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Redox homeostasis is described as the net physiologic balance between inter-convertible oxidized and reduced equivalents within subcellular compartments that remain in a dynamic equilibrium. This equilibrium is impacted by reactive oxygen species (ROS), which are natural by-products of normal cellular activity. Studies have shown that cancer cells have high ROS levels and altered redox homeostasis due to increased basal metabolic activity, mitochondrial dysfunction, peroxisome activity, as well as the enhanced activity of NADPH oxidase, cyclooxygenases, and lipoxygenases. Glioblastoma (GBM) is the most prevalent primary brain tumor in adults with a median survival of 15 months. GBM is characterized by its extreme resistance to therapeutic interventions as well as an elevated metabolic rate that results in the exacerbated production of ROS. Therefore, many agents with either antioxidant or pro-oxidant mechanisms of action have been rigorously employed in preclinical as well as clinical settings for treating GBM by inducing oxidative stress within the tumor. Among those agents are well-known antioxidant vitamin C and small molecular weight SOD mimic BMX-001, both of which are presently in clinical trials on GBM patients. Despite the wealth of investigations, limited data is available on the response of normal brain vs glioblastoma tissue to these therapeutic interventions. Currently, a sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the quantification of a panel of oxidative stress biomarkers: glutathione (GSH), cysteine (Cys), glutathione disulfide (GSSG), and cysteine disulfide in human-derived brain tumor and mouse brain samples; this method will be enriched with additional oxidative stress biomarkers homocysteine (Hcy), methionine (Met), and cystathionine (Cyst). Using this enriched method, we propose to evaluate the thiol homeostasis and the redox state of both normal brain and GBM in mice after exposure with redox-active therapeutics. Our results showed that, compared to normal brain (in intact mice), GBM tissue has significantly lower GSH/GSSG and Cys/CySS ratios indicating much higher oxidative stress levels. Contralateral “normal” brain tissue collected from the mice with intracranial GBM were also under significant oxidative stress compared to normal brains collected from the intact mice. Importantly, normal brain tissue in both studies retained the ability to restore redox homeostasis after treatment with a redox-active therapeutic within 24 hours while glioblastoma tissue does not. Ultimately, elucidating the differential redox response of normal vs tumor tissue will allow for the development of more redox-active agents with therapeutic benefit.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.