YM155 Down-Regulates Survivin and Induces P53 Up-Regulated Modulator of Apoptosis (PUMA)-Dependent in Oral Squamous Cell Carcinoma Cells

Abstract

BACKGROUND YM155, which inhibits the anti-apoptotic protein survivin, is known to exert anti-tumor effects in various cancers. However, there were few reports describing the inhibitory effect of YM155 on human oral squamous cell carcinoma (OSCC) cells that highly express survivin. In this study, we investigated the anti-tumor effects of YM155 on OSCC cells and then examined its molecular mechanisms. MATERIAL AND METHODS SCC9 cells of OSCC were treated with series of concentrations of YM155 (0.01, 0.1, 1, and 10 ng/ml) for 6, 12, and 24 h. The effect of YM155 on survival of SCC9 cells was detected by MTT and colony formation assay. Cell apoptosis was detected by flow cytometric analysis and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assays. Western blot was used to detect the protein expression of survivin, p53, and PUMA. Caspase-3 activity was measured by cleavage of the caspase-3 substrate. To test the role of PUMA and caspase-3 on YM155-induced apoptosis and growth inhibition, the SCC9 cells was transfected with PUMA siRNA or caspase-3 siRNA or control siRNA for 16 h before YM155 (1 and 10 ng/ml) treatment for 24 h. In addition, we also investigated the effect of YM155 in an in vivo xenograft model. RESULTS Treatment of YM155 efficiently reduced survivin expression and increased PUMA expression and caspase-3 activation in the SCC9 cells. YM155 treatment resulted in 18-86% decrease in cell viability, 10-60% decrease in colony numbers, and 8-40% increase in cell apoptosis (p<0.05 and p<0.01). However, the induction of cell apoptosis growth inhibition was reversed by PUMA siRNA or caspase-3 transfection. In addition, animals treated with YM155 showed more than 60% tumor growth inhibition compared to the controls (p<0.05). CONCLUSIONS YM155 is a potent inhibitor of progression of SCC9 cells, which could be due to attenuation of survivin, and activation of the PUMA/caspase-3 cellular signaling processes. This study suggests that YM155 may be a potential molecular target with therapeutic relevance for the treatment of OSCC.

Figure 1

YM155 affects growth and colony formation, and induces apoptosis of SCC9 cells SCC9 cells were treated with 0.01, 0.1, 1, and 10 ng/ml YM155 for 24 h. (A) MTT assay; (B) Colony formation assay; (C) FACS analysis; (D) TUNEL assay. The survival rate and apoptotic rates are the means ±SD of 3 independent experiments vs. 0 ng/ml, * p<0.05, ** p<0.01

Figure 2

Effect of YM155 on survivin, p53, PUMA expression, and caspase-3 activity. SCC9 cells were treated with 0.01, 0.1, 1, and 10 ng/ml YM155 for 6, 12, and 24 h. (A) Survivin, p53, and PUMA protein expression was detected by Western blot assay; (B) Caspase-3 activity was detected by cleavage of the caspase-3 substrate vs. 0 ng/ml, * p<0.05, ** p<0.01 and *** p<0.001.

Figure 3

Effect of PUMA/caspase-3 on YM155-induced apoptosis and growth inhibition of SCC9 cells. SCC9 cells were transfected with PUMA siRNA or caspase-3 siRNA for 16 h, then we treated the transfected cells with YM155 (1 and 10 ng/ml) for 24 h. (A) PUMA protein expression was detected by Western blot assay; (B) Caspase-3 activity was detected by cleavage of the caspase-3 substrate. (C) Cell survival rate was detected by MTT assay; (D) Colony formation assay; (E) Apoptosis was determined by FACS analysis; (F) Apoptosis was determined by TUNEL assay vs. control, * p<0.05.

Figure 4

YM155 inhibits xenograft growth of SCC9 cells. (A) Tumor xenografts were established by s.c. injection of SCC9 cells into the flanks of the mice. Animals bearing SCC9 tumors were subcutaneously administered a 3-day per week continuous infusion for 2 weeks. Tumor size was measured every 2 days. The tumor growth curve is shown. * p<0.05 compared with the control group. (B) Apoptotic cells was detected by TUNEL assay, p<0.01. (C) Survivin was detected by immunohistochemistry staining assay. (D) PUMA was detected by immunohistochemistry staining assay.