CPT11 activation by carboxylesterases in colon cancer

这是响应NCI PA- 01-010“癌症检测、预后和预测的探索性研究”的R21发展资助申请。总体目标是开发一种CPT-11（伊立替康）羧酸酯酶测定方法，可用于预测接受CPT-11治疗的结直肠癌患者的治疗结果和/或毒性。CPT-11是一种半合成的前药，在体内被水解为SN-38。SN-38是一种有效的拓扑异构酶I抑制剂，治疗可抑制细胞生长。另一种重要的代谢物APC也被水解成SN-38。CPT-11和APC水解为SN-38的具体羧酸酯酶尚不清楚。两种人类羧酸酯酶hCE-1和hCE-2在肠道中高表达，这可能与CPT- 11治疗的主要毒性并发症腹泻有关。我们的假设是CPT-1和APC羧酸酯酶的组织和细胞特异性表达可能是APC羧酸酯酶相关治疗结果和毒性的重要决定因素。对GenBank中羧酸酯酶样基因的分析和对肿瘤细胞系的初步观察表明，可能存在其他羧酸酯酶可以催化CPT-11和APC的水解。蛋白质组学和PCR方法将用于筛选羧酸酯酶，CPT-11和APC作为底物将使用分离或表达的酶进行动力学分析。该资助的第二个科学目标是开发和验证使用活性分析、凝胶电泳或PCR方法分析CPT-11治疗患者肿瘤和正常结肠组织中CPT-11羧酸酯酶表达的分析方法。一项初步研究将从印第安纳大学癌症中心诊断为转移性疾病的患者身上收集肿瘤和正常组织进行。术后给予5-氟尿嘧啶、亚叶酸素和CPT-11治疗。对CPT-11治疗的反应和相关毒性将与肿瘤和正常结肠组织中羧酸酯酶的表达进行比较。如果存在正相关，未来将提出多机构研究。

This is a R21 developmental grant application in response to NCI PA- 01-010, "Exploratory studies in cancer detection, prognosis and prediction." The overall goal is to produce a CPT-11 (Irinotecan) carboxylesterase assay that could be used to predict treatment outcome and/or toxicity for patients on CPT-11 therapy for colorectal cancer. CPT-11 is a semi-synthetic pro-drug that is activated by hydrolysis in vivo to SN-38. SN-38 is a potent inhibitor of topoisomerase I and therapy inhibits cell growth. Another important metabolite called APC is also hydrolyzed to SN-38. The specific carboxylesterases responsible for the hydrolytic activation of CPT-11 and APC to SN-38 are not known. Two human carboxylesterases, hCE-1 and hCE-2 is highly expressed in intestine, which may be related to the major toxic complication of CPT- 11 therapy, diarrhea. Our hypothesis is that the tissue and cell-specific expression of CPT-1 and APC carboxylesterases may be an important determinant of the therapeutic outcome and toxicity associated with the APC carboxylesterases. Analysis of carboxylesterase-like genes in GenBank and preliminary observations in tumor cell lines suggest that there may be other carboxylesterases that could catalyze the hydrolysis of CPT-11 and APC. Proteomics and PCR methodologies will be used to screen for carboxylesterases and kinetic analysis with CPT-11 and APC as substrates will be performed with isolated or expressed enzymes. The second scientific aim of the grant is to develop and validate assays employing activity assays, gel electrophoresis or PCR methodologies for analysis of CPT-11 carboxylesterase expression in tumor and normal colon tissue from patients treated with CPT-11. A pilot study will be performed with tumor and normal tissue collected from patients at the Indiana University Cancer Center who presented with metastatic disease at diagnosis. After surgery the patients will be treated with 5- fluorouracil, Leucovorin and CPT-11. The response to CPT-11 therapy and associated toxicity will be compared to carboxylesterase expression in tumor and normal colon tissue. If there is a positive correlation, a future multi-institutional study will be proposed.