Identification of cytochrome P450 enzymes in human colorectal metastases and the surrounding liver: a proteomic approach
Introduction
Cytochrome P450 proteins (CYPs) in the liver are known to be of major importance to the fate of anticancer agents; however their expression and role in tumours has received little attention. The completion of the draft of the human genome revealed the presence of approximately 100 different CYP genes, of atleast which 59 are thought to be expressed based on transcript identification [1]. CYP-mediated metabolism is generally viewed as a route to drug detoxification and increased elimination, although CYP activation of certain anticancer drugs, e.g., cyclophosphamide, dacarbazine and thiotepa, has long been known and the importance of this process as a way of targeting novel anticancer therapy is being explored [2]. CYPs are also responsible for reductive metabolism and can contribute to the activation of tumour hypoxia targeted cytotoxins [3]. The outcome of cancer therapeutic in part relies on the expression profile of an array of different CYP enzymes, especially in the liver, contributing to the metabolic fate of administered drugs. The heterogeneity of the human population means that a CYP profile in any one individual is unique due to a combination of genetic and environmental factors including the plethora of drugs used in treating disease. Determination of the CYP protein expression profile on an individual patient basis prior to cancer chemotherapy could provide important information regarding the fate of the selected drugs and hence outcome of therapy. In addition, the relative activity of liver and tumour drug metabolism will have a major impact in determining therapeutic outcome and in the development of tumour-specific prodrugs. Traditional methods for the detection of CYPs rely on immunoblotting, activity assays and the detection of CYP mRNA. These techniques have significant limitations. Immunoblotting, whilst being very sensitive, relies on the availability of enzyme-specific antibodies. In addition, it is necessary to pre-select which CYPs are to be investigated, and to identify each enzyme in turn. Activity assays that are designed to interrogate the activity of a CYP enzyme invariably require multiple analysis techniques, since different assays must be developed for different target substrates; even then, they may not be totally enzyme-specific. Measurement at the expression level is fraught with uncertainty since the presence and abundance of a particular type of mRNA does not necessarily infer the presence and abundance of the corresponding protein [4], [5], [6]. Mass spectrometry provides an attractive approach for the analysis of expressed proteins, offering uniquely the ability to directly detect low levels of multiple proteins in a single analytical run. Generally, two-dimensional gel electrophoresis (2DE) followed by matrix assisted laser desorption/ionisation–time-of-flight (MALDI–TOF) mass spectrometry and peptide mass fingerprinting (PMF) has become a principal approach for the proteomic profiling of various in vitro and in vivo biological systems [7], [8]. A major drawback of the 2DE approach is its low performance in the separation of membrane proteins including CYPs [9]. We have demonstrated the use of nano-scale reversed phase liquid chromatography (nano-LC), electrospray ionisation tandem mass spectrometry (ESI-MS/MS) in combination with one-dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) for the identification of CYPs in male and female rat liver [10]. The aim of the present study was to adopt a proteomic analysis of CYPs in tumour and liver samples, and to reveal its potential in the assessment of which CYP enzymes could contribute to the outcome of chemotherapy.
Section snippets
Subjects
The protocol for this study was approved by the Ethics Committee of the Royal Free Hospital and University College School of Medicine. Informed patient consent for use of tissues was obtained in all cases. Tissues for the study were accessed from the resected and discarded masses of tumour and surrounding liver, which were removed as part of the surgical treatment for hepatic metastases arising from colon cancers.
Tissue sampling
Samples of normal liver and of tumour (approximately 0.5 g) were taken in
Validation of the method
Initially, we evaluated the performance of the SDS–PAGE nano-LC–ESI-MS/MS procedure for the identification of CYPs by loading known amounts of purified recombinant human CYP enzymes 1A2, 2E1 and 3A4 onto the SDS–PAGE gel and proceeding through the analytical cycle to identify the proteins. It was found that CYPs could be successfully identified down to approximately 1 pmol loaded onto the gel (data not shown). A successful identification was defined as one in which two or more unique peptides
Discussion
The importance of tumour CYP expression in influencing the outcome of chemotherapy through resistance or prodrug activation is poorly understood [2]. Hence, there is a need to profile CYP expression in a way that does not rely on anticipating the CYPs present. This is the first study that investigates CYP enzymes in cancer without pre-selection of the proteins to be interrogated. In total, 14 CYP proteins were identified in colorectal metastases of human liver and matched liver samples. The
Acknowledgements
We thank Mike Cocksedge, Emmanuel Samuel and Amanda Wilgoss for their technical assistance.
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Present address: Department of Chemistry, University of Hull, Hull, UK.