Novel extrahepatic P450 enzymes with emphasis on the tumor specific CYP2W1

Cytochrome P450 is a superfamily of enzymes involved in the metabolism of endogenous and exogenous compounds. The P450 enzymes are expressed at high levels in the liver, but are also found in extrahepatic tissues and in transformed tissues. The expression of P450s in tumors has drawn interest because their presence might influence cancer therapy and since the enzymes may be utilized as drug targets in new cancer therapy strategies. In the current study two novel extrahepatic P450s, CYP2U1 and CYP2W1, were identified and characterized. The expression of CYP2W1 in transformed tissues was investigated, resulting in the proposal for CYP2W1 as a novel potential drug target for colon cancer therapy. CYP2U1 was cloned and identified as a highly conserved P450, having an ortholog in Fugu fish (Takifugu rubripes). The gene contained only 5 exons and the deduced amino acid sequence harbored two unique NH2-terminal stretches, with one of them being proposed to be important for efficient folding. The CYP2U1 cDNA was expressed in HEK293 cells yielding a properly folded enzyme. Antibodies were developed against a human COOH-terminal sequence, and analysis showed that the CYP2U1 mRNA and/or protein from human and rat were found to be expressed especially in brain, in particular in the limbic structures and cortex, and in thymus. It is suggested that the enzyme can be used as a target for drugs in brain. CYP2W1, initially found as a partial clone in HepG2 cells, was cloned and expressed in HEK293 cells yielding an enzyme with a proper folding capable of metabolizing arachidonic acid. Antibodies were developed and showed the presence of three different immunoreactive bands in Western blotting, probably representing different post-translational modifications. In adult human tissues, CYP2W1 mRNA was found to be expressed at no or very low levels. In rat, high mRNA expression was seen in fetal colon, with expression levels increasing by fetal age and then decreasing again after birth, but CYP2W1 was not seen in other adult tissues investigated. CYP2W1 mRNA was expressed in human tumors originating from the adrenals and colon. High CYP2W1 mRNA and protein expression were especially seen in Caco2TC7 cells and colon tumors. The CYP2W1 gene was found to contain CpG islands, and demethylation of a CpG island located in the exon1-intron 1 region appeared to be required for expression in tumor samples. It is concluded that CYP2W1 most probably is a colonic enzyme expressed in fetal life and overexpressed in about 50% of human colon tumors, which suggest the use of the enzyme as drug target in cancer therapy. However, more studies are needed in order to fully understand its role in tumor biology and its potential as drug target. LIST OF PUBLICATIONS I. Karlgren M, Backlund M, Johansson I, Oscarson M, Ingelman-Sundberg M. Characterization and tissue distribution of a novel human cytochrome P450 CYP2U1. Biochem Biophys Res Commun. 2004, 315: 679-685. II. Karlgren M, Miura S, Ingelman-Sundberg M. Novel extrahepatic cytochrome P450s. Toxicol Appl Pharmacol. 2005, 207: S57-S61. III. Karlgren M, Gomez A, Stark K, Svard J, Rodriguez-Antona C, Oliw E, Bernal ML, Ramon y Cajal S, Johansson I, Ingelman-Sundberg M.Tumorspecific expression of the novel cytochrome P450 enzyme, CYP2W1. Biochem Biophys Res Commun. 2006, 341: 451-458. IV. Karlgren M, Ingelman-Sundberg M. Tumour-specific expression of CYP2W1: Its potential as a drug target in cancer therapy. Expert Opin Ther Targets. 2007, 11: 61-67. V. Gomez A*, Karlgren M*, Edler D, Bernal ML, Mkrtchian S, IngelmanSundberg M. Expression of cytochrome P450 2W1 (CYP2W1) in colon tumors; regulation by gene methylation. Manuscript. *these authors contributed equally to this work