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Combined incubation of colon carcinoma cells with phorbol ester and mitochondrial uncoupling agents results in synergic elevated reactive oxygen species levels and increased γ-glutamyltransferase expression

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Abstract

The NADPH oxidase (NOX) is a significant determinant for the expression and activity of γ-glutamyltransferase (GGT), which is frequently upregulated after increased levels of reactive oxygen species (ROS) and oxidative stress. Earlier studies on human colon carcinoma HT-29 cells have shown that treatment with phorbol 12-myristate 13-acetate (PMA) activates NOX thus increasing the intracellular level of ROS and upregulating GGT. Another important source of cellular ROS is the mitochondria, and treatment with the mitochondria uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) results in increased ROS levels. The present study shows that when HT-29 cells were simultaneously treated with both agents, a significant and synergic increase in intracellular ROS was detected. NOX activity contributed at least 50 % of this increase as inhibiting NOX activity with apocynin or downregulating the NOX activity using siRNA against p22 phox reduced the synergic ROS production. The combined FCCP and PMA treatment also provoked highly increased GGT mRNA levels after 24 h whereas only minor and delayed increases in GGT protein and enzyme activity levels were detected. The results strongly indicate that ROS production by both mitochondria and NOX is involved in the regulation of GGT expression in colon carcinoma cells.

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Acknowledgments

This work was supported by The Norwegian Cancer Society, The Erna and Olav Aakre Foundation for Cancer Research and The Northern Norway Regional Health Authorities. We thank Roy Lyså at the Bio Imaging FUGE Core facility, Institute of Medical Biology, The Faculty of Health Sciences, for his assistance with the acquisition of the flow cytometry data.

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Authors and Affiliations

  1. Tumor Biology Research Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, 9037, Tromsö, Norway

    Seila Pandur, Chandra Ravuri, Ugo Moens & Nils-Erik Huseby

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  1. Seila Pandur
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  2. Chandra Ravuri
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  4. Nils-Erik Huseby
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Correspondence to Nils-Erik Huseby.

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Pandur, S., Ravuri, C., Moens, U. et al. Combined incubation of colon carcinoma cells with phorbol ester and mitochondrial uncoupling agents results in synergic elevated reactive oxygen species levels and increased γ-glutamyltransferase expression. Mol Cell Biochem 388, 149–156 (2014). https://doi.org/10.1007/s11010-013-1906-1

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