Division of Head and Neck Oncologic and Microvascular Surgery, Department of Otolaryngology-Head and Neck Surgery, University of Virginia Health System, Charlottesville, VA, USA.
Department of Biochemistry, National Liver Institute, Menoufiya University, Shibin Al Kawm, Egypt.
Drugs R D. 2017 Dec;17(4):545-555. doi: 10.1007/s40268-017-0204-x.
The epidermal growth factor receptor (EGFR) is highly expressed in a variety of solid tumors including oral cavity squamous cell carcinoma (OSCC) and has been implicated in the resistance of these tumors to cisplatin. This study was performed to determine if the EGFR tyrosine kinase inhibitor gefitinib could enhance the cytotoxic effect of cisplatin on OSCC cells in vitro.
The expression of EGFR and the phosphorylation of its downstream signaling to ERK, and AKT pathway were detected by Western blotting. Cell proliferation and survival were determined by AlamarBlue and colony formation assay respectively. Cells apoptosis were determined by Western blotting for cleaved PARP protein and by flowcytometry of cells stained with Annexin V and PI.
Cal27, OSC19, and SCC25 cells treated with gefitinib 1 μM demonstrated reduced phosphorylation of EGFR, AKT, and ERK proteins with very limited inhibition of proliferation. Cisplatin inhibited proliferation of the same cell lines in a dose-dependent manner. The concentration producing 50% inhibition (IC) for cisplatin decreased in the presence of gefitinib 1 μM, and a combination of cisplatin 5 µM and gefitinib 1 µM caused synergistic growth inhibition and synergistic reduction in cell survival. The growth inhibitory effect of the combination was associated with reduced ERK and AKT activation, increased poly ADP ribose polymerase (PARP) cleavage, and increased apoptosis.
Thus, in OSCC cells in vitro, inhibition of EGFR activity with gefitinib enhances the apoptotic effect of cisplatin. This has potential implications for enhancement of cisplatin effectiveness in tumors that over-express the EGFR.
表皮生长因子受体（EGFR）在多种实体瘤中高度表达，包括口腔鳞状细胞癌（OSCC），并与这些肿瘤对顺铂的耐药性有关。本研究旨在确定表皮生长因子受体酪氨酸激酶抑制剂吉非替尼是否能增强顺铂对体外 OSCC 细胞的细胞毒性作用。
通过 Western blot 检测 EGFR 的表达及其下游信号通路 ERK 和 AKT 的磷酸化。通过 AlamarBlue 和集落形成实验分别测定细胞增殖和存活。通过 Western blot 检测裂解的 PARP 蛋白和用 Annexin V 和 PI 染色的流式细胞术检测细胞凋亡。
用 1 μM 吉非替尼处理的 Cal27、OSC19 和 SCC25 细胞显示 EGFR、AKT 和 ERK 蛋白磷酸化减少，增殖受到非常有限的抑制。顺铂以剂量依赖性方式抑制相同细胞系的增殖。在存在 1 μM 吉非替尼的情况下，顺铂产生 50%抑制（IC）的浓度降低，5 μM 顺铂和 1 μM 吉非替尼联合使用可协同抑制生长并协同降低细胞存活率。联合用药的生长抑制作用与 ERK 和 AKT 激活减少、多聚 ADP 核糖聚合酶（PARP）裂解增加和凋亡增加有关。
因此，在体外 OSCC 细胞中，用吉非替尼抑制 EGFR 活性增强了顺铂的凋亡作用。这对增强过度表达 EGFR 的肿瘤中顺铂的有效性具有潜在意义。
