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. 2015 Apr 16:8:845-54.
doi: 10.2147/OTT.S81041. eCollection 2015.

Artesunate inhibits the growth and induces apoptosis of human gastric cancer cells by downregulating COX-2

Ping Zhang  1 , He-Sheng Luo  1 , Ming Li  1 , Shi-Yun Tan  1
Affiliations

Artesunate inhibits the growth and induces apoptosis of human gastric cancer cells by downregulating COX-2

Ping Zhang et al. Onco Targets Ther. .

Abstract

Artesunate, a derivative of artemisinin isolated from Artemisia annua L., has been traditionally used to treat malaria, and artesunate has demonstrated cytotoxic effects against a variety of cancer cells. However, there is little available information about the antitumor effects of artesunate on human gastric cancer cells. In the present study, we investigated the antitumor effect of artesunate on human gastric cancer cells and whether its antitumor effect is associated with reduction in COX-2 expression. The effects of artesunate on the growth and apoptosis of gastric cancer cells were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis of annexin V-fluorescein isothiocyanate/propidium iodide staining, rhodamine 123 staining, and Western blot analysis. Results indicate that artesunate exhibits antiproliferative effects and apoptosis-inducing activities. Artesunate markedly inhibited gastric cancer cell proliferation in a time- and dose-dependent manner and induced apoptosis in gastric cancer cells a dose-dependent manner, which was associated with a reduction in COX-2 expression. Treatment with the selective COX-2 inhibitor celecoxib, or transient transfection of gastric cancer cells with COX-2 siRNA, also inhibited cell proliferation and induced apoptosis. Furthermore, the treatment with artesunate promoted the expression of proapoptotic factor Bax and suppressed the expression of antiapoptotic factor Bcl-2. In addition, caspase-3 and caspase-9 were activated, and artesunate induced loss of mitochondrial membrane potential, suggesting that the apoptosis is mediated by mitochondrial pathways. These results demonstrate that artesunate has an effect on anti-gastric cancer cells. One of the antitumor mechanisms of artesunate may be that its inhibition of COX-2 led to reduced proliferation and induction of apoptosis, connected with mitochondrial dysfunction. Artesunate might be a potential therapeutic agent for gastric cancer.

Keywords: COX-2; apoptosis; artesunate; gastric cancer cells.

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Figures

Figure 1
Figure 1
Artesunate inhibits the proliferation of gastric cancer cells in vitro. Notes: A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the proliferation of gastric cancer cells. (A) Cell viability of three gastric cancer cell lines, BGC-823, HGC-27, and MGC-803, treated with 40 mg/L artesunate for 0, 12, 24, 36, 48, 60, and 72 hours. (B) Cell viability of gastric cancer cell lines treated with various concentrations of artesunate for 48 hours.
Figure 2
Figure 2
Artesunate-induced apoptosis in HGC-27 cells. Notes: HGC-27 cells were treated with the indicated concentrations of artesunate for 48 hours, and the effect of artesunate on cell apoptosis was analyzed by flow cytometry. (A) 0 mg/L. (B) 20 mg/L. (C) 40 mg/L. (D) 80 mg/L. The cells in the D4 quadrant of the histogram represent the number of early apoptotic cells, while those in the D2 quadrant of the histogram represent the cells in late apoptosis. Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide.
Figure 3
Figure 3
Artesunate suppressed the expression of COX-2. Notes: The levels of COX-2 were determined in cell lysates using Western blot analysis. Treatment of HGC-27 cells with artesunate resulted in downregulation of COX-2 expression.
Figure 4
Figure 4
Celecoxib, a selective COX-2 inhibitor, inhibits cell proliferation in HGC-27 cells. Notes: The viability of HGC-27 cells treated with the indicated concentrations of celecoxib for 0, 24, 48, and 72 hours is shown. Treatment with celecoxib caused a dose- and time-dependent inhibition of cell proliferation. *P<0.05 versus 0 μmol/L.
Figure 5
Figure 5
Celecoxib induces apoptosis in HGC-27 cells. Notes: HGC-27 cells were treated with the indicated concentrations of artesunate for 48 hours, and annexin V–FITC/PI staining was performed. (A) 0, (B) 20, (C) 40, and (D) 80 μmol/L celecoxib. The cells in the LR quadrant of each histogram represent the number of early apoptotic cells, while those in the UR quadrant of each histogram represent the cells in late apoptosis. Abbreviations: FITC, fluorescein isothiocyanate; LR, lower right; PI, propidium iodide; UR, upper right.
Figure 6
Figure 6
siRNA knockdown of COX-2 leads to inhibition of cell proliferation and induction of cell apoptosis. Notes: (A) COX-2 expression was analyzed by RT-PCR analysis and Western blotting in HGC-27 cells and COX-2 siRNA-transfected HGC-27 cells. (B) Forty-eight hours after transfection, the proliferation of HGC-27 cells was significantly inhibited. *P<0.05 versus control siRNA-transfected HGC-27 cells. (C) The apoptosis-inducing effect was analyzed by flow cytometry after 48 hours of transfection in control siRNA-transfected cell group (a) and COX-2 siRNA-transfected cell group (b). Abbreviations: FITC, fluorescein isothiocyanate; LR, lower right; PI, propidium iodide; RT-PCR, reverse transcription polymerase chain reaction; UR, upper right.
Figure 7
Figure 7
Effects of artesunate on the expression of proapoptotic and antiapoptotic elements in HGC-27 cells. Notes: HGC-27 cells were treated with 0, 20, 40, and 80 mg/L artesunate for 48 hours. (A) Artesunate promoted the expressions of Bax, but decreased Bcl-2 expression. (B) Analysis of relative caspase-3 and caspase-9 activity in cells. Artesunate increased the activity of caspase-3 and caspase-9. *P<0.05 versus 0 mg/L.
Figure 8
Figure 8
Artesunate reduced the rhodamine 123 fluorescence intensity in HGC-27 cells treated with 0, 20, 40, and 80 mg/L artesunate for 48 hours. Notes: The results suggest artesunate could reduce the mitochondrial membrane potential and artesunate treatment of gastric cancer cells induces apoptosis through the mitochondrial apoptosis pathway. *P<0.05 versus 0 mg/L.
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