Sodium butyrate enhances the cytotoxic effect of cisplatin by abrogating the cisplatin imposed cell cycle arrest

Abstract

Background: Histone deacetylase inhibitors have been proposed as potential enhancers of the cytotoxic effect of cisplatin and other anticancer drugs. Their application would permit the use of lower therapeutic doses and reduction of the adverse side effects of the drugs. However, the molecular mechanisms by which they sensitize the cells towards anticancer drugs are not known in details, which is an obstacle in developing effective therapeutic protocols.

Results: In the present work, we studied the molecular mechanisms by which sodium butyrate sensitizes cancer cells towards cisplatin. HeLa cells were treated with 5 mM butyrate, with 8 microM cis-diaminedichloroplatinum II (cisplatin), or with both. Cells treated with both agents showed approximately two-fold increase of the mortality rate in comparison with cells treated with cisplatin only. Accordingly, the life span of albino mice transfected with Ehrlich ascites tumor was prolonged almost two-fold by treatment with cisplatin and butyrate in comparison with cisplatin alone. This showed that the observed synergism of cisplatin and butyrate was not limited to specific cell lines or in vitro protocols, but was also expressed in vivo during the process of tumor development. DNA labeling and fluorescence activated cell sorting experiments showed that cisplatin treatment inhibited DNA synthesis and arrested HeLa cells at the G1/S transition and early S phase of the cell cycle. Western blotting and chromatin immunoprecipitation revealed that this effect was accompanied with a decrease of histone H4 acetylation levels. Butyrate treatment initially reversed the effect of cisplatin by increasing the levels of histone H4 acetylation in euchromatin regions responsible for the G1/S phase transition and initiation of DNA synthesis. This abrogated the cisplatin imposed cell cycle arrest and the cells traversed S phase with damaged DNA. However, this effect was transient and continued only a few hours. The long-term effect of butyrate was a massive histone acetylation in both eu- and heterochromatin, inhibition of DNA replication and apoptosis.

Conclusion: The study presents evidence that cell sensitization towards cisplatin by sodium butyrate is due to hyperacetylation of histone H4 in specific chromatin regions, which temporarily abrogates the cisplatin imposed cell cycle arrest.

Figure 1

HeLa cells mortality rates . Exponentially growing HeLa cells were treated with 8 μM cisplatin, with 5 mM butyrate, or with both. At days 1, 2 and 3 after the treatment, cells were collected, stained with trypan blue and counted. The numbers of viable cells were expressed as percentage of the number of control cells at day 1. The results are means of three independent experiments and standard deviations are shown with error bars.

Figure 2

FACS analysis of HeLa cells . HeLa cells were treated with 8 μM cisplatin, with 5 mM butyrate, or with both. Twenty four hours after the treatment cells were fixed, stained with propidium iodide and 2 × 10⁴ cells were subjected to FACS analysis.

Figure 3

DNA synthesis in HeLa cells . HeLa cells were treated with 8 μM cisplatin, with 5 mM butyrate, or with both. At the specified times aliquots were withdrawn, 1 μCi/mL of ³H-thymidine was added and 30 min later the cells were collected, washed and counted. ³H incorporation was expressed as percentage of the control. The results are means of three independent experiments and standard deviations are shown with error bars.

Figure 4

Western blotting of acetylated histone H4 . HeLa cells were treated with 8 μM cisplatin, with 5 mM butyrate, or with both. Four and 24 hours after the treatment aliquots were withdrawn, total histone fraction was isolated and equal amount of histones were subjected to SDS-polyacrylamide gel electrophoresis. (A) The proteins were transferred onto nitrocellulose membranes and stained with anti-acetylated histone H4 antibody. Ponceau S staining of H4 is shown as loading control. (B) The acetylated histone H4 was visualized and quantified by the Odyssey scanning system. The acetylation levels are expressed as percentage of the untreated control. The results are means of five independent experiments and standard deviations are shown by error bars.

Figure 5

Acetylation status of chromatin at four DNA sequences: c-myc ori, c-myc gene, β-globin ori and G-globin gene, upon treatment with cisplatin and butyrate . (A) Map of the human c-myc and β-globin loci. The positions of the origins of replication zones (replicators) are shown by solid lines and the genes are shown by arrows. The four sequence-tagged sites (STSs) amplified by PCR are designated as c-myc ori and c-myc gene and β-globin ori and G-globin gene and shown with vertical lines. (B) HeLa cells were treated with 8 μM cisplatin, with 5 mM butyrate, or with both and 4 h or 24 h after the treatment, chromatin was immunoprecipitated with anti-acetylated histone H4 antibody. Aliquots from the immunoprecipitated (ChIP) and input (Input) DNA were subjected to PCR using primers for the respective DNA sequences. The PCR products were separated on 2.5% agarose gels and stained with ethidium bromide. (C) The DNA fractions were scanned and quantified and the ratios ChIP/Input for the four sequences after the different treatments were shown. Results are means of three independent experiments and the standard deviations are shown with error bars.