Increasing the anti-tumor activity of CPT-11 by conversion into SN-38 in the tumor microenvironment
| Autor: | Yuan-Ting Hsieh, 謝源庭 |
|---|---|
| Rok vydání: | 2015 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 CPT-11, also known as Irinotecan, is a prodrug used for the first-line treatment of colorectal cancer. In humans, CPT-11 is hydrolyzed by human carboxylesterase 2 (hCE2) into SN-38. SN-38 is the active form of the drug that to serves as a DNA topoisomerase I inhibitor for killing cancer cells. However, the conversion is slow. Less than 5% of CPT-11 can be converted into SN-38. In addition, the majority of SN-38 is further conjugated with a glucuronide group by uridine-5’-diphosphate glucuronosyltransferase to become SN-38G, which is much less toxic than SN-38. In this study, we examined different strategies to improve CPT-11 therapeutic efficacy. In the first study, we overexpressed E. coli beta-glucuronidase in E. coli to selectively deliver the enzyme to tumors and convert SN-38G into SN-38. Engineered E. coli (lux/βG) expressed functional luciferase and beta-glucuronidase, sensitized cancer cells to SN-38G in vitro and targeted to tumors specifically. However, E. coli (lux/βG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli, E. coli (lux). We found that this phenomenon was caused by the preferential colonization of E. coli in necrotic areas of tumors and the cellular location of beta-glucuronidase in E. coli. These two impediments hindered the E. coli directed glucuronidase therapy efficiency. In the second study, we investigated the effect of hCE2 cellular location on CPT-11 hydrolysis and anticancer activity. Many studies showed that expression of hCE2 in the endoplasmic reticulum (ER) or as a secreted form sensitized cancer cells to CPT-11. However, the preferable cellular location for hCE2 gene therapy is still controversial. In this study, we directly compared hCE2 activity at different cellular locations with similar enzyme expression levels. We coexpressed enhanced green fluorescence protein (eGFP) either with membrane-tethered, secreted, or ER-retained hCE2 in cancer cells via a 2A peptide derived from foot-and-mouth disease virus. We showed that the hCE2 level in the cells correlated with eGFP intensity. We then isolated cancer cells that expressed similar hCE2 amounts by monitoring the green fluorescence intensity by fluorescence-activated cell sorting (FACS). We found that hCE2 expressed on the membrane or in the ER of cancer cells more efficiently hydrolyzed substrates and sensitized cancer cells to CPT-11 as compared to hCE2 secreted from the cells. Therefore, we suggest that ER or membrane expression of hCE2 are preferable locations to improve CPT-11 anticancer therapy. In the third study, we generated hCE2 mutants with higher enzyme activity by directed evolution. Since hCE2 is inefficient to convert CPT-11 to SN-38, expression of hCE2 mutants with higher enzyme activity in tumors might improve CPT-11 antitumor activity. We combined neutral drift and computational design to introduce mutations. Neutral drift induced mutations randomly distribute in the full length of hCE2 whereas hCE2 homology modeling-based computational design focused on 7 residues which interacted with 4-PP, a by-product produced from CPT-11 hydrolysis, in the backdoor region of hCE2. We isolated the hCE2 mutants with higher CPT-11 hydrolysis activity by enzyme cleavable surface tethered all-purpose screening system (ECSTASY). These clones were further shuffled by DNase I to generate a recombinant library combining the positive hot spots from these two libraries. The best clone from this recombinant library improved the CPT-11 hydrolysis activity up to 14 fold greater than wild type hCE2. These high activity hCE2 mutants might be useful for gene or antibody directed CPT-11 therapy in the future. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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