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Updated: May 8, 2025
Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies
Published on: April 22, 2017
Regulation and Function of CCL2 and N-Myc in Retinoic Acid-treated Neuroblastoma Cells
Nanke Murra1, Nina Sophie Pommert1, Berit Schmidt1
1Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.
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.
Materials And Methods:
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.
Results:
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.
Conclusion:
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.
Insights
Retinoic acid (RA) treatment affects neuroblastoma cell viability by influencing N-Myc and CCL2. While both promote survival, their levels don't always correlate with outcomes, especially during apoptosis.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Retinoic acid (RA) typically inhibits neuroblastoma growth and differentiation by suppressing MYCN oncogene expression.
- RA's targeting of protumoral chemokines like CCL2 may induce resistance.
- The interplay between CCL2 and N-Myc in RA-treated neuroblastoma requires further investigation.
Purpose of the Study:
- To investigate the regulation and functional role of CCL2 and N-Myc in neuroblastoma cells treated with retinoic acid.
- To understand how MYCN amplification and CCL2 signaling impact cell viability and response to RA.
Main Methods:
- Quantitative PCR, ELISA, and Western blotting were used to analyze gene and protein expression.
- Cell viability was assessed using cell fitness assays in Kelly and SH-SY5Y neuroblastoma cell lines.
- MYCN and CCL2 signaling pathways were manipulated through overexpression and inhibition.
Main Results:
- MYCN-amplified Kelly cells had lower endogenous CCL2 than MYCN-non-amplified SH-SY5Y cells.
- RA treatment increased CCL2 release but decreased N-Myc levels and cell numbers in Kelly cells.
- Both N-Myc and CCL2 signaling impaired RA-treated cell survival, but their levels were not consistently correlated with cellular outcomes, particularly during apoptosis.
Conclusions:
- CCL2 and N-Myc are crucial mediators promoting the viability of neuroblastoma cells undergoing RA treatment.
- The correlation between CCL2/N-Myc levels and cellular outcomes is complex and not always direct, especially in the context of apoptotic signaling.
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