Abstract 1266: TNF-alpha mediated down-regulation of Na+/H+ exchanger 1 (NHE1) in breast cancer MCF7 cells and its relevance to apoptosis

Abstract

Abstract Intracellular alkalinization is a hallmark of cancer cells shown to confer resistance against caspase mediated apoptosis. Na+/H+ exchanger 1 (NHE1), an ion exchanger important in the maintenance of intracellular pH and cell volume homeostasis had been implicated in this intracellular alkalinization. Increased NHE1 protein level and activity has been found in many cancer cells types where increase in intracellular pH by constitutively active NHE1 confers resistance to apoptosis in these cells. Silencing of NHE1 protein in cancer cells results in reduced cell growth and increased in sensitivity to death triggers. TNF-α can induce H2O2 production in cells to mediate apoptosis with characteristic cell shrinkage. H2O2 had recently been shown to downregulate NHE1 expression and increase cells’ sensitivity to apoptosis. This study was undertaken to study the role of caspases and H2O2 in the regulation of NHE1 protein and gene expression by TNF-α. Results showed that TNF-α had both cytostatic and cytotoxic effects on MCF-7 cells with concurrent decrease in intracellular pH and NHE1 activity. NHE1 was found to be downregulated both at the protein and mRNA level following TNF-α treatment. Gelshift assay showed that transcription factors binding to the promoter of NHE1 could be blocked by pre-incubation with H2O2, suggesting that transcription of NHE1 could be redox-regulated. Indeed, we also show by using a luciferase reporter assay that NHE1 promoter is down-regulated in presence of H2O2. Various caspases were activated in TNF-α treated cells and the inhibition of caspases prevented the downregulation of NHE1. ROS was elevated in MCF-7 within 2 hours of treatment. Incubation of cells with catalase increased their survival in response to TNF-α, thus implicating H2O2 as a mediator of TNF-α induced cell death. Overexpression of catalase in MCF-7 reduced the downregulation of NHE1 by TNF-α. Our results suggest by TNF-α mediate cell death through decreasing NHE1 activity and down-regulating NHE1 expression in a ROS mediated manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1266.