The conformation change of Bcl-2 is involved in arsenic trioxide-induced apoptosis and inhibition of proliferation in SGC7901 human gastric cancer cells

Abstract

Background: Arsenic trioxide has been established as a first-line agent for treating acute promyelocytic leukemia. Experimental data suggest that arsenic trioxide also can have a potential use as chemotherapeutic agent for other malignancies. The precise mechanisms of action of arsenic trioxide have though not been elucidated. As the role of Bcl-2 in arsenic trioxide-mediated cell apoptosis and conformation change of Bcl-2 in response to arsenic trioxide treatment has not been studied. The aim of the present study was to determine whether conformation change of Bcl-2 is involved in the action of arsenic trioxide.

Methods: Human gastric cancer SGC7901 cells were exposed to different concentrations of arsenic trioxide. Proliferation was measured by using the Kit-8 cell counting assay. Analysis of nuclear morphology was observed by DAPI staining. The apoptosis rates of cells treated with arsenic trioxide were analyzed by flow cytometry using Annexin V-FITC staining. The conformation change of Bcl-2 and Bax activation were detected by immunostaining and Western blot analysis. Total expression of Bcl-2 and Bax were examined by Western blot analysis.

Results: Arsenic trioxide inhibited the growth of human gastric cancer SGC7901 cells and induced apoptosis. There were two Bcl-2 phenotypes coexisting in SGC7901 cells and the Bcl-2 cytoprotective phenotype could change into a cytodestructive phenotype following conformational change of Bcl-2, triggered by arsenic trioxide exposure. Bax activation might also be involved in arsenic trioxide-induced Bcl-2 conformational change. Arsenic trioxide did not change levels of total Bcl-2 expression, but up-regulated total Bax expression for the treatment time ranging from 3 to 24 hours.

Conclusion: Arsenic trioxide induces apoptosis through induction of Bcl-2 conformational change, Bax activation and up-regulation of total Bax expression rather than affecting total Bcl-2 expression in human gastric cancer SGC7901 cells. The conformational change of Bcl-2 may be a novel described mechanism of arsenic trioxide-induced apoptosis in cancer cells.

Figure 1

A. Inhibitory effects of arsenic trioxide on SGC7901 cell. B The effects of arsenic trioxide on early and late apoptosis/necrosis of SGC7901 cell. (* represents p < 0.05 compared to the black control group and arsenic trioxide treated groups).

Figure 2

A. Nuclear morphologic changes showing features of apoptosis in SGC7901 cells after treatment with arsenic trioxide for 24 hours. (a) Untreated SGC7901 cells; (b-f) 15 μmol/L arsenic trioxide-treated SGC7901 cells. The cells were stained using DAPI staining. B. Untreated SGC7901 cells stained by anti-Bcl-2 N terminus antibody. C SGC7901 cell stained by anti-Bcl-2 BH3 and anti-Bax(6A7) antibody before and after exposure to 15 μmol/L arsenic trioxide for 12 hours.

Figure 3

A. The expression of BH3 exposed Bcl-2 and activated Bax after exposed to 15 μmol/L arsenic trioxide in SGC7901 cell. B The expression of total Bcl-2 and total Bax after exposure to 15 μmol/L arsenic trioxide in SGC7901 cell. (* represents p < 0.05 between black control group and arsenic trioxide treated groups).