Figure 1.
DCB-3503 down regulated expression of cyclin D1, survivin, β-catenin, p53, and p21.
(A) Structure of DCB-3503. (B) and (C) HepG2 cells and HeLa cells were treated with DCB-3503 for the indicated time and dose. Whole cell lysates were probed with antibodies as indicated. β-Actin blot was used as internal loading control. D. Down-regulation of cyclin D1 by DCB-3503 in Huh7 and MCF-7 cells. B, C, and D are representatives from at least three separate experiments. (* Indicating non-specific band).
Figure 2.
DCB-3503 did not decrease mRNA levels of cyclin D1, survivin, β-catenin, p53, and p21.
A. HepG2 cells were treated with either DCB-3503 or CHX for the indicated time. mRNA levels of cyclin D1, survivin, β-catenin, p53, and p21 were analyzed by real-time RT-PCR using specific primers and probes and were normalized to that of β-actin. Values are depicted as mean ± S.D. from at least three separate experiments and are expressed relative to the basal mRNA level in the corresponding untreated control cells. B. mRNA level of cyclin D1 was analyzed by Northern blot under DCB-3503 treatment. Ribosomal RNA gel image on the bottom panel shows equal loading of total RNA. C. Expressions of cyclin D1, survivin, β-catenin, p53, and p21 under treatment of CHX, DCB-3503, and CHX and DCB-3503 in combination were examined by Western blot analysis in HepG2 cells. β-Actin blot was used as internal loading control. D. (a) 2 µM MG-132 reverses suppression of cyclin D1, survivin, β-catenin, p53, and p21 by DCB-3503 within 4-hour culture, (b) 2 µM MG-132 does not block cytotoxicity of DCB-3503 after 24-hour treatment. B, C, and D are representatives from at least three separate experiments. Numbers marked in C are normalized band intensity. (* Indicating non-specific band).
Figure 3.
DCB-3503 inhibited incorporation of amino acid in a time- and dose-dependent manner.
A. DCB-3503 inhibited [3H]-amino acid incorporation in both time- (a) and dose-dependent (b) manner in HepG2 cells. B. DCB-3503 inhibited [3H]-amino acid incorporation in HeLa cells (a) and PANC-1 cells (b) in a dose-dependent fashion. A and B results from three separate experiments are presented as mean ± S.D. C. Inhibitory effect of DCB-3503 on global protein synthesis and cyclin D1 protein synthesis assessed by [35S]-methionine/cysteine incorporation. This is a representative from three separate experiments.
Figure 4.
DCB-3503 preferentially inhibited [14C]-thymidine incorporation in a time- and dose-dependent manner, but not [14C]-uridine incorporation.
A. and B. The inhibitory effect of DCB-3503 on [14C]-thymidine incorporation was time-dependent and dose-dependent in HepG2 cells. (C) and (D) DCB-3503 showed less than 50% inhibition on [14C]-uridine incorporation in either HepG2 cells followed by different time and dose treatment. Hydroxyurea (Hu) and actinomycin D were used as positive controls. Values are depicted as mean ± S.D. from at least three separate experiments.
Figure 5.
DCB-3503-mediated inhibition of protein synthesis differs from that of CHX and rapamycin.
A. Effects of DCB-3503 and CHX on translation of uncapped and capped poly(A) tail cyclin D1 mRNAs in HeLa cell free lysate system. B. Effects of DCB-3503, CHX, and rapamycin on translation of capped luciferase mRNAs with or without 3'poly(A) tail in HeLa cell free lysate system. Values are depicted as mean ± S.D. from at least three separate experiments. C. Western blot analysis of phosphorylated p70 S6Kinase T389, S6 S235/236, phosphorylated 4EBP1 S65 and T37/46 under DCB-3503 and rapamycin treatment for 2 hours in HepG2 cells. 4EBP1 and β-Actin blots were used as internal loading controls. A and C are representatives from three separate experiments.
Figure 6.
DCB-3503 inhibited the elongation step of translation.
A. DCB-3503 treatment induced accumulation of polysomes in HepG2 and HeLa cells after 1-hour treatment. B. DCB-3503 induced accumulation of mRNAs of cyclin D1 and β-Actin in polysomal fractions in the sucrose gradient in HepG2 cells. A and B were representatives from three separate experiments with similar results.