Rhein induces apoptosis of human gastric cancer SGC-7901 cells via an intrinsic mitochondrial pathway

Abstract

Rhein is a primary anthraquinone found in the roots of a traditional Chinese herb, rhubarb, and has been shown to have some anticancer effects. The aim of the present study was to investigate the effect of rhein on the apoptosis of the human gastric cancer line SGC-7901 and to identify the mechanism involved. SGC-7901 cells were cultured and treated with rhein (0, 50, 100, 150, and 200 µM) for 24, 48, or 72 h. Relative cell viability assessed by the MTT assay after treatment was 100, 99, 85, 79, 63% for 24 h; 100, 98, 80, 51, 37% for 48 h, and 100, 97, 60, 36, 15% for 72 h, respectively. Cell apoptosis was detected with TUNEL staining and quantified with flow cytometry using annexin FITC-PI staining at 48 h after 100, 200 and 300 µm rhein. The percentage of apoptotic cells was 7.3, 21.9, 43.5%, respectively. We also measured the mRNA levels of caspase-3 and -9 using real-time PCR. Treatment with 100 µM rhein for 48 h significantly increased mRNA expression of caspase-3 and -9. The levels of apoptosis-related proteins including Bcl-2, Bax, Bcl-xL, and pro-caspase-3 were evaluated in rhein-treated cells. Rhein increased the Bax:Bcl-2 ratio but decreased the protein levels of Bcl-xL and pro-caspase-3. Moreover, rhein significantly increased the expression of cytochrome c and apoptotic protease activating factor 1, two critical components involved in mitochondrial pathway-mediated apoptosis. We conclude that rhein inhibits SGC-7901 proliferation by inducing apoptosis and this antitumor effect of rhein is mediated in part by an intrinsic mitochondrial pathway.

Figure 1.. Rhein-induced anti-proliferation of SGC-7901 cells. SGC-7901 cells were treated with rhein at doses of 0, 50, 100, 150, or 200 µM for 24, 48, and 72 h. Cell viability was evaluated with the MTT assay and results are reported as relative cell viability (%). All data were normalized to the control group, which was considered to be 100%. The results showed that rhein inhibited proliferation of SGC-7901 cells in a dose- and time-dependent manner. ^*P < 0.05 versus control group (0 µM) (two-way ANOVA followed by the Tukey post hoc test).

Figure 2.. Cell apoptosis observed using TUNEL staining. SGC-7901 cells were treated with rhein (0, 100, 200, or 300 µM) for 48 h. Apoptotic cells exhibited morphological changes in the nuclei typical of apoptosis. Photographs were taken under an inverted microscope (scale bar, 50 µm). Arrows indicate apoptotic cells.

Figure 3.. Rhein-induced apoptosis in SGC-7901 cells was determined by flow cytometry using the annexin FITC-PI staining method. The cells were treated with rhein (0, 100, 200, or 300 µM) for 48 h. The lower right quadrant indicates the percentage of early apoptotic cells (FITC-stained cells) and the upper right quadrant indicates the percentage of late apoptotic cells (FITC-PI-stained cells) (A). The experiment was repeated three times and the percentage of apoptotic cells (means ± SEM) for each treatment group is shown in B. ^*P < 0.05 versus control group (0 µM) (one-way ANOVA followed by the Tukey post hoc test).

Figure 4.. Rhein increased gene expression of caspase-9 and -3 in SGC-7901 cells in a dose-dependent manner. SGC-7901 cells were treated with rhein (0, 100, 200, or 300 µM) for 48 h. The expression of mRNAs was analyzed by real-time quantitative PCR and normalized by GAPDH expression. ^*P < 0.05 versus control group (0 µM) (two-way ANOVA followed by the Tukey post hoc test).

Figure 5.. Rhein decreased the expression of Bcl-2, Bcl-xL and pro-caspase-3 but increased the expression of the pro-apoptotic protein Bax in SGC-7901 cells. SGC-7901 cells were treated with rhein (0, 100, 200, or 300 µM) for 48 h and the expression of proteins in treated cells was determined by Western blot analysis. A, Rhein-induced changes in Bcl-2 and Bax expression and Bax:Bcl-2 ratio. B, Rhein decreased Bcl-xL. C, Rhein decreased pro-caspase-3. Data are reported as the means ± SEM of at least three experiments. ^*P < 0.05 versus control group (0 µM) (two-way ANOVA followed by the Tukey post hoc test).

Figure 6.. Treatment with rhein increased the expression of cytochrome c and Apaf-1 in SGC-7901 cells. SGC-7901 cells were treated with rhein (0, 100, 200, or 300 µM) for 48 h and protein levels of cytochrome c (A) and Apaf-1 (B) were determined by Western blot. Data are reported as the means ± SEM of at least three experiments. ^*P < 0.05 versus control group (0 µM) (one-way ANOVA followed by the Tukey post hoc test).