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 开发拨款申请。总体目标是生产一种 CPT-11（伊立替康）羧酸酯酶测定法，可用于预测接受 CPT-11 治疗结直肠癌的患者的治疗结果和/或毒性。 CPT-11 是一种半合成前药，在体内水解成 SN-38 后被激活。 SN-38 是拓扑异构酶 I 的有效抑制剂，治疗可抑制细胞生长。另一种重要的代谢物 APC 也被水解为 SN-38。负责将 CPT-11 和 APC 水解活化为 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与 APC 羧酸酯酶相关的治疗结果和毒性的重要决定因素。 GenBank中的类羧酸酯酶基因分析和在肿瘤细胞系中的初步观察表明，可能存在其他可以催化CPT-11和APC水解的羧酸酯酶。将使用蛋白质组学和 PCR 方法来筛选羧酸酯酶，并使用 CPT-11 和 APC 作为底物进行动力学分析，并使用分离的或表达的酶进行底物。该资助的第二个科学目标是开发和验证采用活性测定、凝胶电泳或 PCR 方法的测定，用于分析接受 CPT-11 治疗的患者的肿瘤和正常结肠组织中的 CPT-11 羧酸酯酶表达。一项试点研究将从印第安纳大学癌症中心收集的肿瘤和正常组织进行，这些患者在诊断时患有转移性疾病。手术后，患者将接受 5-氟尿嘧啶、亚叶酸和 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的羧酸酯酶表达进行比较。如果存在正相关性，将提出未来的多机构研究。

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.