Epidermal Growth Factor-induced Cell Death and Radiosensitization in the Epidermal Growth Factor Receptor-overexpressing Cancer Cell Lines

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의과대학 의학과
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서울대학교 대학원
EGFEGFR-overexpressing cancer cell lineapoptosisPI3K-Akt-mTOR signaling pathwayradiosensitization
학위논문 (박사)-- 서울대학교 대학원 : 의학과, 2014. 2. 우홍균.
Purpose: The purpose of this study is to suggest the potential mechanisms of epidermal growth factor (EGF)-induced cell death and radiosensitization in epidermal growth factor receptor (EGFR)-overexpressing cancer cell lines.

Methods: Three EGFR-overexpressing cancer cell lines (AMC-HN3, A431, and SQ20B), one EGFR-null cancer cell line (H520) and normal fibroblasts were cultured with 0.01-1000nM of recombinant human EGF (rhEGF), and clonogenic assays were performed. After culturing serum-starved cells with 10 nM rhEGF, the expression patterns of two apoptosis-associated proteins (cleaved caspase-3 and cleaved PARP) and the PI3K/Akt/mTOR signaling pathway were measured using immunoblotting. EGFR and K-ras mutation analyses were performed. In addition, EGFR phosphorylation and p38 expression after rhEGF treatment were measured. The radiosensitizing effect of EGF was also evaluated with a clonogenic assay and γH2AX immunofluorescence.

Results: In the clonogenic assay, the number of colonies was decreased in a dose-dependent manner in EGFR-overexpressing cell lines, whereas rhEGF treatment increased the number of colonies in the normal fibroblast, and there was no change in the number of colonies in the EGFR-null cell line. Significant expression of cleaved caspase-3 and cleaved PARP was induced in EGFR-overexpressing cell lines, whereas there was no expression in the normal fibroblast, and no expression of cleaved caspase-3 or significant temporal increase in the expression of cleaved PARP in EGFR-null cell line. As for the PI3K/Akt/mTOR signaling pathway, rhEGF treatment suppressed the expression of PI3K, Akt, and mTOR in a time-dependent manner in EGFR-overexpressing cell lines. K-ras mutations were not detected on any of the cancer cell lines, whereas exon 20 missense substitution mutation of the EGFR gene was detected on A431. rhEGF treatment induced phosphorylation of EGFR residue Tyr845 in normal fibroblast, whereas EGFR was phosphorylated at Tyr845, Tyr992, Tyr1045 and Tyr1068 in AMC-HN3 and A431 treated with rhEGF. However, there was no change in the p38 expression in AMC-HN3 and A431 after rhEGF treatment. As for the radiosensitizing effect, EGF enhanced the radiosensitivity of AMC-HN3 and A431 cells, and increased radiation-induced γH2AX foci formation.

Conclusion: rhEGF treatment induced cell death in EGFR-overexpressing cancer cell lines, and the mode of cell death was apoptosis. And, the cell death may be associated with the paradoxical suppression of the 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.
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College of Medicine/School of Medicine (의과대학/대학원)Dept. of Medicine (의학과)Theses (Ph.D. / Sc.D._의학과)
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