Figure 1.
Dose- and time- dependent effects of ST on the viability of GES-1 cells.
The cell viability was determined by the MTT assay after the cells were exposed to ST concentrations in the range of 0.03 to 48 µM for 24, 48, and 72 h. The data represent the means ± SD of three independent experiments. *P<0.05 compared with the control group.
Figure 2.
ST induces DNA damage in GES-1 cells.
Cells were treated with 0.06% DMSO or different concentrations of ST (0.075, 0.3, 1.5, and 3 µM) in DMSO and then subjected to the comet assay as described in Section 2. (A) Cells containing DNA strand breakage (with long tails) were observed under an inverted fluorescence microscope and quantified. (200× magnification; n = 3). The data shown are representative of at least three separate experiments. (B) The ST-induced DNA damage was characterized by an increase in the percentage of DNA tail, the Ttail length, and the Olive tail moment in GES-1 cells. The following groups were assayed: (a) solvent control, (b) 0.075 µM ST, (c) 0.3 µM ST, (d) 1.5 µM ST, and (e) 3 µM ST. The data represent the means ± SD. Differences were considered statistically significant if *P<0.01 according to the non-parametric Mann-Whitney U test.
Figure 3.
ATM-Chk2 signaling pathway is activated in ST-treated GES-1 cells.
GES-1 cells were treated with different concentrations of ST (0.075, 0.3, 1.5, and 3 µM) or solvent for 48 h. (A) Representative immunoblots show the effect of ST treatment on the phosphorylation of ATM (Ser-1981), Chk2 (Thr-68), and Cdc25C (Ser-216) and the expression of ATM, Chk2, and Cdc25C. β-actin was used as the normalization control. (B) Intensities of the immunoreactive bands were quantified by densitometric scanning and compared with those of the control (considered “1”). The values shown represent the means ± SD. *P<0.05 compared with the solvent-treated control group.
Figure 4.
The p53-p21 pathway is activated in ST-treated GES-1 cells.
GES-1 cells were treated with different concentrations of ST (0.075, 0.3, 1.5, and 3 µM) or solvent for 48 h. (A) Representative immunoblots show the effect of ST treatment on the phosphorylation of p53 (Ser-15) and the expression of p53 and p21. β-actin was used as the normalization control. (B) Intensities of the immunoreactive bands were quantified by densitometric scanning and compared with those of the control (considered “1”). The values shown represent the means ± SD. *P<0.05 compared with the solvent-treated control group.
Figure 5.
ATM inhibitor (caffeine) attenuates ST-induced G2 arrest in GES-1 cells.
Cells were treated with the indicated agent for 48 h (pretreatment with 5mM caffeine for 2 hours followed by ST treatment). (A) Caffeine blocked the phosphorylation of ATM (Ser-1981), Chk2 (Thr-68), and p53 (Ser-15) and downregulated the expression of p21 stimulated by ST exposure. (C) Caffeine affected the G2/M phase regulatory proteins that were altered by ST treatment. β-actin was used as the loading control. (B, D) Intensities of the immunoreactive bands in “A” and “C” were quantified by densitometric scanning and compared to those of the control (considered “1”). (E) Caffeine effectively prevented the G2 arrest induced by ST, as demonstrated by flow cytometric analysis. The data represent the means ± SD of three independent determinations. *P<0.05, compared with the solvent-treated control group. ▴P<0.05 compared with the ST-treated group.
Figure 6.
Silencing of p53 by specific p53 siRNA inhibited ST-induced G2 arrest.
Cells were either not transfected or transfected with 100 nM p53 siRNA and then treated with 3 µM ST for 48 h. (A) Cells were subjected to immunoblot analysis for p-p53 (Ser15), p53, p21, and (C) the regulators related to G2 arrest. NC: cells transfected with the same concentration of negative control siRNA. β-actin was used as the loading control. (B, D) Intensities of the immunoreactive bands in “A” and “C” were quantified by densitometric scanning and compared with those of the control (considered “1”). (E) The cell cycle phases of the cells were analyzed by FCM. The values shown represent the means ± SD, *P<0.05 compared with the solvent-treated control group. ▴P<0.05 compared with the ST-treated groups. #P<0.05 compared with the p53 siRNA-treated groups.
Figure 7.
ST induces apoptosis in GES-1 cells.
GES-1 cells were treated with the indicated agents for 48 h. (A) Flow cytometric analysis of ST-induced apoptosis using Annexin V-FITC/PI. The living, early apoptotic, late apoptotic/necrotic, and damaged cells are present in the lower left, lower right, upper right, and upper left quadrants, respectively. (B) Fluorescent staining of nuclei in ST-treated and untreated cells by Hoechst 33258. Cells were visualized with a fluorescence microscope. The following groups were assayed: (a) solvent control, (b) 1.5 µM ST, and (c) 3 µM ST. Condensed and fragmented nuclei and apoptotic bodies were observed in the ST-treated cells, but not in the solvent-treated control cells. (C) Western blot analysis of the effect of the ST dosage on mitochondria-dependent apoptosis-related proteins. Representative immunoblots show the effect of ST on the expression of Bcl-2 and Bax and the activation of caspase-3. β-actin was used as the normalization control. (D) Intensities of the immunoreactive bands in “C” were quantified by densitometric scanning and compared with those of the control (considered “1”). The values shown represent the means ± SD, *P<0.05 compared with the solvent-treated control group.
Figure 8.
Effect of ST on DNA damage-induced ATM activation and G2 arrest in GES-1 cells.
In response to ST-induced DNA damage, ATM serves as a signal transducer for the activation of its downstream signaling pathway. Activated ATM simultaneously phosphorylates the Thr-68 and Ser-15 residues of Chk2 and p53, respectively. These phosphorylations lead to the activation of their downstream pathway components, which results in the inhibition of the activation of Cdc25 and an increase in the expression of p21waf1. These steps finally result in the inactivation of the Cyclin B1/Cdc2 complex and the induction of G2 arrest.