Koizumi Fumiaki, Kanzawa Fumihiko, Ueda Yutaka, Koh Yasuhiro, Tsukiyama Shoji, Taguchi Fumiko, Tamura Tomohide, Saijo Nagahiro, Nishio Kazuto
Support Facility of Project Ward, National Cancer Center Hospital, Tokyo, Japan.
Int J Cancer. 2004 Jan 20;108(3):464-72. doi: 10.1002/ijc.11539.
Epidermal growth factor receptor [EGFR (HER1, erbB1)] is a receptor with associated tyrosine kinase activity, and is expressed in colorectal cancers and many other solid tumors. We examined the effect of the selective EGFR tyrosine kinase inhibitor (EGFR-TKI) gefitinib ("Iressa") in combination with the DNA topoisomerase I inhibitor CPT-11 (irinotecan) on human colorectal cancer cells. EGFR mRNA and protein expression were detected by RT-PCR and immunoblotting in all 7 colorectal cancer cell lines studied. Gefitinib inhibited the cell growth of the cancer cell lines in vitro with an IC(50) range of 1.2-160 microM by 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Lovo cells exhibited the highest level of protein and autophosphorylation of EGFR and were the most sensitive to gefitinib. The combination of gefitinib and CPT-11 induced supra-additive inhibitory effects in COLO320DM, WiDR and Lovo cells, assessed by an in vitro MTT assay. Administration of gefitinib and CPT-11 had a supra-additive inhibitory effect on WiDR cells and tumor shrinkage was observed in Lovo cell xenografts established in nude mice, whereas no additive effect of combination therapy was observed in COLO320DM cells. To elucidate the mechanisms of synergistic effects, the effect of CPT-11-exposure on phosphorylation of EGFR was examined by immunoprecipitation. CPT-11 increased phosphorylation of EGFR in Lovo and WiDR cells in time- and dose-dependent manners. This EGFR activation was completely inhibited by 5 microM gefitinib and gefitinib-induced apoptosis was enhanced by combination with CPT-11, measured by PARP activation although no PARP activation was induced by 5 microM CPT-11 alone. These results suggested that these modification of EGFR by CPT-11, in Lovo cells, is a possible mechanism for the synergistic effect of CPT-11 and gefitinib. These findings imply that the EGFR-TKI gefitinib and CPT-11 will be effective against colorectal tumor cells that express high levels of EGFR, and support clinical evaluation of gefitinib in combination with CPT-11, in the treatment of colorectal cancers.
表皮生长因子受体[EGFR(HER1,erbB1)]是一种具有相关酪氨酸激酶活性的受体，在结直肠癌和许多其他实体瘤中均有表达。我们研究了选择性EGFR酪氨酸激酶抑制剂(EGFR-TKI)吉非替尼（“易瑞沙”）与DNA拓扑异构酶I抑制剂CPT-11（伊立替康）联合应用对人结直肠癌细胞的影响。通过RT-PCR和免疫印迹法检测了所研究的所有7种结直肠癌细胞系中的EGFR mRNA和蛋白表达。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-四氮唑溴盐(MTT)法，吉非替尼在体外抑制癌细胞系的细胞生长，IC(50)范围为1.2 - 160 microM。Lovo细胞中EGFR的蛋白水平和自磷酸化水平最高，对吉非替尼最敏感。通过体外MTT法评估，吉非替尼和CPT-11联合应用在COLO320DM、WiDR和Lovo细胞中诱导了超相加抑制作用。给予吉非替尼和CPT-11对WiDR细胞具有超相加抑制作用，并且在裸鼠中建立的Lovo细胞异种移植瘤中观察到肿瘤缩小，而在COLO320DM细胞中未观察到联合治疗的相加作用。为了阐明协同作用的机制，通过免疫沉淀法检测了CPT-11作用对EGFR磷酸化的影响。CPT-11以时间和剂量依赖性方式增加了Lovo和WiDR细胞中EGFR的磷酸化。这种EGFR激活被5 microM吉非替尼完全抑制，并且通过PARP激活检测发现，与CPT-11联合应用增强了吉非替尼诱导的细胞凋亡，尽管5 microM CPT-11单独使用未诱导PARP激活。这些结果表明，在Lovo细胞中CPT-11对EGFR的这种修饰是CPT-11和吉非替尼协同作用的一种可能机制。这些发现意味着EGFR-TKI吉非替尼和CPT-11对表达高水平EGFR的结直肠肿瘤细胞将有效，并支持吉非替尼与CPT-11联合应用治疗结直肠癌的临床评估。
