Combination of Rapamycin and MK-2206 Induced Cell Death via Autophagy and Necroptosis in MYCN-Amplified Neuroblastoma Cell Lines.

Yudi Dong,Wei Gong,Bo Chen,Zhijie Li,Carol J Thiele,Zhongyan Hua,Zhihui Liu,Guifeng Zhao

doi:10.3389/fphar.2020.00031

Abstract

Neuroblastoma (NB) is the most common pediatric malignant extracranial solid tumor. Despite multi-modality therapies, the emergence of drug resistance is an obstacle in the treatment of high-risk NB patients (with MYCN amplification). In our previous study, we found that rapamycin and MK-2206 synergistically induced cell death in MYCN-amplified cell lines but the mechanisms remained unclear. In our present study, either 3-MA or necroatatin-1 blocked the cell death induced by rapamycin and MK-2206, but z-VAD-fmk did not block this cell death. The expressions of autophagy markers (ATG5, ATG7, Beclin-1, LC3 B) and the necroptosis marker RIPK3 increased and another necroptosis marker RIPK1 decreased after the combination treatment of rapamycin and MK-2206, and were accompanied by the morphological characteristics of autophagy and necroptosis. In NB xenograft tumor tissues, the expressions of autophagy and necroptosis markers were consistent with observations in vitro. These data suggested that autophagy and necroptosis contributed to the cell death induced by rapamycin and MK-2206 in NB cells. To understand the role of MYCN in this process, MYCN expression was downregulated in MYCN-amplified cell lines (NGP, BE2) using siRNAs and was upregulated in MYCN non-amplified cell lines (AS, SY5Y) using plasmid. We found the cell death induced by rapamycin and MK-2206 was MYCN-dependent. We also found that the metabolic activity in NB cells was correlated with the expression level of MYCN. This study delineates the role of MYCN in the cell death induced by combination treatment of rapamycin and MK-2206 in MYCN-amplified NB cells.

Highlights

Neuroblastoma (NB) is the most common pediatric malignant extracranial solid tumor and is derived from cells of the neural crest
To investigate whether the cell death induced by rapamycin and MK-2206 was caspase-dependent, NGP and BE2 cells were pretreated with pan-caspase inhibitor z-VAD-fmk before administration of rapamycin and/or MK-2206
Pretreatment of the NB cells with z-VAD-fmk before the administration of rapamycin+MK2206 resulted in a 5.5% difference in NGP cells and 2.5% difference in BE2 cells for the cell viability, indicating that z-VAD-fmk did not block the cell death induced by a combination of rapamycin and MK-2206 (Figure 1E)

Summary

Introduction

Neuroblastoma (NB) is the most common pediatric malignant extracranial solid tumor and is derived from cells of the neural crest. Its incidence is only 7%–8%, it contributes to 15% of all pediatric cancer mortality (Smith et al, 2010; Gatta et al, 2014). The current treatments include chemotherapy, radiotherapy, surgery, biotherapies and immunotherapies. According to the international NB stage system (INSS), patients' clinical stage, histological grade and MYCN gene amplification, NB patients are divided into low, moderate, and high risk groups (Maris et al, 2007). Despite multi-modality therapies, one of the barriers to treatment in high-risk patients with NB is drug resistance. Novel agents for the NB treatment are urgently needed

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Results

Conclusion