Inhibitory Effect of Berberine on Zeste Homolog 2 (Ezh2) Enhancement in Human Esophageal Cell Lines

S Chen,X Chen,F Zhang,X Mo,S Yang,Y Li,Y Ding,Kl Mo

doi:10.4314/tjpr.v14i9.7

Abstract

Purpose: To investigate the inhibitory effect of berberine treatment on enhancement of zeste of homolog 2 (Ezh2) expressions in KYSE450 human esophageal cancer cells.Methods: Transwell motility chambers were used to analyze cell migration and invasion. Bio-Rad protein assay was used for the determination of protein concentration. Chemiluminescence with ECL system was employed for the detection of protein bands as per the manufacturer’s protocol. Staining was carried out with Alexa-Fluor 647 mouse anti-BrdU antibody. Flow cytometry was performed after adding DAPI. Annexin-V/DAPI staining and flow cytometry were used for the quantification of apoptotic cell death. Total RNA was isolated from KYSE450 cells using an RNA isolation kit.Results: Berberine-induced inhibition of Ezh2 expression led to inhibition of cell proliferation by G1 phase cell cycle arrest and induced anti-invasive properties of KYSE450 cells in Boyden chamber assays. There was 92 % reduction in invasive tendency of KYSE450 cells following treatment with berberine. Histone methylation inhibitor, 3-deazaneoplanocin A (DZNep), also led to a similar effect on cell proliferation of KYSE450 cells. Berberine treatment also resulted in strong transcriptional reduction of the AXL receptor kinase. The results of qRT-PCR and FACS analyses showed significant inhibition of AXL mRNA and protein expression in KYSE450 carcinoma cells after treatment with berberine.Conclusion: Berberine may be an effective therapeutic agent in the treatment of esophageal carcinoma.Keywords: Berberine, Histone methylation inhibitor, Anti-invasive, Cell proliferation, Human Esophageal cancer

Highlights

During embryonic development cell fate decision is mainly regulated by polycomb group (PcG) proteins [1,2,3]
All samples were analysed in triplicate and the results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
The expression of enhancement of zeste of homolog 2 (Ezh2) was stronger in perinecrotic areas (Fig. 1C). These results demonstrate that expression of Ezh2 is associated with malignant esophageal gliomas

Summary

INTRODUCTION

During embryonic development cell fate decision is mainly regulated by polycomb group (PcG) proteins [1,2,3]. Berberine can suppress metastasis by enhancing the expression of a metastasis suppression gene, NM23-H1, or by targeting Rho kinasemediated ezrin phosphorylation in NPC 5-8 F cell line [15,17] It enhances the anti-cancer effects of estrogen receptor antagonists on human breast cancer cells (MCF-7) through down-regulating the expression of EGFR, HER2, Bcl-2, and COX-2, as well as upregulating IFN-α and p21 [18]. After incubation the cells were harvested, fixed in ice-cold methanol and treated with a PBS-based buffer containing 0.1 M hydrochloric acid (VWR) and 0.3 % Triton X-100 for 20 min at 4 oC. Cells were incubated for 10 min followed by flow cytometry examination using a BD FACS CANTO II cytometer. A value of p less than 0.05 was considered to be statistically significant

RESULTS

DISCUSSION

CONCLUSION