Calein C, a Sesquiterpene Lactone Isolated From Calea Pinnatifida (Asteraceae), Inhibits Mitotic Progression and Induces Apoptosis in MCF-7 Cells

Breast cancer is the most common cancer in women worldwide. Estrogen receptor-positive (ER+) breast cancer represents approximately 75% of diagnosed cases, while 15–20% of them are triple-negative (TN). Although there have been improvements in the therapeutic approach, the mortality rate remains elevated. Thus, it is necessary to identify new chemotherapeutic agents. The present study aimed to evaluate the effects of calein C, a sesquiterpene lactone isolated from Calea pinnatifida, on breast cancer cell lines MCF-7 (ER+), Hs578T (TN) and MDA-MB-231 (TN). Calein C significantly reduced the viability of all cell lines; however, MCF-7 cells were more responsive than MDA-MB-231 or Hs578T cells. Thus, the MCF-7 cell line was selected for further investigation. We demonstrated that calein C inhibited cell cycle progression in MCF-7 cells at M-phase. Increased frequency of mitosis was observed in calein C-treated samples compared to the control group, especially of the cell population in initial stages of the mitosis. These events were associated with the ability of calein C to modulate expression levels of critical regulators of mitosis progression. We observed a significant reduction in the relative mRNA abundance of PLK1 and AURKB along with a concomitant increase in CDKN1A (p21) in treated samples. In addition, calein C induced apoptosis in MCF-7 cells due to, at least in part, its ability to reduce the BCL2/BAX ratio. Therefore, our data provide evidence that calein C is an important antimitotic agent and should be considered for further in vivo investigations

Genomics and metabolomics study of the fungi Gymnopilus imperialis for the evaluation of its biosynthetic potential for the production of bioative metabolites

Os fungos basidiomicetos, cujos corpos de frutificação são popularmente conhecidos como cogumelos, são organismos que despertam o interesse devido à sua complexidade, funções ecológicas e produção de metabólitos especiais bioativos. Atualmente, em química de produtos naturais, a identificação de metabólitos pode ser realizada por meio de técnicas de desreplicação e metabolômica, onde é possível acessar o perfil metabolômico dos organismos em misturas complexas, sem a necessidade de isolamento e caracterização dos compostos. A ascensão das ferramentas metabolômicas atuais é resultado do avanço das técnicas analíticas como espectrometria de massas de alta resolução, e também de ferramentas de bio e quimioinformática que auxiliam na análise dos dados gerados por essas técnicas. Partindo da necessidade de explorar o potencial químico e biossintético dos basidiomicetos, esse trabalho visou avaliar o perfil metabolômico das espécies Gymnopilus imperialis, Galerina marginata, Neonothopanus gardneri, além de outras espécies do gênero Pleurotus, por meio de análises de cromatografia líquida de alta eficiência acoplada à espectrometria de massas de alta resolução (UHPLC­MS/MS) e desreplicação dos extratos fúngicos com o auxílio da plataforma Global Natural Product Social Molecular Networking (GNPS). Esse estudo também visou avaliar o potencial antitumoral de polissacarídeos de G. imperialis, por meio de avaliação da atividade imunomodulatória das frações contendo β­glicanas. A partir dessas análises foi possível observar um aumento na produção de de óxido nítrico (NO) em macrófagos murino RAW 264.7, induzindo polarização de macrófagos do tipo M1. Por outro lado, a partir das análises de desreplicação e redes moleculares, foi possível a anotação de 62 metabólitos por meio de biblioteca espectral e 24 metabólitos por análise das redes moleculares, sendo a maioria deles, provenientes da espécie G. imperialis. Baseado no grande número de metabólitos anotados, e na escassez de dados da literatura para esta espécie, foi realizado um estudo do potencial biossintético de G. imperialis, integrando a metabolômica e genômica. Para isso, foram utilizadas técnicas de genome mining para prospecção de clusters gênicos biossintéticos (BGCs), permintindo a anotação de 41 BGCs para a espécie, sendo os clusters relacionados a sesquiterpenos sintases (STSs) (29) e policetídeo sintases (PKSs) (2), os de maior destaque para a prospecção de metabólitos bioativos. Análises filogenéticas e de genômica comparativa entre G. imperialis e espécies correlatas, revelaram que, em sua maioria, os BGCs são conservados entre as espécies, o que sugere que produzam metabólitos especiais similares entre si. Logo, clusters biossintéticos conservados entre as espécies correlatas foram expressos heterologamente em organismo modelo Aspergillus oryzae para a produção de PKSs e STSs. A integração das ciências ômicas se mostrou uma importante ferramenta de prospecção e avaliação do potencial biossintético de metabólitos especiais de fungos basidiomicetos, permitindo conhecer os clusters gênicos biossintéticos, sugerir a atribuição de metabólitos desreplicados aos respectivos genes biossintéticos, e ainda conhecer o potencial biossintético de espécies correlatadas do gênero Gymnopilus e Galerina.Basidiomycete fungi, whose fruiting bodies are popularly known as mushrooms, are organisms that arouse interest due to their complexity, ecological functions and production of special bioactive metabolites. Currently, in natural product chemistry, the identification of metabolites can be performed through dereplication and metabolomics techniques, where it is possible to access the metabolomic profile of organisms in complex mixtures, without the need for isolation and characterization of compounds. The rise of current metabolomics tools is a result of advances in analytical techniques such as highresolution mass spectrometry, as well as bio and chemoinformatics tools that aid in the analysis of data generated by these techniques. Based on the need to explore the chemical and biosynthetic potential of basidiomycetes, this work aimed to evaluate the metabolomic profile of the species Gymnopilus imperialis, Galerina marginata, Neonothopanus gardneri, as well as other species of the genus Pleurotus, through coupled highperformance liquid chromatography analysis highresolution mass spectrometry (UHPLCMS/ MS) and dereplication of fungal extracts with the aid of the Global Natural Product Social Molecular Networking (GNPS) platform. This study also aimed to evaluate the antitumor potential of polysaccharides from G. imperialis, by evaluating the immunomodulatory activity of fractions containing βglycans. From these analyzes it was possible to observe an increase in the production of nitric oxide (NO) in RAW 264.7 murine macrophages, inducing polarization of type M1 macrophages. On the other hand, from the analysis of dereplication and molecular networks, it was possible to annotate 62 metabolites by means of a spectral library and 24 metabolites by analysis of molecular networks, most of them coming from the species G. imperialis. Based on the large number of annotated metabolites, and the lack of data in the literature for this species, a study of the biosynthetic potential of G. imperialis was carried out, integrating metabolomics and genomics. For this, genome mining techniques were used to prospect biosynthetic gene clusters (BGCs), allowing the annotation of 41 BGCs for the species, the clusters being related to sesquiterpene synthases (STSs) (29) and polyketide synthases (PKSs) (2), the most prominent for the prospection of bioactive metabolites. Phylogenetic and comparative genomic analyzes between G. imperialis and related species revealed that, for the most part, BGCs are conserved between species, which suggests that they produce special metabolites similar to each other. Therefore, conserved BGCs in all the related species were submitted to heterologous expression, in order to produce the expected PKSs and STSs in Aspergillus oryzae. The integration of omics sciences proved to be an important tool for prospecting and evaluating the biosynthetic potential of special metabolites of basidiomycete fungi, allowing to know the biosynthetic gene clusters, suggesting the attribution of unreplicated metabolites to the respective biosynthetic genes, and even knowing the biosynthetic potential of species correlates of the genus Gymnopilus and Galerina.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP: 2018/25646-4CAPES-PRINT: 88887.572373/20200