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  • br Keto PGE has an electrophilic unsaturated carbonyl


    15-Keto PGE2 has an electrophilic α,β-unsaturated carbonyl group that can covalently modify nucleophilic cysteine residue(s) present in various proteins, regulating their functions/activities [16]. We 
    observed that the α,β-unsaturated carbonyl moiety of 15-keto PGE2 plays an important role in directly blocking the STAT3 activation through covalent modification of Cys259 of STAT3. In support of this notion, STAT3 signaling was not inhibited by the non-electrophilic analogue, 13,14-dihydro-15-keto PGE2 lacking the α,β-unsaturated carbonyl group. The interaction between 15-keto PGE2 and STAT3 was disrupted by the thiol reducing agent, DTT, suggesting that the Michael addition reaction occurs between the α,β-unsaturated carbonyl group of 15-keto PGE2 and cysteine residue(s) in STAT3.
    The STAT3 protein structurally consists of 6 domains [41]. These include N-terminal domain, a coiled-coil domain, a DNA binding do-main, a linker domain, Src-homology 2 (SH2) domain and C-terminal
    E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
    stained tumor tissue sections. Scale bar: 100 μM. Magnification:×4 and ×10. H. Total protein samples from the tumor specimens were subjected to Western blot analysis to measure the levels of P-STAT3Y705 and STAT3. Actin was used as the internal control. ***p < 0.001, N.S, not significant. I. The effect of the higher dose (140 μg/kg) of 15-keto PGE2 on the AMG-176 of P-STAT3Y705 was determined by immunohistochemical analysis (magnification, ×100).
    phosphorylation, dimerization, and activation [50]. Some compounds containing the α,β-unsaturated carbonyls group directly target the cy-steine residues of STAT3, thereby deactivating this transcription factor.
    E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
    For example, eriocalyxin B having two α,β-unsaturated carbonyls di-rectly interacts with STAT3 at the Cys712 of STAT3, which hampers the phosphorylation of STAT3 [47]. A small molecule compound, NSC-368,262 (C48) alkylates Cys468 of STAT3, resulting in suppression of STAT3 DNA binding [42]. Distinct from these compounds, 15-keto PGE2 interacts directly with Cys259 located in the coiled-coil domain of STAT3 to form a covalent linkage. In our previous study, curcumin, a 
    natural polyphenolic compound present in the turmeric, induces apoptosis in MCF10A-ras cells through direct targeting Cys259 of STAT3 [51]. Zhang et al. reported that the coiled-coil domain also in-teracts directly with the gp130 peptide like SH2 domain which is re-quired for STAT3 phosphorylation [52].
    Other prostaglandin metabolites, especially A series of pros-taglandins which also have an α,β-unsaturated carbonyl group,
    E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
    Fig. 8. A proposed mechanism underlying suppression of STAT3 signaling and growth and progression of breast cancer by 15-keto PGE2. 15-Keto PGE2 covalently binds to STAT3, and this hampers the phosphorylation, dimerization, nuclear localization and transcriptional activity of STAT3.
    modulates the intracellular signaling involved cell proliferation and differentiation [53]. For instance, PGA2 acts as an electrophile inducing
    a conformation change of p53, thereby impairing p53-mediated apop-tosis [54]. 15-Deoxy 12,14 prodtaglandin J2 (15d-PGJ2) undergoes nucleophilic addition to Cys136 on PTEN, thereby activating PI3K-Akt signaling [55]. 15d-PGJ2 inhibits IL-6-stimulated phosphorylation of STAT3 in endothelial cells which is attributable to its electrophilic nature [56]. This cyclopentenone prostaglandin also covalently modify the thiol groups of Keap1 [57] and other proteins including PPARγ [16,58].
    The compounds targeting STAT3 inhibit the growth of cells and induces apoptosis in cancers [22,29,48,52]. We observed that sup-pression of STAT3 signaling by 15-keto PGE2 accompanies proliferation of breast cancer cells and tumor growth. There has been no previous study evaluating the antitumor effect of 15-keto PGE2 in an animal model. Our present study demonstrates for the first time that the growth of xenograft tumors is suppressed by subcutaneous adminis-tration of 15-keto PGE2. In conclusion, 15-keto PGE2 inhibits STAT3 signaling through covalent modification, and suppresses breast cancer growth and progression (Fig. 8).