Histone deacetylase inhibitor enhances sensitivity of non-small-cell lung cancer cells to 5-FU/S-1 via down-regulation of thymidylate synthase expression and up-regulation of p21(waf1/cip1) expression

Abstract

It is desirable to find more appropriate therapeutic opportunities in non-small-cell lung cancer (NSCLC) due to the current poor prognosis of affected patients. Recently, several histone deacetylase (HDAC) inhibitors, including suberoylanilide hydroxamic acid (SAHA), have been reported to exhibit antitumor activities against NSCLC. S-1, a novel oral fluorouracil anticancer drug, has been developed for clinical use in the treatment of NSCLC in Japan. Using an MTT assay, we analyzed the growth-inhibitory effect of 5-fluorouracil (5-FU), S-1, and SAHA against three NSCLC cell lines, as well as the breast cancer cell line MCF7 which is known to be highly sensitive to 5-FU. Combined treatment with low-dose SAHA enhanced 5-FU- and S-1-mediated cytotoxicity and resulted in synergistic effects, especially in 5-FU-resistant cells. Both the mRNA and protein expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT), which are associated with 5-FU sensitivity/response, were analyzed in the cells undergoing treatment. 5-Fluorouracil-resistant lung cancer cells displayed high expression of TS mRNA and protein. Suberoylanilide hydroxamic acid down-regulated TS mRNA and protein expression, as well as repressed the rapid induction of this factor during 5-FU treatment, in all examined cell types. We also examined the status of the Rb-E2F1 pathway, with SAHA up-regulating p21(waf1/cip1) expression via promoter histone acetylation; this, in turn, blocked the Rb-E2F1 pathway. We conclude that combination therapy with SAHA and S-1 in lung cancer may be promising due to its potential to overcome S-1 resistance via modulation of 5-FU/S-1 sensitivity-associated biomarker (TS) by HDAC inhibitor.

Figure 1

Effect of 5‐fluorouracil (5‐FU) and suberoylanilide hydroxamic acid (SAHA) on cell growth in vitro. An MTT assay was used to investigate effects on cell viability mediated by 5‐FU and SAHA. Cells were seeded into 96‐well plates and treated with various doses of 5‐FU and SAHA for 72 h, and then incubated with MTT reagent for 4 h. Cell viabilities were determined by measuring absorbance at 560 nm. The IC₅₀ value was defined as the concentration of drug(s) needed for a 50% reduction in absorbance (560 nm) based on cell growth curves. Points, mean of at least three independent experiments; bars, SE. Note: The PC9/f14 cell line is a highly metastatic derivative of PC9 cells established at Nippon Medical School using an artificial metastasis method^{( 26 )}.

Figure 2

Effect of combination therapy with 5‐fluorouracil (5‐FU)/S‐1 and suberoylanilide hydroxamic acid (SAHA) on cell growth in vitro. (a) Indicated non‐small‐cell lung cancer (NSCLC) cells were treated with 5‐FU or S‐1, a compound composed of 5‐FU and 2‐chloro‐2,4‐dihydro‐xypyridine (CDHP) (1 μM), in combination with SAHA (1 μM). MTT assay was performed 72 h after addition of indicated drugs. IC₅₀ was defined as the drug concentration required to inhibit cell proliferation by 50% compared with that of untreated control cells. Data represent the mean value of IC₅₀ ± SE of three independent experiments in triplicate. *P < 0.05 compared to 5‐FU alone. **P < 0.05 compared to S‐1 alone. (b) 5‐Fluorouracil therapy for 72 h after low‐dose SAHA treatment (1 μM) for 24 h, and concurrent therapy for 72 h, was examined in PC9/f14 cells. Each result is expressed as cell viability in treated samples compared with the untreated sample (100%) for 5‐FU alone or compared with the 1 μM SAHA‐treated sample (92.4% and 87.0% of the untreated sample; Fig. S1) for 5‐FU therapy after 1 μM SAHA treatment, and concurrent therapy with 1 μM SAHA treatment. Data represent the mean value of cell viability ± SE of three independent experiments in triplicate; *P < 0.05 compared to 5‐FU mono‐therapy.

Figure 3

Correlation of 5‐fluorouracil (5‐FU) sensitivity with expression of factors related to 5‐FU metabolism in cancer cell lines. The expression of thymidylate synthase (TS), orotate phospho‐ribosyltransferase (OPRT), and dihydropyrimidine dehydrogenase (DPD) mRNA and protein was determined by real‐time RT‐PCR and western blot analysis, respectively, in lung cancer cell lines, as well as MCF7 cells. (a) The level of gene expression was expressed as ratio of the relevant mRNA in a particular sample to the level of GAPDH mRNA in that sample. The ratio was compared to the corresponding expression level observed in MCF7 cells. Points, mean of at least three independent experiments; bars, SE. (b) Expression of TS, OPRT, and DPD proteins were determined by western blot analyses. Quantitative analyses of protein expression ([TS, OPRT, and DPD]/β‐actin) was examined using NIH image software.

Figure 4

Effect of suberoylanilide hydroxamic acid (SAHA) treatment on expression of the factors related to 5‐fluorouracil (5‐FU) metabolism and the expression of thymidylate synthase (TS) in cancer cell lines after 5‐FU monotherapy and SAHA combination therapy. (a) The effect of SAHA treatment for 24 h on TS, orotate phosphoribosyltransferase (OPRT), and dihydropyrimidine dehydrogenase (DPD) mRNA and protein expression in PC9/f14 cells was examined by real‐time RT‐PCR and western blot analyses, respectively. (1) For the different doses of SAHA used, the level of gene expression was expressed as a ratio of the relevant mRNA to the level of GAPDH mRNA. Points, mean of at least three independent experiments; bars, SE. (2) Expression of TS, OPRT, and DPD proteins, following exposure to different doses of SAHA, were determined by western blot analyses. (b) PC9/f14 and MCF7 cells were incubated with various doses of 5‐FU and SAHA (1 μM) for 24 h. Thymidylate synthase expression was determined by western blot analysis. A rapid increase in TS protein expression was observed after the exposure to 5‐FU. Suberoylanilide hydroxamic acid suppressed 5‐FU‐mediated induction of TS protein.

Figure 5

Mechanistic studies relating to down‐regulation of thymidylate synthase (TS) transcription by suberoylanilide hydroxamic acid (SAHA) treatment. (a) PC9/f14 cells were incubated with various doses of SAHA for 24 h. Protein expression levels were determined by western blot analyses. (b) Quantitative analyses of protein expression (E2F1, p53, pRb, p16, p21^waf1/cip1, p27, and cleaved poly‐(ADP‐ribose)polymerase/β‐actin) were examined using NIH image software.

Figure 6

Summary of the proposed mechanism underlying down‐regulation of thymidylate synthase (TS) transcription by suberoylanilide hydroxamic acid (SAHA) treatment. (a) Acetylation of the p21^waf1/cip1 promoter was determined by a ChIP assay. Soluble chromatin was immunoprecipitated with anti‐H3 and H4 antibodies from untreated cells and 1 μM SAHA. Polymerase chain reaction primers for two regions of the p21^waf1/cip1 gene were used to amplify the DNA isolated from immunoprecipitated chromatin. (b) The mechanism underlying the down‐regulation of TS transcription by SAHA treatment is summarized.