| AIM:Sodium butyrate has antitumor effects on colon cancer cells such as inhibiting cell growth and promoting differentiation and apoptosis. But the mechanism that sodium butyrate induces apoptosis of colon carcinoma cells is still indefinite. It’s a focus and difficulty in research on revealing the signal transduction pathway and gene regulation of apoptosis. The aim of this study is to investigate whether sodium butyrate induces apoptosis in human colon cancer cell line HT-29 and to examine the intracellular mechanisms involved, especially the role of caspase activation in the process and the modulation of two important regulator genes:Bcl-2 and Bax.METHODS:HT-29 cells were cultured 24-36 h to logarithmic phase before treatment with sodium butyrate of 5.0 mmol/L, van-caspase inhibitor z-VAD-fmk, and caspase-3,-8,-9 inhibitors z-DEVD-fmk, z-IETD-fmk, z-LEHD-fmk at the concentration of 20μmol/L. The latter were added in the medium ahead of sodium butyrate for 1 h. Then, the stain Annexin V-FITC and PI were used to analyze HT-29 apoptosis and the dye JC-1 was applied to detect mitochondrial membrane potential by flow cytometer. Caspase activity within the cells was measured respectively using a specific caspase activity assay kit and a microplate reader. The expression levels of Bcl-2 and Bax proteins in HT-29 under sodium butyrate and/or caspase-9 inhibitor z-LEHD-fmk intervention were detected by Western blot. The expression levels of mRNA of Bcl-2 and Bax genes were assayed by RT-PCR.RESULTS:Preincubation of HT-29 cells with sodium butyrate significantly increased apoptosis ((35.40±0.70)%) and decreased mitochondrial membrane potential (5.53±0.91). This effect could be blocked when pretreatment were enforced with z-VAD-fmk, z-DEVD-fmk and z-LEHD-fmk, P＜0.001. The apoptosis percentages were (1.33±0.59)%, (1.40±0.53)%. (1.27±0.91)%respectively and mitochondrial membrane potential were 9.80±1.15,10.23±0.50,10.33±1.02 respectively. But z-IETD-fmk was not observed reduction the role, which presented the apoptosis percentage of (32.10±2.33)%, P=0.079 and mitochondrial membrane potential of 5.93±1.31, P>0.05. An enhancement of caspase 3 and 9 activity (2-3-fold) but no changes of caspase 8 activity was confirmed, P=0.993. The mRNA of Bcl-2 was suppressed in HT-29 cells treated with Sodium butyrate contrast to normal cells detected by RT-PCR(0.23±0.02 vs 0.52±0.03, P＜0.001), and Bcl-2 protein was down regulated (0.08±0.01 vs 0.17±0.03, P=0.002) measured by Western blot. The mRNA of Bax was raised in HT-29 cells contrast to normal cells detected by RT-PCR(0.72±0.03 vs 0.45±0.02, P＜0.001), and Bax protein was up regulated 0.35±0.03 vs 0.15±0.02, P＜0.001) measured by Western blot. Neither the mRNA nor the protein of Bcl-2 was alterated in HT-29 cells when both sodium butyrate and z-IETD-fmk were added. The RT-PCR data was 0.46±0.03 vs 0.52±0.03, P=0.096 and the Western blot outcome was 0.13±0.01 vs 0.17±0.03, P=0.084. The similar results were observed in experiment of Bax. The mRNA of Bax was 0.39±0.03 vs 0.45±0.02, P=0.072 and the protein of Bax 0.14±0.01 vs 0.15±0.02, P>0.05.CONCLUSIONS:Apoptosis of HT-29 colon carcinoma cells induced by sodium butyrate is tightly linked to caspase-9 activation via mitochondrial/cytochrome c pathway other than tumor necrosis factor-alpha. Sodium butyrate down regulates the expression of Bcl-2 gene and up regulates the expression of Bax gene in HT-29 cells and accordingly modulates the activation of caspase. Caspase-9 inhibitor z-LEHD-fmk feedback regulates Bcl-2 and Bax genes. Sodium butyrate has the potential to promote apoptosis and thereby may contribute to the progression of colon cancer. |
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