Figure 1.
Expression of SIRT1, SIRT2, and acetylated (Ac)-p53 in gastric cancer cells.
A: The expression of SIRT1, SIRT2, p53, Ac-p53, and phospho-53 in seven gastric cancer cell lines and MRC-5 fibroblasts was examined by Western blotting. Semi-quantitation of Western blotting densitometry involved normalization to β-actin levels. MCF-7*: MCF-7 cells treated with doxorubicin (1 µM, 24 h). B: Expression of SIRT1 mRNA and SIRT2 mRNA in gastric cancer cells. Levels of SIRT1 mRNA and SIRT2 mRNA were determined in gastric cancer cells and MRC-5 fibroblasts by quantitative real-time PCR and normalized to the level of 18S rRNA. mRNA levels are shown relative to those of MRC-5 fibroblasts.
Figure 2.
Tenovin-6-induced acetylation of H3 and α-tubulin, and antitumor effects in gastric cancer cells.
A: Gastric cancer cells were cultured with tenovin-6 (10 µM) for various time periods and analyzed for the levels of SIRT1, SIRT2, Ac-H3, and Ac-α-tubulin by Western blotting. B: Tenovin-6 inhibited the growth of gastric cancer cells regardless of TP53 status. All experiments were assessed by WST-8 assay and carried out in triplicate. Results are expressed as the mean ± SD. C: WST-8 assay was performed in MRC-5 cells to compare the toxicity of tenovin-6 to cancer cell lines. The growth inhibition of tenovin-6 in NUGC-4 cells was significantly higher than that in MRC-5 cells. The significance of differences was evaluated using Student's t-test.
Figure 3.
Induction of apoptotic cell death by tenovin-6 in gastric cancer cells.
A: Time-course analysis of the expression of p53, its downstream molecule p21Waf/Cip1, and apoptosis-related molecules in gastric cancer cells treated with tenovin-6 (10 µM). Relative intensity of the proteins' expression is shown in Figure S1. Tenovin-6 induced the expression of Ac-p53 and p21Waf/Cip1, but not Bcl-2. DR5 expression was strongly induced by tenovin-6. TRAIL, and cleaved PARP were slightly increased, but FADD expression was not affected. A doublet of DR5 shows its precursor (upper band) and mature isoforms (lower band). B: Four cell lines were treated with tenovin-6 (10 µM) or a vehicle control for 72 h, double-stained with FITC-annexin V and PI, and analyzed by flow cytometry. The statistical significance of differences between groups was evaluated using Student's t-test. * p<0.01; ** p<0.05.
Figure 4.
Effects of DR5 knockdown on cell survival and apoptosis in TP53-null KatoIII cells.
A: KatoIII cells were transfected with 1 nM siRNA control or siRNA against DR5 mRNA. Forty-eight hours after transfection, the cells were treated with 5 µM tenovin-6 for 24 h. Western blotting showed the down-regulation of DR5 by siRNA transfection. A doublet of DR5 shows its precursor (upper band) and mature isoforms (lower band). B: Cells were transfected with 1nM siRNA control or DR5 siRNA for 48 h, treated with 0.2, 1, and 5 µM tenovin-6 for 72 h and then subjected to cell viability measurements by WST-8 assay. The statistical significance of differences between groups was evaluated using Student's t-test. * p<0.01; ** p<0.05. C: The effect of DR5 knockdown on tenovin-6 induced apoptosis was analyzed by flow cytometry using PI staining. The results are expressed as the mean ± SD. The statistical significance of differences between groups was evaluated using Student's t-test.
Figure 5.
Expression of proteins associated with ER stress in gastric cancer cell lines treated with tenovin-6.
A: Expression of proteins associated with ER stress in gastric cancer cell lines before and after treatment with 10 µM tenovin-6. Thapsigargin at 3 µM was administered to HEK293 cells as a control. A doublet of DR5 shows its precursor and mature isoforms. B: Relative intensity of expression of CHOP in cell lines tested is shown. The statistical significance of differences between groups was evaluated using Dunnett's test.
Table 1.
Combination index of tenovin-6 plus docetaxel, SN-38, cisplatin, and 5-FU in gastric cancer cells.