Tumor-suppressor Protein p53 Sensitizes Human Colorectal Carcinoma HCT116 Cells to 17α-estradiol-induced Apoptosis via Augmentation of Bak/Bax Activation = 17α-Estradiol에 의한 인체 대장암 세포주 HCT116의 에폽토시스에 수반되는 Bak/Bax의 활성화에 미치는 종양억제단백질 p53의 강화효과

국문 초록 (Abstract)

17α-estradiol (17α-E₂)의 에폽토시스 유도활성에 미치는 종양억제단백질 p53의 조절효과를 조사하고자, 17α-E₂에 의해 유도되는 에폽토시스 현상들을 인체 대장암 세포주 유래 클론인 HCT116 (p53+/+) 및 HCT116 (p53-/-) 세포에서 비교하였다. HCT116 (p53+/+) 및 HCT116 (p53-/-) 세포를 17α-E₂ (2.5～10 μM)로 처리하거나 혹은 HCT116 (p53+/+) 및 HCT116 (p53-/-) 세포를 10 μM 17α-E₂로 시간 별로 처리한 결과, HCT116 (p53+/+)에 있어서는 세포독성과 에폽토시스-관련 sub-G₁ peak의 비율은 처리농도와 시간에 의존적으로 나타났다. 그러나 HCT116 (p53-/-) 세포의 경우는 이러한 현상이 미약하게 나타났다. 17α-E₂에 의해 유도되는 비정상적 유사분열방추사 형성, 중기판 염색체 배열의 미완성, 이에 따른 유사분열정지(G₂/M arrest) 등의 현상은 HCT116 (p53+/+) 및 HCT116 (p53-/-) 세포에서 유사한 수준으로 나타났다. 이에 반해, 17α-E₂에 의해 유도되는 Bak과 Bax의 활성화, 미토콘드리아의 막전위 상실(Δψm loss), 그리고 PARP 분해 등의 현상은 HCT116 (p53-/-) 세포에 비해 HCT116 (p53+/+) 세포에서 훨씬 높은 수준으로 확인되었다. 아울러 17α-E₂로 처리된 HCT116 (p53+/+) 세포에서 확인되는 p53 (Ser-15)의 인산화 및 p53 수준의 증가와 일치하여, 세포 내의 p21및 Bax 수준도 현저히 증가하였다. 이때 17α-E2로 처리된 HCT116 (p53-/-) 세포에서는 p21 및 Bax의 발현수준이 매우 낮았다. 한편, 에폽토시스 억제단백질인 Bcl-2 단백질수준은 HCT116 (p53-/-) 세포에 비해 HCT116 (p53+/+) 세포에서 다소 낮았으나, 이러한 Bcl-2 단백질 수준은 17α-E₂ 처리 후에도 크게 변화하지 않는 것으로 나타났다. 이러한 결과들은 17α-E₂ 처리에 의해 유도되는 에폽토시스 유도 경로의 구성원들의 변화, 즉 비정상적 유사분열방추사 형성 및 이에 따른 유사분열정지(G2/M arrest), 뒤이은 Bak 및 Bax의 활성화, 미토콘드리아의 막전위 상실, 그리고 이에 수반되는 caspase cascade 활성화 및 PARP 분해로 진행되는 에폽토시스 현상들 중에서, Bak 및 Bax의 활성화 단계가 종양억제단백질 p53의 에폽토시스 증진 활성에 의해 양성적으로 조절되는 작용 타켓임을 보여준다.

다국어 초록 (Multilingual Abstract)

The regulatory effect of the tumor-suppressor protein p53 on the apoptogenic activity of 17α-estradiol (17α-E₂) was compared between HCT116 (p53+/+) and HCT116 (p53-/-) cells. When the HCT116 (p53+/+) and HCT116 (p53-/-) cells were treated with 2.5~10 μM 17α-E₂ for 48 h or with 10 μM for various time periods, cytotoxicity and an apoptotic sub-G₁ peak were induced in the HCT116 (p53+/+) cells in a dose- and time-dependent manner. However, the HCT116 (p53-/-) cells were much less sensitive to the apoptotic effect of 17α-E₂. Although 17α-E₂ induced aberrant mitotic spindle organization and incomplete chromosome congregation at the equatorial plate, G₂/M arrest was induced to a similar extent in both cell types. In addition, 17α-E₂-induced activation of Bak and Bax, Δψm loss, and PARP degradation were more dominant in the HCT116 (p53+/+) than in the HCT116 (p53-/-) cells. In accordance with enhancement of p53 phosphorylation (Ser-15) and p53 levels, p21 and Bax levels were elevated in the HCT116 (p53+/+) cells treated with 17α-E₂. The HCT116 (p53-/-) cells exhibited barely or undetectable levels of p21 and Bax, regardless of 17α-E₂ treatment. On the other hand, although the level of Bcl-2 was slightly lower in the HCT116 (p53+/+) than in the HCT116 (p53-/-) cells, it remained relatively constant after the 17α-E₂ treatment. Together, these results show that among the components of the 17α-E₂-induced apoptotic-signaling pathway, which proceeds through mitotic spindle defects causing mitotic arrest, subsequent activation of Bak and Bax and the mitochondria-dependent caspase cascade, leading to PARP degradation, 17α-E₂-induced activation of Bak and Bax is the upstream target of proapoptotic action of p53.

목차 (Table of Contents)

• Introduction
• Materials and Methods
• Results and Discussion
• References
• 초록

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