Acta Pharmacologica Sinica (2010) 31: 66–72; doi: 10.1038/aps.2009.177; published online 28 December 2009

Li-jing YANG^1,#, Yan CHEN^1,#, Qi MA², Jun FANG¹, Jing HE¹, Yi-quan CHENG¹, Qiu-ling WU^1,*

¹Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; ²Vascular Biology Institute, University of Miami, Miller School of Medicine, Miami, Florida 33136, USA

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

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 IC₅₀ value for 24 h was 18.25 μg/mL (95% confidence interval: 15.16 to 27.31 μg/mL).  Cells treated with BA showed increased cell population in G₂/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, G₂/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.

Keywords: betulinic acid; gastric adenocarcinoma AGS cells; Hiwi; cyclin B1; cell cycle; apoptosis