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  • br Further the colony formation


    Further, the colony formation (Fig. 5H) and anchorage-independent MCF10A-ras cell growth (Fig. 5I) were not suppressed by 13,14-di-hydro-15-keto PGE2. Above results suggest that the electrophilic α,β-unsaturated carbonyl group of 15-keto PGE2 is essential for its deacti-vating STAT3.
    E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
    Fig. 5. Comparative effects of 15-keto PGE2 and its non-electrophilic analogue, 13,14-dihydro-15-keto PGE2 on STAT3 activation, and clonogenicity and anchorage-independent growth of MCF10A-ras cells. A. 15-Keto PGE2 is reduced to the 13,14-dihydro-15-keto PGE2 by PTGR2. 15-Keto PGE2 has an α,β- unsaturated carbon which is considered to target nucleophiles whereas 13,14-dihydro-15-keto PGE2 has no such electrophilic moiety. B. MCF10A-ras cells were treated with 20 μM each of 15-keto PGE2 or 13,14-dihydro-15-keto PGE2 for indicated time points. The expression levels of P-STAT3Y705 and STAT3 were measured
    E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
    3.6. 15-Keto PGE2 covalently binds to STAT3 via Michael addition reaction
    STAT3 possesses several cysteines [39] and many studies focus on targeting cysteine residues in the SH2 and DNA binding domains to inhibit the STAT3 signaling [40–43]. We speculated that the electro-philic α,β-unsaturated carbonyl moiety of 15-keto PGE2 could modify the thiol group(s) of STAT3 (Fig. 6A) which might hamper its phos-phorylation. Pretreatment of MCF10A-ras cells with the thiol reducing agent, DTT abolished the inhibitory effect of 15-keto PGE2 on STAT3 
    phosphorylation (Fig. 6B), lending support to the above supposition. To investigate direct interaction between 15-keto PGE2 and STAT3, MCF10A-ras cells were treated with biotinylated 15-keto PGE2, and the immunoprecipitation assay using the biotin-streptavidin system was performed. As shown in Fig. 6C, biotinylated 15-keto PGE2 directly bound to STAT3, and this was abrogated in the presence of DTT. A docking model predicted Cys251 and Cys259 of STAT3 as putative NPS-2143 of 15-keto PGE2 (Fig. 6D).
    To further verify the direct interaction between 15-keto PGE2 and
    E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
    Fig. 6. Covalent modification of STAT3 by15-keto PGE2. A. A proposed interaction between 15-keto PGE2 and a thiol group of STAT3. B. MCF10A-ras cells were pretreated with a thiol reducing agent, DTT (100 μM), for 1 h followed by exposure to 15-keto PGE2 (20 μM) for an additional 24 h. The protein levels of P-STAT3Y705 and STAT3 were determined by Western blot analysis. **p < 0.01, ***p < 0.001. C. The direct binding of 15-keto PGE2 to STAT3 was assessed using the avidin-biotin system. MCF10A-ras cells were pretreated with DTT (100 μM) for 1 h and then treated with biotinylated 15-keto PGE2 (40 μM) for an additional 12 h. The bioti-nylated 15-keto PGE2-STAT3 complex was detected by the immunoprecipitation and Western blot analyses. D. A putative model of 15-keto PGE2 covalently bound to Cys259 or Cys251 of STAT3 as predicted by a docking study. The blue dotted line represents a H-bond. E. PC3 cells were transfected with WT or Cys251 or Cys259-mutated STAT3 followed by treatment with 15-keto PGE2 (40 μM) for 24 h. WT STAT3 and STAT3 mutants were detected by anti-STAT3 antibody. F. Annotated fragment MS/MS spectrum of 15-keto PGE2 in positive ESI mode. The elucidation of fragment ion at m/z 333.2073, 315.1951 from the structure is given in the inset of the spectra. G. Mass spectrum of the STAT3 peptide (RQQIACIGGPPNICLDR) with 15-keto PGE2 binding. Annotated MS/MS spectrum illustrating the binding of 15-keto PGE2 to STAT3 protein at cysteine 259 residues was identified by LC-MS/MS. An exclamation mark indicates a carbamidomethylation on Cys251, and an asterisk indicates 15-keto PGE2 binding to Cys259. The precursor ion [M+3H]3 + for peptide with 15-keto PGE2 binding is m/z 754.0403. The 15-keto PGE2 fragment ions are inserted into the spectrum as a table. Each molecular formula present with b and y ions on the spectrum represents the 15-keto PGE2 fragment ion bound on Cys259. The sample preparation and other experimental details for mass spectral analysis are described in the Materials and methods. Sequence-in-formative fragmentation ions are summarized on the peptide sequence.