Figure 1.
25-OCH3-PPD inhibits the growth of breast cancer xenograft tumors.
25-OCH3-PPD was administered by i.p. injection at doses of 5 or 20 mg/kg/d, 5 days/wk for 6 (MCF7 (A)) or 4 weeks (MDA-MB-468 (B)). The growth of tumors was monitored. The body weights of animals were also monitored as a surrogate marker for toxicity in MCF7 (C) and MDA-MB-468 (D) xenograft models.
Figure 2.
25-OCH3-PPD decreases MDM2 expression in a dose- and time-dependent manner in human breast cancer cells.
MCF7 (p53 wild type) and MDA-MB-468 (p53 mutant) cells were treated with various concentrations of 25-OCH3-PPD for 24 h (A) or with 25 µM 25-OCH3-PPD for various periods (B). (C) 25-OCH3-PPD also inhibits MDM2 expression in vivo. The tumor-bearing animals were treated with 25-OCH3-PPD (20 mg/kg/d, 5 days/wk) and tumors were removed at indicated times. The protein levels of MDM2, p53, and p21 in tumor tissue homogenates were analyzed by Western blotting.
Figure 3.
25-OCH3-PPD inhibits MDM2 transcription.
(A) MCF7 cells were treated with various concentrations of 25-OCH3-PPD for 24 h. MDM2 mRNA was co-amplified with β-actin mRNA. The relative levels of MDM2 were normalized to that of β-actin. (B) MCF7 cells were co-transfected with full-length MDM2 P2 promoter luciferase vectors and a Renilla luciferase reporter, followed by incubation with 25-OCH3-PPD for 24 h. The MDM2 luciferase activity was detected using the Dual-Luciferase Reporter Assay System. All the analyses were performed in triplicate. (C) Structures of full-length and deleted MDM2 P2 promoter. (D) The effects of 25-OCH3-PPD (10 µM) on the activity of various MDM2 luciferase reporters were analyzed using the same procedure as above (B). Luciferase activities were plotted as percentages of the control. * P<0.01.
Figure 4.
25-OCH3-PPD destabilizes MDM2 protein.
(A) MCF7 and MDA-MB-468 cells were treated with DMSO or 25-OCH3-PPD (25 µM) followed by exposure to protein synthesis inhibitor cycloheximide (CHX, 10 µg/mL). MDM2 levels were detected by Western blotting at indicated times after exposure to CHX. (B) MCF7 cells were transfected with MDM2 and ubiquitin plasmids followed by treatment with various concentrations of 25-OCH3-PPD for 24 h. Cells were harvested (left panel) or exposed to proteasome inhibitor MG132 (25 μM) for additional 6 h (right panel). Ubiquitinated MDM2 was detected by immunoblotting. (C) The cell lysates of MCF7 cells treated as above were immunoprecipitated with anti-MDM2 antibody. The ubiquitinated MDM2 was detected using anti-ubiquitin antibody.
Figure 5.
MDM2 overexpression reduces cell response to 25-OCH3-PPD treatment.
MCF7 MDM2-inducible cells were incubated with (Tet+) or without tetracycline (Tet-) for 12 h, followed by exposure to various concentrations of 25-OCH3-PPD for various times: (A) 72 h for analysis of cell viability using MTT assay; (B) 24 h for analysis of cell proliferation using BrdUrd assay; (C) 48 h for apoptosis analysis using Flow Cytometry; and (D) 24 h for cell cycle analysis. (E) 25-OCH3-PPD inhibits MDM2 expression in MCF7 inducible cells without tetracycline. MDM2 overexpression was confirmed in MCF7 inducible cell line treated with tetracycline, which reversed the effect of 25-OCH3-PPD.
Figure 6.
MDM2 knockdown reduces cell response to 25-OCH3-PPD treatment.
MCF7 MDM2-inducible cells were incubated with (Tet+) or without tetracycline (Tet-) for 12 h, followed by exposure to various concentrations of 25-OCH3-PPD for various times: (A) 72 h for analysis of cell viability using MTT assay; (B) 24 h for analysis of cell proliferation using BrdUrd assay; (C) 48 h for apoptosis analysis using Flow Cytometry; and (D) 24 h for cell cycle analysis. (E) 25-OCH3-PPD inhibits MDM2 expression in MCF7 inducible cells with or without tetracycline. MDM2 knockdown was confirmed in MCF7 inducible cell line treated with tetracycline, which reversed the effect of 25-OCH3-PPD.
Figure 7.
MDM2 knockdown reduces cell sensitivity to 25-OCH3-PPD treatment.
PC3 cells and PC3 MDM2 KD cells were treated with various concentrations of 25-OCH3-PPD for various times: (A) 72 h for analysis of cell viability using MTT assay; (B) 24 h for analysis of cell proliferation using BrdUrd assay; (C) 48 h for apoptosis analysis using Flow Cytometry; and (D) 24 h for cell cycle analysis.
Figure 8.
Tetracycline treatment does not affect parent MCF7 cells.
MCF7 cells were incubated with (Tet+) or without tetracycline (Tet-) for 12 h, followed by exposure to 25-OCH3-PPD (25 µM) for various times: (A) 72 h for analysis of cell viability using MTT assay; (B) 24 h for analysis of cell proliferation using BrdUrd assay; (C) 48 h for apoptosis analysis using Flow Cytometry; and (D) 24 h for cell cycle analysis. (E) 25-OCH3-PPD inhibits MDM2 expression in MCF7 cells with or without tetracycline.
Figure 9.
25-OCH3-PPD inhibits cell migration in vitro and metastasis in vivo.
(A) Wound healing assay in MDA-MB-231 cells. After cell growth reached confluence, a scratch was made, and then the cells were incubated with various concentrations of 25-OCH3-PPD for indicated times. The closure of the scratch was imaged. (B) Effects of 25-OCH3-PPD on the expression of EMT markers in human breast cancer cells. Cells were treated with various concentrations of 25-OCH3-PPD for 48 h. The expression of EMT markers was analyzed by Western blotting. (C) In vivo metastasis assay. 1×106 of MDA-MB-231-Luc cells were intravenously injected into a tail vein of nude mice. 25-OCH3-PPD was administered by i.p. injection at a dose of 10 mg/kg/d, 5 days/wk for 2 weeks. The luciferase signals were determined and photographed using IVIS in vivo image system on Days 1, 8, and 15.