Green, downregulated. a physiological cysteine-based redox change in Top1 to mediate their selective toxicity to rapidly proliferating malignancy cells. CPT displays problematic toxicity, instability, and poor bioavailability (Venditto and Simanek, 2010, Martino et al., 2017) but semisynthetic analogs derived from CPT such as topotecan (TPT) and irinotecan (IRN, a pro-drug for the active metabolite SN-38) that conquer these limitations have become core components of common regimens for lung, ovarian, and additional solid tumors (Moukharskaya and Verschraegen, 2012, Delgado et al., 2018, Liang et al., 2019). Structural analysis of CPT and its analogs in ternary complexes Platycodin D with Top1 and DNA founded interfacial inhibition like a mechanism of action, where drug binding stabilizes Top1cc, delaying launch of the 3 DNA strand (Pommier et al., 2015). Nonetheless, the structural diversity of agents found to poison Top1 (Cinelli, 2019) is definitely hard to reconcile with the Lum constraints of interfacial inhibition, suggesting option mechanisms may mediate Top1cc formation results in Michael addition in the active site cysteine C630. Drawing on the paradigm of reversible inactivation of tyrosine phosphatases by LDE changes of catalytic cysteines (Ostman et al., 2011), we propose that changes of C630 by LDEs may block Y723 dephosphorylation, trapping Top1cc by avoiding release of the bound 3 DNA strand. Results Camptothecins induce proteomic signatures of redox stress in tumor cells Reports over the past three decades, including recent studies by our group (Flor et al., 2016, Flor et al., 2017), have explained improved cellular ROS and/or LPO in cells and animals treated with topoisomerase poisons. To directly examine oxidative stress induced by camptothecin medicines, we carried out label-free LC-MS/MS quantitative proteomics analysis of whole cell lysates from A549 human being lung adenocarcinoma cells treated in triplicate with CPT or TPT for 72 h (Fig. 1a). Of 3117 total proteins recognized in samples taken from cells treated with either drug (0.1 M) or vehicle control (VEH; 0.1% DMSO), 1882 (60.3%) were identified across all samples (Fig. 1b). Of proteins significantly down-regulated or up-regulated ( 1.5 fold) in CPT or TPT compared to control, most were similarly changed in the drug-treated samples (Fig. 1c). Open in a separate window Number 1. Camptothecins induce proteomic signatures of redox stress in tumor cells.A549 human lung adenocarcinoma cells were treated in triplicate with vehicle (VEH), camptothecin (CPT) or topotecan (TPT) at 0.1 M for 72 h. Cells were then lysed and proteins separated by PAGE for proteomics analysis. (a) After PAGE, gel lanes Platycodin D were separated and segmented into three sections as demonstrated. Each section was then digested and prepared for LC-MS/MS analysis. (b) Venn diagram indicates total proteins recognized per treatment condition and shared between conditions. Of 3,117 proteins Platycodin D recognized overall, 1882 were detected in all three conditions. (c) Warmth map showing proteome-wide label-free quantitation of relative manifestation among the three conditions. Green, downregulated. Red, upregulated. Black, no significant switch. Overall, patterns of manifestation were related between CPT/VEH and TPT/VEH while the CPT/TPT assessment reveals few significant variations. (d) Volcano plots of proteins from the GO pathway Oxidation-Reduction Process (GO:0055114) quantified in the CPT/VEH and TPT/VEH comparisons, displayed as Log2 collapse change in manifestation the percentage of LPO+ cells for each Top1 poison, linear regression analysis exposed a moderately strong correlation (R2 = 0.85) between ROS and LPO (Fig. 2b). Confirming improved LPO and production of LDEs, cells treated with 0.1 M CPT, TPT, SN-38 or GENZ for 72 h revealed 2.1 to 24.1 fold raises in HNE protein adducts over untreated control (Fig. 2c), and 1.8 to 4.0 fold depletion of GSH (Fig. 2d). A circulation cytometry assay to detect aldehyde dehydrogenase (ALDH) activity with the cell-permeable probe AldeRed 610 exposed improved ALDH activity in cells treated with each Top1 poison (Fig. 2e). Percentage of ALDHHI cells was 5% in vehicle-only treated cells, vs. 53.5% for CPT, 82.5% for TPT, 78.2% for SN-38, and 52.8% for GENZ. Camptothecins induce Top1 changes by HNE The pattern of LPO induced by Top1 poisons raised the question whether the producing lipid derived aldehydes might form adducts to Top1 during drug treatment. After treating A549 cells in triplicate with 0.1 M CPT for 72 h, we used proximity ligation assay (PLA, Fig. 3) to detect HNE-protein adducts on Top1 protein Probing the cells with rabbit anti-Top1 and goat anti-HNE protein adduct antibodies and detection with fluorescent anti-species secondary antibodies revealed unique patterns in.