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Abstract
Irinotecan is a topoisomerase-I (Top-I) inhibitor used for the treatment of colorectal cancer. DNA demethylating agents, including 5-azacytidine (5-aza), display synergistic antitumor activity with several chemotherapy drugs. 5-Aza may enhance irinotecan cytotoxicity by at least one of the following mechanisms: (a) Top-I promoter demethylation, (b) activation of genes involved in Top-I transcriptional regulation (p16 or Sp1), and (c) modulation of the cell cycle and apoptosis after DNA damage. The growth-inhibitory effects of SN38, the active metabolite of irinotecan, 5-aza, and their combinations, were studied in four colorectal cancer cell lines. The effects of treatments on cell cycle were analyzed by flow cytometry, and apoptosis was measured by fluorescence microscopy. Top-I, Sp1, and p53 expression modulated by 5-aza were measured by real-time PCR. Methylation of Top-I, p16, 14-3-3sigma, and hMLH1 promoters before and after 5-aza treatment were measured by MethyLight PCR and DNA bisulfite sequencing. Low-dose 5-aza significantly enhanced the apoptotic effect of irinotecan in all colorectal cancer cells, whereas a synergistic cytotoxic effect was observed only in p53-mutated cells (HT29, SW620, and WiDr). This synergistic effect was significantly correlated with Top-I up-regulation by 5-aza, and coupled to p16 demethylation and Sp1 up-regulation. p16 demethylation was also associated with enhanced cell cycle arrest after irinotecan treatment. In contrast, 5-aza down-regulated Top-I expression in the p53 wild-type LS174T cells in a p53-dependent manner, thereby reducing SN38 cytotoxicity. In conclusion, 5-aza modulates Top-I expression by several mechanisms involving Sp1, p16, and p53. If confirmed in other models, these results suggest that p16 and p53 status affects the 5-aza-irinotecan interaction.
Highlights
For many years, cancer has been viewed as a disease driven by progressive genetic alterations involving oncogenes and tumor suppressor genes, but only recently, it has become apparent that cancer is driven by epigenetic alterations, i.e., changes in gene expression patterns not dependent on changes in DNA sequence
Treatment of colorectal cancer cells with 1 μmol/L of 5aza resulted in a minimal growth inhibition of WiDr and HT29 cells, and in a more relevant effect on SW620 and LS174T cells [i.e., the growth-inhibitory effect ranged from 4.3 ± 3.9% (WiDr) to 23.1 ± 1.0%
For LS174T, a synergistic activity was observed only at low FA, whereas at the most relevant FA levels, the two drugs showed a clear antagonism. These data were confirmed by dose reduction index (DRI) values which showed that 5-aza pretreatment remarkably reduced the SN38 cytotoxic concentration in p53-mutated cells
Summary
Cancer has been viewed as a disease driven by progressive genetic alterations involving oncogenes and tumor suppressor genes, but only recently, it has become apparent that cancer is driven by epigenetic alterations, i.e., changes in gene expression patterns not dependent on changes in DNA sequence. A global genome demethylation is observed, coupled with selective hypermethylation of tumor suppressor, cell cycle regulator, and proapoptotic genes [2]. DNMT inhibitors, including 5-azacytidine (5-aza) and decitabine, may reverse these epigenetic alterations by reactivating silenced genes, blocking cancer cell proliferation, and/or inducing apoptosis. Several studies suggested that demethylating agents might be synergistic with classic chemotherapy drugs, inducing a global change in gene expression and reversing acquired or constitutive drug resistance [6]. Most of these studies did not clarify the relative contribution of apoptosis, growth arrest, and modulation of drug target–genes in determining this synergism
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