RESUMEN
Activation of Toll-like receptor 3 (TLR3) was previously shown to contribute to the generation of epileptic seizures in rodents by evoking a proinflammatory response in the forebrain. This suggests that TLR3 blockade may provide therapeutic effects in epilepsy. We report that brain activation of TLR3 using the synthetic receptor ligand Poly I:C may also result in remarkable dose- and time-dependent inhibitory effects on acute seizures in mice without inducing inflammation. These inhibitory effects are associated with reduced neuronal excitability in the hippocampus as shown by a decrease in the population spike amplitude of CA1 pyramidal neurons following Schaffer collaterals stimulation. TLR3 activation which results in seizure inhibition does not evoke NF-kB-dependent inflammatory molecules or morphological activation of glia, however, it induces the alternative interferon (IFN) regulatory factor (IRF)-3/IFN-ß signaling pathway. IFN-ß reproduced the inhibitory effects of Poly I:C on neuronal excitability in hippocampal slices. Seizure inhibition attained with activation the TLR3-IRF3/IFN-ß axis should be carefully considered when TLR3 are targeted for therapeutic purposes.
Asunto(s)
Factor 3 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Receptor Toll-Like 3/metabolismo , Animales , Antiinflamatorios/farmacología , Anticonvulsivantes/farmacología , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Neuroglía/metabolismo , Poli I-C/farmacología , Receptores de Superficie Celular/metabolismo , Convulsiones/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
This study was designed to investigate the mode of action of trabectedin in myelomonocytic leukemia cells by applying systems biology approaches to mine gene expression profiling data and pharmacological assessment of the cellular effects. Significant enrichment was found in regulons of target genes inferred for specific transcription factors, among which MAFB was the most upregulated after treatment and was central in the transcriptional network likely to be relevant for the specific therapeutic effects of trabectedin against myelomonocytic cells. Using the Connectivity Map, similarity among transcriptional signatures elicited by treatment with different compounds was investigated, showing a high degree of similarity between transcriptional signatures of trabectedin and those of the topoisomerase I inhibitor, irinotecan, and an anti-dopaminergic antagonist, thioridazine. The study highlights the potential importance of systems biology approaches to generate new hypotheses that are experimentally testable to define the specificity of the mechanism of action of drugs.
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Antineoplásicos Alquilantes/uso terapéutico , Leucemia Mieloide/tratamiento farmacológico , Trabectedina/farmacología , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Biología de Sistemas/métodos , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive disease, that can be described as a member of the family of small round blue cell tumors. The molecular diagnostic marker is the t(11;22)(p13;q12) translocation, which creates an aberrant transcription factor, EWS-WT1, that underlies the oncogenesis of DSRCT. Current treatments are not very effective so new active drugs are needed. Trabectedin, now used as a single agent for the treatment of soft tissue sarcoma, was reported to be active in some pre-treated DSRCT patients. Using JN-DSRCT-1, a cell line derived from DSRCT expressing the EWS-WT1 fusion protein, we investigated the ability of trabectedin to modify the function of the chimeric protein, as in other sarcomas expressing fusion proteins. After detailed characterization of the EWS-WT1 transcripts structure, we investigated the mode of action of trabectedin, looking at the expression and function of the oncogenic chimera. METHODS: We characterized JN-DSRCT-1 cells using cellular approaches (FISH, Clonogenicity assay) and molecular approaches (Sanger sequencing, ChIP, GEP). RESULTS: JN-DSRCT-1 cells were sensitive to trabectedin at nanomolar concentrations. The cell line expresses different variants of EWS-WT1, some already identified in patients. EWS-WT1 mRNA expression was affected by trabectedin and chimeric protein binding on its target gene promoters was reduced. Expression profiling indicated that trabectedin affects the expression of genes involved in cell proliferation and apoptosis. CONCLUSIONS: The JN-DSRCT-1 cell line, in vitro, is sensitive to trabectedin: after drug exposure, EWS-WT1 chimera expression decreases as well as binding on its target promoters. Probably the heterogeneity of chimera transcripts is an obstacle to precisely defining the molecular mode of action of drugs, calling for further cellular models of DSRCT, possibly growing in vivo too, to mimic the biological complexity of this disease.
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Tumor Desmoplásico de Células Pequeñas Redondas/tratamiento farmacológico , Dioxoles/farmacología , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/efectos de los fármacos , Tetrahidroisoquinolinas/farmacología , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Tumor Desmoplásico de Células Pequeñas Redondas/metabolismo , Tumor Desmoplásico de Células Pequeñas Redondas/fisiopatología , Dioxoles/uso terapéutico , Humanos , Proteínas de Fusión Oncogénica/genética , Proteína EWS de Unión a ARN , Tetrahidroisoquinolinas/uso terapéutico , Trabectedina , Proteínas WT1RESUMEN
BACKGROUND: Neoadjuvant chemotherapy (NACT) has been recognized as a reliable therapeutic strategy in patients with unresectable advanced epithelial ovarian cancer (EOC). The molecular events leading to platinum (Pt) response in NACT settings have hitherto not been explored. In the present work, longitudinal changes of miRNA expression profile were investigated to identify miRNA families with prognostic role in high-grade serous EOC patients who received the NACT regimen. PATIENTS AND METHODS: One hundred sixty-four matched tumor biopsies taken at initial laparoscopic evaluation and at interval-debulking surgery (IDS) after four courses of Pt-based therapy were selected from 82 stage IIIC-IV high-grade serous-EOC patients that were judged unsuitable for complete primary debulking and subjected the NACT protocol. miRNA profiling by microarray, real-time PCR and immuno-histochemical staining for Smad2 phosphorylation (P-Smad2) were used for data analysis. RESULTS: Analysis revealed that 369 miRNAs were differentially expressed in matched biopsies (referred to as DEMs). DEMs were not scattered across the genome, but clustered into families: miR-199, let-7, miR-30, miR-181 and miR-29. Multivariate analysis showed that miR-199a-3p, miR-199a-5p, miR-181a-5p and let-7g-5p associated with overall and progression-free survival (P < 0.05); miR-199a-3p, miR-199a-5p and miR-181a-5p associated with residual tumor volume and Pt-free interval (P < 0.05). Immuno-histochemical staining confirmed an enrichment of P-Smad2, a marker of transforming growth factor-ß activation, in tumors from patients with shorter PFS and OS, and with high levels of expression of miR-181a-5p (P < 0.05). Kaplan-Meier curves plotting concomitant expression of P-Smad2 and miR-181a-5p show significant differences in PFS and OS compared with those depicting the expression of each biomarker alone (P < 0.001). CONCLUSIONS: This study describes several miRNA families with a prognostic role in the NACT setting. It also confirms that concomitant analysis of P-Smad2 and miR-181a-5p in surgical samples may be capable of identifying those ovarian cancer patients with poor outcome and little chance of response to Pt-based NACT.
Asunto(s)
Cistadenocarcinoma Seroso/tratamiento farmacológico , MicroARNs/biosíntesis , Terapia Neoadyuvante , Neoplasias Ováricas/tratamiento farmacológico , Proteína Smad2/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Biopsia , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , MicroARNs/genética , Persona de Mediana Edad , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Pronóstico , Proteína Smad2/genéticaRESUMEN
BACKGROUND: Clinical and pathological parameters of patients with epithelial ovarian cancer (EOC) do not thoroughly predict patients' outcome. Despite the good outcome of stage I EOC compared with that of stages III and IV, the risk assessment and treatments are almost the same. However, only 20% of stage I EOC cases relapse and die, meaning that only a proportion of patients need intensive treatment and closer follow-up. Thus, the identification of cell mechanisms that could improve outcome prediction and rationalize therapeutic options is an urgent need in the clinical practice. PATIENTS AND METHODS: We have gathered together 203 patients with stage I EOC diagnosis, from whom snap-frozen tumor biopsies were available at the time of primary surgery before any treatment. Patients, with a median follow-up of 7 years, were stratified into a training set and a validation set. RESULTS AND CONCLUSIONS: Integrated analysis of miRNA and gene expression profiles allowed to identify a prognostic cell pathway, composed of 16 miRNAs and 10 genes, wiring the cell cycle, 'Activins/Inhibins' and 'Hedgehog' signaling pathways. Once validated by an independent technique, all the elements of the circuit resulted associated with overall survival (OS) and progression-free survival (PFS), in both univariate and multivariate models. For each patient, the circuit expressions have been translated into an activation state index (integrated signature classifier, ISC), used to stratify patients into classes of risk. This prediction reaches the 89.7% of sensitivity and 96.6% of specificity for the detection of PFS events. The prognostic value was then confirmed in the external independent validation set in which the PFS events are predicted with 75% sensitivity and 94.7% specificity. Moreover, the ISC shows higher classification performance than conventional clinical classifiers. Thus, the identified circuit enhances the understanding of the molecular mechanisms lagging behind stage I EOC and the ISC improves our capabilities to assess, at the time of diagnosis, the patient risk of relapse.
Asunto(s)
Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Pronóstico , Adulto , Anciano , Carcinoma Epitelial de Ovario , Supervivencia sin Enfermedad , Femenino , Humanos , MicroARNs/genética , Análisis por Micromatrices , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patologíaRESUMEN
BACKGROUND: The majority of patients with stage III-IV epithelial ovarian cancer (EOC) relapse after initially responding to platinum-based chemotherapy, and develop resistance. The genomic features involved in drug resistance are unknown. To unravel some of these features, we investigated the mutational profile of genes involved in pathways related to drug sensitivity in a cohort of matched tumors obtained at first surgery (Ft-S) and second surgery (Sd-S). PATIENTS AND METHODS: Matched biopsies (33) taken at Ft-S and Sd-S were selected from the 'Pandora' tumor tissue collection. DNA libraries for 65 genes were generated using the TruSeq Custom Amplicon kit and sequenced on MiSeq (Illumina). Data were analyzed using a high-performance cluster computing platform (Cloud4CARE project) and independently validated. RESULTS: A total of 2270 somatic mutations were identified (89.85% base substitutions 8.19% indels, and 1.92% unknown). Homologous recombination (HR) genes and TP53 were mutated in the majority of Ft-S, while ATM, ATR, TOP2A and TOP2B were mutated in the entire dataset. Only 2% of mutations were conserved between matched Ft-S and Sd-S. Mutations detected at second surgery clustered patients in two groups characterized by different mutational profiles in genes associated with HR, PI3K, miRNA biogenesis and signal transduction. CONCLUSIONS: There was a low level of concordance between Ft-S and Sd-S in terms of mutations in genes involved in key processes of tumor growth and drug resistance. This result suggests the importance of future longitudinal analyses to improve the clinical management of relapsed EOC.
Asunto(s)
Adenocarcinoma de Células Claras/genética , Adenocarcinoma Mucinoso/genética , Cistadenocarcinoma Seroso/genética , Neoplasias Endometriales/genética , Genes Relacionados con las Neoplasias/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación/genética , Neoplasias Ováricas/genética , Adenocarcinoma de Células Claras/mortalidad , Adenocarcinoma de Células Claras/secundario , Adenocarcinoma de Células Claras/terapia , Adenocarcinoma Mucinoso/mortalidad , Adenocarcinoma Mucinoso/secundario , Adenocarcinoma Mucinoso/terapia , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biopsia , Terapia Combinada , Cistadenocarcinoma Seroso/mortalidad , Cistadenocarcinoma Seroso/secundario , Cistadenocarcinoma Seroso/terapia , Resistencia a Antineoplásicos/genética , Neoplasias Endometriales/mortalidad , Neoplasias Endometriales/secundario , Neoplasias Endometriales/terapia , Femenino , Estudios de Seguimiento , Recombinación Homóloga , Humanos , Estudios Longitudinales , Metástasis Linfática , Persona de Mediana Edad , Clasificación del Tumor , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/terapia , Estadificación de Neoplasias , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapia , Pronóstico , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: We describe the first case of a pediatric patient with acute intermittent porphyria and severe chronic porphyric neuropathy treated with givosiran, a small-interfering RNA that drastically decreases delta-aminolevulinic acid production and reduces porphyric attacks' recurrence. CASE REPORT: A 12-year-old male patient with refractory acute intermittent porphyria and severe porphyric neuropathy was followed prospectively for 12 months after givosiran initiation (subcutaneous, 2.5 mg/kg monthly). Serial neurological, structural, and resting-state functional magnetic resonance imaging (MRI) evaluations were performed, including clinical scales and neurophysiological tests. Delta-aminolevulinic acid urinary levels dropped drastically during treatment. In parallel, all the administered neurological rating scales and neurophysiological assessments showed improvement in all domains. Moreover, an improvement in central motor conduction parameters and resting-state functional connectivity in the sensory-motor network was noticed. At the end of the follow-up, the patient could walk unaided after using a wheelchair for 5 years. CONCLUSIONS: A clear beneficial effect of givosiran was demonstrated in our patient with both clinical and peripheral nerve neurophysiologic outcome measures. Moreover, we first reported a potential role of givosiran in recovering central motor network impairment in acute intermittent porphyria (AIP), which was previously unknown. This study provides Class IV evidence that givosiran improves chronic porphyric neuropathy.
Asunto(s)
Acetilgalactosamina/análogos & derivados , Porfiria Intermitente Aguda , Humanos , Masculino , Porfiria Intermitente Aguda/tratamiento farmacológico , Niño , Acetilgalactosamina/uso terapéutico , Ácido Aminolevulínico/análogos & derivados , Ácido Aminolevulínico/orina , Imagen por Resonancia Magnética , Pirrolidinas/uso terapéutico , Uridina/análogos & derivados , Uridina/uso terapéutico , Uridina/administración & dosificación , Recuperación de la Función , Enfermedad Crónica , Resultado del TratamientoRESUMEN
We studied whether pharmacological blockade of the IL-1ß-mediated signaling, rapidly activated in forebrain by epileptogenic injuries, affords neuroprotection in two different rat models of status epilepticus (SE). As secondary outcome, we measured treatment's effect on SE-induced epileptogenesis. IL-1ß signaling was blocked by systemic administration of two antiinflammatory drugs, namely human recombinant IL-1 receptor antagonist (anakinra), the naturally occurring and clinically used competitive IL-1 receptor type 1 antagonist, and VX-765 a specific non-peptide inhibitor of IL-1ß cleavage and release. Antiinflammatory drugs were given 60min after antiepileptic (AED) drug-controlled SE induced by pilocarpine, or 180min after unrestrained electrical SE, for 7days using a protocol yielding therapeutic drug levels in brain. This drug combination significantly decreased both IL-1ß expression in astrocytes and cell loss in rat forebrain. Neuroprotection and the antiinflammatory effect were more pronounced in the electrical SE model. Onset of epilepsy, and frequency and duration of seizures 3months after electrical SE were not significantly modified. Transcriptomic analysis in the hippocampus showed that the combined treatment did not affect the broad inflammatory response induced by SE during epileptogenesis. In particular, the treatment did not prevent the induction of the complement system and Toll-like receptors, both contributing to cell loss and seizure generation. We conclude that the IL-1ß signaling represents an important target for reducing cell loss after SE. The data highlight a new class of clinically tested agents affording neuroprotection after a delayed post-injury intervention. Earlier blockade of this rapid onset inflammatory pathway during SE, or concomitant treatment with antiinflammatory drugs targeting additional components of the broad inflammatory response to SE, or co-treatment with AEDs, is likely to be required for optimizing beneficial outcomes.
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Epilepsia del Lóbulo Temporal/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Interleucina-1beta/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Dipéptidos/uso terapéutico , Modelos Animales de Enfermedad , Estimulación Eléctrica/efectos adversos , Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/patología , Epilepsia del Lóbulo Temporal/prevención & control , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Proteína Antagonista del Receptor de Interleucina 1/sangre , Proteína Antagonista del Receptor de Interleucina 1/líquido cefalorraquídeo , Litio/toxicidad , Masculino , Pilocarpina/toxicidad , Ratas , Ratas Sprague-Dawley , para-Aminobenzoatos/uso terapéuticoRESUMEN
Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and ß, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Dioxoles/farmacología , Liposarcoma Mixoide/genética , Liposarcoma Mixoide/metabolismo , Tetrahidroisoquinolinas/farmacología , Apoptosis , Proteína C-Reactiva/genética , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Metilación de ADN , Metilasas de Modificación del ADN/deficiencia , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN/deficiencia , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Resistencia a Antineoplásicos , Fibronectinas/genética , Humanos , Liposarcoma Mixoide/tratamiento farmacológico , Liposarcoma Mixoide/patología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Regiones Promotoras Genéticas , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Componente Amiloide P Sérico/genética , Transducción de Señal , Temozolomida , Trabectedina , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Rayos UltravioletaRESUMEN
BACKGROUND: The detection of homologous recombination deficiency (HRD) can identify patients who are more responsive to platinum and poly ADP ribose polymerase inhibitors (PARPi). MyChoice CDx (Myriad) is the most used HRD test in ovarian cancer (OC). However, some limitations of commercial tests exist, because of the high rate of inconclusive results, costs, and the impossibility of evaluating functional resistance mechanisms. PATIENTS AND METHODS: Two academic genomic tests and a functional assay, the RAD51 foci, were evaluated to detect HRD. One hundred patients with high-grade OC enrolled in the MITO16A/MaNGO-OV2 trial and treated with first-line therapy with carboplatin, paclitaxel, and bevacizumab were analyzed. RESULTS: The failure rate of the two genomic assays was 2%. The sensitivity in detecting HRD when compared with Myriad was 98.1% and 90.6%, respectively. The agreement rate with Myriad was 0.92 and 0.87, with a Cohen's κ coefficient corresponding to 0.84 and 0.74, respectively. For the RAD51 foci assay, the failure rate was 30%. When the test was successful, discordant results for deficient and proficient tumors were observed, and additional HRD patients were identified compared to Myriad; sensitivity was 82.9%, agreement rate was 0.65, and Cohen's κ coefficient was 0.18. The HRD detected by genomic assays and residual tumor at primary surgery and stage was correlated with progression-free survival at multivariate analysis. CONCLUSIONS: Results suggest the feasibility of academic tests for assessing HRD status that show robust concordance with Myriad and correlation with clinical outcome. The contribution of the functional information related to the RAD51 foci test to the genomic data needs further investigation.
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Mangifera , Neoplasias Ováricas , Femenino , Humanos , Bevacizumab/uso terapéutico , Carboplatino/uso terapéutico , Recombinación Homóloga , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Paclitaxel/uso terapéutico , Platino (Metal)/uso terapéutico , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/uso terapéuticoRESUMEN
In cancer, myeloid cells have tumor-supporting roles. We reported that the protein GPNMB (glycoprotein nonmetastatic B) was profoundly upregulated in macrophages interacting with tumor cells. Here, using mouse tumor models, we show that macrophage-derived soluble GPNMB increases tumor growth and metastasis in Gpnmb-mutant mice (DBA/2J). GPNMB triggers in the cancer cells the formation of self-renewing spheroids, which are characterized by the expression of cancer stem cell markers, prolonged cell survival and increased tumor-forming ability. Through the CD44 receptor, GPNMB mechanistically activates tumor cells to express the cytokine IL-33 and its receptor IL-1R1L. We also determined that recombinant IL-33 binding to IL-1R1L is sufficient to induce tumor spheroid formation with features of cancer stem cells. Overall, our results reveal a new paracrine axis, GPNMB and IL-33, which is activated during the cross talk of macrophages with tumor cells and eventually promotes cancer cell survival, the expansion of cancer stem cells and the acquisition of a metastatic phenotype.
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Fibrosarcoma/patología , Receptores de Hialuranos/metabolismo , Interleucina-33/metabolismo , Neoplasias Pulmonares/patología , Macrófagos/inmunología , Glicoproteínas de Membrana/metabolismo , Células Madre Neoplásicas/patología , Animales , Apoptosis , Proliferación Celular , Fibrosarcoma/etiología , Fibrosarcoma/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Receptores de Hialuranos/genética , Interleucina-33/genética , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos DBA , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Sarcoma Experimental/etiología , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: We evaluated hyperhomocysteinaemia (HHcy) in a cohort of HIV-infected patients in order to assess its relation to cardiovascular risk (CVR) and identify determinants of HHcy variability. METHODS: Cross-sectional observational study. HIV-infected patients on stable highly active antiretroviral therapy (ART) were evaluated for the presence of the metabolic syndrome, lipodystrophy and traditional CVR factors. Plasma homocysteine levels were measured using high-performance liquid chromatography. RESULTS: Five hundred and sixty-seven patients (38% female) with a median age of 44 years were included in the study. Homocysteine (Hcy) was significantly higher in patients with the metabolic syndrome and lipodystrophy. No significant association was found between Hcy levels and the use of ART. However, Hcy was associated with higher blood pressure, waist circumference and waist-to-hip ratio, total lean body mass, visceral adipose tissue (VAT), VAT/total adipose tissue, homeostasis model assessment of insulin resistance (HOMA-IR), triglycerides, high-density lipoprotein cholesterol, apolipoprotein A1, B, and creatinine. All 10-year CVR assessment scores were significantly associated with Hcy. In a multivariate regression model, systolic blood pressure, vitamin supplementation and HOMA-IR were significantly and independently related to Hcy. CONCLUSIONS: Hcy is elevated in HIV-infected patients and is significantly associated with increased CVR. Measurement of Hcy might be useful in identifying particularly high-risk populations at whom therapeutic interventions could be targeted.
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Enfermedades Cardiovasculares/etiología , Infecciones por VIH/complicaciones , VIH-1 , Síndrome de Lipodistrofia Asociada a VIH/complicaciones , Hiperhomocisteinemia/complicaciones , Síndrome Metabólico/complicaciones , Adulto , Antropometría , Terapia Antirretroviral Altamente Activa , Biomarcadores , Enfermedades Cardiovasculares/metabolismo , Estudios Transversales , Femenino , Infecciones por VIH/metabolismo , Síndrome de Lipodistrofia Asociada a VIH/metabolismo , Homocisteína/sangre , Humanos , Hiperhomocisteinemia/metabolismo , Masculino , Síndrome Metabólico/metabolismo , Valor Predictivo de las Pruebas , Estudios Prospectivos , Medición de RiesgoRESUMEN
Coproporphyrin (CP) is one of the main by-products of heme biosynthesis and its abnormal accumulation is associated with different forms of porphyria. Indirect data obtained from animal and human models have suggested a possible role for Multidrug Resistance-associated Protein 2 (MRP2) and other MRPs in hepatocyte excretion of CP. Using normal, MRP2-deficient and a cholestatic rat model, we have assessed the role of MRPs in CP disposition. MRP levels were assayed using immunofluorescence. Biliary and urinary excretion patterns of CP and conjugate bilirubin were measured during equimolar infusions of CP isomers with and without phenoldibromopthalein sulfonate (BSP), a well-known MRP2 substrate. Our results suggest a role for the MRP system as a possible regulator of CP traffic and accumulation in normal and pathological conditions. Alteration in this systems (as observed in cholestatic disease) may play an important role in triggering clinical expression of porphyria in individuals with underlying mutations leading to porphyrin accumulation and may help explain the phenotypic heterogeneity in patients affected by different forms of porphyrias.
Asunto(s)
Bilis/metabolismo , Coproporfirinas/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Bilirrubina/orina , Transporte Biológico , Colestasis/metabolismo , Colestasis/patología , Coproporfirinas/orina , Modelos Animales de Enfermedad , Isomerismo , Ictericia Idiopática Crónica/metabolismo , Ictericia Idiopática Crónica/patología , Masculino , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/deficiencia , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Ratas , Ratas WistarRESUMEN
Variegate Porphyria (VP) is an autosomal dominant disorder found worldwide but is rare in Italy. In this study we provide an overview of clinical, biochemical and genetic background of 33 Italian VP patients diagnosed in the last fifteen years. About 70% of patients had experienced clinical symptoms: 43.4% had photosensivity, 8.7% acute attacks and 47.8% both. Among the 33 patients, 14 different mutations were identified. Of these only 6 defects have been previously described in other countries and 8 are unique having been identified for the first time in Italy. Two of these, the c.851G>T and the c.1013C>G, were found in two and four unrelated families respectively. No mutation has been found in homozygosis and no significant correlation has been observed between specific clinical and biochemical manifestations and the type of mutation. In contrast, normal faecal protoporphyrin excretion was high predictive of silent phenotype. Normal urinary excretion of PBG and ALA, predicted absence of neurovisceral symptoms. This paper represents the first compilation of data on genotype-phenotype relation in Italian patients with VP.
Asunto(s)
Flavoproteínas/genética , Proteínas Mitocondriales/genética , Porfiria Variegata/genética , Protoporfirinógeno-Oxidasa/genética , Población Blanca/genética , Adulto , Anciano , Ácido Aminolevulínico/orina , Femenino , Flavoproteínas/metabolismo , Estudios de Asociación Genética , Genotipo , Humanos , Italia , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/metabolismo , Polimorfismo Genético , Porfobilinógeno/orina , Porfiria Variegata/diagnóstico , Porfirinas/orina , Protoporfirinógeno-Oxidasa/metabolismoRESUMEN
BACKGROUND: P63 belongs to the 'p53 family' whose role in cancer progression has been recently revisited in light of the plethora of splicing variants that are generated. We analyzed the expression of the full-length TAp63 gene and its dominant-negative form deltaNp63 in ovarian cancer biopsies to correlate their expression with clinical outcome. MATERIALS AND METHODS: Real-time RT-PCR analysis was used to determine the levels of TAp63 and deltaNp63 in 83 stage I and in 86 stage III ovarian cancer biopsies and in seven human ovarian cancer cell. RESULTS: TAp63 levels were comparable in stage I and stage III, but deltaNp63 levels increased 77-fold in stage III, independently of the p53 status. Patients with high deltaNp63 expression had the worst overall survival (OS); patients with a deltaNp63/TAp63 ratio >2 had a poor OS. Patients with a high deltaNp63/TAp63 ratio were those with a poor response to platinum-based therapy. CONCLUSIONS: Data indicate a role for deltaNp63 as a potential biomarker to predict patient's outcome and tumor progression in ovarian cancer. This would have particularly clinical relevance in ovarian cancer where the high rate of mortality reflects our lack of knowledge of molecular mechanisms underlying cell progression toward malignancy.
Asunto(s)
Biomarcadores de Tumor/análisis , Proteínas de Unión al ADN/análisis , Neoplasias Ováricas/genética , Neoplasias Ováricas/mortalidad , Transactivadores/análisis , Proteínas Supresoras de Tumor/análisis , Biopsia , Progresión de la Enfermedad , Femenino , Humanos , Persona de Mediana Edad , Mutación , Estadificación de Neoplasias , Neoplasias Ováricas/patología , Análisis de Supervivencia , Factores de TranscripciónRESUMEN
The p73 gene has a complex regulation, which leads to the expression of different isoforms, often with opposite biological effects. We have generated in the human colocarcinoma cell line HCT116, expressing a wild-type p53, an inducible DNp73alpha expressing system. Two clones (HCT116/DN3 and HCT116/DN14), upon doxycycline addition, show a strong expression of DNp73alpha. In vitro the two DNp73alpha overexpressing clones grow at similar rate of the control transfected clone (HCT116/8a) and similarly respond to DNA damage. When injected in mice, HCT116/DN3, HCT116/DN14, and HCT116/8a cells grew similarly in the absence or presence of tetracycline. In HCT116/DN3 and HCT116/DN14 tumors, tetracycline induced a strong expression of DNp73alpha both as mRNA and protein. These results indicate that in this system the overexpression of the DNp73alpha does not induce a more aggressive phenotype and does not seem to be associated with a reduced response of the cells to treatment with anticancer agents.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Doxorrubicina/farmacología , Doxiciclina/farmacología , Expresión Génica , Genes Supresores de Tumor , Células HCT116 , Humanos , Ratones , Neoplasias/genética , Fenotipo , Proteína Tumoral p73 , Proteínas Supresoras de TumorRESUMEN
We examined the consequences of p73alpha overexpression on gene expression and cellular response to anticancer agents in clones from the human ovarian cancer cell line A2780. Using microarray filters, the expression of 588 genes in two clones overexpressing p73alpha (A2780/p73.4 and A2780/ p73.5) in comparison with empty vector-transfected cells was evaluated. There were clear differences in gene expression profiles. Both of the clones showed a marked increase in the expression of genes involved in DNA repair, including genes participating in nucleotide excision repair and mismatch repair. This was confirmed by reverse transcription-PCR and Northern blot analysis and was associated with an increase in the ability of p73alpha-expressing clones to repair two different DDP (cis-dichlorodiammine platinum)-damaged plasmids in a host reactivation assay. p73alpha overexpressing clones were less sensitive than parental cells to alkylating agents treatment or UV radiation but equally sensitive to the topoisomerase I inhibitor topotecan, which indicated that the increase in expression of DNA repair genes has implications for the response to DNA damaging agents.
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Daño del ADN , Proteínas de Unión al ADN/biosíntesis , Proteínas Nucleares/biosíntesis , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , División Celular , Cisplatino/farmacología , Células Clonales , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Resistencia a Antineoplásicos , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Proteínas Nucleares/genética , Neoplasias Ováricas/tratamiento farmacológico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Topotecan/farmacología , Transfección , Células Tumorales Cultivadas , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor , Rayos Ultravioleta/efectos adversosRESUMEN
The activity of 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548), a new alkycycline with high antitumor activity against a broad range of cancer cells, was evaluated in vitro and in vivo in cells selected for resistance to different anticancer agents. Both in vitro and in vivo, PNU-159548 did retain its activity in cells expressing the multidrug resistance (MDR) phenotype, associated to MIDR-1 gene overexpression or with an alteration in the topoisomerase II gene (altered MDR), independently on the drug used for the selection of the resistant cell line. According to these data, the intracellular uptake of PNU-159548 is not influenced by the presence of MDR-1. PNU-159548 was also active, both in vitro and in vivo, against cells showing resistance to various alkylating agents iincluding cisplatin, cyclophosphamide, and melphalan) and topoisomerase I-inhibitors. Cells defective in nucleotide excision repair, which did show hypersensitivity to treatment with UV irradiation and alkylating agents, showed only a marginally increased sensitivity to PNU-159548. Similarly, the activity of the drug was not influenced by the mismatch repair system, as assessed in two different cellular systems deficient in hMLH1 expression and in which hMLH1 activity was restored by chromosome 3 transfer. The results obtained clearly indicate that the new anticancer agent PNU-159548 is able to overcome the classical mechanisms of resistance emerging after treatment with the most clinically used anticancer agents, and it could represent an alternate choice in the treatment of those tumors refractory to conventional therapy.
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Antibióticos Antineoplásicos/farmacología , Daunorrubicina/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Animales , Células CHO , Cricetinae , Daunorrubicina/análogos & derivados , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Concentración 50 Inhibidora , Leucemia L1210/tratamiento farmacológico , Leucemia P388/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBARESUMEN
Hemizygosity and homozygosity mapping studies show that many common sporadic cancers including lung, breast, kidney, cervical, ovarian, and head and neck cancer display deletions on the short arm of chromosome 3. For ovarian cancer, monochromosomal transfer suppression studies have identified three candidate regions for chromosome 3p ovarian cancer tumor suppressor genes (OCTSGs). To accurately map OCTSG candidate regions, we analyzed 70 ovarian tumors for loss of heterozygosity (LOH) at 20 loci on chromosome 3p that were selected to target those regions proposed to contain tumor suppressor genes for common sporadic cancers. All samples were informative for at least five markers. In 33 (52%) tumors without microsatellite instability, LOH was observed for at least one 3p marker. Analysis of 27 ovarian tumors demonstrating both loss and retention of 3p markers enabled us to define four nonoverlapping minimal deletion regions (OCLOHRs): (a) OCLOHR-1 mapped distal to D3S3591 at 3p25-26; (b) OCLOHR-2 mapped between D3S1317 and D3S1259 at 3p24-25; (c) OCLOHR-3 mapped between D3S1300 and D3S1284, an area that includes the FHIT locus at 3p14.2; and (d) OCLOHR-4 mapped between D3S1284 and D3S1274 at 3p12-13, a region known to contain overlapping homozygous deletions in lung and breast tumor cell lines. However, microsatellite markers from the chromosome 3p21.3 interval homozygously deleted in lung cancer cell lines did not identify a distinct OCLOHR. The frequency and extent of 3p LOH correlated with tumor stage such that LOH at two or more OCLOHRs was present in 53% (16 of 30) of stage III tumors but only 26% (5 of 19) of stage I/II tumors (P = 0.08). To determine the relationship between the OCLOHRs and the three candidate ovarian cancer suppression regions (OCSRs) identified previously by monochromosome transfer studies, we performed detailed genetic and physical mapping studies to define the extent of the three candidate OCSRs and to establish YAC contigs covering each region. OCSR-A at 3p25-26 and OCSR-B at 3p24 were shown to overlap with OCLOHR-1 and OCLOHR-2, respectively, providing further evidence for OCTSGs in these regions. We also show that OCSR-C overlaps with a locus at 3p21.3 previously implicated in lung and breast cancer.
Asunto(s)
Ácido Anhídrido Hidrolasas , Cromosomas Humanos Par 3 , Genes Supresores de Tumor , Pérdida de Heterocigocidad , Neoplasias Ováricas/genética , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Femenino , Marcadores Genéticos , Humanos , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Neoplasias Ováricas/sangre , Neoplasias Ováricas/patología , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Proteínas/genéticaRESUMEN
4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548) belongs to a novel class of antitumor compounds (termed alkycyclines) and is currently undergoing Phase II clinical trial. In the present study, we investigated the in vitro and in vivo antitumor activity, the pharmacokinetics, and the toxicological profile of this compound. PNU-159548 showed good cytotoxic activity in murine and human cancer cells growing in vitro, with an average concentration for 50% growth inhibition of 15.8 ng/ml. The drug showed strong antitumor efficacy in vivo after i.v. and p.o. administration against rapidly proliferating murine leukemias and slowly growing transplantable human xenografts. At non-toxic doses, PNU-159548 produced complete regression and cures in ovarian, breast, and human small cell lung carcinomas. Fourteen of 16 models studied, including colon, pancreatic, gastric, and renal carcinomas, astrocytoma and melanoma, were found to be sensitive to PNU-159548. In addition, PNU-159548 was effective against intracranially implanted tumors. Toxicological studies revealed myelosuppression as the main toxicity in both mice and dogs. The maximum tolerated doses, after a single administration, were 2.5 mg/kg of body weight in mice, 1.6 mg/kg in rats, and 0.3 mg/kg in dogs. In the cyclic studies, the maximum tolerated doses were 0.18 mg/kg/day (cumulative dose/cycle: 0.54 mg/kg) in rats and 0.05 mg/kg/day (cumulative dose/cycle: 0.15 mg/kg) in dogs. PNU-159548 showed minimal cardiotoxicity, when compared with doxorubicin in the chronic rat model at a dose level inducing similar myelotoxicity. Animal pharmacokinetics, carried out in mice, rats, and dogs, was characterized by high volumes of distribution, plasma clearance of the same order of the hepatic blood flow, and short terminal half-life. These findings support the conclusion that PNU-159548 is an excellent candidate for clinical trials in the treatment of cancer.