RESUMEN
AIMS: Sex cord stromal tumours (SCSTs) of the ovary encompass several histological tumour subtypes that are defined by characteristic histological features. Some can show morphological overlap with other subtypes of SCSTs, as well as with non-SCSTs. The E-cadherin/catenin complex constitutes the adherens junction, which is well developed in epithelial tissue, but the constituent molecules are also expressed in several non-epithelial tumours. The aim of this study was to determine whether the expression patterns of E-cadherin and catenins in ovarian SCSTs can be of diagnostic utility. METHODS AND RESULTS: We studied the expression of E-cadherin, α-, ß- and γ-catenin in 55 tumours using immunohistochemistry. We found that all tumour subtypes showed nuclear expression of E-cadherin, while only microcystic stromal tumours (MCSTs) displayed a distinct profile, with nuclear localization of all three catenins in almost all cases. CONCLUSIONS: We conclude that the E-cadherin expression profile in SCSTs can assist in distinguishing between SCSTs and non-SCSTs in which there is no nuclear expression of E-cadherin. The nuclear localization of catenins may be of potential use in distinguishing MCST from other subtypes of SCST.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Cadherinas/metabolismo , Neoplasias Ováricas/metabolismo , Tumores de los Cordones Sexuales y Estroma de las Gónadas/metabolismo , Antígenos CD , Desmoplaquinas/metabolismo , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Neoplasias Ováricas/patología , Ovario/metabolismo , Tumores de los Cordones Sexuales y Estroma de las Gónadas/patología , alfa Catenina/metabolismo , beta Catenina/metabolismo , gamma CateninaRESUMEN
Interleukin-8 (IL-8) is a pro-inflammatory cytokine which exerts its effects via binding to 2 receptors, CXCR1 and CXCR2 and is known to promote angiogenesis, mitogenesis and motogenesis in cancer. IL-8 is over expressed in endometrial carcinoma, but the expression of CXCR1 and CXCR2 in endometrial carcinoma has not been previously investigated. The aim of this study was to determine the expression of IL-8 receptors in endometrial carcinoma. The expression of CXCR1 and CXCR2 was studied in endometrial carcinomas and normal endometrium by immunohistochemistry in 101 tumours. IL-8 and IL-8 receptor expression was also studied by Real-time quantitative PCR (RT-qPCR) in 17 tumours in comparison to normal endometrium. The expression profile was correlated to the clinico-pathological features of the tumours. Immunohistochemistry showed CXCR1 and CXCR2 were expressed in all cases of endometrial carcinoma, with CXCR1 showing stronger expression. There was a statistically significant correlation between CXCR2 staining intensity and tumour grade (P=0.012) and disease free survival (P=0.015) independently. On RT-qPCR, 14/17, 15/17 and 16/17 tumours showed significant increase in IL-8, CXCR1 and CXCR2 expression levels in comparison to normal endometrium, with median fold increase of 42-fold, 51-fold and 27-fold, respectively. This is the first report of the expression of IL-8 receptors in endometrial carcinoma and the results show an association between IL-8 and IL-8 receptors and the pathogenesis of endometrial carcinoma, and represent potential prognostic biomarkers and therapeutic targets.
Asunto(s)
Neoplasias Endometriales/genética , Regulación Neoplásica de la Expresión Génica , Interleucina-8/genética , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8B/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Endometriales/patología , Femenino , Humanos , Interleucina-8/metabolismo , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Resultado del TratamientoRESUMEN
The majority of borderline ovarian tumours (BOTs) behave in a benign fashion, but some may show aggressive behavior. The reason behind this has not been elucidated. The epidermal growth factor receptor (EGFR) is known to contribute to cell survival signals as well as metastatic potential of some tumours. EGFR expression and gene status have not been thoroughly investigated in BOTs as it has in ovarian carcinomas. In this study we explore protein expression as well as gene mutations and amplifications of EGFR in BOTs in comparison to a subset of other epithelial ovarian tumours. We studied 85 tumours, including 61 BOTs, 10 low grade serous carcinomas (LGSCs), 9 high grade serous carcinomas (HGSCs) and 5 benign epithelial tumours. EGFR protein expression was studied using immunohistochemistry. Mutations were investigated by Sanger sequencing exons 18-21 of the tyrosine kinase domain of EGFR. Cases with comparatively higher protein expression were examined for gene amplification by chromogenic in situ hybridization. We also studied the tumours for KRAS and BRAF mutations. Immunohistochemistry results revealed both cytoplasmic and nuclear EGFR expression with variable degrees between tumours. The level of nuclear localization was relatively higher in BOTs and LGSCs as compared to HGSCs or benign tumours. The degree of nuclear expression of BOTs showed no significant difference from that in LGSCs (mean ranks 36.48, 33.05, respectively, p=0.625), but was significantly higher than in HGSCs (mean ranks: 38.88, 12.61 respectively, p< 0.001) and benign tumours (mean ranks: 35.18, 13.00 respectively, p= 0.010). Cytoplasmic expression level was higher in LGSCs. No EGFR gene mutations or amplification were identified, yet different polymorphisms were detected. Five different types of point mutations in the KRAS gene and the V600E BRAF mutation were detected exclusively in BOTs and LGSCs. Our study reports for the first time nuclear localization of EGFR in BOTs. The nuclear localization similarities between BOTs and LGSCs and not HGSCs support the hypothesis suggesting evolution of LGSCs from BOTs. We also confirm that EGFR mutations and amplifications are not molecular events in the pathogenesis of BOTs.
Asunto(s)
Cistadenocarcinoma Seroso/genética , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Ováricas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Secuencia de Bases , Cistadenocarcinoma Seroso/patología , Receptores ErbB/metabolismo , Femenino , Amplificación de Genes , Humanos , Mutación/genética , Clasificación del Tumor , Neoplasias Ováricas/patología , Estructura Terciaria de Proteína/genética , Análisis de Secuencia de ADNRESUMEN
In the past decade, the incidence and mortality rates of cholangiocarcinoma (CCA) have been increasing worldwide. The relatively low responsiveness of CCA to conventional chemotherapy leads to poor overall survival. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has emerged as the most promising anti-cancer therapeutic agent since it is able to selectively induce apoptosis of tumor cells but not normal cells. In this study, we aimed to investigate the therapeutic effect of TRAIL in CCA cell lines (M213, M214 and KKU100) compared with the immortal biliary cell line, MMNK1, either alone or in combination with a subtoxic dose of 5-fluorouracil (5-FU). We found that recombinant human TRAIL (rhTRAIL) was a potential agent which significantly inhibited cell proliferation and mediated caspase activities (caspases 8, 9 and 3/7) and apoptosis of CCA cells. The combined treatment of rhTRAIL and 5-FU effectively enhanced inhibition of CCA cell growth with a smaller effect on MMNK1. Our finding suggests TRAIL to be a novel anti-cancer therapeutic agent and advantage of its combination with a conventional chemotherapeutic drug for effective treatment of CCA.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Colangiocarcinoma/tratamiento farmacológico , Fluorouracilo/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Apoptosis/efectos de los fármacos , Caspasas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fluorouracilo/administración & dosificación , Proteínas Ligadas a GPI/genética , Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/metabolismo , Miembro 10c de Receptores del Factor de Necrosis Tumoral/genética , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Receptores Señuelo del Factor de Necrosis Tumoral/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Platinum based drugs are the cornerstone of chemotherapy for ovarian cancer, however the development of chemoresistance hinders its success. IL-8 is involved in regulating several pro-survival pathways in cancer. We studied the expression of IL-8 and IL-8 receptors in platinum sensitive and resistant cell lines. Using qRT-PCR and immunohistochemistry, both platinum sensitive (PEA1, PEO14) and resistant (PEA2, PEO23) show increased expression of IL-8 and IL-8 receptors. IL-8RA shows nuclear and cytoplasmic expression, whilst IL-8RB is present solely in the cytoplasm. Knockdown of IL-8 increased sensitivity to cisplatin in platinum sensitive and reversed platinum resistance in resistant cell lines, decreased the expression of anti-apoptotic Bcl-2 and decreased inhibitory phosphorylation of pro-apoptotic Bad. IL-8 receptor antagonist treatment also enhanced platinum sensitivity. Nuclear localisation of IL-8RA was only detected in platinum resistant tumours. Inhibition of IL-8 signalling can enhance response in platinum sensitive and resistant disease. Nuclear IL-8RA may have potential as a biomarker of resistant disease.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos , Interleucina-8/metabolismo , Neoplasias Quísticas, Mucinosas y Serosas/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Interleucina-8/genética , Clasificación del Tumor , Neoplasias Quísticas, Mucinosas y Serosas/genética , Neoplasias Quísticas, Mucinosas y Serosas/metabolismo , Neoplasias Quísticas, Mucinosas y Serosas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , Receptores de Interleucina-8A/antagonistas & inhibidores , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8B/antagonistas & inhibidores , Receptores de Interleucina-8B/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Proteína Letal Asociada a bcl/metabolismoRESUMEN
Serous endometrial carcinoma is an aggressive type of endometrial carcinoma. Wilms tumor gene 1 (WT-1) is commonly expressed in ovarian serous carcinomas and considered a diagnostic marker of these tumors. However, it is generally believed that WT-1 is rarely expressed by endometrial serous carcinoma. The aim of this study was to evaluate the frequency and significance of WT-1 expression in endometrial serous carcinoma. We studied the expression of WT-1 in formalin-fixed, paraffin-embedded tumor sections from 77 cases of endometrial serous carcinoma. Thirty-four tumors showed positive expression for WT-1 (44%). There was a statistically significant association between the presence of WT-1 expression and disease-free survival (DFS), where patients with tumors expressing WT-1 had a shorter DFS compared with those with no WT-1 expression (P = .031; median DFS, 15 and 38 months, respectively). By multivariate Cox regression analysis, DFS was independent from other clinicopathological data (tumor stage, presence of lymphovascular space invasion, cervical involvement, and extrauterine spread), indicating that WT-1 expression is independently associated with DFS. Our study shows that WT-1 is expressed in a considerable percentage of endometrial serous carcinomas, suggesting a role for WT-1 in the pathology of these tumors. This has therapeutic significance, as WT-1 is an emerging target for immunotherapy. Moreover, our results show that WT-1 has prognostic value, being predictive of DFS. As a potential prognostic marker and therapeutic target, we recommend that WT-1 expression should be included in histopathologic reports of endometrial serous carcinoma.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Endometrioide/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Neoplasias Endometriales/metabolismo , Proteínas WT1/metabolismo , Anciano , Anciano de 80 o más Años , Carcinoma Endometrioide/patología , Estudios de Cohortes , Cistadenocarcinoma Seroso/patología , Supervivencia sin Enfermedad , Neoplasias Endometriales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Persona de Mediana Edad , Adhesión en Parafina , PronósticoRESUMEN
Histone deacetylation mediated by histone deacetylases (HDACs) has been reported as one of the epigenetic mechanisms associated with tumorigenesis. The poor responsiveness of anticancer drugs found with cholangiocarcinoma (CCA) leads to short survival rate. We aimed to investigate mRNA expression of HDACs class I and II, and the effect of HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), in CCA in vitro. Expression of HDACs was studied in CCA cell lines (M213, M214 and KKU-100) and an immortal cholangiocyte (MMNK1) by semi-quantitative reverse transcription-PCR. SAHA and VPA, as well as a classical chemotherapeutic drug 5-fluorouracil (5-FU) were used in this study. Cell proliferation was determined by sulforhodamine assay. IC50 and IC20 were then analyzed for each agent and cell line. Moreover, synergistic potential of VPA or SAHA in combination with 5-FU at subtoxic dose (IC20) of each agent was also evaluated. Statistic difference of HDACs expression or cell proliferation in each experimental condition was analyzed by Student's t-test. The result demonstrated that HDACs were expressed in all studied cell types. Both SAHA and VPA inhibited cell proliferation in a dose-dependent manner. Interestingly, KKU-100 which was less sensitive to classical chemotherapeutic 5-FU was highly sensitive to HDAC inhibitors. Simultaneous combination of subtoxic doses of HDAC inhibitors and 5-FU significantly inhibited cell proliferation in CCA cell lines compared to single agent treatment (P ≤ 0.01), while sequentially combined treatments were less effective. The present study showed inhibitory effects of HDACIs on cell proliferation in CCA cell lines, with synergistic antitumor potential demonstrated by simultaneous combination of VPA or SAHA with 5-FU, suggesting a novel alternative therapeutic strategy in effective treatment of CCA.
Asunto(s)
Neoplasias de los Conductos Biliares/tratamiento farmacológico , Conductos Biliares Intrahepáticos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colangiocarcinoma/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/química , Neoplasias de los Conductos Biliares/enzimología , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos/enzimología , Conductos Biliares Intrahepáticos/patología , Colangiocarcinoma/enzimología , Colangiocarcinoma/patología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Genetic abnormalities of cholangiocarcinoma have been widely studied; however, epigenomic changes related to cholangiocarcinogenesis have been less well characterized. We have profiled the DNA methylomes of 28 primary cholangiocarcinoma and six matched adjacent normal tissues using Infinium's HumanMethylation27 BeadChips with the aim of identifying gene sets aberrantly and epigenetically regulated in this tumor type. Using a linear model for microarray data, we identified 1610 differentially methylated autosomal CpG sites, with 809 hypermethylated (representing 603 genes) and 801 hypomethylated (representing 712 genes) in cholangiocarcinoma versus adjacent normal tissues (false-discovery rate ≤ 0.05). Gene ontology and gene set enrichment analyses identified gene sets significantly associated with hypermethylation at linked CpG sites in cholangiocarcinoma including homeobox genes and target genes of PRC2, EED, SUZ12, and histone H3 trimethylation at lysine 27. We confirmed frequent hypermethylation at the homeobox genes HOXA9 and HOXD9 by bisulfite pyrosequencing in a larger cohort of cholangiocarcinoma (n = 102). Our findings indicate a key role for hypermethylation of multiple CpG sites at genes associated with a stem cell-like phenotype as a common molecular aberration in cholangiocarcinoma. These data have implications for cholangiocarcinogenesis, as well as possible novel treatment options using histone methyltransferase inhibitors.
Asunto(s)
Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos/patología , Biomarcadores de Tumor/genética , Colangiocarcinoma/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Células Madre Neoplásicas/patología , Neoplasias de los Conductos Biliares/patología , Conductos Biliares/metabolismo , Conductos Biliares Intrahepáticos/metabolismo , Colangiocarcinoma/patología , Islas de CpG/genética , Humanos , Células Madre Neoplásicas/metabolismo , Fenotipo , Regiones Promotoras Genéticas/genética , Sulfitos/químicaRESUMEN
OBJECTIVES: To validate methylation-sensitive high resolution melting (MS-HRM) for detection of DNA methylation. DESIGN AND METHODS: Methylation of two independent loci, OPCML and DcR1, was analyzed in cholangiocarcinoma and adjacent normal samples by using MS-HRM, methylation-specific PCR and pyrosequencing. RESULTS: There was significant agreement between methods at both loci. CONCLUSIONS: MS-HRM represents the excellent potential and reliability for quantifying DNA methylation levels in clinical samples.
Asunto(s)
Colangiocarcinoma/genética , Metilación de ADN , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , Neoplasias de los Conductos Biliares/química , Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos/química , Moléculas de Adhesión Celular/genética , Colangiocarcinoma/química , Islas de CpG , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Sitios Genéticos , Genoma Humano , Humanos , Modelos Lineales , Desnaturalización de Ácido Nucleico , Miembro 10c de Receptores del Factor de Necrosis Tumoral , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Receptores Señuelo del Factor de Necrosis Tumoral/química , Receptores Señuelo del Factor de Necrosis Tumoral/genéticaRESUMEN
OBJECTIVES: To verify complete bisulfite modification. DESIGN AND METHODS: Bisulfite modified DNA was screened by PCR with Calponin-specific primer sets prior to detecting methylation status of 14-3-3sigma by methylation-specific PCR. RESULTS: False positive methylation was obtained from incomplete bisulfite modification. The lower detection limit of wild type Calponin-specific primer is approximately 0.375 ng of unmodified DNA. CONCLUSIONS: Verification of complete bisulfite modification by Calponin-specific primer sets can reduce misinterpretation of methylation.