Colorectal Cancer-Specific Cytochrome P450 2W1: Intracellular Localization, Glycosylation, and Catalytic Activity

Abstract

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.

Endoplasmic reticulum anchored heme-oxygenase 1 faces the cytosol

“…However, a cytochrome p450 was recently identified that faces the ER lumen and maintains enzymatic activity, suggesting that electron transfer from CPR can take place to either side of the ER membrane. 17 In addition to CPR, also biliverdin reductase (BVR), the enzyme that catalyzes the formation of biliruy. gottlieb et al…”

Colon Cancer–Specific Cytochrome P450 2W1 Converts Duocarmycin Analogues into Potent Tumor Cytotoxins

“…Clinical studies indicate that increased levels of CYP2W1 expression positively correlate with the degree of tumor malignancy, as well as with decreased 10-year survival in patients with colon cancer (12,13). So far, several groups have reported on the metabolism of a few candidate substrates (i.e., benzopyrene, sterigmatocystin, aflatoxin B1, lysophospholipids, and fluorobenzothiazoles) for CYP2W1 expressed in E. coli or mammalian cells (14)(15)(16)(17)(18)(19). However, the potential of CYP2W1 as a specific target in cancer chemotherapy remains hitherto unexplored.…”

CYP2S1 and CYP2W1 Mediate 2-(3,4-Dimethoxyphenyl)-5-Fluorobenzothiazole (GW-610, NSC 721648) Sensitivity in Breast and Colorectal Cancer Cells

“…Thus, for inoperable CRC, discovery of novel chemotherapeutic agents is imperative. It has recently been reported that novel AhR-inducible cytochrome P450 2W1 (CYP2W1) is overexpressed in human colon cancer tissue (11,12). CYP2W1 expression is tumor-specific and regulated by DNA methylation status, that is, hypomethylation in the body of CYP2W1 results in enhanced protein expression.…”