Abstract

Neuroblastoma is the most frequent malignant extracranial solid tumor of infancy. The overall objective of this work consists of determining the presence of alterations in the p53/MDM2/p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. Firstly, we characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway by analysis of TP53 point mutations, MYCN and MDM2 amplification, and p14ARF methylation, homozygous deletions, and expression. Secondly, we chose SK-N-FI (mutated at TP53) and SK-N-Be(2) (wild-type TP53) cell lines, treated them with chemotherapeutic agents (doxorubicin, etoposide, cisplatin, and melphalan) and with two isomers of retinoic acid (RA): (9-cis and all-trans). Finally, we analyzed the distribution of the cell cycle, the induction of apoptosis, and the expression levels of p53, p21, and Bcl-2 in those two cell lines. P14ARF did not present promoter methylation, homozygous deletions, and protein expression in any of the 10 neuroblastoma cell lines. One TP53 point mutation was detected in the SK-N-FI cell line. MYCN amplification was frequent, while most cell lines did not present MDM2 amplification. Treatment of SK-N-FI and SK-N-Be(2) cells with doxorubicin, etoposide, cisplatin, and melphalan increased apoptosis and blocked the cycle in G2/M, while retinoic acid isomers induced apoptosis and decreased the percentage of cells in S phase in TP53 mutated SK-N-FI cells, but not in TP53 wild-type SK-N-Be(2) cells. Treatment with cisplatin, melphalan, or 9-cis RA decreased p53 expression levels in SK-N-FI cells but not in SK-N-Be (2). The expression of p21 was not modified in either of the two cell lines. Bcl-2 levels were reduced only in SK-N-FI cells after treatment with cisplatin. However, treatments with doxorubicin, etoposide, or 9-cis-RA did not modify the levels of this protein in either of the two cell lines. In conclusion, TP53 mutated SK-N-FI cells respond better to the retinoic isomers than TP53 wild-type SK-N-Be(2) cells. Although these are in vitro results, it seems that deciphering the molecular alterations of the p53/MDM2/p14ARF signaling pathway prior to treating patients of neuroblastoma might be useful for standardizing therapies with the aim of improving survival.

Highlights

  • IntroductionTreatment of neuroblastoma is multidisciplinary and includes surgery, chemotherapy, radiation therapy, and immunotherapy [1,2]

  • Introduction distributed under the terms andTreatment of neuroblastoma is multidisciplinary and includes surgery, chemotherapy, radiation therapy, and immunotherapy [1,2]

  • 1/3 of the mean ratio obtained in the non-doubtful samples, which makes it possible to rule out the presence of P14ARF homozygous deletions in these samples

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Summary

Introduction

Treatment of neuroblastoma is multidisciplinary and includes surgery, chemotherapy, radiation therapy, and immunotherapy [1,2]. Chemotherapy, used for the first time in neuroblastoma in 1946 [3], is indicated in disseminated neuroblastoma and in unresectable localized neuroblastoma, in which it is administered with the aim of facilitating subsequent conditions of the Creative Commons. Pharmaceuticals 2021, 14, 1184 resection of the tumor. In tumors with metastatic spread, chemotherapy is aimed at controlling metastases and reducing the primary tumor to facilitate its subsequent resection. In these cases, the response to induction chemotherapy is consolidated with myeloablative chemotherapy and autologous bone marrow progenitor cell transplantation [1,2].

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