Epigenetic mechanisms of irinotecan sensitivity in colorectal cancer cell lines

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, 14-3-3σ, 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 upregulation 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.

Identification of a potent epigenetic biomarker for resistance to camptothecin and poor outcome to irinotecan-based chemotherapy in colon cancer

“…Some showed that DAC enhanced susceptibility to CPT (22,23,25,29), which is consistent with our data. In our study, DAC achieved 3-fold increase of susceptibility to CPT in the colon cancer cell line, HCT116 cells harboring DEXI methylation.…”

p16Ink4a overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors

“…For example, promoter hypermethylation of p16 Ink4a or cytoplasmic p16 Ink4a sequestration by AE1 has been identified as changes that allow malignant transformation. Therefore, demethylating therapies or inhibition of p16 Ink4a sequestering proteins (AE1) could restore p16 Ink4a and induce premature senescence in cancer cells (Schmitt et al, 2007;Schwabe and Lubbert, 2007;Shen et al, 2007;Crea et al, 2009). It has also been shown that DNA-damaging and differentiation-inducing anticancer therapies can provoke senescence as part of their anticancer effect (Schmitt et al, 2007).…”

Expression of p16 in lymph node metastases of adjuvantly treated stage III colorectal cancer patients identifies poor prognostic subgroups

“…47 Few studies have assessed the potential predictive value of p16 in colorectal cancer in vitro and in vivo. 52,53 However, Kamoshida and colleagues compared p16 and thymidylate synthase expression in preoperative biopsies and postsurgical Original Article…”