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Introduction:

Phosphoinositide-specific phospholipase C (PI-PLC)-γ1 is activated downstream of receptor tyrosine kinases to promote cell motility. Inhibitors of PLC-γ1 are being explored as therapeutic agents for blocking cancer cell invasion and metastasis. The clinical development of these drugs requires the implementation of pharmacodynamic markers of target modulation. In this study we use magnetic resonance spectroscopy (MRS), a technique for non-invasively monitoring metabolism in cells, animals and humans, to explore metabolic biomarkers of PLC-γ1 downregulation in cancer cells with siRNA and novel small molecule inhibitors.

Methods:

The PC3LN3 human prostate carcinoma cell line was treated for 24h with 10 µM of three novel in-house developed PLC-γ1 selective inhibitors (compounds A, B and C) or a synthetic peptide (Pep) that competitively binds to PLC-γ1 and inhibits its activity. For RNAi experiments, stable clones of PC3LN3 cells were generated expressing either an inducible siRNA sequence specific for PLC-γ1 or a non-targeting siRNA sequence. For metabolic analysis, cells were extracted in equal volumes of cold methanol, chloroform and water and the aqueous fractions analysed by 1H and 31P MRS.

Results:

PLC-γ1 knockdown resulted in reduced cell adhesion and migration towards 5% foetal calf serum to about 30-52% relative to controls. Analysis of cell metabolism by 31P MRS indicated that PLC-γ1 knockdown clones exhibited reduced levels of phosphocholine (PCho) down to 34±14% relative to the control clones. Nucleotide-triphosphate levels also decreased by half in the knockdown clones relative to the controls. No significant alterations were recorded in the other detectable metabolites. A decrease in the PCho and total choline signal to 40±16% and 45±18% of controls, respectively, was also observed by 1H MRS analysis. To confirm that these metabolic alterations are a result of PLC-γ1 downregulation, parental PC3LN3 cells were exposed to pharmacologic inhibitors of PLC-γ1. Treatment with compounds A, B, C and Pep resulted in a reduction in the PCho signal to 62±11%, 66±4%, 78±1% and 75±2% respectively as seen by 31P MRS. No other changes were recorded in the 31P MR spectra of treated cells.

Conclusions:

This study has shown that inhibition of cancer cell motility via downregulation of PLC-γ1 with siRNA and small molecule inhibitors causes alterations in choline phospholipid metabolism that can be detected by MRS. These changes may in future serve as pharmacodynamic biomarkers for monitoring the action of PLC-γ1-targetted therapies during clinical trials.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA