Molecular determinants of the antitumor effects of trichostatin A in pancreatic cancer cells

Figure 1 Enhancement of histone H3 acetylation by trichostatin-A (TSA). The indicated pancreatic cancer (PC) cell lines were treated with various concentrations of TSA for 24 h. A: Histone H3 acetylation was analyzed by immunoblotting; B: Reprobing of the blot with an anti-H3 protein-specific antibody revealed no systematic differences of the histone H3 amount among the samples; C: Acetyl-histone H3 levels were further investigated using image analysis software and related to the histone H3 protein level. Therefore, acetyl-histone H3 and H3 protein signal intensities were determined, and the ratio acetyl-histone H3/H3 protein was calculated. A ratio of 100% corresponds to control cells cultured without TSA. The data shown are representative of three independent experiments.

Figure 2 Effects of TSA on the BrdU incorporation of PC cell lines. BxPC-3, AsPC-1 and CAPAN-1 cells were treated with TSA as indicated for 24 h, before DNA synthesis was assessed with the BrdU incorporation assay. 100% BrdU incorporation corresponds to cells cultured without TSA. Data are presented as mean ± SE (n = 6 separate cultures); ^aP < 0.05 vs control cultures, ^cP < 0.05 vs BxPC-3 cells (Wilcoxon’s rank sum test).

Figure 3 Expression and phosphorylation of signal transduction proteins in TSA-treated PC cell lines. BxPC-3, AsPC-1 and CAPAN-1 cells were treated with TSA at concentrations up to 10 × 10^-7 mol/L for 24 h. A: Expression and phosphorylation of the indicated proteins was analyzed by immunoblotting. Phospho-proteins were detected first, followed by a reprobing of the blots with anti-protein specific antibodies. Actin was used as a housekeeping control protein; B-D: Fluorescence signal intensities of phospho (P) proteins and total proteins were quantified using Odyssey^® software version 3.0. Subsequently, the ratios phospho-ERK/ERK protein (B), phospho-AKT/AKT protein (C) and phospho-p38/p38 protein (D) were determined. A ratio of 100% corresponds to control cells cultured without TSA. Data of 8 independent experiments were used to calculate mean values and SE; ^aP < 0.05 vs control cultures, ^cP < 0.05 vs BxPC-3 cells (Wilcoxon’s rank sum test).

Figure 4 SB202190 diminished inhibition of BrdU incorporation by TSA in BxPC-3 cells. The cells were treated with TSA, SB202190 and SB202474 at the indicated concentrations for 24 h, before DNA synthesis was assessed with the BrdU incorporation assay. 100% BrdU incorporation corresponds to untreated cells. Data are presented as mean ± SE (n = 16 separate cultures); ^aP < 0.05 vs control cultures, ^cP < 0.05 vs SB202474-treated cells (identical concentration of TSA) (Wilcoxon’s rank sum test).

Figure 5 Effects of TSA on BAX and p21^Waf1 expression in PC cell lines. A, B: The cell lines were incubated with TSA for 8 h as indicated. The mRNA expression of Bax (A), p21^Waf1 (B) and the housekeeping gene GAPDH (A and B) was analyzed by real time PCR, and relative amounts of target mRNA were determined. 100% mRNA expression of each gene corresponds to cells cultured without TSA. Data of ≥ 6 independent experiments were used to calculate mean values and SE; ^aP < 0.05 vs control cultures, ^cP < 0.05 vs BxPC-3 cells (Mann-Whitney test); C: BxPC-3, AsPC-1 and CAPAN-1 cells were treated with TSA at concentrations up to 10 × 10^-7 mol/L for 24 h. Expression of the p21^Waf1 protein and actin (housekeeping control protein) was analyzed by immunoblotting. Fluorescence signal intensities of p21^Waf1 and actin were quantified, and the ratio p21^Waf1/actin was determined. A ratio of 100% corresponds to control cells cultured without TSA. Data of 8 independent experiments were used to calculate mean values and SE; ^aP < 0.05 vs control cultures, ^cP < 0.05 vs BxPC-3 cells (Wilcoxon’s rank sum test).