Fig 1: Compound 323s reduced the clonogenicity and induced apoptosis. (A) On the 2nd day after seeding, compounds 323–1 and 323–2 were added and DU145, PC3, EPT3M1-STAT3, and 22Rv1 cells were allowed to grow for 2 weeks to form colonies and next stained with 0.4% crystal violet (w/v). (B) Flow cytometric analyses of cell apoptosis induced by treatment with vehicle (DMSO), 323–1, 323–2, cryptotanshinone, or S3I201 at indicated concentrations for 72 h in DU145 cells. Data are representative results of three experiments.
Fig 2: Compounds 323–1 and 323–2 target the IL-6/STAT3 pathway. (A) Chemical structure and nomenclature of compounds 323–1 and 323–2. (B) HEK 293T cells transfected with the STAT3 luciferase reporter were treated with different doses, as indicated of 323–1 or 323–2, S3I-201, or cryptotanshinone (Crypt) in the presence of 20 ng/ml IL-6 for 24 h. All data are represented as the average ±s. e.m. (n = 3).Significance was analyzed by using one-way ANOVA multiple comparison. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (C) LNCaP cells were treated with DMSO, 20 µM 323–1 or 323–2, or 5 µM cryptotanshinone for 24 h before treatment with or without 10 ng/ml IL-6 for the last 15 min. (D) PC3 cells were treated with DMSO, 20 µM 323–1, 20 µM 323–2, 100 µM S3I-201, or 5 µM cryptotanshinone for 24 h before treatment with or without 500 IU/ml IFNɣ for the last 15 min. (E) DU145 cells were treated with indicated doses of 323–1, 323–2, or S3I-201 for 24 h. (F) EPT3M1-STAT3 or LNCaP cells were treated with vehicle (DMSO), 20 µM 323–1, and 20 µM 323–2 for 48 h, respectively. (C–F) Lysates were analyzed by Western blotting and indicated antibodies. Data are shown as the representative results of three separate repeats.
Fig 3: Inhibitory effects of 15-keto PGE2on STAT3 target gene expression and neoplastic transformation of MCF10A-ras cells. A. MCF10A-ras cells were treated with 15-keto PGE2 (20 μM) or vehicle for 24 h. The mRNA levels of two representative STAT3 target genes Cyclin D1 and Bcl2 were determined by real-time PCR. β-Actin was measured to ensure equal amount of cDNA loaded. N.S.: not significant. *p < 0.05, **P < 0.01, N.S, not significant. B. MCF10A-ras cells were seeded in 6 well plates and treated with two different concentrations of 15-keto PGE2 (10 or 20 μM) or vehicle (DMSO) in DMEM/F12 medium containing 5% heat-inactivated horse serum and supplementary agents. The incubation conditions and other experimental details are described in Materials and methods. The colony size >100 μm was counted under a light microscope. Results are the means ± S.D. *p < 0.05, ***P < 0.001. C. MCF10A-ras cells were plated on a 60 mm dish containing 0.5% (down) and 0.33% (up) double layer agar. The cells were treated every day with DMEM/F-12 containing DMSO or 15-keto PGE2. After 3-week of incubation, the colonies were stained with crystal violet, and the number of colonies was counted as described in Materials and methods. Results are the means ± S.D. *p < 0.05, ***P < 0.001.
Fig 4: Inhibition of xenograft tumor growth and STAT3 phosphorylation in BALB/nude mice. A. MDA-MB-231 cells were treated with 15-keto PGE2 (20 μM) for 48 h. The expression levels of P-STAT3Y705 and STAT3 were measured by Western blot analysis. ***p < 0.001. B. MDA-MB-231 cells treated with 15-keto PGE2 (20 μM) for 24 h were subjected to the clonogenic assay. C. The representative shots of human mammary tumor (MDA-MB-231) xenografts in mice treated with vehicle, 15-keto PGE2 (70 μg/kg) and 15-keto PGE2 (140 μg/kg) for 4 weeks (n = 14/group). D, E. The size of tumors was measured with digital calipers. The calculated formula is 0.52 × length x width2. **p < 0.005, ***p < 0.001 (Two-sided t-test). F. The effect of 15-keto PGE2 treatment on body weight. G. H&E 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 expression of P-STAT3Y705 was determined by immunohistochemical analysis (magnification, ×100).
Fig 5: Comparative effects of 15-keto PGE2and its non-electrophilic analogue, 13,14-dihydro-15-keto PGE2on 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 by Western blot analysis. **p < 0.01 , N.S, not significant. C. MCF10A-ras cells were treated with 15-keto PGE2 (20 μM) or the same concentration of 13,14-dihydro-15-keto PGE2 for 12 h. Homo-dimerization of STAT3 was measured by the immunoprecipitation assay as described in Materials and methods. *p < 0.05, N.S., not significant. D. Human prostate cancer PC3 cells were co-transfected with HA-tagged STAT3 and Myc-tagged STAT3 and treated with 15-keto PGE2 or 13,14-dihydro-15-keto PGE2 (20 μM each) for 12 h. The total lysates obtained from the transfected cells were immunoprecipitated with anti-Myc antibody and analyzed by Western blotting with anti-HA antibody to measure the formation of a STAT3 dimer. E. MCF10A-ras cells were incubated with 15-keto PGE2 (20 μM) or 13,14-dihydro-15-keto PGE2 (20 μM) for 24 h. The nuclear extracts were separated and subjected to Western blot analysis. F. After MCF10A-ras cells were treated with 15-keto PGE2 (20 μM) or 13,14-dihydro-15-keto PGE2 (20 μM) for 24 h, the fixed cells were incubated with anti-P-STAT3Y705, which was detected using TRITC red fluorescence-labeled secondary antibody. G. The HeLa/P-STAT3-luc reporter cells were pretreated with 20 μM each of 15-keto PGE2 or 13,14-dihydro-15-keto PGE2 for 24 h followed by treatment with OSM for another 6 h. The cells were analyzed for the luciferase activity using a microplate luminometer. ***p < 0.001. H. MCF10A-ras cells treated with 15-keto PGE2 (20 μM) or 13,14-dihydro-15-keto PGE2 (20 μM) were subjected to the clonogenic assay. The colony size >100 μm was counted under light microscope. Results are the means ± S.D. ***p < 0.001. I. MCF10A-ras cells were plated on a 60 mm dish containing 0.5% (down) and 0.33% (up) double layer agar. The cells were treated every day with DMEM/F-12 containing DMSO, 15-keto PGE2 (20 μM) or 13,14-dihydro-15-keto PGE2 (20 μM). After 3-week of incubation, the colonies were stained with crystal violet, and the number of colonies was counted as described in Materials and methods. ***p < 0.001.
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