Vitamin C in combination with inhibition of mutant IDH1 synergistically activates TET enzymes and epigenetically modulates gene silencing in colon cancer cells

Abstract

Mutations in the enzyme isocitrate dehydrogenase 1 (IDH1) lead to metabolic alterations and a sustained formation of 2-hydroxyglutarate (2-HG). 2-HG is an oncometabolite as it inhibits the activity of α-ketoglutarate-dependent dioxygenases such as ten-eleven translocation (TET) enzymes. Inhibitors of mutant IDH enzymes, like ML309, are currently tested in order to lower the levels of 2-HG. Vitamin C (VC) is an inducer of TET enzymes. To test a new therapeutic avenue of synergistic effects, the anti-neoplastic activity of inhibition of mutant IDH1 via ML309 in the presence of VC was investigated in the colon cancer cell line HCT116 IDH1^R132H/+ (harbouring a mutated IDH1 allele) and the parental cells HCT116 IDH1^+/+ (wild type IDH1). Measurement of the oncometabolite indicated a 56-fold higher content of 2-HG in mutated cells compared to wild type cells. A significant reduction of 2-HG was observed in mutated cells after treatment with ML 309, whereas VC produced only minimally changes of the oncometabolite. However, combinatorial treatment with both, ML309 and VC, in mutated cells induced pronounced reduction of 2-HG leading to levels comparable to those in wild type cells. The decreased level of 2-HG in mutated cells after combinatorial treatment was accompanied by an enhanced global DNA hydroxymethylation and an increased gene expression of certain tumour suppressors. Moreover, mutated cells showed an increased percentage of apoptotic cells after treatment with non-cytotoxic concentrations of ML309 and VC. These results suggest that combinatorial therapy is of interest for further investigation to rescue TET activity and treatment of IDH1/2 mutated cancers.

Keywords: IDH1; TET; Vitamin C; cancer cells; epigenetics.

Figure 1.

Measurement of 2-HG levels in HCT116 wild type IDH1 (IDH1^+/+) and mutant IDH1 (IDH1^R132H/+) cells. (a) Comparison of 2-HG levels in untreated IDH1^+/+ and IDH1^R132H/+ (mean ± SD; **** p < 0.0001; n = 3), (b) Influence of VC (1 mM) and ML309 (10 µM) treatment on 2-HG levels in IDH1^R132H/+ cells after 48 h (mean ± SD; **** p < 0.0001; n = 3)

Figure 2.

Genomic 5-hydroxymethyl-2´-deoxycytidine (5-hmdC) and 5-methyl-2´-deoxycytidine (5-mdC) levels in HCT116 wild type IDH1 (IDH1^+/+) and mutant IDH1 (IDH1^R132H/+) cells. (a) 5-hmdC/dC levels in untreated IDH1^+/+ and IDH1^R132H/+ (mean ± SD; ****p < 0.0001; n = 3), (b) The 5-mdC/dC levels in IDH1^+/+ and IDH1^R132H/+ (mean ± SD; n = 3)

Figure 3.

Impact of VC on genomic 5-hmdC/dC levels in IDH1^+/+ and IDH1^R132H/+ cells. (a,b) Quantification of 5-hmdC/dC levels in IDH1^+/+ and IDH1^R132H/+ treated for 48 h with increasing concentrations of VC as indicated (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 – significant results; error bars = SD; n = 3).

Figure 4.

Impact of ML309 and VC on genomic 5-hmdC/dC levels in IDH1^+/+ and IDH1^R132H/+ cells. (a,b) Quantification of 5-hmdC/dC levels in IDH1^+/+ and IDH1^R132H/+ treated for 48 h with increasing concentrations of ML309 alone or in combination with vitamin C (1 mM) as indicated (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 – significant results; error bars = SD; n = 3).

Figure 5.

Gene expression profile of 25 genes after single or combinatorial treatment with VC and ML309 for 48 h in IDH1^+/+ and IDH1^R132H/+ cells. Heat map of relative mRNA-levels of the differential expressed genes in (a) IDH1^+/+ and (b) IDH1^R132H/+ cells after treatment with VC (1 mM), ML309 (10 µM) and combinatorial treatment. (relative to the HMBS housekeeper gene, using the 2^ΔΔCT method)

Figure 6.

Effect of mutant IDH1 inhibitor ML309 and VC on cell viability of HCT116 IDH1^+/+ or IDH1^R132H/+. HCT116 cells were exposed to increasing concentrations of ML309, as indicated. The incubation was carried out for 24 h in HCT116 IDH1^+/+ (a) or IDH1^R132H/+ (b) with ML309 alone or in combination with VC (1 mM), and cell viabilities assessed by MTT assay. Data are expressed as a percentage of the untreated control (100% are indicated as black line), viable cell levels <75% were taken to indicate cytotoxic induction (error bars = SD; n = 3), dotted line shows 50% viability for determination of IC50 values.

Figure 7.

Measurement of apoptotic cells upon VC and ML309 incubation. (a, c) Representative contour plots of Annexin V/propidium iodide staining in cells treated for 48 h with combination of VC and ML309, as indicated. Q1 – gates for necrotic cells, Q2 – for late apoptosis, Q3 – for early apoptosis and Q4 – for healthy cells. (b, d) Quantitative representation of early and late apoptotic cells after treatment with VC, ML309 or combined with indicated concentrations. (error bars = SD; n = 3; Statistical significance of the treated groups to the untreated control was calculated using 2-way ANOVA and Tukey post-test: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).