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  • Inhibition enhancer of zeste homologue 2 promotes senescence and apoptosis induced by doxorubicin in p53 mutant gastric cancer cells.

Inhibition enhancer of zeste homologue 2 promotes senescence and apoptosis induced by doxorubicin in p53 mutant gastric cancer cells.

Cell proliferation (2014-04-18)
J Bai, M Ma, M Cai, F Xu, J Chen, G Wang, X Shuai, K Tao
ABSTRACT

Enhancer of zeste homologue 2 (EZH2) is crucially involved in epigenetic silencing by acting as a histone methyltransferase. Although EZH2 is overexpressed in many cancers and is involved in malignant cell proliferation and invasion, the role of EZH2 in senescence induced by DNA damage has up to now remained largely unknown. In this study, we sought to explore the outcome of EZH2 depletion along with exposure of doxorubicin (DOX), and related mechanisms, in gastric cancer cells. Here, senescence induced by DNA damage was achieved in gastric cancer cells by DOX treatment. EZH2 was downregulated by transfection with siRNA or treated with (-)-epigallocatechin-3-gallate, a targeted inhibitor. Senescence-associated β galactosidase (SA-β-gal) and formation of senescence-associated heterochromatin foci were used to identify cell senescence. To investigate effects of EZH2 depletion on the cell cycle, apoptosis and proliferation, flow cytometry and MTT analysis were employed. Changes in p53-p21 axis activation were detected by Western blotting. We found that cell proliferative arrest caused by DOX could be promoted by EZH2 depletion. Mechanistically, EZH2 depletion not only worked in coordination with DNA damage during the progression of cell senescence but also promoted apoptosis in p53 mutant cells. However, it had no cooperative relationship with DOX in p53 wild-type cells. These data help unravel a crucial role for EZH2 in senescence and apoptosis in gastric cancer cells and that p53 genomic status was associated with different cell responses to EZH2 silencing.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Doxorubicin hydrochloride, 98.0-102.0% (HPLC)
Sigma-Aldrich
(−)-Epigallocatechin gallate, ≥95%
Sigma-Aldrich
Doxorubicin hydrochloride, suitable for fluorescence, 98.0-102.0% (HPLC)
Sigma-Aldrich
(−)-Epicatechin, ≥90% (HPLC)
Supelco
(+)-Catechin, analytical standard
Doxorubicin hydrochloride, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
(−)-Epigallocatechin gallate, ≥80% (HPLC), from green tea
Sigma-Aldrich
(−)-Epicatechin, ≥98% (HPLC), from green tea
Supelco
Epigallocatechin gallate, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
(−)-Epigallocatechin gallate, analytical standard
Supelco
(−)-Epicatechin, analytical standard
USP
Doxorubicin hydrochloride, United States Pharmacopeia (USP) Reference Standard
Epicatechin, primary reference standard
(±)-Catechin hydrate, primary reference standard
Epigallocatechin gallate, primary reference standard
Sigma-Aldrich
Anti-EZH2 (N-terminal) antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution