DNA-intercalators causing rapid re-expression of methylated and silenced genes in cancer cells

Abstract

Epigenetic inactivation of tumor-suppressor and other regulatory genes plays a critical role in carcinogenesis. Transcriptional silencing is often maintained by DNA methyl transferase (DNMT)-mediated hypermethylation of CpG islands in promoter DNA. Nucleoside analogs including azacytidine and decitabine have been used to inhibit DNMT and re-activate genes, and are clinically used. Their shortcomings include a short half-life and a slow onset of action due to required nucleotide incorporation during DNA replication, which may limit clinical utility. It might be useful to begin to identify lead compounds having novel properties, specifically distinct and fast-acting gene desilencing. We previously identified chemicals augmenting gene expression in multiple reporter systems. We now report that a subset of these compounds that includes quinacrine re-expresses epigenetically silenced genes implicated in carcinogenesis. p16, TFPI2, the cadherins E-cadherin and CDH13, and the secreted frizzle-related proteins (SFRPs) SFRP1 and SFRP5 were desilenced in cancer cell lines. These lead compounds were fast-acting: re-expression occurred by 12-24 hours. Reactivation of silenced genes was accompanied by depletion of DNMT1 at the promoters of activated genes and demethylation of DNA. A model compound, 5175328, induced changes more rapidly than decitabine. These gene desilencing agents belonged to a class of acridine compounds, intercalated into DNA, and inhibited DNMT1 activity in vitro. Although to define the mechanism would be outside the scope of this initial report, this class may re-activate silenced genes in part by intercalating into DNA and subsequently inhibiting full DNMT1 activity. Rapid mechanisms for chemical desilencing of methylated genes therefore exist.

Figure 1. Acridine compounds enhanced nonspecific gene expression

Luciferase assays were performed on CHO AA8-Luc Tet-Off cells 18 hours after treatment with compounds 5175323 (A), 5175328 (B), or quinacrine (C). Cells were plated in quadruplicate for each treatment. Data from representative experiments are shown.

Figure 2. Acridine compounds re-activated methylated and silenced genes in cancer cell lines in a dose-dependent fashion

Cells were treated with an acridine compound for 24 hours or with decitabine for 48 hours with drug and medium replaced 24 hours after the beginning of treatment. RNA extracted from the cells was subjected to RT-PCR analysis. β-actin was used as an internal control. A, Compound 5175323 induced gene re-expression in MiaPaCa2 cells. Re-expressed genes: CDH13, E-cadherin, SFRP1, and TFPI2. 5175323 concentrations (μg/ml): 0.01, 0.1, 1, 1.5, 2, 2.5, 3, 4, and 5. Decitabine concentrations (μM): 1 and 5. B, 5175323 induced gene re-expression in RKO cells. Re-expressed genes: p16, SFRP1, and SFRP5. 5175323 concentrations (μg/ml): 0.01, 0.1, 1, 1.5, 2, 2.5, 3, 4, and 5. Decitabine concentrations (μM): 1 and 5. C, Quinacrine induced gene re-expression in MiaPaCa2 cells. Re-expressed genes: CDH13, E-cadherin, SFRP1, and TFPI2. Quinacrine concentrations (μg/ml): 0.01, 0.1, 1, 2, 3, 5, 8, 10, and 20. Decitabine concentrations (μM): 1 and 5. D, Quinacrine induced gene re-expression in RKO cells. Re-expressed genes: p16, SFRP1, and SFRP5. Quinacrine concentrations (μg/ml): 0.01, 0.1, 1, 2, 3, 5, 8, 10, and 20. Decitabine concentrations (μM): 1 and 5. E, 5175328 induced gene re-expression in RKO cells. Re-expressed genes: SFRP1 and SFRP5. 5175328 concentrations (μg/ml): 0.01, 0.05, 0.1, 0.25, 0.5, 1, 1.5, 2, 5. Decitabine concentrations (μM): 1 and 5. F, 5175328 induced gene re-expression in HCT116 cells. Re-expressed genes: SFRP1, SFRP5, and TFPI2. 5175328 concentrations (μg/ml): 0.01, 0.05, 0.1, 0.25, 0.5, 1, 1.5, 2, 5. Decitabine concentrations (μM): 1 and 5.

Figure 3. Compound 5175328 re-activated gene expression as early as 12 hours after treatment

Re-expressed genes as detected by RT-PCR analysis: CDH13, E-cadherin, SFRP1, and TFPI2.

Figure 4. Compound 5175328-mediated gene re-expression appeared to be accompanied by DNA demethylation and DNMT1 depletion at specific promoters

A, 5175328 appeared to induce DNA demethylation at CDH13, E-cadherin, and SFRP1 promoters in MiaPaCa2 cells in a dose- and time-dependent manner. U, unmethylated allele; M, methylated allele. Normal human lymphocytes (NL), in vitro methylated DNA (IVD), and water were included as controls. B, 5175328 appeared to induce DNA demethylation at the SFRP1 promoter in RKO cells. U, unmethylated allele; M, methylated allele. Normal human lymphocytes (NL), in vitro methylated DNA (IVD), and water were included as controls. C, DNMT1 quantity and histone acetylation in MiaPaCa2 cells after 5175328 treatment, assayed by western blot. D, DNMT1 localization and histone acetylation at CDH13, E-cadherin, and SFRP1 promoters in MiaPaCa2 cells after 5175328 treatment, assessed by ChIP. PCR assays were performed in triplicate. Data from a representative ChIP experiment are shown.

Figure 5. Acridine compounds intercalate into DNA and inhibit DNMT1 activity in vitro

A, DNMT1 activity assay in the presence of compounds 5175328, 5175323, and quinacrine. 5-fluorouracil (5-FU) was a negative control. Each assay was performed in duplicate in every experiment. Data from a representative experiment are shown. Normalized absorbance was calculated by subtracting the 655 nm OD from the 450 nm OD. Full description of each bar: DNMT1 (1), DNMT1+DMSO (2), DNMT1 + 100 μM 5FU (3), 10 μg/ml 5175328 without DNMT1 (4), DNMT1+0.1μg/ml 5175328 (5), DNMT1+0.3μg/ml 5175328 (6), DNMT1+1μg/ml 5175328 (7), DNMT1 + 3 μg/ml 5175328 (8), DNMT1 + 10 μg/ml 5175328 (9), 10 μg/ml 5175323 without DNMT1 (10), DNMT1+0.1μg/ml 5175323 (11), DNMT1+0.3μg/ml 5175323 (12), DNMT1+1μg/ml 5175323 (13), DNMT1 + 3 μg/ml 5175323 (14), DNMT1 + 10 μg/ml 5175323 (15), 100 μg/ml quinacrine without DNMT1 (16), DNMT1+1μg/ml quinacrine (17), DNMT1+3μg/ml quinacrine (18), DNMT1+10μg/ml quinacrine (19), DNMT1 + 30 μg/ml quinacrine (20), DNMT1 + 100 μg/ml quinacrine (21). B, DNA intercalation assay of 5175328, 5175323, and quinacrine. Ethidium bromide was a positive control, and 5238219 a negative control. For each compound the following concentrations were used (μg/ml): 0.1, 1, 5, 50, and 500.