Alantolactone induces gastric cancer BGC-823 cell apoptosis by regulating reactive oxygen species generation and the AKT signaling pathway

Abstract

Alantolactone (ALT), a natural sesquiterpene lactone, has been suggested to exert anti-cancer activities in various cancer cell lines. However, the effects and mechanisms of action of ALT in human gastric cancer remains to be elucidated. In the present study, the effects of ALT on BGC-823 cells were examined and the underlying molecular mechanisms associated with these effects were investigated. Cell viability was detected by using an MTT assay. Cell cycle, cell apoptosis and the level of reactive oxygen species (ROS) were assessed by flow cytometry, and the expression levels of proteins of interest were analyzed by western blot assay. The results demonstrated that ALT triggered apoptosis and induced G0/G1 phase arrest in a dose-dependent manner. Furthermore, the expression level of the anti-apoptosis protein Bcl-2 was downregulated, and expression of the pro-apoptosis proteins Bax and cleaved PARP were significantly upregulated. The cell cycle-associated proteins cyclin-dependent kinase inhibitor 1 and cyclin-dependent kinase inhibitor 1B were also increased, while cyclin D1 was deceased. In addition, ALT induced apoptosis via the inhibition of RAC-alpha serine/threonine-protein kinase (AKT) signaling and ROS generation, which was effectively inhibited by the ROS scavenger, N-acetyl cysteine. Therefore, the results from the present study indicated that the ROS-mediated inhibition of the AKT signaling pathway serves an important role in ALT-induced apoptosis in BGC-823 cells. In conclusion, the results demonstrated that ALT exerted significant anti-cancer effects against gastric cancer cells in vitro.

Keywords: BGC-823 cells; alantolactone; apoptosis; protein kinase B serine/threonine kinase signaling pathway; reactive oxygen species.

Figure 1.

Effects of ALT on the viability of BGC-823 cancer cells. BGC-823 cells were incubated with different concentrations of ALT (0–100 µM) for 24 h. All data are expressed as means ± standard deviation from 3 independent experiments. **P<0.01, ***P<0.01 and ****P<0.0001 vs. the control group. ALT, alantolactone; ctrl, control.

Figure 2.

Effects of ALT on cell cycle distribution and cell cycle-associated proteins in BGC-823 cells. (A) BGC-823 cells were incubated with different concentrations of ALT (0–60 µM) for 24 h. The percentages of cells in G0/G1, S, and G2/M phases were determined by flow cytometry. All data are expressed as means ± standard deviation from 3 independent experiments. (B) BGC-823 cells were treated with ALT (40 µM) for 3, 6, 12 and 24 h. Western blot analysis was used to detect the expression levels of cyclin D1, p21 and p27. GAPDH was used as an internal control. ****P<0.0001 vs. the control group. ALT, ALT, alantolactone; ctrl, control; p21, cyclin-dependent protein kinase 1; p27, cyclin-dependent protein kinase 1B.

Figure 3.

Effects of ALT on cell cycle distribution and cell apoptosis-associated proteins in BGC-823 cells. (A) BGC-823 cells were treated with the indicated concentrations of ALT (0–60 µM) for 24 h and cell apoptosis was examined in BGC-823 cells by flow cytometry. (B) BGC-823 cells treated with ALT (40 µM) for 3, 6, 12 and 24 h. Western blot analysis was used to detect the expression levels of Bax, Bcl-2 and cleaved PARP. GAPDH was used as an internal control. ****P<0.0001 vs. the control group. ALT, alantolactone; Bcl-2, B cell lymphoma 2; Bax, Bcl-2-associated protein; PARP, poly (adenosine 5′diphosphate-ribose) polymerase; PI, propidium iodide.

Figure 4.

ROS are involved in ALT-induced apoptosis. (A) BGC-823 cells were pre-treated with NAC (10 mM) for 1 h, and then ALT (40 µM) was added to the cells for 6 h. Flow cytometry was used to detect ROS levels. (B) BGC-823 cells were pre-treated with NAC (10 mM) for 1 h, and then ALT (40 µM) was added to the cells for 24 h. MTT was used to measure cell viability. (C) BGC-823 cells were incubated with ALT and NAC as aforementioned. Flow cytometry was used to detect apoptosis in BGC-823 cells. (D) BGC-823 cells were pre-treated with NAC (10 mM) for 1 h, and then ALT (40 µM) was added to the cells for 12 h. Western blot analysis was used to detect the expression levels of Bax, Bcl-2 and cleaved PARP. *P<0.05 and **P<0.01 vs. the control group. ROS, reactive oxygen species; ALT, alantolactone; NAC, N-acetyl cysteine; Bcl-2, B cell lymphoma 2; Bax, Bcl-2-associated protein; PARP, poly (adenosine 5′diphosphate-ribose) polymerase; PI, propidium iodide; ctrl, control.

Figure 5.

ALT induces BGC-823 cell apoptosis via reactive oxygen species-mediated AKT signaling. (A) BGC-823 cells were treated with ALT (40 µM) for 3, 6, 12 and 24 h. The levels of p-AKT and AKT were analyzed by western blot analysis. (B) BGC-823 cells were pre-incubated with NAC (10 mM) for 1 h, and then ALT (40 µM) was added to the cells for 12 h. The expression levels of p-AKT and AKT were measured by western blot analysis. ALT, alantolactone; NAC, N-acetyl cysteine; AKT, RAC-alpha serine/threonine-protein kinase; p, phosphorylated; ctrl, control.