In animal models of liver cancer, treatment with almost any chemopreventive agent produces changes in the abundance of hepatic proteins. In the 1990's, structurally dissimilar chemoprotective agents were shown to alter the balance between the metabolic activation and detoxication of many experimental carcinogens. More recently, transgenic animals and oligonucleotide microarray technologies were used to explore transcriptional regulatory pathways through which chemopreventive agents work. Chemopreventive agents (e.g. sulforaphane, sulindac) originally thought to primarily work by altering classically described phase I and II metabolism or directly inhibiting inducible cycloxygenase are being found to work through additional mechanisms that promote apoptosis in cancer cells. Today, new proteomic discovery tools, such as difference in gel electrophoresis (DIGE), enable the detection of changes in the relative abundance of proteins without the need to identify proteins in advance. Although not cancer endpoints themselves, hepatic proteins whose relative abundances are modulated can provide insights into mechanisms of chemopreventive agent action. These hepatic proteins may be exploited to develop serum biomarkers of responsiveness to chemopreventive treatment. Normal human serum contains small amounts of non-secreted proteins that are predominantly located in the nucleus, mitochondrion and cytoplasm. How they arrive in blood is unknown but probably involves necrosis, apoptosis and a poorly defined phenomenon called """"""""tissue leakage"""""""". Conceptually, the serum concentration of a leaked protein could achieve a steady-state value reflecting its total abundance in different organs. Because of the liver's large size, hepatocytes are expected to be major contributors of non-secreted proteins found in normal human serum. The long-term objectives of this work are to identify hepatic protein biomarkers of responsiveness to chemopreventive intervention and to develop mass spectrometric methods for their analysis in serum. Maturation of these assays should ultimately enable the measurement of serum proteins to determine responsiveness to liver cancer chemopreventive interventions in clinical trials. Initial scientific interest in the triterpenoid 1-[2-cyano-3-12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO- Im) was generated by its unusually potent inhibition of nitric-oxide production (Honda 2002). In addition to their anti-inflammatory properties, CDDO congeners are cytoprotective of healthy cells but exert pro-apoptotic and anti-proliferative effects on different cancer cell types. These properties suggested their use as cancer chemopreventive and therapeutic agents (Sporn and Liby 2005;Yore 2006). The mechanisms through which triterpenoids exert their biological effects remain to be completely elucidated. CDDO congeners are currently in clinical phase I cancer trials and CDDO-Im was recently demonstrated to be the most potent chemopreventive agent ever tested in a rat model of hepatic tumorigenesis (Yates 2006). In this proposal, CDDO-Im will be administered to rats to evaluate the feasibility of developing serum proteins as biomarkers of hepatic responsiveness to chemoprotective intervention.
Aim I : To detect candidate biomarkers of liver responsiveness to treatment, DIGE studies will be performed following the treatment of rats with a previously established chemopreventive dosing regimen of CDDO-Im. Differentially expressed hepatic proteins in nuclear, mitochondrial and cytoplasmic sub-cellular fractions will be identified via in-gel enzymatic digestion, mass spectrometric analysis and database searches.
Aim II : To measure the serum concentration of leaked hepatic proteins identified in Aim I, sample preparation methods and quantitative mass spectrometric assays will be developed. Guided by the physical properties of the candidate biomarker proteins, techniques for their extraction from serum will be established. Isotope dilution mass spectrometric assays will be developed to quantitatively measure the serum concentration of non-secreted proteins for use as biomarkers of hepatic responsiveness to treatment. Pilot DIGE data demonstrate CDDO-Im modulates the relative concentration of over forty cytosolic liver proteins. Their mass spectrometric identification revealed a variety of protein classes that include but are not limited to proteosome complex subunits, mitochondrial proteins, dehydrogenases, Se-binding proteins, aldehyde reductases and GSTs. Several GSTs were mass spectrometrically identified in extracted serum where, in a proof-of-concept demonstration, GST @ was conspicuously elevated in CDDO-Im treated rats.Narrative The long-term objectives of this work are to identify hepatic protein biomarkers of responsiveness to cancer chemopreventive intervention and to develop mass spectrometric methods for their quantitative analysis in serum. Maturation of these assays should ultimately enable the measurement of serum proteins to determine hepatic responsiveness to liver cancer chemopreventive interventions in clinical trials.
