The antitumor activity of natural flavonoids has been exhaustively reported. Previously it has been demonstrated that prenylation of flavonoids allows the discovery of new compounds with improved antitumor activity through the activation of caspase-7 activity. The synthesis of twenty-five flavonoids (4–28) with one or more alkyl side chains was carried out. The synthetic approach was based on the reaction with alkyl halide in alkaline medium by microwave (MW) irradiation. The in vitro cell growth inhibitory activity of synthesized compounds was investigated in three human tumor cell lines. Among the tested compounds, derivatives 6, 7, 9, 11, 13, 15, 17, and 18 revealed potent growth inhibitory activity (GI₅₀ < 10 μM), being the growth inhibitory effect of compound 13 related with a pronounced caspase-7 activation on MCF-7 breast cancer cells and yeasts expressing human caspase-7. A quantitative structure-activity relationship (QSAR) model predicted that hydrophilicity, pattern of ring substitution/shape, and presence of partial negative charged atoms were the descriptors implied in the growth inhibitory effect of synthesized compounds. Docking studies on procaspase-7 allowed predicting the binding of compound 13 to the allosteric site of procaspase-7.

The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by a subset of chronically activated memory T cells and plays an important role in their activation and proliferation. Here, we show that primary malignant T cells isolated from patients with Sézary syndrome (SS) express Kv1.3 and are sensitive to potent Kv1.3 inhibitors ShK and Vm24, but not sensitive to a less potent inhibitor [N17A/F32T]-AnTx. Kv1.3 blockade inhibits CD3/CD28-induced proliferation and IL-9 expression by SS cells in a concentration-dependent manner. In parallel, CD3/CD28-mediated CD25 induction is inhibited, whereas Kv1.3 blockade has no effect on apoptosis or cell death as judged by Annexin V and PI staining. In conclusion, we provide the first evidence that malignant T cells in SS express functional Kv1.3 channels and that Kv1.3 blockade inhibits activation-induced proliferation as well as cytokine and cytokine receptor expression in malignant T cells, suggesting that Kv1.3 is a potential target for therapy in SS.

In our quest to discover antitumor agents with novel mechanisms of action, our strategy concerned multiple molecular modifications in a chemical core.

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).