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1.
J Inorg Biochem ; 259: 112666, 2024 Oct.
Article de Anglais | MEDLINE | ID: mdl-39029397

RÉSUMÉ

Here, we designed, synthesized and characterized three new cyclometalated Ru(II) complexes, [Ru(phen)2(1-(4-Ph-Ph)-IQ)]+ (phen = 1,10-phenanthroline, IQ = isoquinoline, RuIQ9), [Ru(phen)2(1-(4-Ph-Ph)-7-OCH3-IQ)]+ (RuIQ10), and [Ru(phen)2(1-(4-Ph-Ph)-6,7-(OCH3)2-IQ)]+ (RuIQ11). The cytotoxicity experiments conducted on both 2D and 3D multicellular tumor spheroids (MCTSs) indicated that complexes RuIQ9-11 exhibited notably higher cytotoxicity against A549 and A549/DDP cells when compared to the ligands and precursor compounds as well as clinical cisplatin. Moreover, the Ru(II) complexes displayed low toxicity when tested on normal HBE cells in vitro and exposed to zebrafish embryos in vivo. In addition, complexes RuIQ9-11 could inhibit A549 and A549/DDP cell migration and proliferation by causing cell cycle arrest, mitochondrial dysfunction, and elevating ROS levels to induce apoptosis in these cells. Mechanistic studies revealed that RuIQ9-11 could suppress the expression of Nrf2 and its downstream antioxidant protein HO-1 by inhibiting Nrf2 gene transcription in drug-resistant A549/DDP cells. Simultaneously, they inhibited the expression of efflux proteins MRP1 and p-gp in drug-resistant cells, ensuring the accumulation of the complexes within the cells. This led to an increase in intracellular ROS levels in drug-resistant cells, ultimately causing damage and cell death, thus overcoming cisplatin resistance. More importantly, RuIQ11 could effectively inhibit the migration and proliferation of drug-resistant cells within zebrafish, addressing the issue of cisplatin resistance. Accordingly, the prepared Ru(II) complexes possess significant potential for development as highly effective and low-toxicity lung cancer therapeutic agents to overcome cisplatin resistance.


Sujet(s)
Antinéoplasiques , Cisplatine , Complexes de coordination , Résistance aux médicaments antinéoplasiques , Facteur-2 apparenté à NF-E2 , Ruthénium , Danio zébré , Humains , Cisplatine/pharmacologie , Facteur-2 apparenté à NF-E2/métabolisme , Danio zébré/embryologie , Résistance aux médicaments antinéoplasiques/effets des médicaments et des substances chimiques , Complexes de coordination/pharmacologie , Complexes de coordination/composition chimique , Complexes de coordination/synthèse chimique , Animaux , Antinéoplasiques/pharmacologie , Antinéoplasiques/composition chimique , Antinéoplasiques/synthèse chimique , Ruthénium/composition chimique , Ruthénium/pharmacologie , Cellules A549 , Apoptose/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Espèces réactives de l'oxygène/métabolisme , Antioxydants/pharmacologie , Antioxydants/composition chimique , Antioxydants/synthèse chimique , Mouvement cellulaire/effets des médicaments et des substances chimiques
2.
Cancers (Basel) ; 15(19)2023 Sep 30.
Article de Anglais | MEDLINE | ID: mdl-37835513

RÉSUMÉ

The incidence of esophageal adenocarcinoma (EAC) has risen rapidly during the past four decades, making it the most common type of esophageal cancer in the USA and Western countries. The NEK (Never in mitosis A (NIMA) related kinase) gene family is a group of serine/threonine kinases with 11 members. Aberrant expression of NEKs has been recently found in a variety of human cancers and plays important roles in tumorigenesis, progression, and drug-resistance. However, the expression of the NEKs in EAC and its precancerous condition (Barrett's esophagus, BE) has not been investigated. In the present study, we first analyzed the TCGA and 9 GEO databases (a total of 10 databases in which 8 contain EAC and 6 contain BE) using bioinformatic approaches for NEKs expression in EAC and BE. We identified that several NEK members, such as NEK2 (7/8), NEK3 (6/8), and NEK6 (6/8), were significantly upregulated in EAC as compared to normal esophagus samples. Alternatively, NEK1 was downregulated in EAC as compared to the normal esophagus. On the contrary, genomic alterations of these NEKs are not frequent in EAC. We validated the above findings using qRT-PCR and the protein expression of NEKs in EAC cell lines using Western blotting and in primary EAC tissues using immunohistochemistry and immunofluorescence. Our data suggest that frequent upregulation of NEK2, NEK3, and NEK7 may be important in EAC.

3.
Front Pharmacol ; 14: 1121796, 2023.
Article de Anglais | MEDLINE | ID: mdl-37332351

RÉSUMÉ

Introduction: Adverse drug reactions (ADR) are directly related to public health and become the focus of public and media attention. At present, a large number of ADR events have been reported on the Internet, but the mining and utilization of such information resources is insufficient. Named entity recognition (NER) is the basic work of many natural language processing (NLP) tasks, which aims to identify entities with specific meanings from natural language texts. Methods: In order to identify entities from ADR event data resources more effectively, so as to provide valuable health knowledge for people, this paper introduces ALBERT in the input presentation layer on the basis of the classic BiLSTM-CRF model, and proposes a method of ADR named entity recognition based on the ALBERT-BiLSTM-CRF model. The textual information about ADR on the website "Chinese medical information query platform" (https://www.dayi.org.cn) was collected by the crawler and used as research data, and the BIO method was used to label three types of entities: drug name (DRN), drug component (COM), and adverse drug reactions (ADR) to build a corpus. Then, the words were mapped to the word vector by using the ALBERT module to obtain the character level semantic information, the context coding was performed by the BiLSTM module, and the label decoding was using the CRF module to predict the real label. Results: Based on the constructed corpus, experimental comparisons were made with two classical models, namely, BiLSTM-CRF and BERT-BiLSTM-CRF. The experimental results show that the F 1 of our method is 91.19% on the whole, which is 1.5% and 1.37% higher than the other two models respectively, and the performance of recognition of three types of entities is significantly improved, which proves the superiority of this method. Discussion: The method proposed can be used effectively in NER from ADR information on the Internet, which provides a basis for the extraction of drug-related entity relationships and the construction of knowledge graph, thus playing a role in practical health systems such as intelligent diagnosis, risk reasoning and automatic question answering.

4.
Cell Rep ; 42(1): 112005, 2023 01 31.
Article de Anglais | MEDLINE | ID: mdl-36681899

RÉSUMÉ

Infection with Helicobacter pylori (H. pylori) is the main risk factor for gastric cancer, a leading cause of cancer-related death worldwide. The oncogenic functions of cyclin-dependent kinase 1 (CDK1) are not fully understood in gastric tumorigenesis. Using public datasets, quantitative real-time PCR, western blot, and immunohistochemical (IHC) analyses, we detect high levels of CDK1 in human and mouse gastric tumors. H. pylori infection induces activation of nuclear factor κB (NF-κB) with a significant increase in CDK1 in in vitro and in vivo models (p < 0.01). We confirm active NF-κB binding sites on the CDK1 promoter sequence. CDK1 phosphorylates and inhibits GSK-3ß activity through direct binding with subsequent accumulation and activation of ß-catenin. CDK1 silencing or pharmacologic inhibition reverses these effects and impairs tumor organoids and spheroid formation. IHC analysis demonstrates a positive correlation between CDK1 and ß-catenin. The results demonstrate a mechanistic link between infection, inflammation, and gastric tumorigenesis where CDK1 plays a critical role.


Sujet(s)
Protéine-kinase CDC2 , Infections à Helicobacter , Helicobacter pylori , Tumeurs de l'estomac , Animaux , Humains , Souris , bêta-Caténine/métabolisme , Carcinogenèse/anatomopathologie , Protéine-kinase CDC2/métabolisme , Transformation cellulaire néoplasique/anatomopathologie , Muqueuse gastrique/métabolisme , Glycogen synthase kinase 3 beta/métabolisme , Infections à Helicobacter/métabolisme , Helicobacter pylori/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Tumeurs de l'estomac/métabolisme , Tumeurs de l'estomac/anatomopathologie
5.
Antioxidants (Basel) ; 11(10)2022 Sep 21.
Article de Anglais | MEDLINE | ID: mdl-36290582

RÉSUMÉ

Esophageal adenocarcinoma (EAC), the predominant type of esophageal cancer in the United States, develops through Barrett's esophagus (BE)-dysplasia-carcinoma cascade. Gastroesophageal reflux disease, where acidic bile salts refluxate into the esophagus, is the main risk factor for the development of BE and its progression to EAC. The NFE2-related factor 2 (NRF2) is the master cellular antioxidant regulator. We detected high NRF2 protein levels in the EAC cell lines and primary tissues. Knockdown of NRF2 significantly enhanced acidic bile salt-induced oxidative stress, DNA damage, and inhibited EAC cell growth. Brusatol, an NRF2 inhibitor, significantly inhibited NRF2 transcriptional activity and downregulated the NRF2 target genes. We discovered that in addition to inducing apoptosis, Brusatol alone or in combination with cisplatin (CDDP) induced significant lipid peroxidation and ferroptosis, as evidenced by reduced xCT and GPX4 expression, two known ferroptosis markers. The combination of Brusatol and CDDP significantly inhibited EAC tumor xenograft growth in vivo and confirmed the in vitro data showing ferroptosis as an important mechanism in the tumors treated with Brusatol or Brusatol and CDDP combination. Our data support the role of NRF2 in protecting against stress-induced apoptosis and ferroptosis in EACs. Targeting NRF2 in combination with platinum therapy can be an effective strategy for eliminating cancer cells in EAC.

6.
Cancers (Basel) ; 14(6)2022 Mar 09.
Article de Anglais | MEDLINE | ID: mdl-35326553

RÉSUMÉ

Unfolded protein response (UPR) protects malignant cells from endoplasmic reticulum stress-induced apoptosis. We report that Aurora kinase A (AURKA) promotes cancer cell survival by activating UPR in esophageal adenocarcinoma (EAC). A strong positive correlation between AURKA and binding immunoglobulin protein (BIP) mRNA expression levels was found in EACs. The in vitro assays indicated that AURKA promoted IRE1α protein phosphorylation, activating prosurvival UPR in FLO-1 and OE33 cells. The use of acidic bile salts to mimic reflux conditions in patients induced high AURKA and IRE1α levels. This induction was abrogated by AURKA knockdown in EAC cells. AURKA and p-IRE1α protein colocalization was observed in neoplastic gastroesophageal lesions of the L2-IL1b mouse model of Barrett's esophageal neoplasia. The combined treatment using AURKA inhibitor and tunicamycin synergistically induced cancer cell death. The use of alisertib for AURKA inhibition in the EAC xenograft model led to a decrease in IRE1α phosphorylation with a significant reduction in tumor growth. These results indicate that AURKA activates UPR, promoting cancer cell survival during ER stress in EAC. Targeting AURKA can significantly reverse prosurvival UPR signaling mechanisms and decrease cancer cell survival, providing a promising approach for the treatment of EAC patients.

7.
Cancers (Basel) ; 12(9)2020 Sep 10.
Article de Anglais | MEDLINE | ID: mdl-32927604

RÉSUMÉ

The incidence of esophageal adenocarcinoma (EAC) has been rising dramatically in the past few decades in the United States and Western world. The N-myc downregulated gene 4 (NDRG4) belongs to the human NDRG family. In this study, we aimed to identify the expression levels, regulation, and functions of NDRG4 in EAC. Using an integrative epigenetic approach, we identified genes showing significant downregulation in EAC and displaying upregulation after 5-Aza-deoxycitidine. Among these genes, likely to be regulated by DNA methylation, NDRG4 was among the top 10 candidate genes. Analyses of TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus) data sets and EAC tissue samples demonstrated that NDRG4 was significantly downregulated in EAC (p < 0.05). Using Pyrosequencing technology for quantification of DNA methylation, we detected that NDRG4 promoter methylation level was significantly higher in EAC tissue samples, as compared to normal esophagus samples (p < 0.01). A strong inverse correlation between NDRG4 methylation and its gene expression levels (r = -0.4, p < 0.01) was observed. Treatment with 5-Aza restored the NDRG4 expression, confirming that hypermethylation is a driving force for NDRG4 silencing in EAC. Pathway and gene set enrichment analyses of TCGA data suggested that NDRG4 is strongly associated with genes related to cell cycle regulation. Western blotting analysis showed significant downregulation of Cyclin D1, CDK4 and CDK6 in EAC cells after overexpression of NDRG4. Functionally, we found that the reconstitution of NDRG4 resulted in a significant reduction in tumor cell growth in two-dimensional (2D) and three-dimensional (3D) organotypic culture models and inhibited tumor cell proliferation as indicated by the EdU (5-ethynyl-2'-deoxyuridine) proliferation assay.

8.
Cancer Lett ; 458: 46-55, 2019 08 28.
Article de Anglais | MEDLINE | ID: mdl-31132430

RÉSUMÉ

Gastroesophageal reflux disease (GERD) is the main risk factor for Barrett's tumorigenesis. In this study, we investigated the role of NRF2 in response to exposure to acidic bile salts (ABS), in conditions that mimic GERD, using Barrett's esophagus cell models. We detected an increase in NRF2 protein levels, following exposure to ABS. We found oxidization of cysteines (cysteines with oxidized thiol groups) in KEAP1 protein with a weaker interaction between NRF2 and KEAP1, following ABS exposure. Treatment with bile salts increased nuclear NRF2 levels, enhancing its transcription activity, as measured by an ARE (antioxidant response element) luciferase reporter assay. The mRNA expression levels of NRF2 target genes, HO-1 and GR, were increased in response to ABS exposure. Using genetic overexpression and knockdown of NRF2, we found that NRF2 has a critical role in suppressing ABS-induced ROS levels, oxidative DNA damage, DNA double strand breaks, and apoptosis. Collectively, our results suggest that transient induction of NRF2 in response to ABS plays a pivotal role in protecting esophageal cells by maintaining the levels of oxidative stress and DNA damage below lethal levels under GERD conditions.


Sujet(s)
Oesophage de Barrett/génétique , Oesophage de Barrett/métabolisme , Altération de l'ADN , Facteur-2 apparenté à NF-E2/métabolisme , Antioxydants/métabolisme , Oesophage de Barrett/induit chimiquement , Oesophage de Barrett/anatomopathologie , Acides et sels biliaires/antagonistes et inhibiteurs , Acides et sels biliaires/pharmacologie , Lignée cellulaire , Reflux gastro-oesophagien/génétique , Reflux gastro-oesophagien/métabolisme , Reflux gastro-oesophagien/anatomopathologie , Cellules HEK293 , Heme oxygenase-1/génétique , Heme oxygenase-1/métabolisme , Humains , Protéine-1 de type kelch associée à ECH/métabolisme , Facteur-2 apparenté à NF-E2/biosynthèse , Stress oxydatif/effets des médicaments et des substances chimiques , Stress oxydatif/physiologie , ARN messager/génétique , ARN messager/métabolisme , Espèces réactives de l'oxygène/métabolisme
9.
Oncotarget ; 8(33): 54345-54356, 2017 Aug 15.
Article de Anglais | MEDLINE | ID: mdl-28903346

RÉSUMÉ

Gastric cancer (GC) is one of the most common cancers in the world, and remains the third leading cause of cancer-related deaths worldwide. Glutathione peroxidase 7 (GPX7) is a member of GPX family which is downregulated in some cancer types. In this study, we investigated the expression, regulation, and molecular function of GPX7 in gastric cancer using 2D and 3D in vitro models and de-identified human tissue samples. Quantitative real-time RT-PCR, immunofluorescence, Western blot, 3D organotypic cultures, and pyrosequencing assays were used. We detected downregulation of GPX7 in all 7 gastric cancer cell lines that we tested and in approximately half (22/45) of human gastric cancer samples, as compared to histologically normal gastric tissues. Quantitative bisulfite pyrosequencing methylation analysis demonstrated DNA hypermethylation (> 10% methylation level) of GPX7 promoter in all 7 gastric cancer cell lines and in 56% (25/45) of gastric cancer samples, as compared to only 13% (6/45) in normal samples (p < 0.0001). Treatment of AGS and SNU1 cells with 5-Aza-2'-deoxycytidine led to a significant demethylation of GPX7 promoter and restored the expression of GPX7. In vitro assays showed that reconstitution of GPX7 significantly suppressed gastric cancer cell growth in both 2D and 3D organotypic cell culture models. This growth suppression was associated with inhibition of cell proliferation and induction of cell death. We detected significant upregulation of p27 and cleaved PARP and downregulation of Cyclin D1 upon reconstitution of GPX7. Taken together, we conclude that epigenetic silencing of GPX7 could play an important role in gastric tumorigenesis and progression.

10.
Sci Rep ; 7: 40729, 2017 01 19.
Article de Anglais | MEDLINE | ID: mdl-28102292

RÉSUMÉ

The incidence of esophageal adenocarcinoma (EAC) is rapidly rising in the United States and Western countries. In this study, we carried out an integrative molecular analysis to identify interactions between genomic and epigenomic alterations in regulating gene expression networks in EAC. We detected significant alterations in DNA copy numbers (CN), gene expression levels, and DNA methylation profiles. The integrative analysis demonstrated that altered expression of 1,755 genes was associated with changes in CN or methylation. We found that expression alterations in 84 genes were associated with changes in both CN and methylation. These data suggest a strong interaction between genetic and epigenetic events to modulate gene expression in EAC. Of note, bioinformatics analysis detected a prominent K-RAS signature and predicted activation of several important transcription factor networks, including ß-catenin, MYB, TWIST1, SOX7, GATA3 and GATA6. Notably, we detected hypomethylation and overexpression of several pro-inflammatory genes such as COX2, IL8 and IL23R, suggesting an important role of epigenetic regulation of these genes in the inflammatory cascade associated with EAC. In summary, this integrative analysis demonstrates a complex interaction between genetic and epigenetic mechanisms providing several novel insights for our understanding of molecular events in EAC.


Sujet(s)
Adénocarcinome/génétique , Épigenèse génétique , Épigénomique , Tumeurs de l'oesophage/génétique , Régulation de l'expression des gènes tumoraux , Réseaux de régulation génique , Génomique , Lignée cellulaire tumorale , Hybridation génomique comparative , Biologie informatique/méthodes , Variations de nombre de copies de segment d'ADN , Méthylation de l'ADN , Analyse de profil d'expression de gènes , Gene Ontology , Humains
11.
J Cancer ; 5(7): 510-7, 2014.
Article de Anglais | MEDLINE | ID: mdl-24963355

RÉSUMÉ

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.

12.
J Cancer ; 5(6): 457-64, 2014.
Article de Anglais | MEDLINE | ID: mdl-24847386

RÉSUMÉ

BACKGROUND: Desmocollin3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and plays an important role in tumor invasion and metastasis. In this study, we investigated the epigenetic mechanism that regulates DSC3 expression in esophageal adenocarcinomas (EACs). METHODS: Expression of DSC3 was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The promoter DNA methylation level of DSC3 was examined using quantitative bisulfite pyrosequencing. RESULTS: The qRT-PCR analysis demonstrated significant down-regulation of the DSC3 mRNA levels in human EAC cell lines and tissue samples (P<.001). In addition, the EAC cell lines and tumor samples have aberrant promoter hypermethylation as compared to normal esophageal samples (P<.001). DSC3 promoter hypermethylation (>10% methylation level) was detected in 97.5% (39/40) of EAC samples whereas none of the normal tissue samples showed hypermethylation (P<.0001). There was a significant inverse correlation between promoter DNA methylation levels and mRNA expression folds for DSC3 (coefficient r=-0.685, P<.0001). Treatment of FLO-1 and SKGT4 EAC cells with 5-Aza-deoxytidine led to a significant reduction in the promoter DNA methylation levels with restoration of the DSC3 expression, suggesting that promoter DNA methylation is a key epigenetic mechanism regulating DSC3 expression. High DSC3 promoter DNA methylation levels were significantly correlated with advanced tumor stage (P<.001) and lymph node metastasis (P<.001). CONCLUSION: Taken together, our results demonstrate that epigenetic silencing of DSC3 is a frequent finding in EAC that is possibly associated with advanced stages.

13.
Carcinogenesis ; 35(7): 1620-8, 2014 Jul.
Article de Anglais | MEDLINE | ID: mdl-24692067

RÉSUMÉ

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.


Sujet(s)
Adénocarcinome/anatomopathologie , Oesophage de Barrett/anatomopathologie , Transformation cellulaire néoplasique/anatomopathologie , Tumeurs de l'oesophage/anatomopathologie , Facteur de transcription NF-kappa B/métabolisme , Peroxidases/métabolisme , Facteur de nécrose tumorale alpha/pharmacologie , Adénocarcinome/génétique , Adénocarcinome/métabolisme , Oesophage de Barrett/génétique , Oesophage de Barrett/métabolisme , Technique de Western , Transformation cellulaire néoplasique/génétique , Transformation cellulaire néoplasique/métabolisme , Cellules cultivées , Tumeurs de l'oesophage/génétique , Tumeurs de l'oesophage/métabolisme , Technique d'immunofluorescence , Glutathione peroxidase , Humains , Facteur de transcription NF-kappa B/génétique , Peroxidases/antagonistes et inhibiteurs , Peroxidases/génétique , ARN messager/génétique , Petit ARN interférent/génétique , Espèces réactives de l'oxygène/métabolisme , Réaction de polymérisation en chaine en temps réel , Récepteur au facteur de nécrose tumorale de type I/génétique , Récepteur au facteur de nécrose tumorale de type I/métabolisme , Récepteur au facteur de nécrose tumorale de type II/génétique , Récepteur au facteur de nécrose tumorale de type II/métabolisme , RT-PCR , Transduction du signal/effets des médicaments et des substances chimiques
14.
Gut ; 63(4): 540-51, 2014 Apr.
Article de Anglais | MEDLINE | ID: mdl-23580780

RÉSUMÉ

OBJECTIVE: To investigate the potential tumour suppressor functions of glutathione peroxidase 7 (GPX7) and examine the interplay between epigenetic and genetic events in regulating its expression in oesophageal adenocarcinomas (OAC). DESIGN: In vitro and in vivo cell models were developed to investigate the biological and molecular functions of GPX7 in OAC. RESULTS: Reconstitution of GPX7 in OAC cell lines, OE33 and FLO-1, significantly suppressed growth as shown by the growth curve, colony formation and EdU proliferation assays. Meanwhile, GPX7-expressing cells displayed significant impairment in G1/S progression and an increase in cell senescence. Concordant with the above functions, Western blot analysis displayed higher levels of p73, p27, p21 and p16 with a decrease in phosphorylated retinoblastoma protein (RB), indicating its increased tumour suppressor activities. On the contrary, knockdown of GPX7 in HET1A cells (an immortalised normal oesophageal cell line) rendered the cells growth advantage as indicated with a higher EdU rate, lower levels of p73, p27, p21 and p16 and an increase in phosphorylated RB. We confirmed the tumour suppressor function in vivo using GPX7-expressing OE33 cells in a mouse xenograft model. Pyrosequencing of the GPX7 promoter region (-162 to +138) demonstrated location-specific hypermethylation between +13 and +64 in OAC (69%, 54/78). This was significantly associated with the downregulation of GPX7 (p<0.01). Neither mutations in the coding exons of GPX7 nor DNA copy number losses were frequently present in the OAC examined (<5%). CONCLUSIONS: Our data suggest that GPX7 possesses tumour suppressor functions in OAC and is silenced by location-specific promoter DNA methylation.


Sujet(s)
Adénocarcinome/enzymologie , Méthylation de l'ADN/physiologie , Tumeurs de l'oesophage/enzymologie , Peroxidases/physiologie , Protéines suppresseurs de tumeurs/physiologie , Adénocarcinome/métabolisme , Adénocarcinome/physiopathologie , Animaux , Cycle cellulaire/physiologie , Lignée cellulaire tumorale , Prolifération cellulaire , ADN tumoral/physiologie , Tumeurs de l'oesophage/métabolisme , Tumeurs de l'oesophage/physiopathologie , Régulation de l'expression des gènes tumoraux/physiologie , Extinction de l'expression des gènes , Glutathione peroxidase , Humains , Souris , Souris nude , Transplantation tumorale , Peroxidases/métabolisme , Protéines suppresseurs de tumeurs/métabolisme
15.
PLoS One ; 7(10): e46214, 2012.
Article de Anglais | MEDLINE | ID: mdl-23071548

RÉSUMÉ

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.


Sujet(s)
Méthylation de l'ADN , Extinction de l'expression des gènes , Glutathione peroxidase/génétique , Métastase lymphatique/génétique , Tumeurs de l'estomac/anatomopathologie , Azacitidine/analogues et dérivés , Azacitidine/pharmacologie , Études cas-témoins , Lignée cellulaire tumorale , Décitabine , Technique d'immunofluorescence , Régulation de l'expression des gènes codant pour des enzymes/effets des médicaments et des substances chimiques , Humains , Régions promotrices (génétique) , ARN messager/génétique , Réaction de polymérisation en chaine en temps réel , RT-PCR , Tumeurs de l'estomac/enzymologie , Tumeurs de l'estomac/génétique
16.
Gut ; 61(9): 1250-60, 2012 Sep.
Article de Anglais | MEDLINE | ID: mdl-22157330

RÉSUMÉ

OBJECTIVE: Exposure of the oesophageal mucosa to gastric acid and bile acids leads to the accumulation of reactive oxygen species (ROS), a known risk factor for Barrett's oesophagus and progression to oesophageal adenocarcinoma (OAC). This study investigated the functions of glutathione peroxidase 7 (GPX7), frequently silenced in OAC, and its capacity in regulating ROS and its associated oxidative DNA damage. DESIGN: Using in-vitro cell models, experiments were performed that included glutathione peroxidase (GPX) activity, Amplex UltraRed, CM-H(2)DCFDA, Annexin V, 8-oxoguanine, phospho-H2A.X, quantitative real-time PCR and western blot assays. RESULTS: Enzymatic assays demonstrated limited GPX activity of the recombinant GPX7 protein. GPX7 exhibited a strong capacity to neutralise hydrogen peroxide (H(2)O(2)) independent of glutathione. Reconstitution of GPX7 expression in immortalised Barrett's oesophagus cells, BAR-T and CP-A led to resistance to H(2)O(2)-induced oxidative stress. Following exposure to acidic bile acids cocktail (pH4), these GPX7-expressing cells demonstrated lower levels of H(2)O(2), intracellular ROS, oxidative DNA damage and double-strand breaks, compared with controls (p<0.01). In addition, these cells demonstrated lower levels of ROS signalling, indicated by reduced phospho-JNK (Thr183/Tyr185) and phospho-p38 (Thr180/Tyr182), and demonstrated lower levels of apoptosis following the exposure to acidic bile acids or H(2)O(2)-induced oxidative stress. The knockdown of endogenous GPX7 in immortalised oesophageal squamous epithelial cells (HET1A) confirmed the protective functions of GPX7 against pH4 bile acids by showing an increase in the levels of H(2)O(2), intracellular ROS, oxidative DNA damage, double-strand breaks, apoptosis, and ROS-dependent signalling (p<0.01). CONCLUSION: The dysfunction of GPX7 in oesophageal cells increases the levels of ROS and oxidative DNA damage, which are common risk factors for Barrett's oesophagus and OAC.


Sujet(s)
Oesophage de Barrett/enzymologie , Altération de l'ADN/physiologie , Oesophage/enzymologie , Glutathione peroxidase/physiologie , Espèces réactives de l'oxygène/métabolisme , Adénocarcinome/enzymologie , Adénocarcinome/physiopathologie , Oesophage de Barrett/physiopathologie , Acides et sels biliaires/effets indésirables , Technique de Western , Lignée cellulaire , Évolution de la maladie , Cellules épithéliales/enzymologie , Tumeurs de l'oesophage/enzymologie , Tumeurs de l'oesophage/physiopathologie , Oesophage/cytologie , Oesophage/anatomopathologie , Cytométrie en flux , Régulation de l'expression des gènes , Glutathione peroxidase/génétique , Glutathione peroxidase/métabolisme , Humains , Peroxyde d'hydrogène/métabolisme , Stress oxydatif/physiologie
17.
PLoS One ; 6(7): e22009, 2011.
Article de Anglais | MEDLINE | ID: mdl-21818286

RÉSUMÉ

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 (n = 64) and normal samples (n = 51), 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 (r = -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 (P = 0.005 and P = 0.0313, 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.


Sujet(s)
Adénocarcinome/génétique , Épigenèse génétique , Tumeurs de l'oesophage/génétique , Régulation de l'expression des gènes tumoraux , Gènes tumoraux/génétique , Protéines de tissu nerveux/génétique , Adénocarcinome/anatomopathologie , Adulte , Sujet âgé , Sujet âgé de 80 ans ou plus , Azacitidine/pharmacologie , Lignée cellulaire tumorale , Ilots CpG/génétique , Méthylation de l'ADN/effets des médicaments et des substances chimiques , Régulation négative/effets des médicaments et des substances chimiques , Régulation négative/génétique , Épigenèse génétique/effets des médicaments et des substances chimiques , Tumeurs de l'oesophage/anatomopathologie , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Histone/métabolisme , Humains , Métastase lymphatique/génétique , Métallothionéine-3 , Adulte d'âge moyen , Stadification tumorale , Régions promotrices (génétique)/génétique , Maturation post-traductionnelle des protéines/effets des médicaments et des substances chimiques , Protéines de répression/métabolisme
18.
Dig Dis Sci ; 56(1): 125-30, 2011 Jan.
Article de Anglais | MEDLINE | ID: mdl-20927589

RÉSUMÉ

BACKGROUND: Somatostatin (SST), a primary inhibitor of gastrin-stimulated gastric acid secretion, has potent antitumor and anti-secretory activity in several human cancers. AIMS: This study was performed to investigate the SST gene expression levels and possible epigenetic mechanisms that regulate expression of SST in gastric adenocarcinomas. METHODS: Quantitative real-time (RT)-PCR and quantitative bisulfite pyrosequencing were used to study primary gastric cancer tissue samples and cell lines. RESULTS: Quantitative RT-PCR analysis revealed down-regulation of the SST transcript in 93% of gastric carcinoma samples (30/32), compared with 21 normal samples (P<0.001). Because of the presence of a large CpG island in the SST promoter, we next examined its promoter DNA methylation levels by use of quantitative bisulfite pyrosequencing. The results revealed a significant increase in SST promoter DNA methylation in tumor samples compared with normal samples (P<0.05). Promoter DNA hypermethylation and silencing of SST was also detected in seven gastric cancer cell lines that we tested. To confirm the role of promoter DNA methylation as an epigenetic mechanism regulating SST expression, AGS gastric cancer cells were treated with 5-Aza-dc. This treatment led to reduction of promoter DNA methylation levels of SST accompanied by restoration of its mRNA expression. CONCLUSIONS: Our results indicate that promoter DNA methylation levels play a critical role in regulating SST expression in gastric cancer. This finding provides a foundation for further studies on the role of SST in gastric carcinogenesis and its potential as a biomarker for gastric cancers.


Sujet(s)
Adénocarcinome/génétique , Adénocarcinome/métabolisme , Épigenèse génétique , Somatostatine/génétique , Somatostatine/métabolisme , Tumeurs de l'estomac/génétique , Tumeurs de l'estomac/métabolisme , Adénocarcinome/anatomopathologie , Antinéoplasiques/pharmacologie , Azacitidine/analogues et dérivés , Azacitidine/pharmacologie , Marqueurs biologiques tumoraux/génétique , Marqueurs biologiques tumoraux/métabolisme , Lignée cellulaire tumorale , Méthylation de l'ADN , Décitabine , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Humains , Acides hydroxamiques/pharmacologie , Méthylation , Valeur prédictive des tests , ARN messager/métabolisme , Reproductibilité des résultats , Tumeurs de l'estomac/anatomopathologie
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