RESUMO
Esophageal adenocarcinoma (EAC) is the most frequent malignancy in the esophagus in the US and its incidence has been rising rapidly in the past few decades. Chronic gastroesophageal reflux disease (GERD), where the esophageal epithelium is abnormally exposed to acid and bile salts, is a pro-inflammatory condition that is the main risk factor for the development of Barrett's esophagus (BE) and its progression to EAC. Glutathione peroxidase 7 (GPX7) is frequently silenced through DNA hypermethylation during Barrett's tumorigenesis. In this study, we investigated the role of GPX7 in regulating the bile salts-induced inflammatory signaling in Barrett's carcinogenesis. Using quantitative real-time PCR (qRT-PCR), we demonstrated a significant induction in the expression levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8) and chemokines (CXCL-1 and CXCL-2) in esophageal cells after exposure to acidic (pH4) or neutral (pH7) bile salts. Western blot analysis showed that exposure to acidic and neutral bile salts increased p-NF-κB-p65 (S536) protein levels independent of ROS. Reconstitution of GPX7 expression in EAC cells abolished the increase of p-p65 (S536) protein levels and mRNA expression of cytokines and chemokines upon treatment with acidic and neutral bile salts. Examination of human primary EAC tissues by qRT-PCR demonstrated significant overexpression of cytokines (TNF-α, IL-1ß and IL-8) in EAC samples, as compared to normal samples, with significant inverse correlation with GPX7 expression level. Taken together, the loss of GPX7 expression promotes bile salt-induced activation of pro-inflammatory cytokines and chemokines; important contributors to GERD-associated Barrett's carcinogenesis.
RESUMO
Esophageal adenocarcinoma (EAC) is a classic example of inflammation-associated cancer, which develops through GERD (gastroesophageal reflux disease)-Barrett's esophagus (BE)-dysplasia-adenocarcinoma sequence. The incidence of EAC has been rising rapidly in the USA and Western countries during the last few decades. The functions of glutathione peroxidase 7 (GPX7), an antioxidant enzyme frequently silenced during Barrett's tumorigenesis, remain largely uncharacterized. In this study, we investigated the potential role of GPX7 in regulating nuclear factor-kappaB (NF-κB) activity in esophageal cells. Western blot analysis, immunofluorescence and luciferase reporter assay data indicated that reconstitution of GPX7 expression in CP-A (non-dysplastic BE cells) and FLO-1 (EAC cells) abrogated tumor necrosis factor-α (TNF-α)-induced NF-κB transcriptional activity (P < 0.01) and nuclear translocation of NF-κB-p65 (P = 0.01). In addition, we detected a marked reduction in phosphorylation levels of components of NF-κB signaling pathway, p-p65 (S536), p-IκB-α (S32) and p-IKKα/ß (S176/180), as well as significant suppression in induction of NF-κB target genes [TNF-α, interleukin (IL)-6, IL-8, IL-1ß, CXCL-1 and CXCL-2] following treatment with TNF-α in GPX7-expressing FLO-1 cells as compared with control cells. We validated these effects by knockdown of GPX7 expression in HET1A (normal esophageal squamous cells). We found that GPX7-mediated suppression of NF-κB is independent of reactive oxygen species level and GPX7 antioxidant function. Further mechanistic investigations demonstrated that GPX7 promotes protein degradation of TNF-receptor 1 (TNFR1) and TNF receptor-associated factor 2 (TRAF2), suggesting that GPX7 modulates critical upstream regulators of NF-κB. We concluded that the loss of GPX7 expression is a critical step in promoting the TNF-α-induced activation of proinflammatory NF-κB signaling, a major player in GERD-associated Barrett's carcinogenesis.
Assuntos
Adenocarcinoma/patologia , Esôfago de Barrett/patologia , Transformação Celular Neoplásica/patologia , Neoplasias Esofágicas/patologia , NF-kappa B/metabolismo , Peroxidases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Western Blotting , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Imunofluorescência , Glutationa Peroxidase , Humanos , NF-kappa B/genética , Peroxidases/antagonistas & inibidores , Peroxidases/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
Gastric cancer remains the second leading cause of cancer-related death in the world. H. pylori infection, a major risk factor for gastric cancer, generates high levels of reactive oxygen species (ROS). Glutathione peroxidase 3 (GPX3), a plasma GPX member and a major scavenger of ROS, catalyzes the reduction of hydrogen peroxide and lipid peroxides by reduced glutathione. To study the expression and gene regulation of GPX3, we examined GPX3 gene expression in 9 gastric cancer cell lines, 108 primary gastric cancer samples and 45 normal gastric mucosa adjacent to cancers using quantitative real-time RT-PCR. Downregulation or silencing of GPX3 was detected in 8 of 9 cancer cell lines, 83% (90/108) gastric cancers samples, as compared to non-tumor adjacent normal gastric samples (P<0.0001). Examination of GPX3 promoter demonstrated DNA hypermethylation (≥ 10% methylation level determined by Bisulfite Pyrosequencing) in 6 of 9 cancer cell lines and 60% of gastric cancer samples (P = 0.007). We also detected a significant loss of DNA copy number of GPX3 in gastric cancers (P<0.001). Treatment of SNU1 and MKN28 cells with 5-Aza-2' Deoxycytidine restored the GPX3 gene expression with a significant demethylation of GPX3 promoter. The downregulation of GPX3 expression and GPX3 promoter hypermethylation were significantly associated with gastric cancer lymph node metastasis (P = 0.018 and P = 0.029, respectively). We also observed downregulation, DNA copy number losses, and promoter hypermethylation of GPX3 in approximately one-third of tumor-adjacent normal gastric tissue samples, suggesting the presence of a field defect in areas near tumor samples. Reconstitution of GPX3 in AGS cells reduced the capacity of cell migration, as measured by scratch wound healing assay. Taken together, the dysfunction of GPX3 in gastric cancer is mediated by genetic and epigenetic alterations, suggesting impairment of mechanisms that regulate ROS and its possible involvement in gastric tumorigenesis and metastasis.
Assuntos
Metilação de DNA , Inativação Gênica , Glutationa Peroxidase/genética , Metástase Linfática/genética , Neoplasias Gástricas/patologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Decitabina , Imunofluorescência , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/genéticaRESUMO
BACKGROUND: Metallothionein 3 (MT3) maintains intracellular metal homeostasis and protects against reactive oxygen species (ROS)-induced DNA damage. In this study, we investigated the epigenetic alterations and gene expression of the MT3 gene in esophageal adenocarcinomas (EACs). METHODS AND RESULTS: Using quantitative bisulfite pyrosequencing, we detected unique DNA methylation profiles in the MT3 promoter region. The CpG nucleotides from -372 to -306 from the transcription start site (TSS) were highly methylated in tumor (nâ=â64) and normal samples (nâ=â51), whereas CpG nucleotides closest to the TSS (-4 and +3) remained unmethylated in all normal and most tumor samples. Conversely, CpG nucleotides in two regions (from -139 to -49 and +296 to +344) were significantly hypermethylated in EACs as compared to normal samples [FDR<0.001, -log10(FDR)>3.0]. The DNA methylation levels from -127 to -8 CpG sites showed the strongest correlation with MT3 gene expression (râ=â-0.4, P<0.0001). Moreover, the DNA hypermethylation from -127 to -8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (Pâ=â0.005 and Pâ=â0.0313, respectively). The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells. Treatment of OE33 cells with 5-Aza-deoxycitidine restored MT3 expression with demethylation of its promoter region and reversal of the histone modifications towards active histone marks. CONCLUSION: In summary, EACs are characterized by frequent epigenetic silencing of MT3. The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.