Erlichman C, Boerner S A, Hallgren C G, Spieker R, Wang X Y, James C D, Scheffer G L, Maliepaard M, Ross D D, Bible K C, Kaufmann S H
Division of Medical Oncology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Cancer Res. 2001 Jan 15;61(2):739-48.
Because the activities of HER family members are elevated and/or aberrant in a variety of human neoplasms, these cell surface receptors are receiving increasing attention as potential therapeutic targets. In the present study, we examined the effect of combining the HER family tyrosine kinase inhibitor CI1033 (PD 183805) with the topoisomerase (topo) I poison 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, in a number of different cell lines. Colony-forming assays revealed that the antiproliferative effects of simultaneous treatment with CI1033 and SN-38 were synergistic in T98G glioblastoma cells and HCT8 colorectal carcinoma cells, whereas sequential treatments were additive at best. In additional studies examining the mechanistic basis for these findings in T98G cells, immunoblotting revealed that the inhibitory effects of CI1033 on epidermal growth factor receptor autophosphorylation were unaffected by SN-38. Likewise, CI1033 had no effect on topo I polypeptide levels, localization, or activity. Nonetheless, CI1033 markedly enhanced the number of covalent topo I-DNA complexes stabilized by SN-38 or the related agent topotecan (TPT). Analysis of intracellular SN-38 levels by high-performance liquid chromatography and intracellular TPT levels by flow microfluorometry revealed that CI1033 increased the steady-state accumulation of SN-38 and TPT by 9.4 +/- 1.9- and 1.8 +/- 0.2-fold, respectively. Further evaluation revealed that the initial rate of TPT uptake was unaffected by CI1033, whereas the rate of efflux was markedly diminished. Additional studies demonstrated that T98G and HCT8 cells express the breast cancer resistance protein (BCRP), a recently cloned ATP binding cassette transporter. Moreover, CI1033 enhanced the uptake and cytotoxicity of SN-38 and TPT in cells transfected with BCRP but not empty vector. Conversely, CI1033 accumulation was diminished in cells expressing BCRP, suggesting that CI1033 is a substrate for this efflux pump. These results indicate that CI1033 can modulate the accumulation and subsequent cytotoxicity of two widely used topo I poisons in cells that have no history of previous exposure to these agents.
由于HER家族成员的活性在多种人类肿瘤中升高和/或异常，这些细胞表面受体作为潜在的治疗靶点正受到越来越多的关注。在本研究中，我们检测了HER家族酪氨酸激酶抑制剂CI1033（PD 183805）与拓扑异构酶（topo）I毒药7-乙基-10-羟基喜树碱（SN-38）（伊立替康的活性代谢产物）联合使用对多种不同细胞系的影响。集落形成试验显示，CI1033和SN-38同时处理对T98G胶质母细胞瘤细胞和HCT8结肠癌细胞的抗增殖作用具有协同性，而序贯处理充其量只是相加作用。在进一步研究T98G细胞中这些发现的机制基础时，免疫印迹显示CI1033对表皮生长因子受体自身磷酸化的抑制作用不受SN-38影响。同样，CI1033对topo I多肽水平、定位或活性也没有影响。尽管如此，CI1033显著增加了由SN-38或相关药物拓扑替康（TPT）稳定的共价topo I-DNA复合物的数量。通过高效液相色谱分析细胞内SN-38水平以及通过流式微荧光法分析细胞内TPT水平发现，CI1033分别使SN-38和TPT的稳态积累增加了9.4±1.9倍和1.8±0.2倍。进一步评估显示，TPT的初始摄取速率不受CI1033影响，而流出速率则明显降低。额外的研究表明，T98G和HCT8细胞表达乳腺癌耐药蛋白（BCRP），这是一种最近克隆的ATP结合盒转运蛋白。此外，CI1033增强了用BCRP转染但未用空载体转染的细胞中SN-38和TPT的摄取及细胞毒性。相反，在表达BCRP的细胞中CI1033的积累减少，这表明CI1033是这种流出泵的底物。这些结果表明，CI1033可以调节两种广泛使用的topo I毒药在既往没有接触过这些药物历史的细胞中的积累及随后的细胞毒性。
