Novel DNA methyltransferase-1 (DNMT1) depleting anticancer nucleosides, 4'-thio-2'-deoxycytidine and 5-aza-4'-thio-2'-deoxycytidine

Abstract

Purpose: Currently approved DNA hypomethylating nucleosides elicit their effects in part by depleting DNA methyltransferase I (DNMT1). However, their low response rates and adverse effects continue to drive the discovery of newer DNMT1 depleting agents. Herein, we identified two novel 2'-deoxycytidine (dCyd) analogs, 4'-thio-2'-deoxycytidine (T-dCyd) and 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) that potently deplete DNMT1 in both in vitro and in vivo models of cancer and concomitantly inhibit tumor growth.

Methods: DNMT1 protein levels in in vitro and in vivo cancer models were determined by Western blotting and antitumor efficacy was evaluated using xenografts. Effects on CpG methylation were evaluated using methylation-specific PCR. T-dCyd metabolism was evaluated using radiolabeled substrate.

Results: T-dCyd markedly depleted DNMT1 in CCRF-CEM and KG1a leukemia and NCI-H23 lung carcinoma cell lines, while it was ineffective in the HCT-116 colon or IGROV-1 ovarian tumor lines. On the other hand, aza-T-dCyd potently depleted DNMT1 in all of these lines indicating that dCyd analogs with minor structural dissimilarities induce different DNMT1 turnover mechanisms. Although T-dCyd was deaminated to 4'-thio-2'-deoxyuridine, very little was converted to 4'-thio-thymidine nucleotides, suggesting that inhibition of thymidylate synthase would be minimal with 4'-thio dCyd analogs. Both T-dCyd and aza-T-dCyd also depleted DNMT1 in human tumor xenografts and markedly reduced in vivo tumor growth. Interestingly, the selectivity index of aza-T-dCyd was at least tenfold greater than that of decitabine.

Conclusions: Collectively, these data show that 4'-thio modified dCyd analogs, such as T-dCyd or aza-T-dCyd, could be a new source of clinically effective DNMT1 depleting anticancer compounds with less toxicity.

Conflict of interest statement

Conflict of interest None.

Figures

Fig. 1

Characterization of T-dCyd and aza-T-dCyd. a Structures of T-dCyd and aza-T-dCyd, b cells were treated with T-dCyd or aza-T-dCyd for 72 h and cell viability was measured using the Cell Titer Glo assay. Mean IC50 values of three experiments in the indicated cancer cell lines are shown (SD < 10 %)

Fig. 2

T-dCyd and aza-T-dCyd induce DNMT1 depletion in leukemia cells and in solid tumor cells. Exponentially growing leukemia cells (a CCRF-CEM, upper panel and KG1a, lower panel) or solid tumor cells (b NCI-H23 lung carcinoma, HCT-116 colon carcinoma or IGROV-1 ovarian carcinoma cells) were exposed to indicated concentrations of either T-dCyd or aza-T-dCyd for 96 h. Cell lysates were fractionated on SDS-PAGE gels and were analyzed by Western blotting using antibodies against DNMT1 or GAPDH

Fig. 3

Effect of T-dCyd and aza-T-dCyd on p15 CpG methylation and its expression, a MSP analysis: Exponentially growing KG1a were exposed to 3 µM T-dCyd, 1 µM aza-T-dCyd, 1 µM aza-dCyd or 0.3 µM T-araC for 96 h. Genomic DNA was isolated and amplification of bisulfite-treated genomic DNA was accomplished as described in the text. Primer sets used for amplification of DNA are designated as unmethylated (U) and methylated (M). Amplified DNA was run in ethidium bromide stained gels, b RT-PCR analysis of pl 5 expression. Exponentially growing KG1a cells were exposed to 3 µM T-dCyd, 1 µM aza-T-dCyd, 1 µM aza-dCyd or 0.3 µM T-araC for 96 h and total RNA was prepared and reverse transcribed. RT-PCRs for p15 detection was accomplished with first-strand cDNA samples (or negative controls that lacked RT, reverse transcriptase) in ethidium bromide stained agarose gels

Fig. 4

T-dCyd and aza-T-dCyd deplete DNMT1 protein levels in tumors implanted in nude mice, a Mice-bearing subcutaneous CCRF-CEM tumors were injected i.p with T-dCyd for 9 days (qld × 9) at a dose of 9 mg/kg/dose or with aza-T-dCyd at 5 mg/kg/dose. b Aza-T-dCyd or aza-dCyd was injected i.p. at doses of 5, 2.5 or 1.25 mg/ kg/dose (qld × 9) (left panel). In the right panel aza-T-dCyd was injected i.p. at doses of 1.25, 0.625 or 0.3125 mg/kg/dose (qld × 9) and aza-dCyd was injected at doses of 0.625 or 0.3125 mg/kg/dose (qld × 9). Tumors were harvested 24 h after last treatment, and lysates were fractionated on SDS-PAGE gels and were analyzed by Western blotting using the indicated antibodies, c T-dCyd depletes DNMT1 protein levels in NCI-H23 lung tumors implanted in nude mice. Mice implanted with subcutaneous NCI-H23 tumors were injected i.p with T-dCyd for 3 days (qld × 3) at a dose of 25 mg/kg/ dose. Tumors were harvested 3 h after last treatment, and lysates were fractionated on SDS-PAGE gels and were analyzed by Western blotting using the indicated antibodies

Fig. 5

T-dCyd and aza-T-dCyd are effective at slowing growth of NCI-H23 lung tumors implanted in nude mice, a Mice were treated i.p. with vehicle (saline) or T-dCyd at 1.3 and 0.9 mg/kg/dose three times a day, 4 h apart, daily for 9 days, b Mice were treated i.p. with vehicle (saline) (qld × 5, qlw × 5), 1 mg/kg/dose aza-dCyd (qld × 5, qlw × 3) or 5 mg/kg/dose T-dCyd (qld × 5, qlw × 5). c Aza-T-dCyd is effective at slowing growth of NCI-H23 lung tumors implanted in nude mice. Mice were treated i.p. with vehicle (saline) or aza-T-dCyd at 10 and 6.7 mg/kg/dose daily for 9 days (qld × 9)