Modulation of DNA methylation by a sesquiterpene lactone parthenolide

Abstract

Hypermethylation of 5'-cytosine-guanosine islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys(38) of p65 to inhibit nuclear factor-kappaB activation and exhibit anti-tumor activity in human malignancies. In this article, we report that parthenolide 1) inhibits DNA methyltransferase 1 (DNMT1) with an IC(50) of 3.5 microM, possibly through alkylation of the proximal thiolate of Cys(1226) of the catalytic domain by its gamma-methylene lactone, and 2) down-regulates DNMT1 expression possibly associated with its SubG(1) cell-cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in vitro and in vivo global DNA hypomethylation. Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.

Fig. 1.

Human DNMT1 homology model. Alignment of the catalytic domain of human DNA methyltransferase I (hDNMT1) against that of the bacterial DNA methyltransferase M.HhaI (5MHT). The figure shows an alignment of 327 AA of M.HhaI with 478 AA of hDNMT1. Conserved amino acids of these two regions are highlighted in green and red, respectively. There is a 25% identical sequence and 41% similar sequence in the N-terminal 178 AA (37.2% of entire sequence) and a 36% identical sequence and 58% similar sequence in the C-terminal 46 AA (9.6% of entire sequence) of the two proteins. The middle sequence has little homology between these two proteins.

Fig. 2.

Structure of the DNMT1 catalytic domain and its docking with several DNMT1 inhibitors. A, docking of parthenolide into the DNMT1 catalytic site. The DNMT1 catalytic domain is represented by the ribbon model. Docked parthenolide and catalytic Cys¹²²⁶ are shown in ball and stick models. Docked cofactor SAM and flipped cytosine are shown in red and blue lines, respectively. B, superimposed docking of parthenolide, EGCG, and RG-108 into the DNMT1 catalytic site with catalytic cysteine and parthenolide in red, EGCG in green, and RG-108 in blue shown in ball and stick models. DNMT1 active site is represented as a topographic surface is in ball and stick.

Fig. 3.

The DNA methylation inhibition effects of parthenolide (A) and its analog parthenolideA (B) on M.SssI. The double strand oligonucleotide substrate (10 nM) was treated with M.SssI (2 U), SAM without parthenolide or its analog parthenolideA (0, 0.01, 0.03, 0.3, 1, 10, 30, and 100 μM), followed by cutting with HpaII and the addition of Attophos substrate solution to generate fluorescence at excitation 430/emission 560 (filter = 550 nm). The inhibition effect of parthenolide was reflected as decrease in fluorescence intensity and expressed as an IC₅₀.

Fig. 4.

Parthenolide inhibits protein expressions of DNMT1 in MV4-11 (left) and Kasumi-1 (right) cells for 24 h in a dose-dependent manner. MV4-11 and Kasumi-1 cells were incubated with the indicated concentrations of parthenolide (0, 1, 3, and 10 μM). DNMT1 protein levels were detected by Western blot.

Fig. 5.

Effect of parthenolide on cell-cycle distribution in MV4-11 cells. MV4-11 cells were placed in serum-free medium to synchronize the cell cycle for 24 h. Cells were then treated with parthenolide (0, 3, and 10 μM) for 24 h. Cell-cycle distribution was determined by flow cytometry. A, representative sets of histogram for MV4-11 cells (A) and 3 μM (B) and 10 μM (C) parthenolide-treated cells. Percentages of cells in SubG₁, G₀/G₁, S, and G₂/M phase are shown as insets for each experiment.

Fig. 6.

Down-regulation of DNMT1 is associated with depletion of Sp1 in parthenolide-treated MV4-11 cells. A, reduced Sp1 protein expression in MV4-11 cells treated with the indicated concentrations of parthenolide for 24 h. B, parthenolide abolishes Sp1 binding to DNMT1 promoter. EMSA was performed with total cell lysate (left) and nuclear extracts (right) prepared from MV4-11 cells treated without parthenolide or decitabine. C, ChIP of DNMT1 gene promoter with Sp1 shows the dissociation of the transcriptional activator Sp1 from the DNMT1 gene promoter. D, parthenolide decreases RNA levels of DNMT1, DNMT3a, and DNMT3b in MV4-11 cells for 24 h. Ct, untreated; DAC, decitabine (2.5 μM); Par, parthenolide (10 μM).

Fig. 7.

Induction of global DNA hypomethylation by parthenolide in vitro and in vivo and its anti-tumor growth activity. A, global DNA methylation was found to be decreased in K562 and MV4-11 cells, each treated with 5, 10 or 30 μM parthenolide for 24 h. B, the in vivo hypomethylation effect of parthenolide in tumors from MV4-11 xenograft mice following treatment with a single intravenous bolus dose (10 mg/kg) of parthenolide formulated in polyethylene glycol 400, ethanol, and PBS. The global DNA methylation level of the treated tumor tissue is approximately 70% of the control group (p = 0.05), as well as the anti-tumor growth activity of parthenolide on MV4-11-engrafted tumor in nu/nu mice at day 7, after five times daily dosing at 4 mg/kg i.p. (C). The tumor size is approximately 63% of that in control group (p = 0.006). D, the in vivo down-regulation of protein levels of DNMT1 in MV4-11-engrafted tumor tissues collected from nude mice in the anti-tumor growth activity study. DNMT1 protein levels were detected by Western blot.

Fig. 8.

Parthenolide induces promoter hypomethylation methylation-silenced genes and their re-expression. A, perturbation of HIN-1 promoter methylation in MCF-7 cells exposed to indicated concentrations of parthenolide. DNA (1 μg) from parthenolide-treated or untreated MCF-7 cells were treated with bisulfite followed by PCR amplification, M.SssI methylation, and digestion to nucleosides. 5mdC and 2dC in the hydrolysates were measured using the LC-MS/MS method, and the ratio of 5mdC to 2dC is used as a DNA methylation indicator. B, hypomethylation activities of parthenolide (10 and 30 μM) and decitabine (0.75 μM) on HIN-1 promoter detected by bisulfite-sequencing. A 15% decrease of the promoter methylation in parthenolide-treated group was found. The solid circles represent methylated CpG loci, and the open circles represent nonmethylated CpG loci. C, parthenolide increased expression of HIN-1 gene in MCF-7 cells treated with parthenolide for the indicated time points and dosages. HIN-1 gene expression was measured by real-time RT-PCR. Conc, untreated; DAC, decitabine (0.75 μM); Par, parthenolide.