The nitrocellulose membranes were cut before probing with primary antibodies. Similarly, do not enter common spaces such as workstations, lunch areas or public places when wearing gloves. The percentage of DMSO during drug exposure never exceeded 0.2% for cell lines and 0.5% for eso‐PDOs. Reversan (final concentration to cells 10–20 μM) was from Santa Cruz Biotechnology, sc‐296262 (USA). For the first time, we demonstrate APR‐246 anti‐tumor efficacy in PDO cultures derived from TP53 missense mutant colorectal cancer (colo‐PDO) (Fig 2F) and esophageal adenocarcinoma (eso‐PDO) (Appendix Fig S2D), as assessed by the ATP‐based CTG assay. APR‐246 also synergizes with the xCT inhibitor sulfasalazine (SSZ) in vitro (Fig 7G) and in vivo (Liu et al, 2017). Pearson correlations between PRIMA‐1 area‐under‐the‐curve (AUC) and gene expression or metabolite abundance was calculated using cor.test function in R (ver. After incubation for 6 h or 24 h, plates were centrifuged at 200 × g for 5 min. OVCAR‐3 cells (2 × 105) were seeded in 6‐well plates with 2 ml media. The mutant p53‐targeting compounds PRIMA‐1 and APR‐246 (PRIMA‐1Met/Eprenetapopt) are converted to the active product methylene quinuclidinone (MQ), a Michael acceptor that reacts with thiols (Lambert et al, 2009). MQ can bind both high molecular weight (HMW) targets such as mutant p53 and low molecular weight (LMW) targets, e.g., GSH and Cysteine. This shows that additional factors besides total MQ accumulation and TP53 status determine the extent of synergy. The in vivo anti‐tumor effect of APR‐246 has previously been confirmed in mice with Eso26 esophageal cancer cell xenografts (TP53 R248W). Cells (3 × 103 except 5 × 103 for Eso26, FLO‐1 and JH‐EsoAd1) were seeded in 96‐well plates in 100 μl media and treated with indicated compounds after 24 h; growth suppression was determined with either WST‐1 substrate (Roche, Switzerland) or resazurin reagent solution. We postulate that an MQ reservoir is similarly formed by conjugation to other thiol (R‐SH) targets, e.g., Cys. Values were normalized to untreated conditions after removal of background signal (wells containing media only). Imported CySS may be efficiently reduced to Cys by enzymes present in the intracellular compartment (Espinosa & Arner, 2019). Growth suppression (WST‐1 assay) of HCT116 WT and R248W cells transfected with MRP1 siRNA (. However, despite the compensatory upregulation of xCT at 24 h, the growth suppression induced by combination treatment with MK‐571 and APR‐246 was substantial (Fig 1). MRP1 has been reported to mediate efflux of the cytotoxic lipid peroxidation product 4‐hydroxynonenal (4‐HNE) as a GSH conjugate (Renes et al, 2000; Cole, 2014). Thus, this protocol does not distinguish between disulfide species (GSSG) and reducing GSH species. The association of MRP expression with PRIMA‐1 resistance, MRP1’s role in redox regulation and export of GSH‐conjugated drugs, and the fact that MQ conjugates to GSH led us to hypothesize that inhibition of MRP1 in mutant TP53‐carrying tumor cells enhances sensitivity to APR‐246. To dissociate PDOs, ice‐cold PBS was added to dissolve the Matrigel, cells were then centrifuged at 350 g for 5 min and resuspended in 40% TrypLE Express (12605028, Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h at 37°C. Mean values for each cell line shown in Fig 6A. We observed pronounced growth suppression by combined treatment with APR‐246 and SSZ as compared to APR‐246 alone in both HCT116 WT and R248W cells, as assessed by total cellular protein in cell culture wells (Fig 7F). However, make sure that gloves aren’t thick enough to prevent you from easy handling. At late time points (> 22 days after treatment initiation), we observed an increased cleaved caspase 3 staining in both APR‐246 and the combination treatment group (Fig 2D and E). Membranes were visualized using Ponceau S (Bio‐Rad, USA) in order to assess loading migration pattern. Throw after every use. Data are represented as mean ± SEM. Field measurements are highly desirable for other parameters (e.g. We thank Rebecca Fitzgerald (R.C.F.) Cells were treated with indicated concentrations of APR‐246 +/− MK‐571 or Sulfasalazine. Organoid segmentation based on bright‐field images and quantification of organoid area, number, Hoechst and PI texture, and mean Hoechst and PI intensity were performed using CellProfiler software (Version 3.0.0). Interestingly, assessment using ThiolTracker™, a dye that reacts with thiols in intact cells, showed that the observed difference in total GSH + GSSG (Fig 5C and D) is not reflected in the global thiol status of the cell (Fig 5E). ... Doxycycline and chemicals described hereafter were … We thank Peter Chumakov (Engelhardt Institutet of Molecular Biology) for H1299 cells, Bert Vogelstein (Johns Hopkins Oncology Center) for HCT116 cells, and Rolf Kiessling (Karolinska Institutet) for KADA cells. IC50 values in cell lines were calculated using Origin software (OriginLab) based on the mean WST‐1 data (highest treatment was below 30 µM APR‐246) and resazurin assay for esophageal cell lines (up to 100 µM APR‐246). Representative images of hematoxylin/eosin (H&E) staining and immunostaining of Ki67, cleaved caspase 3 and p53, post‐treatment/late timepoint (> 22 days after treatment initiation) with APR‐246 (50 mg/kg) +/− MK‐571 (50 mg/kg). Port Manteaux churns out silly new words when you feed it an idea or two. Thus, GS‐MQ serves as an intracellular drug reservoir. Apart from the appropriate face and headgear, it is also important to put on gloves for protecting your hands. In summary, blocking MRP1 transporter activity using two chemically distinct inhibitors, MK‐571 or Reversan, or siRNA‐mediated downregulation of MRP1, significantly potentiates APR‐246‐induced cancer cell death. Updated world stock indexes. Our time course experiment showed that the Michael reaction was rapidly reversible (Fig 4C and D) which provides a molecular explanation for the previously observed reversible inhibition of thioredoxin and glutaredoxin (Haffo et al, 2018). Representative bright‐field and PI/Hoechst staining images of colo‐PDO1 after 72‐h treatment with APR‐246 +/− MK‐571. Take A Sneak Peak At The Movies Coming Out This Week (8/12) Better days are here: celebrate with this Spotify playlist If data did not pass the Shapiro–Wilk test, a non‐parametric test was applied; for unpaired data, the Mann–Whitney test was used; for paired data, the Wilcoxon test was applied. ABN 47621340878. APR‐246 was dissolved in either DMSO or water. HRP‐conjugated secondary antibodies against mouse (61‐6520, Invitrogen), rat (NA935V, GE Healthcare, UK), or rabbit (65‐6120, Invitrogen) IgG were diluted 1:10,000 and incubated for 1 h at room temperature and then visualized using SuperSignal West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific, USA) in a Fuij Film developer (Fuji Film, Japan). Decreased intracellular Cys levels were observed upon combination treatment with APR‐246 and MK‐571, suggesting that increased intracellular Cys is consumed, modified, or generated to a lesser extent during the combination treatment (Fig 6G). The reversibility of MQ adduct formation is a key aspect of the mechanism of action of APR‐246 (Fig 8) which may also in part account for the benign safety profile reported from clinical studies of APR‐246 (Lehmann et al, 2012). She's also a YouTube star.…” OVCAR‐3 cells (2 × 105) were seeded in 6‐well plates with 2 ml media. Experiments were performed 1 week after thawing of cells up to 8 weeks in culture at the latest unless otherwise specified. PDOs recapitulate tumor heterogeneity and are useful pre‐clinical tools for evaluation of precision therapy (Li et al, 2018). In blue nuclear Hoechst 33342 staining. Accordingly, we also assessed the relative levels of xCT in our cell line panel (Appendix Fig S7F). MRP1 blockade potentiates APR‐246 efficacy by increasing the intracellular pool of APR‐246 active product MQ bound to glutathione (GS‐MQ conjugates) and shifting the intracellular thiol‐disulfide homeostasis. is funded by a Core Programme Grant from the Medical Research Council (RG84369). This is associated with altered cellular thiol status and increased intracellular glutathione‐conjugated MQ (GS‐MQ). Proteins were precipitated by adding 5‐sulfosalicylic acid to a final concentration of 1%. The rapid reversibility and hence inherent instability of the MQ conjugates make detection technically challenging, which may explain why we were not able to detect Cys‐MQ conjugates. This is consistent with results showing that xCT can promote resistance to APR‐246 in mutant TP53‐harboring tumor cells (Appendix Fig S7D and E) (Liu et al, 2017). We would like to show you a description here but the site won’t allow us. Due to the reversible binding of MQ, GS‐MQ conjugates can form an intracellular drug reservoir for the targeting of mutant p53. Detailed information about source, origin, TP53 status, and culturing conditions for all cell lines is provided in Appendix Table S5. For correlation analysis, the compound activity profile of PRIMA‐1, gene expression, and metabolite abundance data was obtained from Cancer Dependency Map (https://www.depmap.org) to determine potential associations. Clustering analysis divided the cell lines into high and low sensitivity to APR‐246 groups where most lines in the high sensitivity group carry mutant TP53 (Appendix Fig S1B). AllStars Negative Control siRNA (Qiagen, Germany) and siGENOME Non‐Targeting siRNA (Dharmacon, USA) were used as negative controls. Simultaneous xCT knockdown and MRP1 inhibition by MK‐571 caused a three‐ and fourfold increase in 14C accumulation (Fig 7A and Appendix Table S4) and increased APR‐246‐induced growth suppression (Appendix Fig S7B). MQ, the active conversion product of APR‐246, reversibly binds the major antioxidant glutathione (GSH). We wrote a customized R script based on the “Synergyfinder” package (https://doi.org/10.18129/B9.bioc.synergyfinder). Samples were washed in PBS and spun down (5 min at 300 × g). K.G.W. It is conceivable that a higher intracellular concentration of MQ in the context of diminished GSH levels will allow more extensive modification of key mutant p53 cysteines or other protein thiols, and thus more efficient mutant p53 activation and subsequent tumor cell killing. However, combination treatment with MK‐571 significantly enhanced APR‐246 anti‐tumor activity (Fig 2A, and Appendix Fig S2A and B) and survival (Fig 2B) in MRP1‐expressing Eso26 xenografts (Appendix Fig S2C), consistent with the in vitro data on esophageal cell lines (Fig EV1B). It has a role as a plant metabolite and a fragrance. Synergy landscape plots over dose matrix were generated by SynergyFinder. Cells were treated with APR‐246 and/or MK‐571 the next day. Primary antibodies used were against Ki67 (1:100, ab16667, Abcam), cleaved caspase 3 (1:100, 9664, Cell Signaling Technology), MRP1 (D5C1X) (1:200, 72202, Cell Signaling Technology), and p53 (1:100, NCL‐L‐p53‐DO7, Leica Biosystems). However, our data suggest that the association between TP53 status and GSH concentration is cell context‐dependent (Fig 5C and D, and Appendix Fig S5A–C). and you may need to create a new Wiley Online Library account. Total concentration of GSH + GSSG was calculated based on a GSH standard curve. For instance, Also Read: Tips For Building Optimized and Efficient Labs. Hence, cells that accumulate more APR‐246/MQ are also more sensitive, irrespective of TP53 status. Detailed information regarding culturing media of the PDOs is included in Appendix Table S2. Melbourne Convention and Exhibition Trust R91. LC‐MS analysis showed that this was a result of increased accumulation of GS‐MQ, whereas no significant accumulation of APR‐246 was observed (Fig 4A and B). And cause harm to the decreased concentration of GS‐MQ increased ( Fig and... Culled at ethical endpoints prior to treatments Kaplan–Meier curves g ) empty vector ( EV ) pcDNA3.1 ( + purchased. 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