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61	Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry Seibert, C., Davidson, B.R., Fuller, B.J., Patterson, Laurence H., Griffiths, W.J., Wang, Y. January 2009 (has links) No / Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total, 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labeled tryptic peptide and analyzed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labeled tryptic peptides and their natural unlabeled analogues quantification could be performed over the range of 0.1-1.5 pmol on column. Liver microsomes from four individuals were analyzed for CYP2E1 giving values of 88-200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 to 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP isoforms in a single sample. Amino acid sequence Chromatography Liquid Methods Cytochrome P-450 enzyme system Analysis Dilution mass spectrometry Electrophoresis Gel Two-dimensional Humans Label-free quantification LC-MS/MS 2D-LC-MS/MS Liver Metabolism Microsomes Molecular sequence data Stable isotope Tandem mass spectrometry REF 2014
62	Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins Travica, S., Pors, Klaus, Loadman, Paul, Shnyder, Steven, Johansson, I., Alandas, Mohammed N., Sheldrake, Helen M., Mkrtchian, S., Patterson, Laurence H., Ingelman-Sundberg, M. January 2013 (has links) No / 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. Animals Bystander effect Cell line Colonic neoplasms Cytochrome P-450 enzyme system Cytotoxins DNA damage Drug resistance Female Gene expression Heterocyclic compounds Humans Indoles Mice Tissue distribution Tumor burden Xenograft model antitumor assays REF 2014 Tumor 3-Ring Neoplasm Drug effects Pharmacokinetics Toxicity Metabolism Enzymology Genetics Pathology

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Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry

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