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Targeting neuroblastoma with a new inhibitor of the TAp73 interaction with MDM2 and mutant p53
Liliana Raimundo * 1 , Sara Gomes 1 , Joana Soares 1 , Joana B. Loureiro 1 , Mariana Leão 1 , Helena Ramos 1 , Madalena N. Monteiro 1 , Juliana Calheiro 1 , Nair Nazareth 1 , Joana Almeida 1 , Agostinho Lemos 2 , Joana Moreira 2 , Madalena Pinto 2 , Petr Chlapek 3, 4 , Renata Veselsk 3, 4 , Emilia Sousa * 2 , Lucília Saraiva * 5
1  LAQV/REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal
2  CIIMAR, Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal
3  Laboratory of Tumor Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
4  International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic.
5  LAQV/REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal.

Published: 30 October 2019 by MDPI AG in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5
10.3390/ECMC2019-06305
Abstract:

TAp73 is a key tumour suppressor protein, regulating the transcription of unique and shared p53 target genes with crucial roles in tumorigenesis and therapeutic response. As such, in tumours with impaired p53 signalling, like neuroblastoma (NBL), TAp73 activation represents an encouraging strategy, alternative to p53 activation, to suppress tumour growth and chemoresistance [1]. Actually, NBL represents one of the most common childhood solid cancers but despite the available treatments, the high-risk patients are still characterized by low survival rates, making the search of new therapeutic options an urgent need [2].

In this work, we report the synthesis and biological evaluation of a new TAp73-activating agent, the 1-carbaldehyde-3,4-dimethoxyxanthone (LEM2), as a potent antitumour agent either alone or in combination with conventional drugs [3]. LEM2 anticancer activity was evaluated in the wild-type p53 expressing human colon adenocarcinoma HCT116 cells (HCT116 p53+/+) and in the respective p53-null isogenic derivative cells (HCT116 p53-/-), by sulforhodamine B assay. Results showed that LEM2 had potent p53-independent tumour growth inhibitory effect, with similar IC50values in p53+/+(0.98 ± 0.12 μM) and p53−/−(0.68 ± 0.08 μM) HCT116 cells. The antiproliferative effect of LEM2 on the growth of human tumour cells expressing distinct mutant p53 forms was also investigated, with IC50values around 1–3 μM. LEM2 antitumor effect was associated with enhanced TAp73 transcriptional activity, cell cycle arrest, and apoptosis in p53-null and mutant p53-expressing tumour cells. Importantly, the LEM2 antiproliferative effect was not associated with genotoxicity. Mechanistically, LEM2 was able to disrupt the TAp73 interaction with MDM2 and mutant p53, both in yeast and in human tumour cells. Additionally, by cellular thermal shift assay (CETSA), it was verified that LEM2 induced thermal stabilization of TAp73α but not of MDM2 or mutant p53, suggesting the potential interaction of LEM2 with TAp73α. LEM2 also displayed potent antitumour activity against immortalized and patient-derived NBL cells [4,5], consistent with an activation of the TAp73 pathway. LEM2 alcohol and carboxylic acid derivativeswere also tested to support that the LEM2 biological activity was due to the molecule itself and not to its potential derivatives. Indeed, both the alcohol 1-(hydroxymethyl)-3,4-dimethoxy-9H-xanthen-9-one (LEMred) and the carboxylic acid 3,4-dimethoxy-9-oxo-9H-xanthene-1-carboxylic acid (LEMox) presented a much lower activity when compared to LEM2 and were unable to inhibit the TAp73-MDM2 interaction.

The antiproliferative effect of LEM2, alone and in combination with doxorubicin and cisplatin, was analysed by MTT assay and the synergistic effect was evaluated through determination of combination and dose reduction index values. The results showed a potent TAp73-dependent cytotoxic activity of LEM2, superior to that of nutlin-3a (a known TAp73 activator), associated with induction of cell cycle arrest and apoptosis and upregulation of TAp73 target genes, in NBL cells. Also, we observed a pronounced synergistic effect between doxorubicin/cisplatin and LEM2, which may result from enhancing TAp73 activation through alternative pathways.

In conclusion, besides its relevant contribution to the advance of TAp73 pharmacology, LEM2 may pave the way to improved therapeutic alternatives against NBL, both alone and in combination with conventional chemotherapeutics.

1 – Wolter J et al. Future Oncol., 2010; 6: 429-444; 2- Nakagawara A et al. Jpn. J. Clin. Oncol., 2018; 48:214-24; 3 – Gomes S et al. Cancer letters., 2019; 0304-3835; 4 - Veselska R et al.Cancer, 2006 6: 32; 5 - Veselska R et al. BMC Cancer, 2008; 8: 300

This work received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through grant UID/QUI/50006/2019. This work received financial support from the European Union (FEDER funds through the Operational Competitiveness Program (COMPETE) POCI-01-0145-FEDER-006684/POCI-01-0145-FEDER-007440 and (3599-PPCDT) PTDC/DTP-FTO/1981/2014 – POCI-01-0145-FEDER-016581) and the FCT grants PTDC/QUIQOR/29664/2017, POCI-01-0145-FEDER-028736 (PTDC/SAU-PUB/28736/2017). We thank FCT and ESF (European Social Fund) through POCH (Programa Operacional Capital Humano) for: L. Raimundo PhD grant ref. SFRH/BD/117949/2016; J. Loureiro PhD grant ref SFRH/BD/128673/2017; H. Ramos PhD grant ref SFRH/BD/119144/2016. J. Calheiro thanks ICETA for her grant ref. ICETA2019-71. M. Monteiro thanks ICETA for her grant ref. ICETA2019-70 We thank (POCH), specifically the BiotechHealth Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences; PD/00016/2012).

Keywords: p73; Carbaldehydic xanthone; Anticancer therapy
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