消癌平注射液联合吉非替尼对耐药非小细胞肺癌H460和H1975裸鼠移植瘤的抑制作用

摘要

目的：研究消癌平注射液（Xap）联合吉非替尼（Gef）对耐药非小细胞肺癌（NSCLC）细胞H460和H1975裸鼠移植瘤的抑制作用及机制。方法 BALB/c裸鼠皮下接种人NSCLC的H460或H1975细胞，待肿瘤体积达到50~100 mm3，分别ip给予Xap 5 g · kg-1、ig给予Gef 50 mg · kg-1或Xap+Gef（Xap+Gef组），连续给药21 d。每7 d测量肿瘤体积2~3次，绘制肿瘤增殖曲线，计算肿瘤增殖率，并记录体质量。取肿瘤组织，称取瘤重，计算肿瘤抑制率。免疫组化法和Western蛋白印迹法检测肿瘤组织中血管生成标志物血管内皮生长因子（VEGF）和内皮糖蛋白（CD105）及耐药相关蛋白磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）、哺乳动物西罗莫司（雷帕霉素）靶蛋白（mTOR）和磷酸化胞外信号调节激酶（ERK）的表达。结果与模型对照组相比，单用Xap或Gef对H460和H1975裸鼠移植瘤的生长无显著影响，而两药联合能显著抑制裸鼠的瘤体积〔H460组：（1103±340）vs（3020±450）mm3，H1975组：（487±153）vs（1269±161）mm3，P<0.05〕、降低瘤重〔H460组：（1.20±0.52） vs （2.78±0.93）g，H1975组：（0.52±0.32） vs （0.92±0.42）g，P<0.05〕，肿瘤增殖率和抑制率分别为42.1%，43.5%（H460，P<0.01）和43.0%,52.5%（H1975，P<0.01）。与单用Xap或Gef相比，联合用药组能显著性地降低瘤重和肿瘤增殖率，提高肿瘤抑制率（P<0.05）。免疫组化结果表明，单用Xap或Gef对肿瘤血管新生标志物VEGF和CD105及耐药相关蛋白p-PI3K，p-ERK，p-Akt和p-mTOR的表达无影响，而联合用药却能明显降低H460和H1975肿瘤组织中VEGF，CD105，p-ERK，p-Akt和p-mTOR的表达。Western蛋白印迹结果表明，与模型对照组相比，联合用可显著抑制p-PI3K及其下游蛋白p-Akt，p-ERK和p-mTOR蛋白的表达（P<0.01），并且优于单用Xap或Gef（P<0.05）。结论联合Xap可显著提高Gef在H460和H1975裸鼠移植瘤的疗效，该协同作用部分是通过抑制肿瘤血管新生和PI3K及其下游信号分子等耐药相关蛋白的表达实现的。%OBJECTIVE To study the antitumor activity and underlying mechanisms of Xiaoaiping injection(Xap)combined with gefitinib(Gef)in nude mice bearing resistant non-small cell lung cancer (NSCLC) cells H460 or H975. METHODS BALB/c nude mice were inoculated with human NSLCL cells H460 or H1975 and the drug treatment did not start until the tumor volume reached 50-100 mm3. The tumor bearing mice were divided into four groups:control group,Xap group(5 g · kg-1,ip),Gef group (50 mg · kg-1,ig),and Xap plus Gef group. All the administration lasted for 21 d continuous. Tumor volumes were measured two or three times per week,and the body weight of animals was re-corded. At the end of the test,tumors were weighed after the sacrifice of mice. Tumor inhibition rate and relative tumor proliferation rate were calculated based on tumor weight and tumor volume. The related biomarkers and proteins in tumor tissues were detected by immunohistochemistry and Western blot assay, respectively. RESULTS Compared with the control group,no significant effect was observed on the growth of H460 and H1975 xenografts in groups of Xap or Gef alone in nude mice. However,the two-drug combination significantly suppressed tumor volume,with (1103 ± 340) versus (3020 ± 450) mm3 for H460 and(487 ± 153)versus(1269 ± 161)mm3 for H1975,respectively(P<0.05). The combined Xap and Gef application also significantly reduced the tumor weight,with(1.20±0.52)versus(2.78± 0.93)g for H460 and(0.52 ± 0.32)versus(0.92 ± 0.42)g for H1975,respectively(P<0.05). The relative tumor proliferation rate and inhibition rate in the combination group was 42.1%and 43.5%for H460(P<0.01),43.0%and 52.5% for H1975(P<0.01). Compared with Xap and Gef drug alone,their combination showed significant difference in reducing tumor weight,suppressing tumor proliferation rate and increasing tumor inhibition rate(P<0.05). Immunohistochemistry results showed that each drug alone had no effect on tumor angiogenesis markers of vascular endothelial growth factor(VEGF)and CD105 expression,or on drug resistance related proteins of p-ERK,p-Akt and p-mTOR,whereas the combination of Xap and Gef obviously reduced the expression of these biomarkers in H460 and H1975 tumor tissues. The decreased drug resistance related proteins of p-PI3K and its downstream molecules p-Akt,p-ERK and p-mTOR by the two-drug combination were also confirmed by Western blot results(P<0.01,compared with control), and showed significant difference compared with each single treatment(P<0.05). CONCLUSION The addition of Xap significantly improves the antitumor activity of Gef in H460 and H1975 xenografts,and this synergistic effect may be ascribed to the inhibition of tumor angiogenesis,the down-regulation of PI3K and its downstream signaling molecules which are associated with drug resistance.