Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma
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Ana-Maria Florea1, Elizabeth Varghese2, Jennifer E. McCallum2, Safa Mahgoub2, Irfan Helmy2, Sharon Varghese2, Neha Gopinath2, Steffen Sass3, Fabian J. Theis3, Guido Reifenberger1, Dietrich Büsselberg2
1Institute of Neuropathology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
2Weill Cornell Medicine in Qatar, Qatar Foundation-Education City, Doha, Qatar
3Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, Germany
Ana-Maria Florea, email: [email protected]
Keywords: neuroblastoma, cisplatin, topotecan, calcium signaling, chemotherapy
Received: September 20, 2016 Accepted: January 27, 2017 Published: February 11, 2017
Neuroblastoma (NB) is a pediatric cancer treated with poly-chemotherapy including platinum complexes (e.g. cisplatin (CDDP), carboplatin), DNA alkylating agents, and topoisomerase I inhibitors (e.g. topotecan (TOPO)). Despite aggressive treatment, NB may become resistant to chemotherapy. We investigated whether CDDP and TOPO treatment of NB cells interacts with the expression and function of proteins involved in regulating calcium signaling. Human neuroblastoma cell lines SH-SY5Y, IMR-32 and NLF were used to investigate the effects of CDDP and TOPO on cell viability, apoptosis, calcium homeostasis, and expression of selected proteins regulating intracellular calcium concentration ([Ca2+]i). In addition, the impact of pharmacological inhibition of [Ca2+]i-regulating proteins on neuroblastoma cell survival was studied. Treatment of neuroblastoma cells with increasing concentrations of CDDP (0.1−10 μM) or TOPO (0.1 nM−1 μM) induced cytotoxicity and increased apoptosis in a concentration- and time-dependent manner. Both drugs increased [Ca2+]i over time. Treatment with CDDP or TOPO also modified mRNA expression of selected genes encoding [Ca2+]i-regulating proteins. Differentially regulated genes included S100A6, ITPR1, ITPR3, RYR1 and RYR3. With FACS and confocal laser scanning microscopy experiments we validated their differential expression at the protein level. Importantly, treatment of neuroblastoma cells with pharmacological modulators of [Ca2+]i-regulating proteins in combination with CDDP or TOPO increased cytotoxicity. Thus, our results confirm an important role of calcium signaling in the response of neuroblastoma cells to chemotherapy and suggest [Ca2+]i modulation as a promising strategy for adjunctive treatment.
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