Gefitinib (ZD1839) is an EGFR first-generation inhibitor with oral activity that inhibits the EGFR 19 Del and L858R mutations. Gefitinib has antitumor activity and is used for the treatment of EGFR-mutated non-small-cell lung cancers. Gefitinib administration RESULTS in the development of the EGFR C797S resistance mutation.

Tyr992:57 nM (NR6W cells), Tyr992:37 nM (NR6wtEGFR cells) , Tyr1173:26 nM (NR6W cells), Tyr1173:37 nM (NR6wtEGFR cells)

方法：23 种肿瘤细胞用 Gefitinib 处理 72 h，使用 MTT 方法检测细胞活力。
结果：只有 PC9 细胞系的 IC50<1 μmol/L (高度敏感)，14 个细胞系的 IC50>10 μmol/L (抗性)，其余 8 个细胞系具有 1-10 μmol/L 的 IC50 (中等敏感）。[1]
方法：肿瘤细胞 HT29、KB、Du145 和 A549 用 Gefitinib (0.032-50 μM) 处理 2 h，在细胞裂解前五分钟加入 EGF (0.1 μg/mL)，使用 Western Blot 方法检测靶点蛋白表达水平。
结果：Gefitinib 在所有肿瘤细胞系中产生 EGFR 自磷酸化的剂量依赖性抑制。[2]

方法：为检测体内抗肿瘤活性，将 Gefitinib (3.125-200 mg/kg in 0.5% polysorbate 80) 口服给药给携带肿瘤 A431、 Du145 或 A549 的 nude 小鼠，每天一次，持续七-十五天。
结果：Gefitinib 剂量依赖性方式抑制 A431、 Du145 或 A549 肿瘤生长。[2]
方法：为检测体内抗肿瘤活性，将 Gefitinib (40 mg/kg，每天一次) 或 Gefitinib (200 mg/kg，每五天一次) 灌胃给药给携带人肺癌肿瘤 H3255 的 athymic nude 小鼠，持续两周。
结果：每周治疗显示出比每天治疗更好的抑制作用。与每日给药方案相比，每周给药方案对 p-EGFR、p-ERK 和 p-AKT 的抑制作用更强。[3]

The human NSCLC H1299, H1975, A549, H460, GLC82, H460, and CALU-3 cell lines were provided by the American Type Culture Collection and maintained in RPMI-1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO2. CALU-3 GEF-R is a cell line obtained in vitro as previously described. Briefly, over a period of 12 months, human CALU-3 lung adenocarcinoma cells were continuously exposed to increasing concentrations of gefitinib. The starting dose was the dose causing the inhibition of 50% of cancer cell growth (IC50; gefitinib, 1 μmol/L). The drug dose was progressively increased to 15 μmol/L in approximately 2 months, to 20 μmol/L after other 2 months, to 25 μmol/L after additional 2 months, and, finally, to 30 μmol/L for a total of 12 months. The established resistant cancer cell lines were then maintained in continuous culture with the maximally achieved dose of each TKI that allowed cellular proliferation (30 μmol/L for each drug) [2].

Four- to 6-week old female balb/c athymic (nu+/nu+) mice were purchased from Charles River Laboratories. Mice were acclimatized for 1 week before being injected with cancer cells and injected subcutaneously with 107 H1299 and CALU-3 GEF-R cells that had been resuspended in 200 μL of Matrigel. When established tumors of approximately 75 mm3 in diameter were detected, mice were left untreated or treated with oral administrations of metformin (200 mg/mL metformin diluted in drinking water and present throughout the experiment), gefitinib (150 mg/kg daily orally by gavage), or both for the indicated time periods. Each treatment group consisted of 10 mice. Tumor volume was measured using the formula π/6 × larger diameter × (smaller diameter)2. Tumor tissues were collected from the xenografts and analyzed by Western blotting for the expression and activation of EGFR, AMPK, mitogen-activated protein kinase (MAPK), and S6 [2].