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KMID : 0545120140240121654
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Journal of Microbiology and Biotechnology
2014 Volume.24 No. 12 p.1654 ~ p.1663
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Tumor Suppressor Protein p53 Promotes 2-Methoxyestradiol-Induced Activation of Bak and Bax, Leading to Mitochondria-Dependent Apoptosis in Human Colon Cancer HCT116 Cells
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Lee Ji-Young
Jee Su-Bean
Park Won-Young
Choi Yu-Jin
Kim Bo-Kyung
Kim Yoon-Hee
Jun Do-Youn
Kim Young-Ho
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Abstract
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To examine the effect of tumor suppressor protein p53 on the antitumor activity of 2- methoxyestradiol (2-MeO-E2), 2-MeO-E2-induced cell cycle changes and apoptotic events were compared between the human colon carcinoma cell lines HCT116 (p53+/+) and HCT116 (p53-/-). When both cell types were exposed to 2-MeO-E2, a reduction in the cell viability and an enhancement in the proportions of G2/M cells and apoptotic sub-G1 cells commonly occurred dose-dependently. These 2-MeO-E2-induced cellular changes, except for G2/M arrest, appeared to be more apparent in the presence of p53. Immunofluorescence microscopic analysis using anti-α-tubulin and anti-lamin B2 antibodies revealed that after 2-MeO-E2 treatment, impaired mitotic spindle network and prometaphase arrest occurred similarly in both cell types. Following 2-MeO-E2 treatment, only HCT116 (p53+/+) cells exhibited an enhancement in the levels of p53, p-p53 (Ser-15), p21WAF1/CIP1, and Bax; however, the Bak level remained relatively constant in both cell types, and the Bcl-2 level decreased only in HCT116 (p53+/+) cells. Additionally, mitochondrial apoptotic events, including the activation of Bak and Bax, loss of Δψm, activation of caspase-9 and -3, and cleavage of lamin A/C, were more dominantly induced in the presence of p53. The Bak-specific and Bax-specific siRNA approaches confirmed the necessity of both Bak and Bax activations for the 2-MeO-E2-induced apoptosis in HCT116 cells. These results show that among 2-MeO-E2-induced apoptotic events, including prometaphase arrest, up-regulation of Bax level, down-regulation of Bcl-2 level, activation of both Bak and Bax, and mitochondria-dependent caspase activation, the modulation of Bax and Bcl-2 levels is the target of the pro-apoptotic action of p53.
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KEYWORD
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2-methoxyestradiol, p53, prometaphase arrest, Bak and Bax activation, apoptosis
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