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  • br Material and methods br

    2024-05-25


    Material and methods
    Results
    Discussion Didox was originally created asa cytostatic drug to inhibit cancer cell proliferation by antagonizing RNR [11]. When used to target highly proliferative cells, Didox has extensive activity in vitro and in vivo. It has also been employed in clinical studies for cancer with minimal toxicity at therapeutic levels [11], [14], [16], [36], [37]. In addition to antiproliferative effects, Didox possesses antioxidant and anti-inflammatory activity with significant therapeutic potential to treat diseases associated with oxidative stress and inflammation [17], [18], [38]. We have recently published Didox suppression of IL-33 mediated mast cell activation [19], however IgE-mediated mast cell function has not been studied. Here we investigated Didox effects in vitro and in vivo and the implications they may have for treating allergic inflammation. Time-course and dose-response experiments showed that Didox optimally suppresses the IgE-mediated mast cell inflammatory response when given 6h prior to mast cell activation, with a drug concentration of 100μM that does not affect cell viability or receptor expression. Specifically, Didox suppresses mRNA synthesis and protein secretion of inflammatory cytokines and select chemokines, while attenuating mast cell degranulation. These results corroborate previous research, where Didox treatment antagonized pro-inflammatory responses of anti-CD3-activated T apomorphine and LPS-stimulated macrophages in allotransplant and macrophage inflammatory disease models, respectfully [17], [18]. Additionally, these data agree with Didox effects on IL-33-mediated mast cell activation [19]. Collectively, these results indicate Didox may have broad anti-inflammatory effects. Didox was designed to improve on HU-mediated RNR inhibition and reduce unwanted side-effects [11], [37], [39]. The enzymatic activity of RNR is dependent upon the association of two subunits, R1 and R2, to form an active heterodimeric tetramer. R2 is the iron-binding subunit, while R1 is the substrate-binding subunit. R2 needs a free tyrosyl radical to be active; iron is a cofactor for the generation and stabilization of this free radical. Didox is able to inhibit the association of R1 and R2, and thus suppress RNR activity, through its ability to scavenge free radicals and chelate iron [40], [41]. Despite its design as an RNR inhibitor, Didox’s free radical scavenger capabilities are important to its broader utility, reducing oxidative stress associated with cardiotoxicity, dementia in HIV patients, and inflammation [13], [17], [18], [42]. To elucidate which of the known Didox biochemical effects explain its ability to suppress IgE-mediated signaling, we used the clinically-approved drugs HU and NAC. The inability of the RNR inhibitor HU to reduce inflammatory cytokines was not surprising, since FceRI-mediated signaling is not closely tied to DNA synthesis or proliferation [43], [44], [45]. Interestingly, HU suppressed degranulation following activation, albeit at 100-fold lower concentrations than Didox. HU has been used in numerous studies to treat mast cell activation syndrome and mastocytomas, reducing both mast cell load and symptoms of mast cell activation potentially by affecting mediator expression or release [46], [47], [48]. However, no studies have examined the effect of HU in healthy, normal mast cells. Our current data suggest that while future studies should examine the mechanisms linking ribonucleotide reductase inhibition to reduced degranulation, they also indicate that Didox may be more efficacious than HU. In addition to HU, the antioxidant NAC antagonized mast cell activation, but only when used at a 100-fold greater concentration. NAC suppressed cytokine production and degranulation, two pathways that have been linked to oxidative stress and reactive oxygen species production [30], [49], [50], [51]. Furthermore, Didox suppressed DCFH-DA fluorescence, indicating reduced oxidative stress [29], and Didox increased SOD1 and catalase expression, further increasing antioxidant potential. These results support those observed by Matsebatlela and colleagues, who reported that Didox reduced iNOS expression and NO production, while enhancing SOD1 and catalase activity [18]. Collectively, these data show the potential role for Didox in modulating oxidative stress in inflammatory diseases, and indicate that Didox may be more potent and efficacious than NAC. Future studies should compared NAC and Didox in other inflammatory disease models, since NAC is in clinical use.