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.
Footnotes
S.M. and M.I.-S. share last authorship.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the Swedish Cancer Foundation; The Swedish Research Council; and the Knut and Alice Wallenberg Foundation [Grant KAW2008.0149].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.067652.
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ABBREVIATIONS:
- P450
- cytochrome P450
- ER
- endoplasmic reticulum
- Endo H
- endoglycosidase H
- PNGase F
- N-glycosidase F
- HEK
- human embryonic kidney
- β2-AR
- β2-adrenergic receptor
- HPLC
- high-performance liquid chromatography
- A4QN
- 1,4-bis{[2-(dimethylamino-N-oxide) ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione.
- Received July 21, 2010.
- Accepted August 26, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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