Enhancement of antineoplastic action of 5-aza-2???-deoxycytidine by zebularine on L1210 leukemia

Abstract

Tumor suppressor genes that have been silenced by aberrant DNA methylation are potential targets for reactivation by novel chemotherapeutic agents. The potent inhibitor of DNA methylation and antileukemic agent, 5-aza-2'-deoxycytidine (5-AZA-CdR, Decitabine), can reactivate silent tumor suppressor genes. One hindrance to the curative potential of 5-AZA-CdR is its rapid in vivo inactivation by cytidine deaminase (CD). An approach to overcome this obstacle is to use 5-AZA-CdR in combination with zebularine (Zeb), a potent inhibitor of CD. Zeb also possesses independent antineoplastic activity due to its inhibition of DNA methylation. We tested the capacity of 5-AZA-CdR and Zeb alone and in combination to inhibit growth and colony formation of different leukemic cell lines. 5-AZA-CdR and Zeb in combination produced a greater inhibition of growth against murine L1210 lymphoid leukemic cells, and a greater reduction in colony formation by L1210 and human HL-60 myeloid leukemic cells, than either agent alone. The ability of these agents to reactivate the tumor suppressor gene, p57KIP2, was also tested using RT-PCR. The combination produced a synergistic reactivation of p57KIP2 in HL-60 leukemic cells. A methylation-specific PCR assay showed that this combination also induced a significantly greater demethylation level of the p57KIP2 promoter than either drug alone. The in vivo antineoplastic activity of the agents was evaluated in mice with L1210 leukemia. A greater increase in survival time of mice with L1210 leukemia was observed with the combination than with either agent alone using three different dose schedules. The enhanced activity observed with 5-AZA-CdR plus Zeb in both murine and human leukemic cells lines provides a rationale for the clinical investigation of these drugs in patients with advanced leukemia. The probable mechanism of this drug interaction involves inhibition of CD by Zeb and the complementary inhibition of DNA methylation by both agents.

The DNA methyltransferase inhibitors azacitidine, decitabine and zebularine exert differential effects on cancer gene expression in acute myeloid leukemia cells

“…In a previous report on preclinical testing of DNMT inhibitor combinations, the authors note that the utility of Zeb may not lie so much in its own DNMT-inhibiting properties but rather in its potential to enhance the antileukemic activity of DAC by slowing the intracellular degradation of the latter. 20 This is in good agreement with our results; we also observed no DNA-hypomethylating effect of low-dose Zeb.…”

“…This is in line with previous publications reporting CDKN1C gene induction after treatment with DAC. 20,21 In parallel, we noted a small but significant decrease in CDKN1C promoter DNA methylation after DAC treatment (Figure 5a, left). The HOXA9 homeobox gene, which also has a 5 0 -CpG island, is an activated oncogene in a subset of AML 22 and MLL-rearranged leukemias, 23 but is specifically repressed in AML1-ETO-positive AML.…”

“…This is, however, in agreement with bisulfite sequencing and with a previous report where Zeb at low toxic concentration caused a 25% reduction in clonogenicity of HL-60 cells but did not decrease DNA methylation. 20 The analysis of LINE1 sequences confirmed on a global genomic level that treatment with DAC resulted in more pronounced hypomethylation than 5AC treatment; again, Zeb had no discernible effect (Figure 3b, bottom). Taken together, the data indicate that DAC is a more potent DNA-hypomethylating agent in Kasumi-1 cells than 5AC at equitoxic concentration, and both agents are more effective than Zeb.…”

Hypermethylation of the nel-like 1 gene is a common and early event and is associated with poor prognosis in early-stage esophageal adenocarcinoma

“…The involvement of CpG island hypermethylation in silencing of the NELL1 gene is also supported by the current study's finding that NELL1 mRNA levels in primary EACs with unmethylated NELL1 promoters were significantly higher than those in EACs with hypermethylated NELL1 promoters (P ¼ 0.02). Furthermore, 5-Aza-dC or its derivatives have shown potential as therapeutic anticancer drugs (Lemaire et al, 2005;Momparler, 2005), and thus NELL1 represents a novel potential target for molecular-based EAC therapies involving demethylation.…”

DNA Methylation

“…Hence administration of cytidine deaminase inhibitors like zebularine should theoretically potentiate therapeutic effects of 5-azaDc by slowing its degradation and stabilizing activity. Indeed, the combination of 5-aza-Dc and zebularine produced greater inhibition in cell proliferation and clonogenicity than either drug alone in leukemic L1210 and HL-60 cell lines [147]. Similarly, treatment of the AML-193 acute myeloid leukemic cell line, which has a densely methylated p15INK4B CpG island, with zebularine followed by the HDAC inhibitor, trichostatin-A, synergistically enhanced p15INK4B expression [134].…”