One of the most important goals that NCI has established in the area of cancer treatment has been for the conversion of cancer from an acute and lethal disease to a chronic condition that can be managed long term. Two requirements are needed to achieve this goal: 1) the development of new anticancer drugs and treatment regimens utilizing these drugs, and 2) the ability to accurately and non-invasively assess the effectiveness of these treatment regimens. Differentiating agents are an attractive possibility in this area of cancer treatment, for they are agents that can either cause reversion of the malignant phenotype or the triggering of apoptosis. Our goal in this project is to delineate the mechanisms by which phenylacetate (PA) and phenylbutyrate (PB), two differentiating agents currently under Phase II clinical trials, inhibit the proliferation of cancer cells. We will concentrate on the changes in phosphatidylcholine (PtdCho) metabolites that can be observed using in vivo magnetic resonance spectroscopy (MRS) with the goal of determining the spectroscopic indicators that can reliably be used as an index for the inhibition of proliferation and induction of apoptosis by PA and PB. This could provide a basis for clinical monitoring of response to differentiation therapy by 1H MRS. In vivo animal tumor studies will be combined with mechanistic studies on perfused cells, using MR spectroscopy with fluorescence microscopy, flow cytometry and molecular biology techniques to identify key checkpoints in PtdCho catabolism associated with differentiation therapy. Our preliminary studies indicate that PA and PB induce changes in MR-visible phospholipid metabolite levels that correlate with the induction of apoptosis. Using fluorescent phospholipase-activated phospholipid analogues, we have identified two different phospholipase activities in prostate cancer cells: one that is constitutive and primarily nuclear, and one that is cytoplasmic and inducible by PA or PB. Thus, the aims of this application are to i) measure in vitro and in vivo phospholipase activation induced by differentiating agents, ii) identify the phospholipase isoform contributing to spectral changes in PBinduced apoptosis; iii) examine the effects of phospholipase inhibition on the MR-visible metabolites in tumor cells undergoing apoptosis and iv) to investigate the potential of MR-visible resonances as a marker for response to differentiation therapy in murine tumor models. These studies provide a mechanistic underpinning for the changes induced in phospholipid metabolism by differentiation therapy and therefore constitute an important advance in the understanding of choline metabolism for the interpretation of in vivo MR spectra of tumors.
