Mechanisms of acquired resistance to iodonium-class NADPH oxidase inhibitors in human colorectal cancer

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Recent studies suggest that reactive oxygen species (ROS) generated by a membrane-bound, non-mitochondrial flavin dehydrogenase, NADPH oxidase 1 (NOX1), play a critical role in the control of human colorectal cancer growth by modulating signal transduction through the Wnt pathway as well as ROS-mediated angiogenesis (Proc. Amer. Assoc. Cancer Res. 2005; 46: [Abstract # 6108; Abstract # 2283]). We demonstrated previously that the iodonium-class NOX inhibitors diphenylene iodonium (DPI) and Di-2-thienyliodonium (DTI) at their IC₅₀ concentrations downregulate cyclin D1 leading to a G₁/S block; drug exposure also produces a significant decrease in ROS production as well as decreased phosphorylation of beta-catenin. To examine the mechanism(s) of action of DPI and DTI further, HT-29, HCT-15, SW-620, and KM-12 human colon cancer lines resistant to these iodonium analogs were developed by stepwise exposure to increasing DPI or DTI concentrations over a period of at least 6 months. Individual DPI- and DTI-resistant variants of each of the four model systems were found to be from 4 to 5-fold resistant to DPI and 4 to18-fold resistant DTI by comparison of their respective IC₅₀ values. Flow cytometric analysis with the fluorescent probe H₂DCF revealed that in both DPI- and DTI-resistant variant cell lines baseline levels of reactive oxygen production were increased by > 3-fold compared to parental cells. Furthermore, expression levels of phospho-beta catenin, c-myc, CDK4, cyclin D1, and the transcription factor TCF-4, that are significantly decreased following exposure to DPI and DTI in parental cells, were either unchanged or upregulated in the resistant cell variants. In summary, DPI and DTI resistance in human colon cancer cells is associated with a significant increase in reactive oxygen setpoint, and a compensatory overexpression of oxidant-sensitive signal transduction components of the Wnt pathway. These studies suggest that reactive oxygen homeostasis plays a critical regulatory role in the growth of colon cancer cells, and thus, may be an important target for the development of novel colon cancer therapeutics.