CYP2W1 polymorphism: functional aspects and relation to risk for colorectal cancer.

AIM: This study aims to investigate the possible association between the risk of colorectal cancer (CRC) and allelic variants of CYP2W1 and their functional properties. MATERIALS & METHODS: The distribution of three different CYP2W1 alleles (CYP2W1*1, CYP2W1*2 and CYP2W1*6) in 1785 CRC patients and 1761 healthy blood donors was determined using the TaqMan(®) (Applied Biosystems, CA, USA) allelic discrimination assay or allele-specific amplification. Corresponding gene products (CYP2W1.1, CYP2W1.2 and CYP2W1.6) were expressed in human colon cancer SW480 cells and their activities towards two different substrates, the duocarmycin analogs ICT2706 and ICT2726, were monitored. RESULTS: No significant differences in the distribution of CYP2W1*1, CYP2W1*2 and CYP2W1*6 alleles were found between CRC patients and controls. The CYP2W1.1, CYP2W1.2 and CYP2W1.6 variant enzymes were expressed at the similar levels in the transfected SW480 cells and had comparable kinetics in terms of the metabolism of the duocarmycin ICT2726, as well as in the bioactivation of ICT2706 into a cytotoxic product. CONCLUSION: These epidemiological data obtained from a large population of CRC patients and controls cannot confirm the previously suggested decreased risk for CRC among carriers of CYP2W1*2. On the molecular level, this conclusion is further supported by the similar catalytic characteristics of the CYP2W1.1, CYP2W1.2 and CYP2W1.6 variants of CYP2W1.

Re-engineering of the duocarmycin structural architecture enables bioprecursor development targeting CYP1A1 and CYP2W1 for biological activity.

A library of duocarmycin bioprecursors based on the CPI and CBI scaffolds was synthesized and used to probe selective activation by cells expressing CYP1A1 and 2W1, CYPs known to be expressed in high frequency in some tumors. Several CPI-based compounds were pM-nM potent in CYP1A1 expressing cells. CYP2W1 was also shown to sensitize proliferating cells to several compounds, demonstrating its potential as a target for tumor selective activation of duocarmycin bioprecursors.

Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins.

PURPOSE: Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, whereas its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706, could be activated by CYP2W1 into potent antitumor agents. EXPERIMENTAL DESIGN: The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on severe combined immunodeficient mice bearing CYP2W1-positive colon cancer xenografts. RESULTS: Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism shown in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue. CONCLUSION: Our findings suggest a novel approach for treatment of colon cancer that uses a locoregional activation of systemically inactive prodrug by the tumor-specific activator enzyme CYP2W1.

Colorectal cancer-specific cytochrome P450 2W1: intracellular localization, glycosylation, and catalytic activity.

Cytochrome P450 2W1 (CYP2W1) is expressed at high levels in colorectal cancer cells. Moreover, we have shown previously that a higher tumor expression is associated with less survival. In this study, we characterize post-translational modification, inverted endoplasmic reticulum (ER) topology, and catalytic activity of CYP2W1. The analysis of colorectal normal and cancer tissues and CYP2W1 overexpressing human embryonic kidney (HEK) 293 cells showed that a fraction of CYP2W1 is modified by N-glycosylation. Bioinformatic analysis identified Asn177 as the only possible glycosylation site of CYP2W1, which was supported by the inability of an N177A mutant to be glycosylated in HEK 293 cells. Analysis of the membrane topology indicated that unlike other cytochromes P450, CYP2W1 in HEK 293-transfected cells and in nontransfected Caco2TC7 and HepG2 cells is oriented toward the lumen of the ER, a topology making CYP2W1 available to the ER glycosylation machinery. Immunofluorescence microscopy and cell surface biotinylation experiments revealed approximately 8% of the CYP2W1 on the cell surface. Despite the reverse orientation of CYP2W1 in the ER membrane, apparently making functional interactions with NADPH-cytochrome P450 reductase impossible, CYP2W1 in HEK 293 cells was active in the metabolism of indoline substrates and was able to activate aflatoxin B1 into cytotoxic products. The study identifies for the first time a cytochrome P450 enzyme with a luminal ER orientation and still retaining catalytic activity. Together, these results suggest the possibility of using CYP2W1 as a drug target in the treatment of colon cancer using antibodies and/or specific CYP2W1 activated prodrugs.