Background and Purpose: Multiple members of the thiol isomerase (TI) family of enzymes are present in and released by platelets. Inhibition of these enzymes results in diminished platelet responses, aggregation, adhesion and thrombus formation.Recently, the therapeutic potential of TI inhibition has been recognised and drugdevelopment technologies were used to identify selective small molecule inhibitors.To date, few pan-TI inhibitors have been characterised and the most studied, bacitracin, is known to be nephrotoxic, which prohibits its systemic therapeutic usage.Experimental Approach: We therefore sought to identify novel broad-spectrum inhibitors of these enzymes and test their effects in vivo. A total of 3,641 compounds were screened for inhibitory effects on the redox activity of ERp5, protein disulphide isomerase (PDI), ERp57, ERp72 and thioredoxin in an insulin turbidity assay. Of the lead compounds identified, zafirlukast was selected for further investigation.
The thiol isomerase, protein disulfide isomerase (PDI), plays important intracellular roles during protein folding, maintaining cellular function and viability. Recent studies suggest novel roles for extracellular cell surface PDI in enhancing cellular activation and promoting their function. Moreover, a number of food-derived substances have been shown to regulate cellular PDI activity and alter disease progression. We hypothesized that PDI may have similar roles during mast cell-mediated allergic responses and examined its effects on IgE-induced mast cell activity during cell culture and food allergy. Mast cells were activated via IgE and antigen and the effects of PDI inhibition on mast cell activation were assessed. The effects of PDI blockade in vivo were examined by treating mice with the irreversible PDI inhibitor, PACMA-31, in an ovalbumin-induced model of food allergy. The role of dietary PDI modulators was investigated using various dietary compounds including curcumin and quercetin-3-rutinoside (rutin). PDI expression was observed on resting mast cell surfaces, intracellularly, and in the intestines of allergic mice. Furthermore, enhanced secretion of extracellular PDI was observed on mast cell membranes during IgE and antigen activation. Insulin turbidimetric assays demonstrated that curcumin is a potent PDI inhibitor and pre-treatment of mast cells with curcumin or established PDI inhibitors such as bacitracin, rutin or PACMA-31, resulted in the suppression of IgE-mediated activation and the secretion of various cytokines. This was accompanied by decreased mast cell proliferation, FcεRI expression, and mast cell degranulation. Similarly, treatment of allergic BALB/c mice with PACMA-31 attenuated the development of food allergy resulting in decreased allergic diarrhea, mast cell activation, and fewer intestinal mast cells. The production of TH2-specific cytokines was also suppressed. Our observations suggest that PDI catalytic activity is essential in the regulation of mast cell activation, and that its blockade may benefit patients with allergic inflammation.
High levels of thiol isomerases in cancer patients have been correlated with increased tumor growth and an increased risk of cancer-induced thrombosis. Inhibition of major extracellular thiol isomerases, which include the enzymes PDI, ERp57, ERp72 and ERp5 in both cancer and thrombosis, have strong therapeutic potential to treat cancer while also inhibiting cancer-induced thrombosis. In this study, we explored the effects of two thiol isomerase inhibitors, isoquercetin (IsoQ) and zafirlukast (Zafi) against OVCAR8 ovarian cancer cells and a xenograft mouse model created from those cells. To explore the potential of these drugs to inhibit tumor growth and cancer-induced thrombosis, multiple in vitro and in vivo experiments were performed. Alterations of cellular OVCAR8 thiol isomerase activity were measured using a DI-E-GSSG assay where, after drug treatment separately with both drugs, DTT and the PDI specific fluorescent probe were added to OVCAR8 cells and then monitored kinetically at 520nm/550nm. Further, the ability of the drugs to inhibit the coagulative potential of OVCAR8 cells was measured by monitoring Factor Xa generation as a marker for tissue factor release. Cells were seeded at a concentration of 100,000 cells, then monitored for Factor Xa generation after addition of a fluorescent probe that is a Factor Xa substrate, Factor VIIa, calcium and Factor X. To explore the effects of IsoQ and Zafi in vivo, an OVCAR8 xenograft mouse model was created. Tumors were allowed to grow to an average of 30mm2 and mice were then treated with 10 mg/kg IsoQ, 30 mg/kg IsoQ or 30 mg/kg Zafi for 46 days via oral gavage. Blood was collected on Day 46, plasma was isolated and examined for alterations in p-selectin expression via ELISA and alterations in thiol isomerase activity by the DI-E-GSSG assay. Cell surface thiol isomerase activity on OVCAR8 cells was inhibited by 32% after with Zafi and by 35% after treatment with IsoQ. OVCAR8 cell induced Factor Xa generation was significantly reduced by 79% after Zafi treatment and by 35% after IsoQ treatment. In the xenografts, 10mg/kg of IsoQ, 30 mg/kg of IsoQ, and 30 mg/kg of Zafi were all separately able to suppress tumor growth by approximately 85% compared to controls. P-selectin expression decreased by approximately 50% and thiol isomerase activity was decreased by approximately 40% under all 3 treatment conditions. These results demonstrate the potential of thiol isomerase inhibitors to not only treat cancer-induced thrombosis but also the cancer itself, as cancer induced thrombosis markers and tumor size were both significantly reduced in these experiments. Citation Format: Justine A. Gelzinis, Daniel R. Kennedy. The role of zafirlukast and isoquercetin in thiol isomerase inhibition in relation to cancer and its complications. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4948.
Thiol isomerases, including PDI, ERp57, ERp5, and ERp72, play important and distinct roles in cancer progression, cancer cell signaling, and metastasis. We recently discovered that zafirlukast, an FDA-approved medication for asthma, is a pan-thiol isomerase inhibitor. Zafirlukast inhibited the growth of multiple cancer cell lines with an IC 50 in the low micromolar range, while also inhibiting cellular thiol isomerase activity, EGFR activation, and downstream phosphorylation of Gab1. Zafirlukast also blocked the procoagulant activity of OVCAR8 cells by inhibiting tissue factor-dependent Factor Xa generation. In an ovarian cancer xenograft model, statistically significant differences in tumor size between control vs treated groups were observed by Day 18. Zafirlukast also significantly reduced the number and size of metastatic tumors found within the lungs of the mock-treated controls. When added to a chemotherapeutic regimen, zafirlukast significantly reduced growth, by 38% compared with the mice receiving only the chemotherapeutic treatment, and by 83% over untreated controls. Finally, we conducted a pilot clinical trial in women with tumor marker-only (CA-125) relapsed ovarian How to cite this article: Gelzinis JA, Szahaj MK, Bekendam RH, et al. Targeting thiol isomerase activity with zafirlukast to treat ovarian cancer from the bench to clinic.
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