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  • br E J Lee et al Redox Biology xxx

    2019-09-16


    E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
    the cysteine residues of STAT3, we conducted site-directed mutagensis studies in which cysteine 251 and 259 were replaced by alanine. PC3 Pyocyanin were transfected with WT or cysteine 251/259-mutated STAT3 followed by treatment with 40 μM of biotinylated 15-keto PGE2.. 15-Keto PGE2 barely bound to cysteine mutated STAT3 (Fig. 6E).
    To further verify the binding site of 15-keto PGE2 on STAT3 protein, MS/MS analysis of recombinant STAT3 protein treated with 15-keto PGE2 was performed. First, the 15-keto PGE2 was analyzed by DIMS in the positive mode. A fragmentation pattern was obtained by HCD of the precursor ion corresponding to 15-keto PGE2. The MS/MS spectrum of 15-keto PGE2 (m/z 351.2170) shows the fragmentation information during HCD (Fig. 6F). Secondly, tryptic peptides of STAT3 protein treated with 15-keto PGE2 were analyzed by LC-MS/MS. Based on the fragmentation pattern, we were able to identify 15-keto PGE2 bound tryptic peptide derived from STAT3 protein as shown in Fig. 6G. From the MS scan, the molecular ion corresponding to [M+3H]3+ (m/z 754.0403) of 246-RQQIACIGGPPNICLDR-262 was identified as 15-keto PGE2 bound STAT3 peptide at the Cys259 residue. The fragment ions matched to the 15-keto PGE2 bound STAT3 peptide were assigned and confirmed manually. These data support the assignment of Cys259 as the primary binding site of 15-keto PGE2 on STAT3.
    3.7. 15-Keto PGE2 attenuates tumor growth and suppresses STAT3 phosphorylation in a MDA-MB-231 xenograft model
    Treatment of human breast cancer MDA-MB-231 cells with 10 or
    20 μM of 15-keto PGE2 significantly decreased the phosphorylation of STAT3 (Fig. 7A). 15-Keto PGE2 also suppressed colony formation of MDA-MB-231 cells (Fig. 7B). We Pyocyanin then examined whether 15-keto PGE2 could inhibit the growth of MDA-MB-231 cells transplanted to BALB/c 
    nude mice. The mice xenografted with MDB-MB-231 cells were sub-cutaneously injected with two different doses of 15-keto PGE2 for 4 weeks. The tumor growth was significantly retarded in mice injected with 15-keto PGE2 at a dose of 70 μg/kg and 140 μg/kg (Fig. 7C and D). The average tumor volume in 15-keto PGE2-treated mice was drama-tically reduced in a dose-dependent manner (Fig. 7E). However, there were no body weight loss and other signs of toxicity in mice treated with 15-keto PGE2 (Fig. 7F). Histopathological analysis of the tumor samples stained for H&E showed that tumor density was decreased in 15-keto PGE2-treated mice compared to the control animals (Fig. 7G). Furthermore, the phosphorylation of STAT3 was markedly decreased in the 15-keto PGE2 treated group (140 μg/kg) compared to the control group (Fig. 7H). Immunohistochemical analysis of tumor samples ver-ified the significantly reduced expression of P-STAT3Y705 in the group treated with the higher dose (140 μg/kg) of 15-keto PGE2 (Fig. 7I), supporting the Western blotting data.
    4. Discussion
    STAT3 as a transcription factor is involved in tumor formation, cancer cell proliferation and survival. Therefore, targeting STAT3 has been considered as a practical approach for anti-cancer therapy [22,24]. Many STAT3 signaling inhibitors developed so far target the upstream kinases of STAT3, particularly JAK2 [44–46]. However, a JAK2 inhibitor, cucurbitacin Ⅰ also induces a phenotypic change inter-fering connective tissue growth in non-tumor cells [46]. Therefore, specific inhibitors of STAT3, especially those targeting the down-stream events, are necessary for developing novel chemotherapeutic drugs. Eriocalyxin B [47], cryptotanshinone [48], S3I-201 [49], an NCI com-pound NSC-368,262 (C48) [42], and galiellalactone [43] have been
    E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
    reported as molecules targeting STAT3 directly. These compounds were found to suppress the phosphorylation, dimerization, nuclear translo-cation, and transcriptional activity of STAT3. For instance, eriocalyxin B covalently binds Cys712 of STAT3 and blocks phosphorylation and subsequent activation of STAT3 [47]. We found that 15-keto PGE2 di-rectly inhibited STAT3 phosphorylation without affecting the JAK2 activity and the expression of SOCS3. As a result, the dimerization, nuclear translocation and transcriptional activity of STAT3 were re-pressed.