Effects of Pharmacological Modulation of Nrf2 Signalling on Chemical Carcinogenesis in Renal Epithelial Cells

Cancer is a major cause of morbidity and mortality globally. There were 23.6 million cancer cases and 10 million cancer deaths globally in 2019 and cases are estimated to reach 28.5 million in 2040. Around 30-50 % of cancer cases can be prevented by avoiding risk factors and implementation of prevention strategies. Exposure to chemical carcinogens can increase a person’s risk of developing cancer in their lifetime. Chemical carcinogens are substances which directly induce malignant tumour formation, increase tumour incidence, or decrease the time taken for a tumour to form at an increased rate compared to background. Carcinogens Aristolochic acid (ARAI) and Ochratoxin A (OTA) are major contaminants of herbal medicines and foodstuffs globally. The kidneys are one of the principal organs affected by carcinogens due to their function in maintaining tissue homeostasis. Renal cell carcinoma arises from the epithelium of the nephrons of the kidney which are disproportionately affected by chemical carcinogens due to their role in filtering plasma for removal of waste products and xenobiotics. In most eukaryotic organisms, oxygen is essential for normal cellular functions. During cellular respiration and metabolism, by-products such as reactive nitrogen species and reactive oxygen species (ROS) are produced. Antioxidants remove free radicals and help restore cellular balance. Oxidative stress occurs when excess free radicals are produced in cells which overwhelms the normal antioxidant capacity. This can be caused by both intrinsic (e.g., metabolism) and extrinsic (e.g., xenobiotics) factors. Excess ROS can damage DNA, proteins and lipids and is associated with many pathophysiological processes including carcinogenesis. The Nuclear Factor Erythroid 2-related factor (Nrf2) pathway is an intrinsic, inducible stress mitigation response which regulates antioxidant response genes to reduce oxidative stress. Bardoxolone methyl (CDDO-Me), a synthetic triterpenoid induces Nrf2 through inhibition of the endogenous repressor Keap-1. Several human carcinogens elicit tumourigenic effects by modulating associated oxidative stress pathways including the Nrf2 pathway. For this research the normal (RPTEC/TERT1) cell line was exposed to carcinogens ARAI and OTA, pre-treated with CDDO-Me. Several effects associated with cellular transformation were characterised including loss of junctional protein expression and barrier function, lipid peroxidation and effects on the primary cilium. Pre-treatment with CDDO-Me affected these endpoints and elicited a spectrum of effects dependent on cell type and the carcinogen in question. To further characterise and contextualise the effects observed, whole cell proteome analysis and systems biology modelling was performed. In summary, the current research suggests that the beneficial effects of Nrf2 induction are highly dependent on the nature of the carcinogenic insult applied. In some cases, Nrf2 enhancement abrogated some of the deleterious effects whilst in others, it appeared to potentiate the cellular damage caused.