) We showed that 13-cis-retinoic acid (13-cis-RA) can inhibit cell growth and decrease expression of the MYCN oncogene in neuroblastoma(NBL) cells in vitro at drug levels obtained in patients in a phase I trial. Our randomized phase Ill trial showed that high- dose, pulse 13-cis-RA significantly improved survival for high-risk NBL when given after intensive chemotherapy, but not all patients benefitted from 13-cis-RA, and some NBL cell lines derived from patients who were clinically resistant to 13-cis-and trans-RA in vitro. We discovered that the RA derivative N-(4-hydroxyphenyl) retinamide (fenretinide; 4-HPR) is highly cytotoxic for NBL cell lines that are resistant to 13-cis-RA, and that 4-HPR caused large increases involved in the levels of ceramide (a lipid involved in apoptotic signaling) in NBL cell lines. Goal: To develop an optimal strategy of using 4-HPR to improve the survival of high-risk NBL patients by eradicating tumor cells resistant to 13-cis-RA. Hypothesis. We hypothesize that modulators of ceramide metabolism will synergistically enhance the anti-tumor activity of 4-HPR.
Specific Aims : We will (1) Determine if generation of ceramide is a key event leading to cytotoxicity for NBL and the mechanism by which 4-HPR induces ceramide. (2) Develop cell lines resistant to 4-HPR and study mechanisms of resistance. (3) Determine if agents which modulate ceramide metabolism enhance 4-HPR activity. (4) Determine cross-resistance between 4-HPR and alkylating agents, etoposide, and 13-cis- RA. Research Design. To determine if cytotoxicity mediated by high-dose 4-HPR is due to increased ceramide we will transfect a NBL cell line with an inducible vector containing glucosylceramide synthase, which will stunt ceramide in 4-HPR treated cells to a non-toxic form. Cell lines will be identified and developed that are resistant to 4-HPR to determine if 4-HPR resistance is mediated by a failure to increase ceramide in response to 4-HPR or by """"""""shunting"""""""" the ceramide to non-toxic forms. Agents that inhibit ceramide shunting will be evaluated for their ability to enhance the cytotoxicity of 4-HPR for NBL cell lines. 4-HPR and enhancing agents will be evaluated for their efficacy against NBL cell lines that are resistant to 13- cis-RA and chemotherapeutic drugs. Methods. We will measure cytotoxicity using the DIMSCAN assay and measure ceramide levels by thin-layer chromatography. Combinations of agents with 4-HPR that have enhanced cytotoxicity against NBL cell lines in vitro will be tested for general toxicity against fibroblasts and myeloid progenitors, and for toxicity and efficacy against NBL. This project, in combination with our ongoing clinical trials of 4-HPR, will provide data to optimize clinical trial design employing 4-HPR against NBL.
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