RESUMEN
While there is no reliable serum biomarker for the diagnosis and monitoring of patients with gastric cancer, we tested the potential diagnostic and prognostic values of detecting methylation changes in the serum of gastric cancer patients. DNA was extracted from the pretherapeutic serum of 60 patients with confirmed gastric adenocarcinoma and 22 age-matched noncancer controls. Promoter hypermethylation in 10 tumour-related genes (APC, E-cadherin, GSTP1, hMLH1, MGMT, p15, p16, SOCS1, TIMP3 and TGF-beta RII) was determined by quantitative methylation-specific PCR (MethyLight). Preferential methylation in the serum DNA of gastric cancer patients was noted in APC (17%), E-cadherin (13%), hMLH1 (41%) and TIMP3 (17%) genes. Moreover, patients with stages III/IV diseases tended to have higher concentrations of methylated APC (P = 0.08), TIMP3 (P = 0.005) and hMLH1 (P = 0.03) in the serum. In all, 33 cancers (55%) had methylation detected in the serum in at least one of these four markers, while three normal subjects had methylation detected in the serum (specificity 86%). The combined use of APC and E-cadherin methylation markers identified a subgroup of cancer patients with worse prognosis (median survival 3.3 vs 16.1 months, P = 0.006). These results suggest that the detection of DNA methylation in the serum may carry both diagnostic and therapeutic values in gastric cancer patients.
Asunto(s)
Adenocarcinoma/genética , ADN de Neoplasias/genética , Neoplasias Gástricas/genética , Adenocarcinoma/sangre , Adenocarcinoma/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Metilación de ADN , ADN de Neoplasias/sangre , Femenino , Genes Supresores de Tumor/fisiología , Humanos , Masculino , Persona de Mediana Edad , Oncogenes/fisiología , Pronóstico , Regiones Promotoras Genéticas , Neoplasias Gástricas/sangre , Neoplasias Gástricas/diagnósticoRESUMEN
The interleukin-mediated Janus kinase (JAK)/STAT pathway plays a crucial role in carcinogenesis. Recently, increased STAT3 activity was found in hepatocellular carcinoma and multiple myeloma in which there was silencing of SOCS-1 (suppressor of cytokine signalling-1) by gene promoter hypermethylation. We investigated the expression level of interleukin-6 (IL-6) and SOCS-1 in gastric cancer cell lines. Expression of SOCS-1 correlated with IL-6 level in most of the cell lines, except for AGS cells in which SOCS-1 was absent despite a high level of IL-6 production. Methylation analysis by methylation-specific polymerase chain reaction and bisulphite sequencing revealed that CpG island of SOCS-1 was densely methylated in AGS cells. Demethylation treatment by 5'aza-deoxycytidine restored SOCS-1 expression and also suppressed constitutive STAT3 phosphorylation in AGS cells. Moreover, methylation of SOCS-1 was detected in 27.5% (11 of 40) of primary gastric tumours samples, 10% (one of 10) of adjacent noncancer tissues but not in any (zero of nine) normal gastric mucosa. Methylation of SOCS-1 also correlated with the loss of mRNA expression in some primary gastric cancers. In conclusion, this is the first report to demonstrate that hypermethylation of SOCS-1 led to gene silencing in gastric cancer cell line and primary tumour samples. Downregulation of SOCS-1 cooperates with IL-6 in the activation of JAK/STAT pathway in gastric cancer.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/farmacología , Interleucina-6/farmacología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Tirosina Quinasas/farmacología , Proteínas Represoras/metabolismo , Neoplasias Gástricas/patología , Transactivadores/farmacología , Proteínas Portadoras/biosíntesis , Transformación Celular Neoplásica , Metilación de ADN , Regulación hacia Abajo , Humanos , Janus Quinasa 1 , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Proteínas Represoras/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3 , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Células Tumorales CultivadasRESUMEN
BACKGROUND AND AIMS: Although peroxisome proliferator activated receptor gamma (PPARgamma) agonists have been implicated in differentiation and growth inhibition of cancer cells, the potential therapeutic and chemopreventive effects on gastric cancer are poorly defined. We examined the in vitro and in vivo effects of PPARgamma ligands on growth of gastric cancer, and the effect of PPARgamma activation on expression of cyclooxygenase 2 (COX-2) and cancer related genes. METHODS: Gastric cell lines (MKN28 and MKN45) were treated with two specific PPARgamma ligands: ciglitazone and 15-deoxy-Delta(12,)(14)-prostaglandin J(2). Cell growth was determined by bromodeoxyuridine incorporation assay and apoptosis was measured by DNA fragmentation. Expression of COX-2 was determined by western blot and real time quantitative polymerase chain reaction (PCR). Expression profiles of cancer related genes were screened with cDNA array. In vivo growth of implanted MKN45 cells in nude mice was monitored after oral treatment with rosiglitazone. RESULTS: PPARgamma ligands suppressed the in vitro growth of MKN45 cells in a dose dependent manner whereas prostacyclin, a PPARdelta agonist, had no growth inhibitory effect. Growth inhibition was more pronounced in MKN45 cells, which was accompanied by DNA fragmentation and downregulation of COX-2. Screening by cDNA microarray showed that PPARgamma ligand treatment was associated with upregulation of bad and p53, and downregulation of bcl-2, bcl-xl, and cyclin E1 in MKN45 cells, which was confirmed by quantitative real time PCR. In contrast, MKN28 cells with lower PPARgamma and COX-2 expression levels had lower growth inhibitory responses to PPARgamma ligands. Microarray experiments only showed induction of the bad gene in MKN28 cells. In vivo growth of MKN45 cells in nude mice was retarded by rosiglitazone. Mean tumour volume in rosiglitazone treated mice was significantly lower than controls at six weeks (p = 0.019) and seven weeks (p = 0.001) after treatment. CONCLUSIONS: PPARgamma ligands suppress both in vitro and in vivo growth of gastric cancer and may play a major role in cancer therapy and prevention.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Neoplasias/fisiología , Prostaglandina D2/análogos & derivados , Neoplasias Gástricas/patología , Factores de Transcripción/fisiología , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Ciclooxigenasa 2 , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , Ligandos , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Coactivadores de Receptor Nuclear , Prostaglandina D2/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Rosiglitazona , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Tiazolidinedionas/farmacología , Tiazolidinedionas/uso terapéutico , Factores de Transcripción/agonistas , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
BACKGROUND: Overexpression of cyclooxygenase 2 (COX-2) is frequently detected in gastric cancer and is believed to play a crucial role in gastric carcinogenesis. AIM: We examined the chemopreventive effect of a COX-2 inhibitor in an animal model of stomach carcinogenesis. METHODS: Eighty six male Wistar rats were divided into six different treatment groups: group A, water alone (n = 5); group B, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG 100 micro g/ml) (n = 16); group C, indomethacin (3 mg/kg/day) (n = 16); group D, celecoxib (5 mg/kg/day) (n = 17); group E, celecoxib (10 mg/kg/day) (n = 16); and group F, celecoxib (20 mg/kg/day) (n = 16). Group B-F animals were treated with 10% sodium chloride (in the initial six weeks) and MNNG in drinking water to induce adenocarcinoma in the stomach. All animals received treatment for 40 weeks, and were sacrificed after death or at 48 weeks. Gastric neoplasm was evaluated by histology. RESULTS: The incidences of gastric cancer were 0% in group A, 75% in group B, 68.8% in group C, 70.6% in group D, 18.8% in group E, and 31.3% in group F (p = 0.002, ANOVA). Compared with MNNG controls, treatment with celecoxib 10 mg/kg/day also showed lower tumour multiplicity (0.19 (0.40) v 1.00 (0.73); p = 0.004) and lower mean tumour volume (2.4 v 2805 mm(3); p = 0.02). Although tumours had significantly higher COX-2 expression than their adjacent normal tissues (p<0.02), there was no significant difference in COX-2 levels among tumours in the different treatment groups. The lowest tumour prostaglandin E(2) level was found in the indomethacin treated group, suggesting that the chemopreventive effect of celecoxib may be mediated by a COX independent pathway. CONCLUSION: While treatment with indomethacin had no significant effect on tumour development, treatment with celecoxib reduced gastric cancer incidence and growth in rats.
Asunto(s)
Inhibidores de la Ciclooxigenasa/administración & dosificación , Neoplasias Gástricas/prevención & control , Sulfonamidas/administración & dosificación , Análisis de Varianza , Animales , Celecoxib , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/uso terapéutico , Dinoprostona/análisis , Esquema de Medicación , Indometacina/uso terapéutico , Isoenzimas/análisis , Masculino , Metilnitronitrosoguanidina , Modelos Animales , Prostaglandina-Endoperóxido Sintasas/análisis , Pirazoles , Ratas , Ratas Wistar , Cloruro de Sodio , Sulfonamidas/uso terapéuticoRESUMEN
Expression of cyclin D2 is absent in 30-70% of gastric cancers. We investigated the role of promoter hypermethylation in the transcriptional silencing of cyclin D2 in five gastric cell lines and 47 primary gastric carcinomas. CpG island methylation status of the cyclin D2 gene was studied by methylation-specific polymerase chain reaction and bisulphite sequencing. RNA and protein expression was analysed by reverse transcription-PCR and Western blot, respectively. Dense methylation of cyclin D2 was detected in three cell lines (KATOIII, AGS and NCI-N87), which also lacked cyclin D2 mRNA and protein expression. Bisulphite DNA sequencing revealed that loss of cyclin D2 expression was closely associated with the density of methylation in the promoter region. Treatment with DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the cyclin D2 expression level in methylated gastric cells. Among the 47 primary gastric cancers, cyclin D2 hypermethylation was detected in 23 (48.9%) cases. None of the 23 normal gastric biopsies from noncancer patients showed hypermethylation. Hypermethylation was associated with loss of mRNA (P&<0.001) and protein (P=0.006) expressions. Our study showed that cyclin D2 hypermethylation is associated with loss of cyclin D2 expression in a subset of gastric cancers, which may suggest an alternative gastric carcinogenesis pathway in the absence of cyclin D2 expression.