Effects of iodonium-class flavin dehydrogenase inhibitors on growth, reactive oxygen production, cell cycle progression, NADPH oxidase 1 levels, and gene expression in human colon cancer cells and xenografts

Abstract

Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explain the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remain an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1 [Nox1]). We found that diphenylene iodonium (DPI), di-2-thienyliodonium (DTI), and iodoniumdiphenyl inhibited the growth of Caco2, HT-29, and LS-174T colon cancer cells at concentrations (10–250 nM for DPI, 0.5–2.5 μM for DTI, and 155 nM to 10 μM for iodoniumdiphenyl) substantially lower than for DU145 human prostate cancer cells that do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H2O2 production and diminished intracellular ROS levels, lasting up to 24 hr, following short-term (1-hr) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G1/S interface in both LS-174T and HT-29 cells exposed to either DPI or DTI; and the G1 block was produced, for LS-174T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H2O2. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174T human tumor xenografts, at dose levels that produced peak plasma concentrations similar to those utilized for our in vitro experiments. These findings suggest that iodonium analogs have therapeutic potential for NADPH oxidase-containing human colon cancers in vivo, and that at least part of their antineoplastic mechanism of action may be related to targeting Nox1.

Characterization of potent and selective iodonium-class inhibitors of NADPH oxidases

“…2A–C), a tumor cell line that depends on NOX activity, and not reactive nitrogen species, for its proliferative potential [10, 24]. In this cell line, the iodonium analogs produced an inhibitory effect on cell proliferation, similar to that observed following genetic inhibition (knockdown) of NOX1 activity [43].…”

“…DPI and DTI inhibit the growth of NOX1-expressing colon cancer cells without altering mitochondrial respiration, if studied at nanomolar concentrations, so that antiproliferative effects may be attributed, at least in part, to a cyclin D 1 -dependent G 1 block that is secondary to inhibition of NOX function [23, 24]. However, these inhibitors lack specificity for NOX enzymes as they have been shown to disrupt the activity of various flavoproteins [21, 25, 26], which limits the precision with which they can be utilized in a therapeutic context.…”

The Quest for Selective Nox Inhibitors and Therapeutics: Challenges, Triumphs and Pitfalls

“…These off-target effects nullify its potential as a therapeutic candidate. That said, in a recent study, DPI and its analog di-2-thienyliodonium (DTI) were shown, in the nanomolar range, to exhibit antineoplastic properties, decreasing colon cancer cell proliferation by blocking cell cycle progression at the G1/S interface and not just to decrease ROS levels but also Nox1 expression (44). Perhaps the emphasis here should be placed on the use of very low concentrations of the iodoniums that may prove relatively selective for Nox versus other flavoproteins.…”

NADPH Oxidases as Therapeutic Targets in Chronic Myelogenous Leukemia

“…Each NADPH oxidase seems to have a particular pattern of expression, and most cells express different members of this family (20). The importance of these enzymes in cancer is beginning to be recognized (21) and, interestingly, it has recently been shown that NADPH oxidase inhibitors are efficient at preventing the growth of colon cancer xenografts (22). This suggests that NADPH oxidases could be exploited as therapeutic targets in the cancer treatment setting.…”