Expression and Characterization of a Human Carboxylesterase 2 Splice Variant

Abstract

(1-piperidino)-1-piperidino] carbonyloxycamptothecin or Irinotecan] is a carbamate prodrug that is activated in vivo by carboxylesterase (CES)-2 to SN-38 (7-ethyl-10-hydroxycamptothecin), a potent topoisomerase I inhibitor. There is high interindividual variation when CPT-11 is used in the treatment of colorectal cancer. Several splice variants of CES2 are reported in the expressed sequence tag database. Real-time polymerase chain reaction was used to determine the abundance of the CES2 and splice variant of human carboxylesterase 2 (CES2⌬ 458 -473 ) transcripts in 10 paired samples of human tumor and normal colon tissue. The results showed that the CES2⌬ 458 -473 transcript accounts for an average of 6% of total CES2 transcripts in colon tissue, and there is large interindividual variation in CES2 expression in both tumor and normal colon samples. The carboxylesterase activity of the colon samples was determined by 4-methylumbelliferyl acetate hydrolysis assays and nondenaturing polyacrylamide gel electrophoresis followed by activity staining. Significant, positive correlations were found between CES2 expression levels and both measures of carboxylesterase activity. We cloned and expressed the CES2⌬ 458 -473 protein in Sf9 insect cells. The purification profiles and preliminary characterization of the CES2⌬ 458 -473 protein indicated that the expressed protein is folded and glycosylated like CES2. However, in vitro assays show that the CES2⌬ 458 -473 protein lacks 4-methylumbelliferyl acetate and irinotecan hydrolase activities. In conclusion, we found that the CES2⌬ 458 -473 protein is an inactive splice variant of CES2 and that its transcript is spliced at a relatively constant rate in tumor and normal colon tissue.CPT-11 is a semisynthetic camptothecin derivative that is approved for the treatment of metastatic colorectal cancer. Currently, several studies are evaluating the efficacy of CPT-11 in the treatment of solid tumors (Conroy et al., 2005;Delord et al., 2005;Reardon et al., 2005;Stathopoulos et al., 2005). Metabolism of CPT-11 is complex with the involvement of several enzymatic reactions (Rivory, 2000;Slatter et al., 2000). CYP3A4-dependent oxidation of CPT-11 results in the formation of APC and NPC (Haaz et al., 1998;Sai et al., 2001); carboxylesterase-dependent hydrolysis of CPT-11 and NPC results in the formation of the active metabolite SN-38 (Humerickhouse et al., 2000;Khanna et al., 2000;Sanghani et al., 2004) and UGT1A1 glucuronidates SN-38 to form the inactive SN-38-glucuronide (Hanioka et al., 2001). The key step in the metabolism of CPT-11 is its activation by carboxylesterases. There are three known human carboxylesterases, CES1, CES2, and CES3. CES2 has 100-and 2000-fold greater catalytic efficiency for irinotecan hydrolysis than CES1 and CES3, respectively . Therefore, CES2, which is predominantly expressed in the liver, small intestines, and colon (Satoh et al., 2002), is the most important carboxylesterase for the activation of CPT-11. Clinical data, reported by Xu et al....

Simultaneous Absolute Protein Quantification of Carboxylesterases 1 and 2 in Human Liver Tissue Fractions using Liquid Chromatography-Tandem Mass Spectrometry

“…For CES2, two isoforms (isoforms 1 and 2) are also available in the UniProtKB with isoform 1 being the canonical sequence. The sequence EASQAALQK is common to both CES2 isoforms; however, because the mRNA level of isoform 2 in the total CES2 transcript was very low (Schiel et al, 2007), the above sequence was assumed to be a specific fragment representing the function of CES2. CESs are known as glycoproteins (Kroetz et al, 1993) and therefore may complicate MS-based protein quantification because of posttranscriptional modifications such as glycosylation.…”

CES2, ABCG2, TS and Topo-I Primary and Synchronous Metastasis Expression and Clinical Outcome in Metastatic Colorectal Cancer Patients Treated with First-Line FOLFIRI Regimen

“…It has been shown that 5-FU significantly induces CES2 both in vitro and in vivo [ 8 ]. In particular, CES2 is highly expressed in colon tumors, playing a key role in the metabolic activation of CPT-11 [ 9 , 10 ]. Recent results have shown that the expression of CES2 is related to tumor staging [ 11 ].…”

Alternative splicing: An important regulatory mechanism in colorectal carcinoma

“…CPT‐11p, 7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino] (carbonyloxycamptothecin or irinotecan), is a carbamate prodrug used to treat metastatic CRC, whose essential metabolic step is to activate CPT‐11 via carboxylesterase 1 (CES1), including CES1, CES2, and CES3 150 . Schiel et al 151 discovered the CES2Delta (458–473) protein, which lacks irinotecan hydrolase activity. Its transcripts were spliced at a relatively constant rate in tumor and normal colon tissues.…”