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BH4-deficiency induced changes in NO-redox balance reveals a novel role for BH4 in the mitochondria

ABSTRACT: The nitric oxide synthase (NOS) co-factor, tetrahydrobiopterin (BH4), is a redox-active molecule that regulates nitric oxide (NO) and reactive oxygen species (ROS) production by NOS, and is an example of redox-dependent signalling in the endothelium that underlies both the maintenance of vascular homeostasis and the development of cardiovascular disease (CVD). Loss of endothelial BH4 is observed in CVD states and results in decreased NO production and increased ROS generation by endothelial NO synthase (uncoupling). Genetic mouse models of augmented endothelial BH4 synthesis have shown proof of concept that endothelial BH4 can alter CVD pathogenesis. However, clinical trials of BH4 therapy in vascular disease have been limited by systemic oxidation and limited endothelial uptake of BH4, highlighting the need to explore the wider roles of BH4 in order to find novel therapeutic targets. In this study we aim to elucidate the effects of BH4 depletion on mitochondrial function and bioenergetics using targeted knockdown of the BH4 synthetic enzyme GTPCH in vitro. Knockdown of GTPCH (and, therefore, intracellular BH4 levels) by >90% lead to a striking induction of ROS generation in the mitochondria of murine endothelial cells. This effect was likewise observed in BH4 depleted GCH fibroblasts devoid of NOS, indicating a novel NOS-independent role for BH4 in mitochondrial redox signalling. Furthermore, this BH4-dependent, mitochondrial-derived ROS oxidized mitochondrial BH4 and is seen alongside distinct changes in the thioredoxin and glutathione antioxidant pathways, revealed by mass spectrometry using an unbiased proteomic approach. These changes are accompanied by a modest increase in mitochondrial size, attenuated respiratory function, and marked changes in the cellular metabolic profile; including succinate accumulation. Taken together, these data reveal a novel NOS-independent role for BH4 in the regulation of mitochondrial redox signalling and metabolism.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Endothelial Cell

DISEASE(S): Cutaneous Mastocytosis

SUBMITTER: Roman Fischer

LAB HEAD: Roman Fischer

PROVIDER: PXD004004 | Pride | 2018-07-16

REPOSITORIES: Pride

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Project description:Introduction: The flavonoid myricetin has been shown to induce cell cycle arrest and mitochondrial-dependent apoptosis in preclinical cancer models. We hypothesised that myricetin-derived flavonoids with redox properties and improved physicochemical attributes that enhance cell uptake and mitochondrial targeting might have increased potential as antitumour agents, since mitochondria are the main site of production of reactive oxygen species (ROS) and redox status is known to regulate the development and advancement of certain cancers. In this study we assessed a small library of novel flavonoids, then focussed on the lead compound, second-generation analogue OncamexTM, its mechanism of action and structure-activity relationships as an anti-proliferative agent. Methods: With this aim, we studied the effect of OncamexTM in a panel of 7 breast cancer cell lines using proliferation, cytotoxicity and apoptosis assays. The redox properties and mitochondrial delivery of the drug were studied using cyclic voltammetry and fluorescence microscopy, respectively. The mechanism of action was further studied using western blotting, gene expression analysis and immunohistochemistry (IHC) of treated xenograft tissue from in vivo mice models. Results: Sulforhodamine B (SRB) proliferation assays demonstrated strong, anti-proliferative properties of OncamexTM, with IC50 values in the low micromolar range. Treatment for 8 h exerted concentration-dependent reduction in cell viability and induction of cytotoxicity and apoptosis, with increased caspase activation. Microarray analysis suggested that OncamexTM regulates changes in cell cycle and apoptosis at gene expression level. Fluorescence microscopy was used to investigate mitochondrial targeting and ROS regulation in treated cells. OncamexTM was found to induce production of superoxide at concentrations which exerted anti-proliferative effects. Initial in vivo studies in mice implanted with a MDA-MB-231 breast cancer xenograft showed that OncamexTM inhibited tumour growth, reducing tissue viability and Ki-67 proliferation, with no overall systemic toxicity. Conclusions: OncamexTM is a novel flavonoid capable of specific delivery to the mitochondria and induction of ROS production. We have shown its antitumor activity in preclinical models of breast cancer, both in vitro and in initial in vivo models, where tumour growth was arrested without inducing toxicity, support the potential of this novel drug for its continued development as an anticancer agent. Total RNA was obtained from 5 breast cancer cell lines subjected to 6 hours of 10µM OncamexTM and matched untreated controls (1% DMSO)

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BH4-deficiency induced changes in NO-redox balance reveals a novel role for BH4 in the mitochondria

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