Enhancing Retinoic Acid-mediated Effects Through Inhibition of CYP26A1, CYP26B1 and HGF Signaling in Neuroblastoma Cells.

Abstract

Background/aim: Retinoic acid (RA) induces tumor cell differentiation in diseases like acute promyelocytic leukemia or high-risk neuroblastoma. However, the formation of resistant cells, which results from dysregulation of different signaling pathways, limits therapy success. The present study aimed to characterize basic regulatory processes induced by the application of RA in human neuroblastoma cells, to identify therapeutic targets independent of the often amplified oncogene MYCN.

Materials and methods: In MYCN-amplified Kelly and MYCN non-amplified SH-SY5Y cells, different assays were employed to quantify the viability and cytotoxicity, while RA-mediated expression changes were examined using genome-wide gene expression analysis followed by quantitative PCR. Enzyme-linked immunoabsorbent assays (ELISA) and western blots were used to determine the levels or activation of the examined proteins.

Results: In Kelly cells, treatment with 5 μM RA for 3 days significantly reduced the cell number due to attenuated proliferation, while SH-SY5Y cells were less responsive. An up-regulation of the RA-metabolizing enzymes CYP26A1 and CYP26B1 was observed in both cell lines, and co-treatment with the selective CYP26 inhibitor talarozole markedly decreased cell viability. When RA and ketoconazole, which inhibits CYP26 as well as RA-degrading CYP3A enzymes, were co-administered, not only cell survival was impaired in both cell lines, but also the release of hepatocyte growth factor (HGF). Accordingly, co-application of the c-Met inhibitor tepotinib and RA or ketoconazole substantially decreased cell viability.

Conclusion: Independent of MYCN amplification, inhibitors of RA metabolism or HGF signaling might prevent the emergence of RA-resistant neuroblastoma cells when co-applied with RA.