CRC cell lines HCT116 (ATCC^® CCL-247™, KRAS^mut and TP53^wt) and SW620 (ATCC^® CCL-227TM, KRAS^mut and TP53^mut R273H) were obtained from American Type Culture Collection and tested for mycoplasma contamination and STRs were confirmed. The characteristics of SW620 cells have been previously defined in relevant studies (19,20) and on the ATCC website (https://www.atcc.org/products/all/CCL-227.aspx#characteristics). Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin at 37˚C under 5% CO₂.

All chemotherapeutic agents were purchased from Selleck Chemicals LLC and the following stock solutions were prepared in dimethyl sulfoxide (DMSO) or PBS: 1 M nicotinamide (NAM), 50 mM EX527, 10 mM AGK2, 2 mg/ml cisplatin, 25 mg/ml 5-fluorouracil (5-FU), 10 mM irinotecan, 10 mg/ml oxaliplatin, 10 mg/l paclitaxel, 10 µM gefitinib, 5 mg/ml LY294002, 2 M dichloroacetate (DCA) and 1.5 M metformin. All drugs were freshly added to the medium for the experiments.

HCT116 and SW620 cells were seeded at a density of 1x10⁴ cells/well in 96-well plates and allowed to adhere for 24 h. The cells were then treated with drugs in triplicate at the indicated concentrations for 72 h. After the medium was discarded, fresh medium containing 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc.) was added to each well. The absorbance values at 450 nm were measured and cell viability was calculated as the ratio of the absorbance values between the drug-treated and equal dose vehicle (up to 0.5% DMSO in PBS)-treated cells. IC₅₀ values of the different drugs were determined using inhibition dose-response curves with variable slopes, as previously described (21).

The synergistic or antagonistic effects of various drugs were analyzed according to the Chou-Talalay method (22) using the CompuSyn software program (Version 1.0.1; ComboSyn, Inc.). The combination index (CI) was calculated as D₁/D_x1 + D₂/D_x2, wherein D₁ or D₂ are the inhibitory concentrations of the individual drugs and D_x1 or D_x2 the inhibitory concentration of the drugs when used in combination. A CI<1 and >1 indicate synergistic and antagonistic effects, respectively (22). The -log₁₀ of the CI value was used to define chemo-sensitization (positive value) or antagonism (negative value). At least three independent experiments were performed.

CRC cell lines were treated with vehicle (0.1% DMSO in PBS), cisplatin (2 and 0.2 µg/ml), NAM (3 and 5 mM) or a combination of these drugs for 72 h at 37˚C. The treated cells were fixed in 70% ethanol for at least 12 h at -20˚C, followed by incubation with 500 µl propidium iodide/RNase Staining Buffer Solution (BD Pharmingen; BD Biosciences) for 15 min at room temperature. The stained cells were assessed by flow cytometry using a FACSMelody Flow Cytometer (BD Biosciences) and the proportion of cells in the different cell cycle stages were analyzed using the Modfit LT software (version 3.1; Verity Software House).

HCT116 and SW620 cells were treated with 5 mM NAM or vehicle (PBS) and lysed on ice with RIPA buffer (Nanjing KeyGen Biotech Co., Ltd.) containing protease inhibitors. The lysates were centrifuged at 15,000 x g, 4˚C for 20 min to remove the cell debris and the concentration of total protein was determined using the BCA Protein Quantification kit [Yeasen Biotechnology (Shanghai) Co., Ltd.]. 20 µg (5-20 µl volume) protein in each lane were separated by SDS-PAGE (7.5% separating gel) and transferred to PVDF membranes (GE Healthcare). The membranes were sequentially incubated with the primary antibodies overnight at 4˚C and secondary antibodies for 1 h at room temperature and the bands were detected using ECL HRP substrate (Sigma-Aldrich; Merck KGaA) in the bioanalytical imaging system c300 (Azure Biosystems, Inc.). The following primary antibodies were used: anti-p53 (cat. no. 10442-1-AP; ProteinTech Group, Inc.), anti-histone H3K9 acetylation (cat. no. A7255; ABclonal, Inc.), anti-histone H3 (cat. no. 17168-1-AP; ProteinTech Group, Inc.), anti-phospho-p53 (cat. no. 9284; Cell Signaling Technology, Inc.), anti-p21 (cat. no. 10355-1-AP; ProteinTech Group, Inc.), anti-SIRT1 (cat. no. 60303-1-Ig; ProteinTech Group, Inc.) and anti-GAPDH (cat. no. G9545; Sigma-Aldrich; Merck KGaA). The anti-GAPDH antibody was used as the reference antibody. HRP-labeled goat anti-rabbit (cat. no. KGAA35; Nanjing KeyGen Biotech Co., Ltd.) and goat anti-mouse (cat. no. KGAA37; Nanjing KeyGen Biotech Co., Ltd.) secondary antibodies were used.

Gene expression profiles of TP53^wt and TP53^mut CRC and other tumor cell lines (GSE41258, GSE57343) were downloaded from the Gene Expression Omnibus (GEO) database (accessed on April 22nd, 2019) (23,24). The GSE41258 dataset included the expression data of the TP53^wt lines HTB39 (GSM1012660), LNCaP (prostate cancer cell line, GSM1012661) and LOVO (GSM1012662). The datasets also contained the TP53^mut lines DLD1 (S241F, GSM1012656), HCT15 (S241F P153A, GSM1012657), HT29 (R273H, GSM1012659), SW1116 (A159D, GSM1012665), SW620 (R273H, GSM1012666) and WiDr (R273H, GSM1012667). The GSE57343 dataset included SW620 (GSM1380254-GSM1380259) and HCT116 (GSM1380296-GSM1380301) cell lines. GSEA (version 4.0.3; Broad Institute, Inc.) was performed using the above datasets to explore potential Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and protein acetylation or deacetylation-related Gene Ontology (GO) gene sets using the Molecular Signatures Database (MsigDB, version 7.0; Broad Institute, Inc.). Heat maps of differentially expressed genes (DEGs) were drawn using GraphPad Prism 7 (GraphPad Software, Inc.), as demonstrated in a previous study (25).

GraphPad Prism 7 was used for statistical analysis of cell cycle data. One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used to analyze the differences between each two groups in the cell cycle assays. Data are expressed as the mean ± SD of three independent experiments and P<0.05 was considered statistically significant.

The HCT116 (KRAS^mut and TP53^wt) and SW620 (KRAS^mut and TP53^mut) cells were treated with NAM (a broad spectrum SIRT inhibitor), EX527 (a SIRT1 inhibitor) or AGK2 (a SIRT2 inhibitor) and the respective IC₅₀ values were calculated (Fig. 1). Similarly, the IC₅₀ values of cisplatin, 5-FU, irinotecan, oxaliplatin, paclitaxel, EGFR inhibitor gefitinib, PI3K inhibitor LY294002, pyruvate dehydrogenase kinase inhibitor DCA and the gluconeogenesis inhibitor metformin were determined (Fig. 2). These findings suggested that, SIRT inhibitors and chemotherapeutic agents had tumor inhibitory effects on CRCs.

Inhibition curves were used to predict the 30% inhibitory concentration of each agent in both the HCT116 and SW620 cells (Table I), followed by the calculation of the CI and the -log₁₀ CI. In the TP53^wt HCT116 cells, cisplatin, 5-FU, oxaliplatin, gefitinib, LY294002 and metformin were antagonistic to the SIRT inhibitors, whereas irinotecan and paclitaxel acted synergistically (Fig. 3A-C). In the TP53^mut SW620 cells, the majority of the chemotherapeutic agents showed a weak synergism with the SIRT inhibitors. Cell cycle analysis also confirmed that when used in combination, cisplatin and NAM were not more effective for preventing the G1-S transition of HCT116 cells compared with cisplatin alone (Fig. 3D). However, the G1-S transition was significantly reduced with a combination of cisplatin and NAM when compared to the use of each drug individually in the SW620 cells (Fig. 3E). These results suggested that SIRT inhibitors may antagonize chemotherapeutic drugs in TP53^wt CRC cells, while inhibition of SIRTs may make TP53^mut cells more sensitive to other chemotherapeutic agents in this assay.

TP53 is frequently mutated into a proto-oncogene in tumor cells (26), which encodes a highly acetylated protein that is unstable and degrades easily (27). Therefore, the present study aimed to determine whether similar p53 protein levels and acetylation status existed in HCT116 and SW620 cells. In the present study the level of the deacetylase SIRT1 was significantly higher in HCT116 cells when compared to SW620 cells, with high levels of wt p53. In SW620 cells, the level of the deacetylase SIRT1 was lower than that in HCT116 cells, which may lead to increased levels of acylated and easily-degraded p53 mut (Fig. 3F). Treatment with NAM reduced the levels of the wt p53 protein, activated p-p53Ser15 and its downstream target p21 in HCT116 cells. This effect was not observed in the SW620 cells with a mutated p53 protein (Fig. 3G).

The transcriptome data of TP53^wt and TP53^mut cancer cell lines was extracted from the RNA-Seq GSE41258 dataset and submitted to GSEA for enrichment analysis. KEGG pathway analysis did not reveal any significant differences in the enrichment of genes related to the term ‘p53 signaling pathway’ between the cell lines (Fig. 4A). However, TP53^mut cells were enriched in genes related to the GO-term ‘protein acetylation’ (Fig. 4B), which were also differentially expressed compared to that in the TP53^wt cells (Fig. 4C). Consistent with this, genes associated with the GO term ‘protein deacetylation’ were enriched in the TP53^wt cells (Fig. 4D and E). GSEA of the HCT116 and SW620 transcriptomes (from the GSE7343 dataset) similarly showed a downregulation of genes associated with the KEGG term ‘p53 signaling pathway’ and the GO-term ‘protein deacetylation’ in SW620 cells (Fig. 4F-H). Taken together, these results suggested that the protein deacetylation machinery may be more activated in the TP53^wt compared to TP53^mut CRC cells. With the blockage of the SIRTs inhibitors, the stable wt p53 was significantly reduced, which may antagonize the action of SIRT inhibitors in combination with a chemotherapeutic drug.