Murra Nanke, Pommert Nina Sophie, Schmidt Berit, Issa Reema Sami, Kaehler Meike, Bruckmueller Henrike, Tim Vera, Cascorbi Ingolf, Waetzig Vicki
Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.
Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
Cancer Genomics Proteomics. 2025 Jan-Feb;22(1):90-102. doi: 10.21873/cgp.20490.
BACKGROUND/AIM: Treatment with retinoic acid (RA) often promotes neuroblastoma differentiation and growth inhibition, including the suppression of the expression of the MYCN oncogene. However, RA also targets protumoral chemokines, such as CCL2, which may contribute to the development of resistance. The present study aimed to investigate the regulation and function of CCL2 and N-Myc in RA-treated neuroblastoma cells.
In Kelly or SH-SY5Y cells, viability was quantified by cell fitness assays. Expression was analyzed using quantitative PCR and the regulation of proteins using enzyme-linked immunoabsorbent assays (ELISA) or western blots.
In MYCN-amplified Kelly cells, endogenous CCL2 levels were significantly lower compared to MYCN non-amplified SH-SY5Y cells. Treatment with 5 μM RA increased CCL2 release in both cell lines, but reduced N-Myc levels and cell numbers in Kelly cells. Over-expression of MYCN enhanced viability in SH-SY5Y cells, but did not affect RA-induced CCL2 release, while supplementation of CCL2 in Kelly cells did not prevent RA-mediated growth reduction. Impaired N-Myc or CCL2 signaling reduced the survival of all RA-treated cells and inhibition of N-Myc also decreased CCL2 levels. However, attenuated survival signaling was not generally associated with reduced levels of N-Myc or CCL2. Co-application of RA and the growth factor receptor inhibitors cediranib or crizotinib decreased N-Myc levels only in Kelly cells, while CCL2 release was dependent on the cell type and stimulus.
CCL2 and N-Myc promote the viability of RA-treated cells, although the levels of these mediators were not consistently correlated with cellular outcomes, especially during apoptotic signaling.
背景/目的：视黄酸（RA）治疗通常可促进神经母细胞瘤分化并抑制其生长，包括抑制MYCN癌基因的表达。然而，RA也靶向促肿瘤趋化因子，如CCL2，这可能导致耐药性的产生。本研究旨在探讨CCL2和N-Myc在RA处理的神经母细胞瘤细胞中的调控及功能。
在Kelly或SH-SY5Y细胞中，通过细胞适应性测定法对细胞活力进行定量。使用定量PCR分析基因表达，使用酶联免疫吸附测定（ELISA）或蛋白质印迹法分析蛋白质调控情况。
在MYCN扩增的Kelly细胞中，内源性CCL2水平显著低于MYCN未扩增的SH-SY5Y细胞。用5μM RA处理可增加两种细胞系中CCL2的释放，但降低Kelly细胞中N-Myc的水平和细胞数量。MYCN的过表达增强了SH-SY5Y细胞的活力，但不影响RA诱导的CCL2释放，而在Kelly细胞中补充CCL2并不能阻止RA介导的生长减少。N-Myc或CCL2信号受损会降低所有经RA处理细胞的存活率，抑制N-Myc也会降低CCL2水平。然而，生存信号减弱通常与N-Myc或CCL2水平降低无关。联合应用RA和生长因子受体抑制剂西地尼布或克唑替尼仅在Kelly细胞中降低N-Myc水平，而CCL2的释放则取决于细胞类型和刺激因素。
CCL2和N-Myc可促进经RA处理细胞的活力，尽管这些介质的水平与细胞结果并不始终相关，尤其是在凋亡信号传导过程中。
