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Metformin's Effects on Apoptosis of Esophageal Carcinoma Cells and Normal Esophageal Epithelial Cells: An In Vitro Comparative Study.

ABSTRACT: The effect of metformin on human esophageal normal and carcinoma cells remains poorly understood. We aim to investigate the different antiproliferation effects and underlying distinct molecular mechanisms between these two types of cells. Human esophageal squamous cell carcinoma cell line, EC109, and normal esophageal epithelial cell line, HEEC, were used in the experiment. The cell survival rate was determined by cell counting kit-8 (CCK-8). Cell apoptosis was analyzed by flow cytometry. The mRNA and protein levels of signal transducer and activator of transcription 3 (Stat3) were detected by real-time quantitative PCR and western blot. Interleukin-6 (IL-6) was added to activate Stat3. The level of intracellular reactive oxygen species (ROS) was assessed by a DCFH-DA fluorescent probe. Metformin had more significant inhibitory effects on cell proliferation in EC109 cells than HEECs. Metformin induced apoptosis of EC109 cells in a dose-dependent manner instead of HEECs. The expression of Stat3 in both mRNA and protein levels was higher in EC109 cells than HEECs. Further study revealed that metformin may attenuate the phosphorylation of the Stat3 and the Bcl-2 expression, which was restored by IL-6 partly in EC109 cells but not HEECs. On the contrary, metformin increased the level of ROS in both the cell lines, but this intracellular ROS variation had no effect on apoptosis. Metformin has different functional roles on the apoptosis in esophageal carcinoma cells and normal esophageal cells. Therefore, the Stat3/Bcl-2 pathway-mediated apoptosis underlies the cell-type-specific drug sensitivity, suggesting metformin possesses a therapeutic activity and selectivity on esophageal cancer.

SUBMITTER: Peng J

PROVIDER: S-EPMC7104266 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Metformin's Effects on Apoptosis of Esophageal Carcinoma Cells and Normal Esophageal Epithelial Cells: An In Vitro Comparative Study.

Peng Jianwei J Jing Xubin X Wu Jialing J Hong Danmian D Hu Xi X Wang Qinjia Q Hu Hui H Cai Xianbin X

BioMed research international 20200318

The effect of metformin on human esophageal normal and carcinoma cells remains poorly understood. We aim to investigate the different antiproliferation effects and underlying distinct molecular mechanisms between these two types of cells. Human esophageal squamous cell carcinoma cell line, EC109, and normal esophageal epithelial cell line, HEEC, were used in the experiment. The cell survival rate was determined by cell counting kit-8 (CCK-8). Cell apoptosis was analyzed by flow cytometry. The mR ...[more]

PMID: 32258099

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Project description:BackgroundEpidermal growth factor receptor (EGFR) plays a pivotal role in the pathophysiology of esophageal squamous cell carcinoma (ESCC). However, the clinical effects of EGFR inhibitors on ESCC are controversial. This study sought to identify the factors determining the therapeutic efficacy of EGFR inhibitors in ESCC cells.MethodsImmortalized-human esophageal epithelial cells (EPC2-hTERT), transformed-human esophageal epithelial cells (T-Epi and T-Mes), and ESCC cells (TE-1, TE-5, TE-8, TE-11, TE-11R, and HCE4) were treated with the EGFR inhibitors erlotinib or cetuximab. Inhibitory effects on cell growth were assessed by cell counting or cell-cycle analysis. The expression levels of genes and proteins such as involucrin and cytokeratin13 (a squamous differentiation marker), E-cadherin, and vimentin were evaluated by real-time polymerase chain reaction or western blotting. To examine whether mesenchymal phenotype influenced the effects of EGFR inhibitors, we treated T-Epi cells with TGF-?1 to establish a mesenchymal phenotype (mesenchymal T-Epi cells). We then compared the effects of EGFR inhibitors on parental T-Epi cells and mesenchymal T-Epi cells. TE-8 (mesenchymal-like ESCC cells)- or TE-11R (epithelial-like ESCC cells)-derived xenograft tumors in mice were treated with cetuximab, and the antitumor effects of EGFR inhibitors were evaluated.ResultsCells were classified as epithelial-like or mesenchymal-like phenotypes, determined by the expression levels of E-cadherin and vimentin. Both erlotinib and cetuximab reduced cell growth and the ratio of cells in cell-cycle S phase in epithelial-like but not mesenchymal-like cells. Additionally, EGFR inhibitors induced squamous cell differentiation (defined as increased expression of involucrin and cytokeratin13) in epithelial-like but not mesenchymal-like cells. We found that EGFR inhibitors did not suppress the phosphorylation of EGFR in mesenchymal-like cells, while EGFR dephosphorylation was observed after treatment with EGFR inhibitors in epithelial-like cells. Furthermore, mesenchymal T-Epi cells showed resistance to EGFR inhibitors by circumventing the dephosphorylation of EGFR signaling. Cetuximab consistently showed antitumor effects, and increased involucrin expression in TE-11R (epithelial-like)-derived xenograft tumors but not TE-8 (mesenchymal-like)-derived xenograft tumors.ConclusionsThe factor determining the therapeutic effects of EGFR inhibitors in ESCC cells is the phenotype representing the epithelial-like or mesenchymal-like cells. Mesenchymal-like ESCC cells are resistant to EGFR inhibitors because EGFR signaling is not blocked. EGFR inhibitors show antitumor effects on epithelial-like ESCC cells accompanied by promotion of squamous cell differentiation.

Project description:Esophageal squamous cell carcinoma (ESCC) is one of the 10 most incident cancer types in the world, and it is mainly associated with tobacco and alcohol consumption. ESCC mortality rates stand very close to its incidence, which is a direct consequence of a late diagnosis and an inefficient treatment. Although this scenery is quite alarming, the major molecular alterations that drive this carcinogenesis process remain unclear. We have previously shown through the first ESCC methylome analysis that TFF1 promoter is frequently hypermethylated in ESCC. Here, to evaluate TFF1 methylation as a potential biomarker of early ESCC diagnosis, we investigated the status of TFF1 promoter methylation and its expression in ESSC and histologically normal tumor surrounding tissue of ESCC patients in comparison to healthy esophagus of non-cancer individuals.Analysis of TFF1 promoter methylation, and gene and protein expression in 65 ESCC patients and 88 controls revealed that TFF1 methylation levels were already increased in histologically normal tumor surrounding tissue of ESCC patients when compared to healthy esophagus of non-cancer individuals. This increase in DNA methylation was followed by the reduction of TFF1 mRNA expression. Interestingly, TFF1 expression was capable of distinguishing tumor surrounding normal tissue from normal mucosa of healthy individuals with 92% accuracy. In addition, TFF1 protein was undetectable both in tumor and surrounding mucosa by immunohistochemistry, while submucosa glands of the healthy esophagus showed positive staining. Furthermore, treatment of TE-1 and TE-13 ESCC cell lines with decitabine led to a reduction of promoter methylation and consequent upregulation of TFF1 gene and protein expression. Finally, using TCGA data we showed that TFF1 loss is observed in ESCC, but not in esophageal adenocarcinoma, highlighting the different molecular mechanisms involved in the development of each histological subtype of esophageal cancer.This study shows that TFF1 expression is silenced in early phases of ESCC development, which seems to be mediated at least in part by promoter hypermethylation, and provides the basis for the use of TFF1 expression as a potential biomarker for early ESCC detection.

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Metformin's Effects on Apoptosis of Esophageal Carcinoma Cells and Normal Esophageal Epithelial Cells: An In Vitro Comparative Study.

Publications

Metformin's Effects on Apoptosis of Esophageal Carcinoma Cells and Normal Esophageal Epithelial Cells: An In Vitro Comparative Study.

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