Carboxylesterases expressed in human colon tumor tissue and their role in CPT-11 hydrolysis.
OBJECTIVE
The purpose is to develop new analytical methods to study the expression profile of CPT-11 carboxylesterases and topoisomerase I in colon tumor samples and understand the impact of their expression on CPT-11 metabolism in chemotherapy.


METHODS
We investigated 24 colon tumors for expression of carboxylesterases CES1A1, CES2, CES3, hBr-3, and topoisomerase I genes by real-time PCR and correlated the gene expression with activity assays. The relative abundance of the carboxylesterase isoenzymes and topoisomerase I genes was determined by real-time PCR. Activity assays performed on colon tumor extracts included CPT-11 hydrolase, 4-methylumbelliferyl acetate hydrolase, and topoisomerase I activity assays. Additionally, nondenaturing activity gel electrophoresis with activity staining showed the distribution of carboxylesterases.


RESULTS
We detect the expression of CES1A1, CES2, and CES3 carboxylesterase genes in human colon tumors. We were unable to detect the hBr-3 (also called hCE-3) in human liver, colon, or brain. We find large interindividual variation, >/=150-fold, for both CES1A1 and CES3 genes, 23-fold for CES2, and 66-fold for topoisomerase I. Only CES2 gene expression correlated with the carboxylesterase activity assays (P < 0.01) with CPT-11 and 4-methylumbelliferyl acetate as substrates. Nondenaturing activity gel electrophoresis showed that CES2 was the most predominant activity. Topoisomerase I gene expression significantly correlated with topoisomerase I activity (P < 0.01) in the colon tumors, but interindividual variation was very high.


CONCLUSIONS
We conclude that CES2 is the most abundant carboxylesterase in colon tumors that is responsible for CPT-11 hydrolysis. This pilot study reinforces the hypothesis that there is a large interindividual variation in expression of carboxylesterases that may contribute to variation in therapeutic outcome and/or toxicity of CPT-11 therapy for colon cancer.
Sanghani
Sonal P
SP
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Quinney
Sara K
SK
Fredenburg
Tyler B
TB
Sun
Zejin
Z
Davis
Wilhelmina I
WI
Murry
Daryl J
DJ
Cummings
Oscar W
OW
Seitz
David E
DE
Bosron
William F
WF
D015152:Blotting, Northern; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000378:metabolism*; D043182:Carboxylesterase / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000378:metabolism; D045744:Cell Line, Tumor; D003106:Colon / Q000378:metabolism; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D006868:Hydrolysis; D000077146:Irinotecan; D008099:Liver / Q000378:metabolism; D008297:Male; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014468:Umbelliferones / Q000378:metabolism
D014468:Umbelliferones; C032078:4-methylumbelliferyl acetate; D012313:RNA; D000077146:Irinotecan; D006867:Hydrolases; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin