Tributyrin inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

Abstract

Aim: To evaluate the effects of tributyrin, a pro-drug of natural butyrate and a neutral short-chain fatty acid triglyceride, on the growth inhibition of human gastric cancer SGC-7901 cell.

Methods: Human gastric cancer SGC-7901 cells were exposed to tributyrin at 0.5, 1, 2, 5, 10 and 50 mmol/L(-1) for 24-72 h. MTT assay was applied to detect the cell proliferation. [(3)H]-TdR uptake was measured to determine DNA synthesis. Apoptotic morphology was observed by electron microscopy and Hoechst-33258 staining. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect tributyrin-triggered apoptosis. The expressions of PARP, Bcl-2 and Bax were examined by Western blot assay.

Results: Tributyrin could initiate growth inhibition of SGC-7901 cell in a dose- and time-dependent manner. [(3)H]-TdR uptake by SGC-7901 cells was reduced to 33.6 % after 48 h treatment with 2 mmol/L(-1) tributyrin, compared with the control (P<0.05). Apoptotic morphology was detected by TUNEL assay. Flow cytometry revealed that tributyrin could induce apoptosis of SGC-7901 cells in dose-dependent manner. After 48 hours incubation with tributyrin at 2 mmol/L(-1), the level of Bcl-2 protein was lowered, and the level of Bax protein was increased in SGC-7901, accompanied by PARP cleavage.

Conclusion: Tributyrin could inhibit the growth of gastric cancer cells effectively in vitro by inhibiting DNA synthesis and inducing apoptosis, which was associated with the down-regulated Bcl-2 expression and the up-regulated Bax expression. Therefore, tributyrin might be a promising chemopreventive and chemotherapeutic agent against human gastric carcinogenesis.

Figure 1

Effect of tributyrin on cell growth in SGC-7901 cells. The cells were treated with various concentrations of tribu-tyrin for 24, 48 and 72 h. The antiproliferative effect was mea-sured by MTT assay. Results were expressed as the means ± SD from three independent determinations.

Figure 2

Inhibitory effect of tributyrin on DNA synthesis in-corporated with ³H-TdR. With the concentration of 2 mmol·L^-1, tributyrin inhibited the [³H]-TdR uptake by SGC-7901 cells in a time-dependent manner. Results were expressed as means ± SD from three independent determinations.

Figure 3

Ultrastructural changes of the gastric cancer cells treated with 2 mmol·L^-1 tributyrin for 48 h. (A) The control SGC-7901 cell; (B) the experimental cell showing early changes of apoptosis in which nuclear chromatin condensation and cell shrinkage were observed (B).

Figure 4

Tributyrin-induced apoptosis in SGC-7901 cells stained with Hoechst-33258. A: the control SGC-7901 cells; B: the experimental cells showing nuclear shrinkage or fragmentation.

Figure 5

Tributyrin-induced apoptosis by TUNEL assay. A: the positive control cells; B: the negative control cells; C: the experi-mental cells treated with tributyrin at 2 mmol·L^-1 for 2 d.

Figure 6

Tributyrin-induced apoptosis in SGC-7901 cells by flow cytometry. (A) The control cells, (B)-(D) The experimental cells treated with tributyrin at 0.5, 2 and 10 mmol·L^-1 respectively for 48 h. (E) Apoptosis rates in SGC-7901 cells treated with 0.5, 2 and 10 mmol·L^-1 or without tributyrin for the indicated times.

Figure 7

The expression of Bcl-2, Bax protein and PARP cleav-age in tributyrin-treated SGC-7901 cells. (1) The control cells; (2)-(4) The experimental cells treated with tributyrin for 48 h at 0.5, 2 and 10 mmol.L^-1, respectively.