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PRIMA-1MET-induced neuroblastoma cell death is modulated by p53 and mycn through glutathione level.

ABSTRACT: BACKGROUND:Neuroblastoma is the most common extracranial solid tumor in children. This cancer has a low frequency of TP53 mutations and its downstream pathway is usually intact. This study assessed the efficacy of the p53 activator, PRIMA-1MET, in inducing neuroblastoma cell death. METHODS:CellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1MET. Real-time PCR and western blot were used to assess the most common p53 transactivation targets. Induction of p53 and Noxa, and inhibition of Cas3/7, were used to assess impact on cell death after PRIMA-1MET treatment. Flow cytometry was used to analyze cell cycle phase and induction of apoptosis, reactive oxygen species, and the collapse of mitochondrial membrane potential. RESULTS:Neuroblastoma cell lines were at least four times more susceptible to PRIMA-1MET than were primary fibroblasts and keratinocyte cell lines. PRIMA-1MET induced cell death rapidly and in all cell cycle phases. Although PRIMA-1MET activated p53 transactivation activity, p53's role is likely limited because its main targets remained unaffected, whereas pan-caspase inhibitor demonstrated no ability to prevent cell death. PRIMA-1MET induced oxidative stress and modulated the methionine/cysteine/glutathione axis. Variations of MYCN and p53 modulated intracellular levels of GSH and resulted in increased/decreased sensitivity of PRIMA-1MET. PRIMA-1MET inhibited thioredoxin reductase, but the effect of PRIMA-1MET was not altered by thioredoxin inhibition. CONCLUSIONS:PRIMA-1MET could be a promising new agent to treat neuroblastoma because it demonstrated good anti-tumor action. Although p53 is involved in PRIMA-1MET-mediated cell death, our results suggest that direct interaction with p53 has a limited role in neuroblastoma but rather acts through modulation of GSH levels.

SUBMITTER: Mlakar V

PROVIDER: S-EPMC6373164 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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PRIMA-1<sup>MET</sup>-induced neuroblastoma cell death is modulated by p53 and mycn through glutathione level.

Mlakar Vid V Jurkovic Mlakar Simona S Lesne Laurence L Marino Denis D Rathi Komal S KS Maris John M JM Ansari Marc M Gumy-Pause Fabienne F

Journal of experimental & clinical cancer research : CR 20190212 1

<h4>Background</h4>Neuroblastoma is the most common extracranial solid tumor in children. This cancer has a low frequency of TP53 mutations and its downstream pathway is usually intact. This study assessed the efficacy of the p53 activator, PRIMA-1<sup>MET</sup>, in inducing neuroblastoma cell death.<h4>Methods</h4>CellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1<sup>MET</sup>. Real-time PCR and western blot were used to assess the most common p53 transa ...[more]

PMID: 30755224

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Project description:P63 is a major transcription factor regulating skin development and homeostasis. It controls many genes involved in cell proliferation, adhesion, and early differentiation. P63 is mutated in several rare syndromes called p63-related ectodermal dysplasia syndromes (ED). The main forms are EEC and AEC syndromes due to p63 missense mutations on the DBD and SAM domains, respectively. ED patients display many developmental defects, including ectrodactyly, clef/lip palate, and ectodermal dysplasia, while AEC patients suffer from severe skin erosions that not always heal. We have previously showed that ED-derived iPSC display altered epidermal commitment. P63 belongs to the p53 gene family sharing similar structural domains. We found that ED-iPSC epidermal commitment can be rescued by a p53-reactivating compounds called PRIMA-1MET, also named APR-246 and currently used in anticancer clinical trials. Here, we established primary epidermal culture from two AEC children (S.F. and Y.M.) suffering from persistent skin erosions at age of 9 and 15, respectively. These patients carry missense mutations on the SAM domain (I576T and I537T). We found that primary keratinocytes (KCs) isolated from these AEC patients underwent altered epidermal differentiation that was rescued by PRIMA-1MET treatment. It prompted us to formulate the compound onto a cream that was topically applied on the right hand of one patient and on the scalp of the second patient. In both cases, the daily treatment allowed re-epithelialization of the eroded skin and a drastic loss of pain after few weeks, improving quality of life. Normally, mutant p63 exerts a dominant-negative effect, mainly through the formation of aggregate with WT p63 and p73. PRIMA-1MET did not reduce protein aggregation while enhancing cell differentiation, suggesting that PRIMA-1MET targets cell differentiation and not p63 activity directly. In conclusion, we propose that repurposing of the antitumoral PRIMA-1MET compound could become a general treatment of AEC skin erosions.

Dataset Information

PRIMA-1MET-induced neuroblastoma cell death is modulated by p53 and mycn through glutathione level.

Publications

PRIMA-1<sup>MET</sup>-induced neuroblastoma cell death is modulated by p53 and mycn through glutathione level.

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