Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Kiel, Germany.
Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Kiel, Germany
Anticancer Res. 2024 Oct;44(10):4189-4202. doi: 10.21873/anticanres.17249.
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.
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.
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.
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.
背景/目的：维甲酸（RA）可诱导急性早幼粒细胞白血病或高危神经母细胞瘤等疾病中的肿瘤细胞分化。然而，不同信号通路的失调导致耐药细胞的形成，从而限制了治疗的成功。本研究旨在描述 RA 应用于人类神经母细胞瘤细胞中诱导的基本调控过程，以确定独立于经常扩增的癌基因 MYCN 的治疗靶点。
在 MYCN 扩增的 Kelly 和 MYCN 非扩增的 SH-SY5Y 细胞中，采用不同的测定方法来量化细胞活力和细胞毒性，而 RA 介导的表达变化则通过全基因组基因表达分析结合定量 PCR 进行检测。酶联免疫吸附测定（ELISA）和 Western blot 用于测定所检测蛋白的水平或激活情况。
在 Kelly 细胞中，用 5 μM RA 处理 3 天可显著减少细胞数量，因为细胞增殖受到抑制，而 SH-SY5Y 细胞的反应较小。在两种细胞系中均观察到 RA 代谢酶 CYP26A1 和 CYP26B1 的上调，并且用选择性 CYP26 抑制剂 talarozole 共同处理可显著降低细胞活力。当 RA 和酮康唑（同时抑制 CYP26 和降解 RA 的 CYP3A 酶）共同给药时，不仅两种细胞系的细胞存活受到损害，而且肝细胞生长因子（HGF）的释放也受到损害。因此，c-Met 抑制剂 tepotinib 与 RA 或酮康唑的共同应用可显著降低细胞活力。
无论 MYCN 扩增如何，RA 代谢或 HGF 信号抑制剂与 RA 联合应用时可能会防止出现对 RA 耐药的神经母细胞瘤细胞。
