结肠癌K-ras突变与对西妥昔单抗耐药的实验研究

目的：研究结肠癌K-ras基因突变导致对西妥昔单抗耐药的可能机制。方法：通过基因测序及免疫细胞化学染色,筛选表皮生长因子受体（EGFR）表达阳性,同时K-ras基因呈突变型及野生型的结肠癌细胞各1株,应用细胞增殖实验（CCK8方法）、流式细胞技术（Annexin V标记）,检测西妥昔单抗（C225）在体外对结肠癌细胞增殖和凋亡的影响。同时用K-ras呈野生型和突变型的结肠癌细胞株,分别建立裸鼠皮下移植瘤模型,分成HT29-C225、HT29-NS、SW620-C225及SW620-NS组,给予西妥昔单抗（每3天注射1次,每次0.5mL,共4周）。治疗后,绘制肿瘤生长曲线,用TDT介导的缺口末端标记技术（TUNEL）检测细胞凋亡,用定量PCR、免疫组织化学染色（IHC）及蛋白印迹（Western-blot）等技术检测移植瘤标本中EGFR信号转导通路中AKT及其磷酸化蛋白的表达。结果：体外增殖及凋亡实验显示,西妥昔单抗对不同K-ras基因型的结肠癌细胞均未能显示细胞毒作用。而体内研究发现,西妥昔单抗能明显抑制K-ras野生型结肠癌裸鼠皮下移植瘤的生长,但对K-ras突变型者抑制作用较差。对K-ras突变型结肠癌裸鼠皮下移植瘤的定量PCR检测结果显示,西妥昔单抗治疗组裸鼠的AKT基因表达率高于对照组。IHC及Western-blot显示,西妥昔单抗治疗组与对照组间AKT蛋白的表达率无明显差异,但西妥昔单抗治疗组裸鼠的p-AKT表达率高于对照组。结论：K-ras突变状态与西妥昔单抗的疗效相关,K-ras突变型者EGFR信号通路中的衔接蛋白AKT呈自主激活状态,该基因突变可能是导致对西妥昔单抗耐药的机制之一。

Exprimental research on K-ras mutation and the resistance to cetuximab

Objective To investigate the possible mechanism of K-ras mutation induced resistance to cetuximab. Methods Two colon cancer cell lines, HT29 and SW620, which were epidermal growth factor receptor (EGFR) positive, but separately with K-ras wild type and mutant type. were screened. The cytotoxic effect and apoptosis-inducing effect of cetuximab in vitro were evaluated via the cell proliferation assay (CCK8) and flow cytomety (Annexin V labeled). Subcutaneous xenograft nude mice model was established, and divided into 4 groups : HT29-C225, HT29-NS, SW620-C225 and SW620-NS; the inhibitory effects of cetuximab on these 2 colon cancer cell lines in vivo were observed. The EGFR expression, K-ras status. its down-stream signal protein AKT and its phospho-protein in EGFR pathway were detected by immunohistochemical staining and Westem-blot. Results To colon cancer xenograft models, cetuximab significantly inhibited the growth of colon cancer xenograft with the K-ras wild-type; yet, in colon cancer xenograft with the K-ras mutant-type, cetuximab did not inhibit the tumor growth. Detailed molecular detection demonstrated that the expression of phospho-AKT was elevated in the K-ras mutant colon cancer xenograft. Conclusions The K-ras status might influence the effects of cetuximab treatment. K-rass mutation might lead to the spontaneous activation of its down-stream signal adaptor protein AKT, thus in turn trigger the EGFR pathway activation and cause cetuximab resistance.