Effect of betulinic acid on the regulation of Hiwi and cyclin B1 in human gastric adenocarcinoma AGS cells

Abstract

Aim: To investigate the effect of betulinic acid (BA) on the proliferation, apoptosis and cell cycle of gastric adenocarcinoma cell AGS in vitro and the underlying mechanism.

Methods: The effect of BA on the proliferation of AGS cells was measured by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was analyzed by using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of BA on cell cycle of AGS cells was tested by PI staining. Both FCM and reverse transcription-PCR (RT-PCR) technologies were applied to detect the expression of Hiwi and Cyclin B1.

Results: BA exhibited significant cell proliferation inhibition, as well as its potency of inducing apoptosis in AGS cells in vitro in a time- and dose-dependent manner. The IC(50) value for 24 h was 18.25 microg/mL (95% confidence interval: 15.16 to 27.31 microg/mL). Cells treated with BA showed increased cell population in G(2)/M phase, with decreased S phase population. The expression of Hiwi and Cyclin B1 was down-regulated in BA-treated AGS cells in a dose-dependent manner.

Conclusion: BA exerted potent effect on growth inhibition, G(2)/M cell cycle arrest and induction of apoptosis in AGS cells in vitro, possibly associated with the down-regulation of Hiwi and its downstream target Cyclin B1 expression. The potent antitumor capacity of BA suggested that it could be a promising new experimental anticancer agent in human gastric adenocarcinoma treatment.

Figure 1

Inhibition of proliferation by BA in AGS cells. Cells were treated in vitro with various concentrations of BA (9, 12, 15, 18 and 21 μg/mL) for 24, 36 and 48 h, respectively, while PBMCs treated with BA for 24 h were served as control. Growth inhibition was detected by MTT assay and shown as an inhibition ratio. Data were mean±SD of three independent experiments.

Figure 2

Effect of BA on apoptosis rate of AGS cells. Apoptosis ratio was detected with FCM using Annexin V-FITC/PI double staining. Q2 quadrant represented late apoptosis, while Q4 quadrant represented early apoptosis. (A) 0 μg/mL; (B) 9 μg/mL; (C) 12 μg/mL; (D) 15 μg/mL; (E) 18 μg/mL. The figures were representative of three separate experiments. ^bP<0.05 vs control group.

Figure 3

Effect of BA on apoptosis of AGS cells. Morphological changes of AGS cells treated with 15 μg/mL BA for 24 h which were stained with Hoechst33258 and detected using a fluorescence microscope. Fragmented or condensed nuclei indicative of apoptosis could be observed in the BA-treated groups as the arrows indicated (B), while the untreated cells showed normal cell nuclei morphology (A). White bars: 25 μm (Magnification×400).

Figure 4

Effect of BA on cell cycle of AGS cells. Cells were incubated with various concentrations of BA for 24 h, and the distribution of cell cycle was detected by PI assay. (A) 0 μg/mL (control group); (B) 9 μg/mL; (C) 12 μg/mL; (D) 15 μg/mL; (E) 18 μg/mL. n=3 experiments. Mean±SD. ^bP<0.05 vs control group; ^eP<0.05 vs 9 μg/mL BA group.

Figure 5

The expression of Hiwi and Cyclin B1 protein in the AGS cells treated with various concentrations of BA. Untreated cells served as controls. Mean fluorescence intensity represented the expression levels of Hiwi and Cyclin B1. Panel A: The graphs of flow cytometry of Hiwi protein in AGS cells treated with various concentration of BA. (A1) negative control; (A2) 0 μg/mL (control); (A3) 9 μg/mL; (A4) 12 μg/mL; (A5) 15 μg/mL; (A6) 18 μg/mL. Panel B: Mean fluorescence intensity of Hiwi protein in AGS cells. Panel C: Mean fluorescence intensity of Cyclin B1 protein. n=3 experiments. Mean±SD. ^bP<0.05 vs control group; ^dP>0.05 vs 15 μg/mL BA-treated group.

Figure 6

Effect of BA on mRNA expression of Hiwi (panel A) and Cyclin B1 (panel B) in AGS cells. M: marker, 9: 9 μg/mL, 12: 12 μg/mL, 15: 15 μg/mL, 18: 18 μg/mL, 21: 21 μg/mL, control: 0 μg/mL (control group). n=3 experiments. Mean±SD. ^aP>0.05, ^bP<0.05 vs control group; ^dP>0.05 vs 18 μg/mL BA-treated group.