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Updated: May 26, 2025
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
Novel Epigenetics Control (EpC) Nanocarrier for Cancer Therapy Through Dual-Targeting Approach to DNA
Risa Mitsuhashi1, Kiyoshi Sato1, Hiroyoshi Kawakami1
1Department of Applied Chemistry for Environment, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji 192-0397, Tokyo, Japan.
Background/Objectives:
Aberrant hypermethylation in the promoter regions of tumor suppressor genes facilitates the pathogenesis and progression of cancer. Therefore, inhibitors targeting DNA methyltransferase (DNMT) have been tested in clinical studies. However, the current monotherapy of DNMT inhibitors shows limited efficacy. Furthermore, the mechanism of action of DNMT inhibitors is DNA replication-dependent. To address these limitations, we developed a novel core-shell-type "epigenetics control (EpC) nanocarrier" that encapsulated decitabine (5-aza-dC) in the PLGA core nanoparticle and hybridized TET1 gene-encoding pDNA on the lipid shell surface. This study aimed to evaluate whether the dual delivery of DNMT inhibitors and pDNA of TET1 could synergistically enhance tumor suppressor gene expression and induce cell cycle arrest and/or apoptosis in cancer cells. Herein, we demonstrate the potential of the EpC carrier in HCT116 human colon cancer cells to upregulate tumor suppressor gene expression and rapidly achieve cell cycle arrest.
Methods:
PLGA core nanoparticles were prepared by the W/O/W double emulsion method. The formation of core-shell nanoparticles and complexation with pDNA were investigated and optimized by dynamic light scattering, zeta potential measurement, and agarose gel electrophoresis. The cellular uptake and transfection efficiency were measured by confocal laser scanning microscopy and a luciferase assay, respectively. The expression of p53 protein was detected by Western blotting. The anti-tumor effects of the EpC nanocarrier were evaluated by cell cycle analysis and an apoptosis assay.
Results:
The EpC nanocarrier delivered the DNMT inhibitor and TET gene-encoding pDNA into HCT116 cells. It promoted the expression of the tumor suppressor protein p53 and induced rapid cell cycle arrest in the G2/M phase in HCT116 cells.
Conclusions:
Our findings suggest that the dual-targeting of DNMT and TET enzymes effectively repairs aberrant DNA methylation and induces growth arrest in cancer cells, and the dual-targeting strategy may contribute to the advancement of epigenetic cancer therapy.
Insights
This study introduces an epigenetics control (EpC) nanocarrier that delivers both a DNA methyltransferase (DNMT) inhibitor and TET1 gene to cancer cells. The EpC nanocarrier effectively upregulated tumor suppressor genes and induced cell cycle arrest in colon cancer cells.
Area of Science:
- Cancer Biology
- Nanomedicine
- Epigenetics
Background:
- Aberrant DNA hypermethylation of tumor suppressor genes drives cancer progression.
- Current DNA methyltransferase (DNMT) inhibitor monotherapy has limited efficacy due to replication-dependent mechanisms.
- A novel core-shell Epigenetics Control (EpC) nanocarrier was developed to overcome these limitations.
Purpose of the Study:
- To evaluate the synergistic effects of co-delivering a DNMT inhibitor and TET1 gene via an EpC nanocarrier.
- To assess the potential of this dual-delivery system to enhance tumor suppressor gene expression and induce cancer cell death.
- To demonstrate the efficacy of the EpC nanocarrier in HCT116 human colon cancer cells.
Main Methods:
- Fabrication of PLGA core-shell nanocarriers encapsulating decitabine (DNMT inhibitor) and surface-hybridized with TET1 pDNA.
- Characterization of nanocarrier properties using dynamic light scattering, zeta potential, and gel electrophoresis.
- Assessment of cellular uptake, transfection efficiency, p53 protein expression, cell cycle arrest, and apoptosis in HCT116 cells.
Main Results:
- The EpC nanocarrier successfully delivered both the DNMT inhibitor and TET1 pDNA into HCT116 colon cancer cells.
- Significant upregulation of the tumor suppressor protein p53 was observed.
- Rapid induction of cell cycle arrest in the G2/M phase was achieved.
Conclusions:
- Dual-targeting of DNMT and TET enzymes via the EpC nanocarrier effectively corrects aberrant DNA methylation.
- This strategy induces cancer cell growth arrest, showing potential for advancing epigenetic cancer therapy.
- The EpC nanocarrier demonstrates promise for synergistic cancer treatment by combining epigenetic modulation and gene delivery.
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