Wogonoside promotes apoptosis in gastric cancer AGS and SGC-7901 cells through induction of mitochondrial dysfunction and endoplasmic reticulum stress

Abstract

Wogonoside (Wg), a natural flavonoid, has anticancer effects against several human cancers. The purpose of the present study was to investigate the antitumor effects and underlying mechanisms of Wg on gastric cancer (GC) cell lines. We report that Wg treatment inhibited cell viability and induced apoptosis in human GC cell lines AGS and SGC-7901, and also retarded GC tumor growth in xenograft mice in vivo. We also found that the Wg exerted its antitumor effects against GC cells via induction of reaction oxygen species accumulation, mitochondrial dysfunction, and endoplasmic reticulum stress. Furthermore, C/EBP homologous protein knockdown inhibited apoptosis and increased the viability of Wg-treated GC cells. Our findings may facilitate the development of novel therapeutic agents for the treatment of GC.

Keywords: endoplasmic reticulum stress; gastric cancer; mitochondrial dysfunction; wogonoside.

Figure 1

Wg inhibits GC cell viability. (A) Chemical structure of Wg. (B) Impaired viability of Wg‐treated AGS and SGC‐7901 cells, measured by MTT assay. (C) Nearly unchanged viability of Wg‐treated GES‐1 cells, measured by MTT assay. (D) Impaired clonogenic growth abilities of Wg‐treated AGS and SGC‐7901 cells, measured by colony formation assay. Data are expressed as mean ± SD from three independent experiments. Differences between groups were analyzed using ANOVA test followed by Tukey's post hoc test. *P < 0.05 versus DMSO‐treated cells.

Figure 2

Wg inhibits GC tumor growth in vivo. (A) Tumor growth was measured in three groups of mice (n = 5/group) every 3 days, and tumor growth curves were plotted. (B) Tumors were excised, and tumor weight was measured at the end of experiments. Differences between groups were analyzed using ANOVA test followed by Tukey's post hoc test. Data are expressed as mean ± SD from three independent experiments. *P < 0.05 versus control group.

Figure 3

Wg induces GC cell apoptosis. (A) Increased apoptosis of Wg‐treated AGS and SGC‐7901 cells, measured by flow cytometric analysis. (B) Western blot analysis of Bcl‐2 and Bax protein expression levels in Wg‐treated AGS and SGC‐7901 cells. Data are expressed as mean ± SD from three independent experiments. Differences between groups were analyzed using ANOVA test followed by Tukey's post hoc test. *P < 0.05 versus DMSO‐treated cells.

Figure 4

Wg induces ROS accumulation and mitochondrial dysfunction in GC cells. (A) Increased ROS accumulation of Wg‐treated AGS and SGC‐7901 cells, measured by flow cytometric analysis. (B) Impaired MMP of Wg‐treated AGS and SGC‐7901 cells, measured by flow cytometric analysis. (C) Western blot analysis of mitochondrial and cytoplasmic cytochrome c expression levels in Wg‐treated AGS and SGC‐7901 cells. Data are expressed as mean ± SD from three independent experiments. Differences between groups were analyzed using ANOVA test followed by Tukey's post hoc test. *P < 0.05 versus DMSO‐treated cells.

Figure 5

Wg induces ER stress in GC cells. Western blot analysis of ER stress‐associated protein expression levels in Wg‐treated AGS and SGC‐7901 cells. Data are expressed as mean ± SD from three independent experiments. Differences between groups were analyzed using ANOVA test followed by Tukey's post hoc test. *P < 0.05 versus DMSO‐treated cells.

Figure 6

CHOP knockdown inhibits the apoptosis of Wg‐treated GC cells. (A) Western blot analysis of CHOP protein expression levels in Wg‐treated AGS and SGC‐7901 cells after transfection with si‐CHOP. (B) Effects of CHOP knockdown on the apoptosis of Wg‐treated AGS and SGC‐7901 cells, measured by flow cytometric analysis. (C) Effects of CHOP knockdown on the viability of Wg‐treated AGS and SGC‐7901 cells, measured by MTT assay. Data are expressed as mean ± SD from three independent experiments. Differences between groups were analyzed using ANOVA test followed by Tukey's post hoc test. *P < 0.05 versus si‐NC‐transfected cells.