Cytotoxic activity of difluoromethylornithine compared with fenretinide in neuroblastoma cell lines

Background: Maintenance therapy with 13-cis-retinoic acid and immunotherapy (given after completion of intensive cytotoxic therapy) improves outcome for high-risk neuroblastoma patients. The synthetic retinoid fenretinide (4-HPR) achieved multiple complete responses in relapse/refractory neuroblastoma in early-phase clinical trials, has low systemic toxicity, and has been considered for maintenance therapy clinical trials. Difluoromethylornithine (DFMO, an irreversible inhibitor of ornithine decarboxylase with minimal single-agent clinical response data) is being used for maintenance therapy of neuroblastoma. We evaluated the cytotoxic activity of DFMO and fenretinide in neuroblastoma cell lines.

Procedure: We tested 16 neuroblastoma cell lines in bone marrow-level hypoxia (5% O₂ ) using the DIMSCAN cytotoxicity assay. Polyamines were measured by HPLC-mass spectrometry and apoptosis by transferase dUTP nick end labeling (TUNEL) using flow cytometry.

Results: At clinically achievable levels (100 μM), DFMO significantly decreased (P < 0.05) polyamine putrescine and achieved modest cytotoxicity (<1 log (90% cytotoxicity). Prolonged exposures (7 days) or culture in 2% and 20% O₂ did not enhance DFMO cytotoxicity. However, fenretinide (10 μM) even at a concentration lower than clinically achievable in neuroblastoma patients (20 μM) induced ≥ 1 log cell kill in 14 cell lines. The average IC₉₀ and IC₉₉ of fenretinide was 4.7 ± 1 μM and 9.9 ± 1.8 μM, respectively. DFMO did not induce a significant increase (P > 0.05) in apoptosis (TUNEL assay). Apoptosis by fenretinide was significantly higher (P < 0.001) compared with DFMO or controls.

Conclusions: DFMO as a single agent has minimal cytotoxic activity for neuroblastoma cell lines.