In Vitro Assessment of the Role of p53 on Chemotherapy Treatments in Neuroblastoma Cell Lines

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.

TRAF inhibition drives cancer cell apoptosis and improves retinoic acid sensitivity in multiple cancers models

“…In this study, we noticed a decrease in p53 levels in the SK-OV-3 and MeWo cell lines following RA treatment, and the mechanism behind this effect is currently unknown. It is possible that the impact of retinoic acid treatment on p53 expression levels varies between cell lines, as a previous study reported that RA treatment reduced p53 expression levels in the SK-N-FI neuroblastoma cells but not in another neuroblastoma cell line, SK-N-Be (2) [ 47 ]. In addition, TRAF4, TRAF5, and TRAF6 possess E3 ubiquitin ligase activities that are involved in the degradation of many signaling molecules through ubiquitination events.…”

Acne Transcriptomics: Fundamentals of Acne Pathogenesis and Isotretinoin Treatment

“…Via binding to cellular retinoic-acid-binding protein 2 (CRABP2), ATRA is transported into the nucleus, where the transcription of p53 is upregulated in an RAR-dependent fashion [221]. The ATRA/RAR-mediated upregulation of the expression of p53 was also reported in other benign and malignant cells [222][223][224][225][226][227][228]. Preferential CRABP2 expression in the suprabasal layers of SGs is mandatory for ATRA-induced transcriptomic changes, resulting in sufficient sebum suppression [150].…”

Preclinical Models of Neuroblastoma—Current Status and Perspectives

“…Moreover, the neuroblastoma cell lines have been used in a wide spectrum of studies on genetic, biochemical, functional, and structural neuroblastoma characteristics, for example on the interaction of neuroblastoma cells with Schwann cells, the synthesis of neurotransmitters, differentiation and transdifferentiation processes, chromosomal structure, the role of MYCN expression, the importance of neuropeptide Y and hypoxia, and many others [ 57 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 ]. A recent CRISPR/Cas9 screen performed in a panel of neuroblastoma cell lines has led to the identification of EZH2 as a molecular driver of the MYCN -amplified neuroblastomas, paving the way for similar studies in the future [ 84 ], importantly, in 2D cell lines, potential chemotherapy [ 85 , 86 , 87 , 88 , 89 ], radiation [ 90 ], and immunotherapy [ 91 , 92 , 93 , 94 ] screening. Furthermore, they are utilized to determine the role of genetic and chromosomal alterations in tumor growth, development, and the possible prognosis of the disease [ 95 , 96 , 97 , 98 ].…”