? In response to the RFA (EB-03-007), we will develop methods to image the expression and activity of phospholipases in vivo. A multi-modality approach with sequential magnetic resonance imaging/spectroscopy (MRI/MRS) and optical imaging using near infrared (NIR) fluorophores will be employed to obtain information that is not available from either technique separately. The key hypothesis to be addressed is that in vivo activation of phospholipase A2 can be measured by determining the changes in MR-visible lipid metabolite levels together with measuring fluorescence released by enzymatic hydrolysis of a self-quenching NIR phospholipid fluorophore. We will compare and validate the MR spectroscopy with results obtained using NIR optical imaging in a tumor model to which the lipid response to differentiating agents is well known. Since phospholipase A2 (PLA2) has been implicated in a wide range of pathologies including cancer, arthritis and Alzheimer's disease, this study will supply critical in vivo data on the specific biochemical pathways involved in these diseases. The hypothesis will be tested by the achievement of the following specific aims:
Aim 1) : Design of lipid-based self-quenching fluorophores containing a stable pyropheophorbide NIR fluorophore, coupled to a black hole quencher. The fluorophore will be attached to the sn-1 or sn-2 position of the glycerol backbone to interrogate phospholipase A1 and A2 activity. The quencher will be attached at different points in the head group designed to distinguish between phospholipase types.
Aim 2) : Demonstration that the self-quenching fluorophores can be delivered to a cultured human prostate cancer cell line, DU145, and that activation of phospholipases by treatment with the differentiating agent phenylbutryate (PB) will lead to increased NIR fluorescence in the prostate tumor cells Aim 3): Demonstration of in vivo delivery and subsequent activation of phospholipases accompanying treatment of prostate tumor xenografts with the differentiating agent PB. These investigations will provide a basis for future in vivo measurement of phospholipase activity in cancer, inflammatory and autoimmune diseases. ? ?
| Mawn, Theresa M; Popov, Anatoliy V; Beardsley, Nancy J et al. (2011) In vivo detection of phospholipase C by enzyme-activated near-infrared probes. Bioconjug Chem 22:2434-43 |
| Milkevitch, Matthew; Jeitner, Thomas M; Beardsley, Nancy J et al. (2007) Lovastatin enhances phenylbutyrate-induced MR-visible glycerophosphocholine but not apoptosis in DU145 prostate cells. Biochim Biophys Acta 1771:1166-76 |
| Milkevitch, Matthew; Shim, Hyunsuk; Pilatus, Ulrich et al. (2005) Increases in NMR-visible lipid and glycerophosphocholine during phenylbutyrate-induced apoptosis in human prostate cancer cells. Biochim Biophys Acta 1734:1-12 |
| Delikatny, Edward J; Poptani, Harish (2005) MR techniques for in vivo molecular and cellular imaging. Radiol Clin North Am 43:205-20 |
