Kim Kyubo, Wu Hong-Gyun, Jeon Sang-Rok
Department of Radiation Oncology, Seoul National University College of Medicine, Seoul, Republic of Korea.
Department of Radiation Oncology, Seoul National University College of Medicine, Seoul, Republic of Korea Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea Institute of Radiation Medicine, Medical Research Center, Seoul National University, Seoul, Republic of Korea
Anticancer Res. 2015 Jan;35(1):245-53.
BACKGROUND/AIM: The aim of the present study was to suggest potential mechanisms of epidermal growth factor (EGF)-induced cell death and radiosensitization in EGF receptor (EGFR)-overexpressing cancer cell lines.
Two EGFR-overexpressing cancer cell lines (AMC-HN3, and A431), one EGFR-null cancer cell line (H520) and normal fibroblasts were cultured with 0.01-1000 nM of recombinant human EGF (rhEGF), and a clonogenic assay was performed. After culturing serum-starved cells with 10 nM rhEGF, the expression patterns of two apoptosis-associated proteins (cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP)) and the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway were measured using immunoblotting. The radiosensitizing effect of EGF was also evaluated with a clonogenic assay and phosphorylated H2AX (γH2AX) immunofluorescence.
In the clonogenic assay, the number of colonies was decreased in a dose-dependent manner in EGFR-overexpressing cancer cell lines. The expression of cleaved caspase-3 and cleaved PARP was significantly induced in EGFR-overexpressing cancer cell lines. As for the PI3K/AKT/mTOR signaling pathway, EGF paradoxically suppressed the expression of PI3K, AKT and mTOR in a time-dependent manner. As for the radiosensitizing effect, EGF enhanced the radiosensitivity of AMC-HN3 and A431 cells.
rhEGF treatment induced cell death in the two EGFR-overexpressing cancer cell lines, and the mode of cell death was apoptosis. Moreover, cell death might be associated with the paradoxical suppression of PI3K/AKT/mTOR signaling pathway. rhEGF in combination with radiation augmented the radiation effect in EGFR-overexpressing cancer cell lines via inhibition of DNA damage repair.
背景/目的：本研究旨在揭示表皮生长因子（EGF）诱导表皮生长因子受体（EGFR）过表达癌细胞系发生细胞死亡及放射增敏的潜在机制。
将两种EGFR过表达癌细胞系（AMC-HN3和A431）、一种EGFR缺失癌细胞系（H520）及正常成纤维细胞用0.01 - 1000 nM重组人EGF（rhEGF）进行培养，并进行克隆形成试验。用10 nM rhEGF培养血清饥饿细胞后，采用免疫印迹法检测两种凋亡相关蛋白（裂解的半胱天冬酶-3和裂解的聚（ADP-核糖）聚合酶（PARP））的表达模式以及磷酸肌醇3-激酶/蛋白激酶B/雷帕霉素哺乳动物靶蛋白（PI3K/AKT/mTOR）信号通路。同时，通过克隆形成试验和磷酸化H2AX（γH2AX）免疫荧光检测评估EGF的放射增敏作用。
在克隆形成试验中，EGFR过表达癌细胞系中的集落数量呈剂量依赖性减少。EGFR过表达癌细胞系中裂解的半胱天冬酶-3和裂解的PARP的表达显著诱导。至于PI3K/AKT/mTOR信号通路，EGF反常地以时间依赖性方式抑制PI3K、AKT和mTOR的表达。至于放射增敏作用，EGF增强了AMC-HN3和A431细胞的放射敏感性。
rhEGF处理诱导两种EGFR过表达癌细胞系发生细胞死亡，且细胞死亡模式为凋亡。此外，细胞死亡可能与PI3K/AKT/mTOR信号通路的反常抑制有关。rhEGF联合辐射通过抑制DNA损伤修复增强了EGFR过表达癌细胞系的辐射效应。
