R. A. Clark1, S. Lee1, J. Qiao1, D. H. Chung1 1University Of Texas Southwestern Medical Center,Pediatric Surgery,Dallas, TX, USA
Introduction:
Pralatrexate is a folate analogue inhibitor of dihydrofolate reductase exhibiting high affinity for reduced folate carrier-1 (RFC-1) with antineoplastic and immunosuppressive activities that are similar to methotrexate. Pralatrexate is approved in the US for the treatment of relapsed or refractory peripheral T-cell lymphoma and is being investigated in various malignancies. Despite intense research advances, the overall survival for children with high-risk neuroblastoma has failed to improve. Therefore, the search continues for the best single agent or combination of chemotherapy drugs to treat these tumors with high relapse rate. Previous studies have demonstrated that the gene encoding RFC1, SLC19A1, is associated with MYCN amplification in neuroblastoma, suggesting a potential role for Pralatrexate. Herein, we sought to assess the inhibitory role of pralatrexate in neuroblastoma cell growth.
Methods:
The IC50 values of select chemotherapeutic agents were measured using four human neuroblastoma cell lines, and they were calculated using GeneData Screener Condoseo software. Antiproliferative effects of pralatrexate were evaluated using the Cell Titer Glo or CCK8 assay. Adherent and non-adherent colony formation assays with pralatrexate were used to determine in vitro cell growth measures. Cell cycle arrest and cellular apoptosis were demonstrated by flow cytometry. Myc expression was examined by western blot, and SLC19A1 mRNA was examined by Q-pcr.
Results:
Treatment with pralatrexate in all four human neuroblastoma cell lines blocked cell growth in 2D and 3D culture conditions, and inhibited cell viability in a time-dependent manner. Pralatrexate-induced apoptosis was confirmed by caspase-3/7 activation and PARP cleavage. The IC50 of pralatrexate was 10-fold lower in all 4 neuroblastoma cell lines when compared to the IC50 values of methotrexate, another folate analog metabolic inhibitor. SLC19A1 mRNA expression did not change under pralatrexate treatment, whereas Myc expression was affected by pralatrexate in neuroblastoma cells.
Conclusion:
Pralatrexate demonstrated effective inhibition of neuroblastoma cell growth and viability. Further, the IC50 of pralatrexate was significantly lower than methotrexate, a drug with high levels of toxicity in neuroblastoma patients, suggesting pralatrexate may be a safer alternative to methotrexate. Our findings support the further clinical development of pralatrexate, alone or in combination with certain chemotherapy drugs and targeted agents, as a potential therapeutic strategy for the treatment of high-risk neuroblastoma.