Portuguese propolis antitumoral activity in melanoma involves ROS production and induction of apoptosis

Melanoma is the most aggressive and life-threatening skin cancer type. The melanoma genome is the most frequently mutated, with the BRAF mutation present in 40–60% of melanoma cases. BRAF-mutated melanomas are characterized by a higher aggressiveness and progression. Adjuvant targeted treatments, such as BRAF and MEK inhibitors, are added to surgical excision in BRAF-mutated metastatic melanomas to maximize treatment effectiveness. However, resistance remains the major therapeutic problem. Interest in natural products, like propolis, for therapeutic applications, has increased in the last years. Propolis healing proprieties offer great potential for the development of novel cancer drugs. As the activity of Portuguese propolis has never been studied in melanoma, we evaluated the antitumoral activity of propolis from Gerês (G18.EE) and its fractions (n-hexane, ethyl acetate (EtOAc), and n-butanol) in A375 and WM9 melanoma cell lines. Results from DPPH•/ABTS• radical scavenging assays indicated that the samples had relevant antioxidant activity, however, this was not confirmed in the cell models. G18.EE and its fractions decreased cell viability (SRB assay) and promoted ROS production (DHE/Mitotracker probes by flow cytometry), leading to activation of apoptotic signaling (expression of apoptosis markers). Our results suggest that the n-BuOH fraction has the potential to be explored in the pharmacological therapy of melanoma.Foundation for Science and Technology (FCT)—projects UIDB/04050/2020, UDBI/04033/2020, UIDB/50026/2020, UIDP/50026/2020, UIDB/50006/2020, UIDP/50006/2020, and by the project NORTE-01-0145-FEDER-000055, supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). S.P.C., C.B.M. and A.S.F. are recipients of FCT grants (2020.05779.BD, SFRH/BD/145955/2019, and PD/BD/128276/2017, respectively

3-bromopyruvate boosts the effect of chemotherapy in acute myeloid leukemia by reducing cell antioxidant defense

Single Cause Single Cure Foundation. Fundação para a Ciência e Tecnologia (FCT) through the PhD grant (SFR/BD/146065/2019)NewG Lab Pharm

Estudo da função e influência terapêutica do gene supressor tumoral SPINT2 em Melanoma

Dissertação de mestrado em Ciências da SaúdeInvasion and metastasis are the leading causes of cancer mortality. That is related to the deregulation of proteases involved in the maintenance of the extracellular matrix and activation of advantageous signaling pathways in solid tumors. SPINT2 is a serine protease inhibitor identified as a tumor suppressor gene involved in HGF/MET signaling and epithelial to mesenchymal transition mechanism. SPINT2 promoter is hypermethylated in various solid tumors, leading to its downregulation, including in melanoma, the deadliest skin cancer. BRAF inhibitors are used for melanoma treatment, although survivors ultimately develop resistance. Therefore, evaluating the functional role and therapeutic potential of SPINT2 in melanoma are the main objectives of this project. For that, we produced stable transfectants of three melanoma cell lines to modulate SPINT2 expression and assessed cellular viability, migration, proliferation, and spheroids growth using two and three-dimensional cell culture models. Effect of SPINT2 deregulation in some mediators and pathways was evaluated to understand SPINT2 influence. Besides that, SPINT2 influence in tumor response to different drugs used in melanoma treatment as well MET inhibitors was evaluated. In vitro analysis showed that SPINT2 expression resulted in a decrease of the cell migration, cell viability, cell proliferation and tumor spheroid growth. Besides that, higher levels of E-cadherin and lower levels of N-cadherin were observed with SPINT2 presence. Moreover, SPINT2 expression seemed to correlate with a decrease of p-STAT3, pERK and p-p90rsk levels. Results regarding melanoma cell sensitivity to targeted therapies available, demonstrated that SPINT2 expression increased the sensibility to BRAF and MEK inhibitors, in BRAF V600E mutated cell lines and decreased to MET inhibitors. In summary, our results confirmed the functional role of SPINT2 as a tumor suppressor gene in melanoma. Also, a correlation of SPINT2 absence and epithelial to mesenchymal transition markers was seen, as with proteins related to tumor progression. Our results showed that SPINT2 absence in melanoma is related to a more aggressive tumor phenotype and the potential for SPINT2 as a biomarker of melanoma therapy. Moreover, the use of MET inhibitors for melanoma treatment with SPINT2 downregulation can be a way to overcome resistance. Nevertheless, in future studies it will be important to clarify the therapeutic influence of SPINT2 in melanoma cells, investigate the tumorigenic role and therapeutic influence of SPINT2 in vivo, as well as address these questions in studies of cohorts of human melanoma samples.Invasão e metástases são as principais causas de mortalidade relacionadas com o cancro. Estas são consequentes da desregulação de proteases envolvidas na manutenção da matriz extracelular e ativação de vias de sinalização vantajosas aos tumores sólidos. O SPINT2 é um inibidor de proteases de serina identificado como um gene supressor tumoral envolvido na via HGF/MET e na transição celular epitelial a mesenquimal. O promotor do SPINT2 encontra-se metilado em vários tumores sólidos, provocando a sua sub-expressão, inclusive em melanoma, o cancro de pele mais letal. Os inibidores de BRAF têm sido utilizados para o tratamento de melanoma, no entanto os pacientes acabam por desenvolver resistência. Desta forma, avaliar o papel funcional e o potencial terapêutico do SPINT2 em melanoma são os principais objetivos deste trabalho. Para isso, transfectamos três linhas celulares de melanoma para inverter a sua expressão de SPINT2 e avaliamos a viabilidade celular, migração, proliferação e crescimento de esferoides através de cultura celulares tri-dimensionais e bi-dimensionais. O efeito da desregulação do SPINT2 em alguns mediadores e vias de sinalização também foi avaliado. A influência do SPINT2 na resposta tumoral a diferentes drogas usadas em melanoma e inibidores de MET, foi avaliado. Análises in vitro relacionaram a expressão de SPINT2 a diminuição do crescimento de esferoides, migração, viabilidade e proliferação celular. Níveis mais elevados de E-caderina e mais baixos de N caderina foram observados na presença de SPINT2. Expressões proteicas de p-STAT3, pERK e p-p90rsk parecem diminuir com a expressão deste gene. Resultados sobre a sensibilidade das linhas celulares às terapias moleculares dirigidas mostraram que a expressão do SPINT2 aumenta a sensibilidade aos inibidores de BRAF e MEK, em linhas celulares com mutação BRAF V600E, e diminui em relação aos inibidores de MET. Em resumo, os resultados confirmaram o papel funcional do SPINT2 como um gene supressor tumoral. Mostraram uma correlação entre o SPINT2 e marcadores de transição celular epitelial a mesenquimal e proteínas relacionadas com a progressão tumoral. Os nossos resultados mostraram, que a ausência de SPINT2 em melanoma está relacionada com um fenótipo tumoral mais agressivo, o potencial de SPINT2 como um biomarcador terapêutico de melanoma, e que o uso de inibidores de MET no tratamento de melanomas com sub-expressão de SPINT2 pode ser uma forma de superar a resistência. Nos próximos avanços deste projecto, seria importante esclarecer a influência terapêutica do SPINT2 em melanoma, investigar o papel tumorigénico e a influência terapêutica do SPINT2 in vivo, bem como estudar estas correlações em séries de amostras de doentes com melanoma.The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), Minho University. Financial support was provided by FEDER funds through the Operational Programme Competitiveness Factors - COMPETE and National Funds through FCT - Foundation for Science and Technology under the project POCI-01-0145-FEDER-007038; and by the project NORTE-01-0145-FEDER 000013, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)

Phenolic Compounds Contribution to Portuguese Propolis Anti-Melanoma Activity

Melanoma is the deadliest type of skin cancer, with about 61,000 deaths annually worldwide. Late diagnosis increases mortality rates due to melanoma’s capacity to metastasise rapidly and patients’ resistance to the available conventional therapies. Consequently, the interest in natural products as a strategy for drug discovery has been emerging. Propolis, a natural product produced by bees, has several biological properties, including anticancer effects. Propolis from Gerês is one of the most studied Portuguese propolis. Our group has previously demonstrated that an ethanol extract of Gerês propolis collected in 2018 (G18.EE) and its fractions (n-hexane, ethyl acetate, and n-butanol) decrease melanoma cell viability. Out of all the fractions, G18.EE-n-BuOH showed the highest potential as a melanoma pharmacological therapy. Thus, in this work, G18.EE-n-BuOH was fractioned into 17 subfractions whose effect was evaluated in A375 BRAF-mutated melanoma cells. The subfractions with the highest cytotoxic activity were analysed by UPLC-DAD-ESI/MSn in an attempt to understand which phenolic compounds could account for the anti-melanoma activity. The compounds identified are typical of the Gerês propolis, and some of them have already been linked with antitumor effectiveness. These results reaffirm that propolis compounds can be a source of new drugs and the isolation of compounds could allow its use in traditional medicine