Colorectal carcinoma cell lines SW480 and HCT116 were obtained from the American Type Culture Collection (LGC Promochem, Wesel, Germany). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) including 10% fetal bovine serum (FBS), sodium pyruvate, L-glutamine and non-essential amino acids (PAN Biotech, Aidenbach, Germany) at 37°C in a 7.5% CO₂ humidified chamber. Cells were treated with 1 μM trichostatin A (TSA; Sigma, Munich, Germany) or 5 μM suberoylanilide hydroxamic acid (SAHA; Cayman Chemicals, Ann Arbor, MI, USA) dissolved in ethanol and 1 mM valproic acid (VPA; Sigma) dissolved in PBS. Vehicle treatment with either ethanol or PBS served as internal control. The proteasome inhibitors MG132 and Epoxomicin (both from Calbiochem, Darmstadt, Germany) were dissolved in DMSO.

Cell growth and viability were evaluated by using bromodeoxyuridine incorporation (BrdU) and methyl thiazol tetrazolium bromide reduction (MTT) assays, respectively. Cells were plated in triplicates in 96-well plates. After treatment with HDIs and control substances for 24, 30 and 48 h or 24 and 48 h after siRNA treatment, MTT or BrdU assay (BrdU cell proliferation ELISA; Roche, Mannheim, Germany) was carried out. The mean absorbance values of HDI-treated cells were normalized to the mean absorbance values of vehicle-treated cells. To detect apoptotic cells, the Apo-One homogeneous Caspase-3/7 assay (Promega, Madison, WI, USA) was carried out according to the manufacturer’s instructions.

For cell cycle analysis, cells were trypsinized after 24 h HDI treatment or 48 h after siRNA transfection. The cell cycle was measured via propidium iodide incorporation on a LSRII flow cytometer and analyzed with FACS DiVa software (Becton Dickinson, Franklin Lakes, NJ, USA). Data evaluation was performed with FlowJo software (TreeStar Inc., Ashland, OR, USA).

HDAC knockdown was performed with siGENOME SMARTpool siRNA from Dharmacon for the genes HDAC6 (M-003499-00), HDAC10 (M-004072-00) and HDAC11 (M-004258-00). Transfection was carried out with DharmaFECT1 (Dharmacon, Lafayette CO, USA) at a ratio of 1:40 in HCT116 and 1:50 in SW480 cells according to the manufacturer’s instructions. Experiments were performed 24 to 72 h after siRNA transfection. Specific target knockdown was measured on mRNA level by quantitative real-time PCR after 24 or 48 h.

RNA isolation was carried out with the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany), cDNA synthesis was performed with RevertAID H Minus First Strand cDNA Synthesis kit (Fermentas, St. Leon-Rot, Germany). The ABsolute QPCR SYBR-Green ROX Mix (Abgene, Epsom, UK) was used for quantitative real-time PCRs on a GeneAmp 5700 Sequence detection system (Applied Biosystems, Foster City, CA, USA). Primer sequences are stated in Table I. Real-time PCR data was analyzed using the ΔΔCt method whereby the relative expression change of a treated sample over a control sample is: fold change = 2^−ΔΔCt.

Total cell lysates were made from HCT116 or SW480 cells after treatment with HDIs for 3, 6 and 16 or 20 h. After SDS gel electrophoresis, western blot analysis was used to determine the relative protein levels of TCF7L2 and β-catenin in total cell lysates. GAPDH was used as an internal control. The antibodies were as follows: TCF7L2 (Cell Signaling Technology, Danvers, MA, USA) 1:2,500 in 5% BSA/TBST; β-catenin (BD Biosciences, Franklin Lakes, NJ, USA) 1:10,000 in 0.5% SlimFast/TBST; GAPDH (Abcam) 1:30,000 in 5% milk/TBST. Band intensities were determined using AIDA software (Raytest, Straubenhardt, Germany). The band intensity (BI) of the analyzed protein was referred to the BI of GAPDH to give the normalized BI. The normalized BI of control treated cells was set to 100%.

For cytosolic and nuclear fractionation of cells, HCT116 cells were grown in 10-cm dishes and HDI treated for 16 h. Cells were washed with ice-cold PBS, scraped into a microcentrifuge tube and centrifuged for 5 min at 1,600 rpm. The cell pellet was resuspended in 100 μl membrane lysis buffer [10 mM HEPES, pH 8.0; 10 mM KCl; 1.5 mM MgCl₂; 1 mM DTT; 1X Complete Protease Inhibitor Cocktail (Roche)] and incubated for 15 min on ice. NP-40 was added to a final concentration of 1%. After vortexing for 10 sec, the sample was centrifuged for 3 min at 13,000 rpm. The supernatant is the cytoplasmic fraction. The pellet containing the nuclei was washed 3 times with 500 μl membrane lysis buffer and centrifuged for 3 min. Then the pellet was resuspended in 100 μl ice cold nuclear envelope lysis buffer [20 mM HEPES, pH 8.0; 1.5 mM MgCl₂; 25% glycerol; 0.42 M NaCl; 0.2 mM EDTA; 1 mM DTT; 1X Complete Protease Inhibitor Cocktail (Roche)] and vortexed for 30 sec. After rotation at 4°C for 30 min, samples were centrifuged for 15 min at 13,000 rpm. The protein concentration of the supernatant was determined by Bradford assay. Concentrations of HDI- and vehicle-treated samples were adjusted to 3 μg/μl. Lysates were used immediately or stored in aliquots at −80°C until use.

For immunoprecipitation, protein G coupled magnetic beads (Dynabeads, Invitrogen) were used according to the manufacturer’s instructions. In brief, 50 μl of beads were loaded with 5 μg of antibody. After washing, the loaded beads were incubated with cellular lysates for 10 to 20 min. After several washing steps, the beads were resuspended in 5X SDS loading buffer and heated to 70°C for 10 min.

To test the acetylation status of β-catenin, immunoprecipitations were carried out with an anti-β-catenin antibody (BD Biosciences) followed by western blot analysis with an anti-β-catenin antibody or an anti-acetylated β-catenin antibody (Cell Signaling Technology).

Statistical calculations were carried out with the GraphPad QuickCalcs online calculator (www.graphpad.com/quickcalcs) and STATISTICA 8 software (StatSoft, Hamburg, Germany) using different t-tests as indicated in the text. A p-value of <0.05 was considered to indicate statistical significance. Significant results are indicated by ^*p<0.05; ^**p<0.01 or ^***p<0.001 in the figures.

We analyzed the effects of the HDIs SAHA, TSA and VPA on the growth of the colorectal carcinoma cell lines HCT116 and SW480. Both cell lines possess an activated Wnt signaling pathway due to mutations in β-catenin or APC, respectively. SAHA and TSA belong to the chemical class of hydroxamic acids; whereas VPA is a short-chain fatty acid. Treatment with hydroxamic acids led to a strong reduction in the number of viable cells, while VPA had no, or only little effect on viability even after 48 h as measured by MTT assay (Fig. 1A). Furthermore, a BrdU assay revealed a pronounced decrease in the cell proliferation rate of SAHA- or TSA-treated cells as opposed to VPA-treated cells (Fig. 1B). To gain further insights into the underlying mechanisms we carried out an apoptosis detection assay. After treatment with hydroxamic acids for 24 and 30 h, the number of apoptotic cells strongly increased (Fig. 1C). After 48 h, the relative apoptotic rate dropped due to the low number of living cells. In contrast, VPA treatment induced no significant changes in the apoptosis rate. These findings were further underlined by flow cytometric cell cycle analysis revealing an increase in the apoptotic cell fraction and an induction of a G2/M-phase cell cycle arrest in response to TSA and SAHA (Fig. 2). In contrast, treatment with VPA led to little, or no changes in the cell cycle.

In order to explore how HDIs influence Wnt regulated genes we measured the expression of endogenous Wnt target genes by quantitative real-time PCR after treatment with HDIs (Fig. 3). Analyzed Wnt target genes included MYC, CCDN1 (encoding cyclin D1), VEGF, MMP7, survivin and the two transcription factors LEF1 and TCF7L2 which are integral parts of the pathway. We observed downregulation of all analyzed genes upon 20 h treatment with hydroxamic acids whereas VPA treatment left the expression of most genes unchanged (Fig. 4). In HCT116 cells, the expression of CCDN1, MYC, VEGF, MMP7, survivin and TCF7L2 was significantly downregulated after 16 to 20 h exposure to SAHA or TSA, but not to VPA. In SW480 cells, the expression levels of CCDN1, MYC, survivin and LEF1 were significantly downregulated by SAHA or TSA, but not by VPA treatment. Only MYC expression, which is known to be regulated by a variety of mechanisms (18), was significantly downregulated by VPA treatment in HCT116.

Based on recent findings that the β-catenin protein can be acetylated, we questioned if a modification of this central player of the Wnt pathway may be involved in the transcriptional downregulation of target genes in response to HDI treatment. Immunoprecipitation experiments with anti-acetylated β-catenin antibody and nuclear lysates from HCT116 cells after 16 h TSA treatment revealed no difference in the amount of precipitated acetylated β-catenin between TSA- and vehicle-treated cells (Fig. 5). Similarly, there was no change in the total amount of β-catenin protein in total cell lysates.

We investigated if TCF7L2 might be a non-histone target of HDACs by analyzing the relative TCF7L2 protein level in total HCT116 cell lysates after HDI treatment. We found a strong depletion of TCF7L2 protein after 20 h SAHA and TSA, but not after VPA treatment (p<0.001) (Fig. 6). This TCF7L2 depletion could not be attributed to transcriptional downregulation alone, as, according to the N-end rule (19), the estimated half-life of the TCF7L2 protein is 30 h. Hence, TCF7L2 depletion might be partly caused by proteasomal degradation. In this case, co-treatment with proteasome inhibitors should attenuate the effect of the HDI treatment. We treated HCT116 cells with TSA for 16 h and added one of the proteasome inhibitors Epoxomicin or MG132 4 or 6 h before the end of the treatment, respectively. As control we treated the cells with the proteasome inhibitors alone. Co-treatment with HDIs and proteasome inhibitors indeed attenuated the downregulation of TCF7L2 protein level (Fig. 6C and D). Treatment with proteasome inhibitors alone did not alter the TCF7L2 level substantially (Fig. 6E), which showed that TCF7L2 was not subjected to constant proteasomal degradation under normal conditions. Thus we demonstrated that HDIs of the hydroxamate class induce a strong proteasome-dependent depletion of TCF7L2.

The previous experiments showed that the hydroxamic acids SAHA and TSA, which inhibit all known HDACs, induced apoptotic cell death of colon carcinoma cells and attenuation of Wnt signaling. In contrast, treatment with VPA, which inhibits only some HDAC classes, led to little, or no change, in the colon carcinoma cell lines. We reasoned that the different effects of the HDIs might be caused by the differences in their HDAC inhibition profiles. Therefore, we carried out siRNA experiments to analyze the effects of the knockdown of HDACs 6, 10 and 11, which are inhibited by hydroxamic acids but not by VPA, on Wnt signaling and colon cancer cell proliferation.

We controlled for a proper gene knockdown by measuring the mRNA expression level of the three HDACs 24 and 48 h after siRNA transfection. All three HDAC siRNAs reduced the respective HDAC expression to at least 40% after 48 h treatment. By using MTT, BrdU, apoptosis assay and flow cytometric analysis, we found that none of the analyzed HDAC siRNAs induced any significant changes in proliferation or apoptosis of HCT116 and SW480 cells. This indicated that the knockdown of a single HDAC is not sufficient to induce major changes in cell proliferation of the analyzed colon carcinoma cell lines.

Next we examined whether siRNA mediated HDAC knockdown had an effect on Wnt target gene expression. Therefore, we performed quantitative real-time PCR of the endogenous Wnt target genes CCDN1, MYC, survivin, LEF1 and TCF7L2 48 h after HDAC siRNA transfection.

In HCT116 cells, HDAC 6 or 10 knockdown reduced the expression of several Wnt target genes, whereas HDAC11 knockdown had an effect on all analyzed genes (Fig. 7). In SW480 cells, the transfection with HDAC10 siRNA led to a significant decrease in all analyzed Wnt target genes, while the siRNAs for HDAC 6 or 11 reduced only the expression of two of the five analyzed genes (MYC and LEF1 or LEF1 and TCF7L2, respectively) (Fig. 7).

Western blot analysis was performed to determine the effects of HDAC knockdown on the TCF7L2 protein level. The knockdown of HDACs 6 or 10 led to a reduction of the TCF7L2 protein level to approximately 50% of the initial protein level after 72 h (p<0.001 and p<0.01) (Fig. 8); while HDAC11 knockdown only slightly decreased the TCF7L2 protein level. Thus, HDACs 6 and 10 may be involved in the SAHA and TSA induced proteasomal degradation of TCF7L2 leading to the reduced expression of Wnt target genes.