Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma.

Ana-Maria Florea,Steffen Sass,Neha Gopinath,Safa Mahgoub,Jennifer E Mccallum,Sharon Varghese,Dietrich Büsselberg,Elizabeth Varghese,Fabian J Theis,Guido Reifenberger,Irfan Helmy

doi:10.18632/oncotarget.15283

Abstract

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.

Highlights

Neuroblastoma (NB) accounts for approximately 7% of pediatric malignancies and is responsible for more than 10% of cancer-related mortality in children [1]
In human neuroblastoma cell lines we tested the sensitivity to cisplatin (CDDP) and topotecan (TOPO) (Figure 1A and 1B)
TOPO (0.1 nM to 1 μM) demonstrated a stronger cytotoxic effect compared to CDDP in all neuroblastoma cell lines tested and cell viability was significantly reduced in SH-SY5Y cell after 24 h, 48 h and 72 h of exposure (Figure 1Ai)

Summary

Introduction

Neuroblastoma (NB) accounts for approximately 7% of pediatric malignancies and is responsible for more than 10% of cancer-related mortality in children [1]. Approximately 20% of neuroblastoma patients show an inadequate response to induction therapy and/ or experience disease progression following initial therapy. Standard North American Children Oncology Group (COG) induction regimens include combinations of anthracyclines, DNA alkylating agents, platinum compounds and topoisomerase II inhibitors delivered every 21 days for 5 to 7 cycles. The International Society of Pediatric Oncology Europe Neuroblastoma uses a more rapid regimen in which cycles are delivered every 10 days and that demonstrated superior 5-year event-free www.impactjournals.com/oncotarget survival of 30%, compared with 18% for standard interval chemotherapy [1, 3]. The topoisomerase I inhibitor topotecan has demonstrated efficacy in recurrent NB and has recently been incorporated into the COG induction regimens [4, 1]. Cisplatin and topotecan are currently used in the treatment of patients with recurrent neuroblastoma [1, 3, 4]

Methods

Results

Conclusion