enhances the drug-DNA interaction, leading to a reinforced inhibition of topoisomerase II. The spermine tail of F14512 serves as a cell delivery vehicle as well as a DNA anchor, and this property translates at the cellular level into a distinct pharmacologic profile. Twenty-nine human solid or hematologic cell lines were used to characterize the high cytotoxic potential of F14512 (median IC50 of 0.18 micromol/L). Finally, the potent antitumor activity of F14512 in vivo was evidenced with a MX1 human breast tumor xenograft model, with partial and complete tumor regressions. This work supports the clinical development of F14512 as a novel targeted cytotoxic drug and sheds light on the concept of selective delivery of drugs to tumor cells expressing the PTS.
Asunto(s)
Poliaminas Biogénicas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Podofilotoxina/análogos & derivados , Inhibidores de Topoisomerasa II , Animales , Unión Competitiva , Poliaminas Biogénicas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Daño del ADN , ADN-Topoisomerasas de Tipo II/biosíntesis , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Neoplasias/metabolismo , Sistemas de Liberación de Medicamentos , Etopósido/farmacología , Femenino , Humanos , Leucemia L1210/tratamiento farmacológico , Leucemia L1210/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Neoplasias/enzimología , Neoplasias/genética , Podofilotoxina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antimitotic drugs targeting the microtubules, such as the taxanes and vinca alkaloids, are widely used in the treatment of neoplastic diseases. Development of drug resistance over time, however, limits the efficacy of these agents and poses a clinical challenge to long-term improvement of patient outcomes. Understanding the mechanism(s) of drug resistance becomes paramount to allowing for alternative, if not improved, therapeutic options that might circumvent this challenge. Vinflunine, a novel microtubule inhibitor, has shown superior preclinical antitumor activity, and displays a different pattern of resistance, compared with other agents in the vinca alkaloid class.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Resistencia a Antineoplásicos , Microtúbulos/efectos de los fármacos , Moduladores de Tubulina/uso terapéutico , Vinblastina/análogos & derivados , Actinas/metabolismo , Antineoplásicos Fitogénicos/antagonistas & inhibidores , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteómica/métodos , Tubulina (Proteína)/genética , Alcaloides de la Vinca/farmacologíaRESUMEN
The ubiquitin-proteasome pathway plays a critical role in the degradation of proteins involved in tumor growth and has therefore become a target for cancer therapy. In order to discover novel inhibitors of this pathway, a cellular assay reporter of proteasome activity was established. Human DLD-1 colon cancer cells were engineered to express a 4 ubiquitin-luciferase (DLD-1 4Ub-Luc) reporter protein, rapidly degraded via the ubiquitin-proteasome pathway and designed DLD-1 4Ub-Luc cells. Following treatment with reference proteasome inhibitors, the 4Ub-Luc protein accumulated in DLD-1 4Ub-Luc cells and a 80-fold increase in luciferase-produced bioluminescence signal was measured, as compared to untreated cells. The screening of over 30,000 compounds using this DLD-1 4Ub-Luc assay led to the identification of physalin B as a novel inhibitor of the ubiquitin-proteasome pathway. Indeed, physalin B induced an increase in bioluminescence from DLD-1 4Ub-Luc cells, at concentrations also producing an accumulation of ubiquitinated proteins and inhibiting TNFalpha-induced NF-kappaB activation. Physalin B did not inhibit catalytic activities of purified proteasome and interfered with cellular proteasomal catalytic activities at 4- to 8-fold higher concentrations than that required to induce significant increase in bioluminescence and accumulation of ubiquitinated proteins in DLD-1 4Ub-Luc cells. Furthermore, physalin B proved to be cytotoxic, triggered apoptosis in DLD-1 4Ub-Luc cells and induced the proapoptotic protein NOXA, characteristic of the proteasome signaling pathway. Therefore, the use of the DLD-1 4Ub-Luc assay allowed the identification of a novel inhibitor of the ubiquitin-proteasome pathway that might interfere with proteasome functions in a different way from reference proteasome inhibitors.
Asunto(s)
Apoptosis/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Secoesteroides/farmacología , Ubiquitina/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Extractos Vegetales/química , Inhibidores de Proteasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Transducción de SeñalRESUMEN
Novel derivatives of the marine alkaloid bengacarboline have been synthesized. The seco derivatives 11 and 12 were evaluated for topoisomerase inhibition, DNA damages, cytotoxicity and cell cycle perturbation. The two synthetic analogs are more potent cytotoxic agents than bengacarboline and they both induce an accumulation of cells in the S phase of DNA synthesis. They do not function as topoisomerase inhibitors but trigger DNA damages in cells.
Asunto(s)
Alcaloides/síntesis química , Alcaloides/farmacología , Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Carbolinas/síntesis química , Carbolinas/farmacología , Inhibidores de Topoisomerasa II , Alcaloides/química , Animales , Antineoplásicos Fitogénicos/química , Apoptosis/fisiología , Carbolinas/química , Ciclo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Biología Marina , Estructura Molecular , Urocordados/químicaRESUMEN
Cisplatin resistance has been associated with altered K+ fluxes. Here, we focused our investigations on the detection of K+ channels in a series of cisplatin-resistant (CP-r) cells with increasing resistance and on the functional relationship of these K+ channels to resistance. Microarray analysis and confocal microscopy indicated that there was overexpression of the ether-a-gogo gene (HERG) and the inwardly rectifying potassium channel gene (TWIK) in a human epidermal KB and human liver BEL-7404 carcinoma cell line series selected for cisplatin resistance. With increased resistance, the plasma membrane potential, but not the mitochondrial membrane potential, also increases in these two series. For these reasons, we conducted cell proliferation studies in the presence of either antibodies directed against the detected K+ channels, omeprazole (a H+ pump inhibitor) or a specific inhibitor of the HERG channel (WAY-123398-A-5). The antibodies and omeprazole influenced cell growth only very slightly. The specific K+ channel blocker did not alter cisplatin resistance. We also observed that manipulation of K+ fluxes with antibodies and the H+ pump with omeprazole resulted in opposite effects on cisplatin resistance in these two cell lines. We conclude that K+ and H+ homeostasis are not critical factors in cisplatin resistance since they affect cisplatin resistance differently in KB and BEL-7404 cells.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Canales de Potasio Éter-A-Go-Go/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Membrana Celular/fisiología , Resistencia a Antineoplásicos , Canales de Potasio Éter-A-Go-Go/biosíntesis , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Expresión Génica , Humanos , Membranas Intracelulares/fisiología , Células KB , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Potenciales de la Membrana/fisiología , Mitocondrias/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Omeprazol/farmacología , Consumo de Oxígeno , Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacologíaRESUMEN
ATP-binding cassette (ABC) transporters play a pivotal role in physiology and pathology. We identified and cloned two novel mRNA isoforms (ABCB 5alpha and ABCB 5beta) of the ABC transporter ABCB 5 in human melanoma cells. The deduced ABCB 5alpha protein appears to be an altered splice variant containing only a putative ABC, whereas the ABCB 5beta isoform shares approximately 70% similarity with ABCB1 (MDR1) and has a deduced topological arrangement similar to that of the whole carboxyl terminal half of the ABCB1 gene product, P-glycoprotein, including an intact ABC. Northern blot, real-time PCR, and conventional RT-PCR were used to verify the expression profiles of ABCB 5alpha/beta. We found that the melanomas included among the NCI-60 panel of cell lines preferentially expressed both ABCB 5alpha and ABCB 5beta. However, ABCB 5alpha/beta expression was undetectable in two amelanotic melanomas (M14 and LOX-IMVI). The expression profile of ABCB 5alpha/beta in all of the other melanomas of the panel was confirmed both by RT-PCR and by sequencing. Neither ABCB 5alpha nor ABCB 5beta expression was found in normal tissues such as liver, spleen, thymus, kidney, lung, colon, small intestines or placenta. ABCB 5alpha/beta mRNAs were also expressed in normal melanocytes and in retinal pigment epithelial cells, suggesting that ABCB 5alpha/beta expression is pigment cell-specific and might be involved in melanogenesis. Our findings indicate that expression of ABCB 5alpha/beta might possibly provide two novel molecular markers for differential diagnosis of melanomas and constitute potential molecular targets for therapy of melanomas.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Melanocitos/metabolismo , ARN Mensajero/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Células Cultivadas , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Perfilación de la Expresión Génica , Biblioteca de Genes , Humanos , Melanoma , Datos de Secuencia Molecular , Especificidad de ÓrganosRESUMEN
Discovery of the multidrug resistance protein 1 (MDR1), an ATP-binding cassette (ABC) transporter able to transport many anticancer drugs, was a clinically relevant breakthrough in multidrug resistance research. Although the overexpression of ABC transporters such as P-glycoprotein/ABCB1, MRP1/ABCC1, and MXR/ABCG2 seems to be a major cause of failure in the treatment of cancer, acquired resistance to multiple anticancer drugs may also be multifactorial, involving alteration of detoxification processes, apoptosis, DNA repair, drug uptake, and overexpression of other ABC transporters. As a tool for the study of such phenomena, we designed and created a microarray platform, the ABC-ToxChip, to evaluate relative levels of transcriptional activation among genes involved in the various mechanisms of resistance. In the ABC-ToxChip, a comprehensive set of genes important in toxicological responses (represented by 2200 cDNA probes) is complemented with probes specifically matching ABC transporters as well as oligonucleotides representing 18,000 unique human genes. By comparing the transcriptional profiles of KB-3-1 and DU-145 parental cells with resistant derivatives selected in colchicine (KB-8-5), and 9-nitro-camptothecin (RCO.1), respectively, we demonstrate that ABC transporters (ABCB1/MDR1 and ABCC2/MRP2, respectively) show dramatic overexpression, whereas the glutathione S-transferase gene GST-Pi shows the strongest decrease in expression among the 20,000 genes studied. The results were confirmed by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. The custom-designed ABC-Tox microarray presented here will be helpful to elucidate mechanisms leading to anticancer drug resistance.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Resistencia a Múltiples Medicamentos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Transportadoras de Casetes de Unión a ATP/metabolismo , Apoptosis , Transporte Biológico , Línea Celular , Sondas de ADN , Reparación del ADN , Exones , Humanos , Proteína 2 Asociada a Resistencia a Múltiples MedicamentosRESUMEN
For analysis of multidrug resistance, a major barrier to effective cancer chemotherapy, we profiled mRNA expression of the 48 known human ABC transporters in 60 diverse cancer cell lines (the NCI-60) used by the National Cancer Institute to screen for anticancer activity. The use of real-time RT-PCR avoided artifacts commonly encountered with microarray technologies. By correlating the results with the growth inhibitory profiles of 1,429 candidate anticancer drugs tested against the cells, we identified which transporters are more likely than others to confer resistance to which agents. Unexpectedly, we also found and validated compounds whose activity is potentiated, rather than antagonized, by the MDR1 multidrug transporter. Such compounds may serve as leads for development.