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Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes
Published on: April 13, 2018
Chidamide-induced ROS accumulation and miR-129-3p-dependent cell cycle arrest in non-small lung cancer cells
Yueh-Feng Wu1, Chu-Chyn Ou2, Peng-Ju Chien3
1Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan; Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan.
Background:
Epigenetic therapy is a promising popular treatment modality for various cancers. Histone modification and miRNA should not be underestimated in lung cancer. This study aimed to investigate whether chidamide, a histone deacetylase inhibitor (HDACi), which inhibits telomerase activity and induces cell cycle arrest, influences ROS and miRNA production in non-small cell lung cancer (NSCLC) cells.
Methods:
H1355 and A549 were treated with chidamide. The analysis of DNA content was measured by FACSCalibur equipped with a 488 nm laser. H1355 cells were transfected with miR-129-3p mimic by Lipofectamine2000. Telomerase activity was performed on the telomeric repeat amplification protocol (TRAP) assay. Detection of thymidylate synthase (TS), p21, p53, pRB, and β-actin, were performed by western blot analysis.
Results:
Our data showed that expression of TS, p21, and pRB were altered in the presence of chidamide by PCR and western blot. Using BrdU-incorporation analysis, we found that chidamide induced G1 arrest through the regulation of the TS gene by miR-129-3p. Chidamide was shown to suppress telomerase activity in the TRAP assay and reduced the expression of human telomerase reverse transcriptase (hTERT) by PCR and q-PCR in H1355 and A549 cells. Chidamide increased the generation of reactive oxygen species (ROS) by flow cytometry. N-acetyl cysteine (NAC), a ROS scavenger, attenuated chidamide-induced telomerase activity inhibition.
Conclusion:
Chidamide repressed telomerase activity through ROS accumulation and cell cycle arrest by miR-129-3p upregulation in both H1355 and A549 cells. This is the first study to demonstrate that chidamide induces miR-129-3p upregulation and ROS accumulation, leading to cell cycle arrest.
Insights
Chidamide, a histone deacetylase inhibitor, suppresses telomerase activity and induces cell cycle arrest in non-small cell lung cancer cells by upregulating miR-129-3p and increasing reactive oxygen species (ROS). This epigenetic therapy shows promise for lung cancer treatment.
Area of Science:
- Oncology
- Molecular Biology
- Epigenetics
Background:
- Epigenetic therapy, including histone modification and miRNA, is a key strategy in cancer treatment.
- Non-small cell lung cancer (NSCLC) warrants further investigation into epigenetic mechanisms.
- Histone deacetylase inhibitors (HDACi) like chidamide show potential in cancer therapy by affecting telomerase and cell cycle.
Purpose of the Study:
- To investigate the effects of chidamide on reactive oxygen species (ROS) and miRNA production in NSCLC cells.
- To explore the role of chidamide in regulating telomerase activity and cell cycle arrest in NSCLC.
- To elucidate the specific miRNA, miR-129-3p, and ROS involvement in chidamide's anti-cancer effects.
Main Methods:
- Treatment of H1355 and A549 NSCLC cells with chidamide.
- Analysis of DNA content, cell cycle (G1 arrest), and BrdU incorporation.
- Measurement of telomerase activity using the TRAP assay.
- Western blot analysis for TS, p21, p53, and pRB expression.
- Detection of ROS generation via flow cytometry and assessment of ROS scavenger (NAC) effects.
- Transfection of H1355 cells with miR-129-3p mimic.
Main Results:
- Chidamide altered the expression of TS, p21, and pRB.
- Chidamide induced G1 arrest, regulated by miR-129-3p through the TS gene.
- Chidamide suppressed telomerase activity and reduced hTERT expression.
- Chidamide increased ROS generation, and NAC attenuated its effect on telomerase activity.
Conclusions:
- Chidamide represses telomerase activity via ROS accumulation and miR-129-3p-mediated cell cycle arrest in NSCLC cells.
- This study is the first to demonstrate chidamide's role in inducing miR-129-3p upregulation and ROS accumulation for cell cycle arrest.
- Chidamide represents a potential epigenetic therapeutic agent for NSCLC by targeting telomerase and cell cycle pathways.
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