Unknown,Transcriptomics,Genomics,Proteomics

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Procyanidin-induced apoptosis of esophageal adenocarcinoma cells is mediated via stress-activated protein kinase activation of JUN-dependent transcription.

ABSTRACT: Epidemiologic studies have shown a significant inverse correlation between fruit and vegetable consumption and incidence of esophageal adenocarcinoma. Procyanidins are polymeric flavanols found in many fruits and vegetables, and have been shown to possess anti-carcinogenic/chemopreventive properties. We previously showed that an oligomeric procyanidin extracted from apples with an average degree of polymerisation of 3.9 induced cell cycle arrest and apoptosis in the esophageal adenocarcinoma cell line OE33. In order to understand the mechanism of action of this procyanidin we determined genome-wide transcriptomic changes induced by procyanidin treatment of OE33 cells. Pathway analysis of these data implicated the MAP kinase signalling pathways in eliciting these responses. An investigation into the role of these pathways showed that procyanidin specifically induced the activation of the stress-activated protein (SAP) kinases JNK1/2 and p38-? and M-^V? leading to the increased expression of JUN and the phosphatases DUSP1 and -10. Gene-specific knockdown of the expression of JNK1, JNK2, p38-?, p38-? or JUN diminished procyanidin-induced effects on apoptosis demonstrating a clear role for these pathways. JUN is a component of the transcription factor AP-1 and AP-1 binding sites are over-represented in the promoters of procyanidin-induced genes, which together with the demonstration that JUN occupies several such promoters highlight the importance of this transcription factor in mediating the cellular response to procyanidin. These data provide a mechanistic understanding of how procyanidin specifically targets distinct pathways involved in the induction of apoptosis in esophageal adenocarcinoma cells and will inform future studies investigating its use as a chemopreventive/therapeutic agent.

ORGANISM(S): Homo sapiens

SUBMITTER: Michiel Adriaens

PROVIDER: E-MEXP-2993 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Esophageal cancers (ECs) are highly aggressive tumors with poor prognosis and few treatment options. This study investigated the possibility of treating esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells by inhibitors of broad and specific histone deacetylases (HDACi; SAHA, MS-275, FK228) and/or of DNMT (Azacytidine, AZA). Drug targets (HDAC1,2,3 and DNMT1) were present in non-neoplastic (HET-1A), ESCC (OE21) and EAC (OE33) cell lines. All cell lines responded to HDACi by reduced HDAC activity and increased histone acetylation as well as to AZA by up-regulation of p21. Expression of drug targets remained largely unaffected by HDACi and AZA treatment. Importantly, cell viability, apoptosis, cell cycle dynamics and DNA damage were only affected by HDACi and/or AZA in ESCC and EAC, but not the non-neoplastic cells. This was specifically seen for the combination of MS-275 and AZA, leading to enhanced cancer cell selectivity and drug efficiency. By transcriptome analyses of MS-275, AZA and MS-275/AZA treated cells, known (e.g. p21) as well as novel regulated genes significantly associated with the cellular effects post HDACi and/or AZA treatment in ESCC and EAC cells were identified. Finally, human EC tissue specimens frequently expressed the actionable drug targets HDAC1/2/3 and DNMT1. In summary, a combined HDACi (MS-275)/AZA treatment is cancer cell selective and efficient in vitro. Since the majority of ECs express the drug targets in situ, this paves the way for further investigations of HDACi/AZA treatment in esophageal cancer cells and their translation into a clinico-pathological setting. To elucidate the transcriptome response to HDAC inhibitors of normal esophageal cells and esophageal tumor cells, total RNA was isolated from non-neoplastic esophageal epithelial cells (Het1A cells) a well as from two esophageal tumor cell lines (OE21 and OE33), respectively. Cells were treated with either MS-275, Azacytidine (AZA) or in combination of both. DMSO treatment was used as control in each case. Total RNA was isolated from cells 24 h after treatment and experiments were performed in biological triplicates.

Project description:Signal transducer and activator of transcription 3 (STAT3) is altered in several epithelial cancers and represents a potential therapeutic target. Here, STAT3 expression, activity and cellular functions were examined in two main histotypes of esophageal carcinomas. In situ, immunohistochemistry for STAT3 and STAT3-Tyr705 phosphorylation (P-STAT3) in esophageal squamous cell carcinomas (ESCC) and BarrettM-bM-^@M-^Ys adenocarcinomas (BAC) revealed similar STAT3 expression in ESCCs and BACs, but preferentially activated P-STAT3 in ESCCs. In vitro, strong STAT3 activation was seen by EGF-stimulation in OE21 (ESCC) cells, whilst OE33 (BAC) cells showed constitutive weak STAT3 activation. STAT3 knockdown significantly reduced cell proliferation of OE21 and OE33 cells and reduced cell migration in OE33, but not in OE21 cells. Transcriptome analysis identified STAT3-knockdown associated down-regulation of cell cycle processes and the selective down-regulation of cyclins and cyclin dependent kinaes associated genes in both OE21 and OE33 cells. Moreover, the transcriptome response showed changes in cell migration/invasion related genes that correlated with the associated phenotype measurements. This study demonstrates the importance of STAT3 expression and activation in esophageal carcinomas, whereby the extent differs between ESCCs and BACs. STAT3 knockdown significantly reduces cell proliferation in both types of esophageal cancer cells and inhibits migration in BAC cells. Thus, STAT3 may be further exploited as potential novel therapeutic target for esophageal cancers. The effect of STAT3 knock-down in OE33 and OE21 cells was calculated from three biologically independent experiments. 3x10e4 cells were seeded in triplicate in 24 well-plates and were transfected with twice 100nM STAT3 siRNA (pool of 4 STAT3 sequences, siGENOMEM-BM-.SMARTpoolM-BM-., Dharmacon RNAi Technologies, Thermo Fisher Scientific, Lafayette, USA) or SilencerM-BM-. negative siRNA control (Invitrogen/Life Technologies GmbH, Darmstadt, Germany) using 1M-BM-5l DharmaFECT (Dharmacon RNAi Technologies, Thermo Fisher Scientific, Lafayette, USA) transfection reagent for OE33 cells or siPORTTM NeoFXTM (Invitrogen/Life Technologies GmbH, Darmstadt, Germany) transfection reagent for OE21 cells. Differential gene regulation was quantified 72 hours after the first transfection by comparing separately for the OE21 and OE33 cells the STAT3 siRNA replicates and the respective cell lines containing the scrambled siRNA vector.

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Procyanidin-induced apoptosis of esophageal adenocarcinoma cells is mediated via stress-activated protein kinase activation of JUN-dependent transcription.

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