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Abstract
Purpose
The causes of colon cancer can be divided into genetic and environmental components. A high-fiber diet is known to reduce the risk of colon cancer. Dietary fiber is converted to short chain fatty acid, butyrate, in the colon by bacteria. Butyrate is used as an energy source for the colonic epithelial cells, and is known to induce apoptosis in colon cancer cell lines. Survivin, a recently discovered member of the IAP (inhibitor of apoptosis) family, is known to suppress apoptosis. Not only does it suppress cell apoptosis, but it also has a protective effect from disabling G2/M phase of the cell cycle by attaching to the microtubule of the mitotic spindle. The purpose of this study is to evaluate the effect of butyrate on the expression of survivin, in HCT116 colon cancer cell lines.
Methods
Cytotoxicity of butyrate was measured by MTS method. Cell cycle phase and apoptosis was analyzed by flowcytometry. Protein expression of survivin was evaluated by Western blot analysis, and the mRNA expression by RT-PCR.
Results
Butyrate can induce apoptosis in HCT116 colon cancer cell line at a concentration of 6 mM. Butyrate suppressed the expression of survivin mRNA and also the expression of cytosolic and nuclear survivin. In flowcytometric analysis, the apoptotic portion was increased and the proportions of S and M phase were decreased when cultured with butyrate.
Figures and Tables
Fig. 1
Cytotoxicity to HCT116 colon cancer cell by butyrate. HCT116 cells were incubated with different concentrations of butyrate for 72 hours. Cytotoxicity was measured by MTS method.
Fig. 2
G1/S phase synchronization was induced by serum withdrawal for 3 days. The cell cycle phases were evaluated by flowcytometry. M1 = subG1 phase; M2 = G0/G1 phase; M3 = S phase fraction; M4 = M phase.
Fig. 3
DNA fragmentation by butyrate. The HCT116 cells were treated with or without butyrate. DNA fragmentation was induced only in 6 mM butyrate treated cells, which indicates cellular apoptosis. M = DNA marker; 1 = without butyrate, 0 hr; 2 = without butyrate, 72 hr; 3 = with 6 mM butyrate, 0 hr; 4 = with 6 mM butyrate, 72 hr incubation.
Fig. 4
Expression of survivin, Bcl-2, and Bax proteins. HCT116 cells were incubated with 6 mM butyrate for 72 hr. The expression of survivin, Bcl-2, and Bax protein was evaluated by Western blotting. Immunoblot analysis revealed that survivin completely lost its expression by butyrate, but on the contrary, Bcl-2 expression was increased by butyrate.
Fig. 5
Expression of cytoplasmic and nuclear survivin protein after 72 hour incubation with or without butyrate (6 mM). Butyrate can inhibit both cytoplasmic and nuclear survivin expression.
Fig. 6
Expression of apoptosis-related gene mRNA induced by butyrate. There was no difference in expression of Bcl-2, Bax mRNA transcripts by butyrate. But survivin mRNA transcript was reduced by butyrate, indicating that the absent expression of survivin protein by butyrate is due to suppression of mRNA transcription.
Fig. 7
Cell cycle analysis with flowcytometry. HCT116 cells were incubated for 1, 2, and 3 days with 6 mM butyrate. (A) 0 hr, without butyrate, (B) incubated for 1 day without butyrate, (C) incubated for 1 day with 6 mM of butyrate, (D) incubated for 2 day without butyrate, (E) incubated for 2 day with 6 mM of butyrate, (F) incubated for 3 day without butyrate, (G) incubated for 3 day with 6 mM of butyrate.
Fig. 8
Apoptosis analysis with flowcytometry. HCT116 cells were incubated for 1, 2, and 3 days with 6 mM butyrate. (A) incubated for 1 day without butyrate, (B) incubated for 1 day with 6 mM of butyrate, (C) incubated for 2 days without butyrate, (D) incubated for 2 days with 6 mM of butyrate, (E) incubated for 3 days without butyrate, (F) incubated for 3 days with 6 mM of butyrate. The apoptotic proportion of cell cycle was greatly increased by butyrate (from 3.13% to 47.86% on 2nd day of incubation, from 0.70% to 18.8% on 3rd day of incubation with butyrate).
References
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