A Novel Small Molecular Inhibitor of DNMT1 Enhances the Antitumor Effect of Radiofrequency Ablation in Lung Squamous Cell Carcinoma Cells

Abstract

Radiofrequency ablation (RFA) is a relatively new and effective therapeutic strategy for treating lung squamous cell carcinomas (LSCCs). However, RFA is rarely used in the clinic for LSCC which still suffers from a lack of effective comprehensive treatment strategies. In the present work, we investigate iDNMT, a novel small molecular inhibitor of DNMT1 with a unique structure. In clinical LSCC specimens, endogenous DNMT1 was positively associated with methylation rates of miR-27-3p's promoter. Moreover, endogenous DNMT1 was negatively correlated with miR-27-3p expression which targets PSEN-1, the catalytic subunit of γ-secretase, which mediates the cleavage and activation of the Notch pathway. We found that DNMT1 increased activation of the Notch pathway in clinical LSCC samples while downregulating miR-27-3p expression and hypermethylation of miR-27-3p's promoter. In addition of inhibiting activation of the Notch pathway by repressing methylation of the miR-27-3p promoter, treatment of LSCC cells with iDNMT1 also enhanced the sensitivity of LSCC tumor tissues to RFA treatment. These data suggest that iDNMT-induced inhibition of DNMT-1 enhances miR-27-3p expression in LSCC to inhibit activation of the Notch pathway. Furthermore, the combination of iDNMT and RFA may be a promising therapeutic strategy for LSCC.

Keywords: DNA methyltransferase 1; Notch pathway; lung squamous cell carcinoma; radiofrequency ablation; small molecular inhibitor.

FIGURE 1

miR-27-3p/PSEN-1 axis in LSCC. (A) PSEN-1 expression in LSCC clinical specimens or the paired non-tumor tissues are illustrated as scatter-plots. (B) miRNA expression potentially targeting to PSEN-1 in LSCC or in the paired non-tumor tissues is shown as histograms. (C) Wild-type or the mutated sequences of miR27-3p-targeting sites located in the 3′UTR of PSEN-1. (D) miR-27-3p expression in LSCC clinical specimens is shown as a scatter-plot. (E) Correlation between PSEN-1 and miR-27-3p is depicted as a scatter-plot (n = 35). *p < 0.05.

FIGURE 2

miR-27-3p hypermethylation. (A) Methylation rates of mR-27-3p in LSCC clinical specimens or the paired non-tumor tissues are shown as scatter-plots. (B) Correlation between miR-27-3p and DNMT-1 is shown as scatter-plot images. (C) Correlation between the methylation of miR-27-3p and DNMT-1 is shown as a scatter-plot. (D) Promoter region of miR-27-3p with CpG island. (E) DNMT-1 expression in LSCC or the paired non-tumor tissues is shown as a scatter-plot. (F) Correlation between PSEN-1 and DNMT-1 is shown as scatter-plots. (G) Correlation between N-cadherin and DNMT-1 is shown as scatter-plot (n = 35). *p < 0.05.

FIGURE 3

Leading inhibitory DNMT-1 compound. (A) Structure of the leading compound. (B,C) R1 and R2 positions for leading compound substitutions.

FIGURE 4

Predicted mode of iDNMT/DNMT-1 binding.

FIGURE 5

iDNMT inhibits activation of the Notch pathway. (A) Effect of iDNMT on the expression of Notch pathway-related factors is depicted as a heat-map. (B) Effect of iDNMT on Notch protein cleavage is shown as cellular subfraction.

FIGURE 6

Oral administration of iDNMT inhibits the subcutaneous proliferation of H226 LSCC cells in nude mice. (A) Images of tumor tissues. (B, C) Volumes and weights of tumor tissues are displayed as histograms. (D) Effect of iDNMT on the expression of Notch pathway-related factors is displayed as a heat-map. Comparison between the iDNMT group and control group. *p < 0.05.

FIGURE 7

RFA treatment inhibits subcutaneous growth of H226 LSCC cells in nude mice. (A) Images of tumor tissues. (B) Tumor growth curves. (C,D) Volumes and weights of the tumor tissues are displayed as histograms. (E) Effect of RFA on the expression of Notch pathway-related factors is shown as a heat-map. Comparison between the iDNMT group and control group. *p < 0.05.

FIGURE 8

RFA + iDNMT inhibits the subcutaneous proliferation of H226 LSCC cells in nude mice. (A) Images of tumor tissues. (B) Tumor growth curves. (C,D) Volumes and weights of tumor tissues are displayed as histograms. (E) Effect of RFA or iDNMT on the expression of Notch pathway-related factors is displayed as a heat-map. Comparison between the iDNMT group and control group. *p < 0.05.

FIGURE 9

Proposed model of the present work. (A) In LSCC cells, DNMT-1 may induce cleavage and methylation of the miR-27-3p promoter, thereby inhibiting expression of miR-27-3p. Low miR-27-3p expression leads to upregulated PSEN-1 expression, which activates the Notch pathway by cleaving the Notch protein. (B) Treating LSCC cells with iDNMT inhibits DNMT-1 activity, downregulates methylation of the miR-27-3p promoter region, upregulates miR-27-3p expression, downregulates PSEN-1 expression, and ultimately inhibits Notch pathway activity.