Arina Lázaro Rochetti

Constitutive Androstane Receptor Ligands Modulate the Anti-Tumor Efficacy of Paclitaxel in Non-Small Cell Lung Cancer Cells

Background Lung tumors are the leading cause of cancer deaths worldwide and paclitaxel has proven to be useful for patients with lung cancer, however, acquired resistance is a major problem. To overcome this problem, one promising option is the use of Constitutive Androstane Receptor (CAR) ligands in combination with chemotherapeutics against cancer cells. Therefore, we wish to elucidate the effects of CAR ligands on the antineoplastic efficacy of paclitaxel in lung cancer cells. Methodology/Principal Findings Our results from cell viability assays exposing CAR agonist or inverse-agonist to mouse and human lung cancer cells modulated the antineoplastic effect of paclitaxel. The CAR agonists increased the effect of Paclitaxel in 6 of 7 lung cancer cell lines, whereas the inverse-agonist had no effect on paclitaxel cytotoxicity. Interestingly, the mCAR agonist TCPOBOP enhanced the expression of two tumor suppressor genes, namely WT1 and MGMT, which were additively enhanced in cells treated with CAR agonist in combination with paclitaxel. Also, in silico analysis showed that both paclitaxel and CAR agonist TCPOBOP docked into the mCAR structure but not the inverse agonist androstenol. Paclitaxel per se increases the expression of CAR in cancer cells. At last, we analyzed the expression of CAR in two public independent studies from The Cancer Genome Atlas (TCGA) of Non Small Cell Lung Cancer (NSCLC). CAR is expressed in variable levels in NSCLC samples and no association with overall survival was noted. Conclusions/Significance Taken together, our results demonstrated that CAR agonists modulate the antineoplastic efficacy of paclitaxel in mouse and human cancer cell lines. This effect was probably related by the enhanced expression of two tumor suppressor genes, viz. WT1 and MGMT. Most of NSCLC cases present CAR gene expression turning it possible to speculate the use of CAR modulation by ligands along with Paclitaxel in NSCLC therapy.

Antileishmanial activity of verbascoside: Selective arginase inhibition of intracellular amastigotes of Leishmania (Leishmania) amazonensis with resistance induced by LPS plus IFN-γ

Graphical abstract Figure. No Caption available. ABSTRACT Verbascoside is the main component of the traditional medicinal plants that were used against protozoa parasites that cause malaria and leishmaniasis. Previously, we have described verbascoside inhibition of Leishmania amazonensis arginase as well as its antileishmanial action against extracellular promastigotes. In this study, we have assessed arginase parasite inhibition in intracellular amastigotes. In addition, we verified whether verbascoside can influence the host defense against the parasite by measuring gene expression of cytokines IL‐1b, IL‐10, IL‐18, TNF‐&agr; and murine macrophage arginase as well as nitric oxide synthase enzymes. Our results show that verbascoside acts on intracellular amastigotes of L. amazonensis (EC50 = 32 &mgr;M) by selectively inhibiting the parasite arginase. Verbascoside did not affect the expression of cytokines or enzymes by murine macrophages. However, verbascoside was active against L. (L.) amazonensis amastigotes that were resistant to treatment with LPS and IFN‐&ggr;. Verbascoside action on L. amazonensis can be associated with reduction of the protective oxidative mechanism of the parasite leading to impaired trypanothione synthesis that is induced by the parasite arginase inhibition.

Expression of NR1I3 in mouse lung tumors induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanone

Nuclear receptor subfamily 1, group I, member 3 (NR1I3) is reported to be a possible novel therapeutic target for some cancers, including lung, brain and hematopoietic tumors. Here, we characterized expression of NR1I3 in a mouse model of lung carcinogenesis induced by 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanone (NNK), the most potent tobacco carcinogen. Lung tumors were collected from mice treated with NNK (400 mg/kg) and euthanized after 52 weeks. Benign and malignant lesions were formalin-fixed and paraffin-embedded for histology and immunohistochemistry, with samples snap-frozen for mRNA analysis. Immunohistochemically, we found that most macrophages and type I and II pneumocytes expressed NR1I3, whereas fibroblasts and endothelial cells were NR1I3−. Compared with benign lesions, malignant lesions had less NR1I3+ tumor cells. Gene expression analysis also showed an inverse correlation between NR1I3 mRNA expression and tumor size (P=0.0061), suggesting that bigger tumors expressed less NR1I3 transcripts, in accordance with our immunohistochemical NR1I3 tests. Our results indicate that NR1I3 expression decreased during progression of malignant lung tumors induced by NNK in mice.

Transcriptomic profile reveals molecular events associated to focal adhesion and invasion in canine mammary gland tumour cell lines

The prevalence of cancer in animals has increased significantly over the years. Mammary tumours are the most common neoplasia in dogs, in which around 50% are presented in the malignant form. Hence, the development and characterization of in vitro models for the study of canine tumours are important for the improvement of cancer diagnosis and treatment. Thus, the aim of this study was to characterize cell lines derived from canine mammary gland neoplasias which could be further used for basic and applied oncology research. Samples of canine mammary carcinomas were taken for cell culture and 2 cell lines were established and characterized in terms of cell morphology, tumourigenicity and global gene expression. Both cell lines presented spindle-shape morphology and shown common malignant features as in vitro invasion potential and expression of epithelial and mesenchymal proteins. Also, we found gene expression patterns between the 2 cell cultures in comparison to the normal mammary gland tissue. Cells from M25 culture showed a higher invasion and in vivo tumourigenic potential, associated to the overexpression of genes involved in focal adhesion and extracellular matrix communication, such as FN1, ITGA8 and THBS2. The phenotypic characterization of these cells along with their global gene expression profile potentially determine new therapeutic targets for mammary tumours.

Comparative genomic survey of Bacillus cereus sensu stricto isolates from the dairy production chain in Brazil

Abstract The genomes of 262 Bacillus cereus isolates were analyzed including 69 isolates sampled from equipment, raw milk and dairy products from Brazil. The population structure of isolates showed strains belonging to known phylogenetic groups II, III, IV, V and VI. Almost all the isolates obtained from dairy products belonged to group III. Investigation of specific alleles revealed high numbers of isolates carrying toxin‐associated genes including cytK (53.62%), hblA (59.42%), hblC (44.93%), hblD (53.62%), nheA (84.06%), nheB (89.86%) and nheC (84.06%) with isolates belonging to groups IV and V having significant higher prevalence of hblACD and group IV of CytK genes. Strains from dairy products had significantly lower prevalence of CytK and hblACD genes compared to isolates from equipment and raw milk/bulk tanks. Genes related to sucrose metabolism were detected at higher frequency in isolates obtained from raw milk compared to strains from equipment and utensils. The population genomic analysis demonstrated the diversity of strains and variability of putative function among B. cereus group isolates in Brazilian dairy production, with large numbers of strains potentially able to cause foodborne illness. This detailed information will contribute to targeted interventions to reduce milk contamination and spoilage associated with B. cereus in Brazil. Figure. No Caption available.

ZEB1 and ZEB2 transcription factors are potential therapeutic targets of canine mammary cancer cells

Mammary tumours are the most frequent in female dogs as in women and half are malignant. Tumorigenicity and invasiveness are important acquired characteristics for the development and progression of cancers and could be regulated by transcription factors associated with epithelial-mesenchymal transition (EMT) as ZEB1, ZEB2, SNAI1, SLUG and STAT3. Thus, here, we evaluate the expression of EMT-associated transcription factors in canine mammary cancer (CMC) cell lines characterized for invasiveness and tumorigenicity to determine if these could be considered good targets for future development of therapies. Five CMC cell lines were characterized regarding their morphology, doubling time and expression of intermediate and actin filaments. In addition, gene expression of SLUG, STAT3, ZEB1, ZEB2 and CDH1, tumorigenicity and invasiveness were assessed. Two of these cells presented an epithelial-like morphology (E20 and E37) and three a mesenchymal-like morphology (M5, M25 and CF41.Mg). M25 and CF41.Mg presented higher invasiveness. Furthermore, only mesenchymal-like cells formed tumorspheres and CF41.Mg made more and larger tumorspheres. The mesenchymal-like cells are more malignant than the epithelial-like cells being the CF41.Mg the most malignant. This cell presented higher ZEB1 and ZEB2 and lower CDH1 gene expression. Finally, our results revealed that there is a positive correlation between ZEBs and the tumorsphere number and size. In conclusion, these findings support ZEB1 and ZEB2 as potential therapeutic targets for CMC cells, demonstrating a great potential of canine models for comparative and translational studies.

Abstract 819: Transcriptomic profiling of two canine mammary cancer cell lines for translational oncology studies

Dogs develop spontaneous cancers as humans and shares a variety of features including histology, genomic alterations, molecular targets, biological behavior and response to conventional therapies. Although dogs can be considered good models for translation of new cancer treatments, there is a lack of established preclinical in vitro models of canine cancers. Therefore, our aim here was to establish two canine mammary cancer cell lines and characterize their transcriptomic profile to speed the translation of new cancer treatments from dogs to humans. Mammary cancer samples were obtained from female dogs that underwent surgery. Pieces of the tumors were FFPE for histopathology and the other fragments were washed with PBS, minced and dissociated with hyaluronidase and collagenase. Cells were filtered and put in culture with DMEM-F12, 5%FBS and 1% pen-strep. After 5 passages, FBS was increased to 10%. After passage 15, cells were characterized for doubling time, karyotype, immunocytochemistry for cytokeratin, vimentin and α-smooth muscle actin, matrigel-invasion assay and tumor growth in nude mice. At last, RNA-seq was performed on total RNA extracted with TRIZOL for functional enrichment analysis of differentially expressed (DE) genes from cancer cell lines. From 49 tumor samples, we characterized 2 cell lines named M5 and M25, originally from a comedocarcinoma and a mixed carcinoma, respectively. Both cell cultures showed spindle-shape cells and were positive for cytokeratin, vimentin and α-smooth actin, suggesting myoepithelial origin. The doubling time was 26.0h for M5 and 42.8h for M25 cells. Karyotyping analysis showed moderate aneuploidy for both cell cultures. M25 cell line presented significant higher invasion potential than M5 cells and formed tumors in mice but the latter didn’t. Histopathology of these tumors resembled the original tumor in the dog. An average of 16.2 million paired-end 100 bp reads were sequenced per replicate (three for each cell culture) with an average of 81.1% alignment to the reference genome. A total of 11,260 and 11,476 genes with more than 1 count per million were detected in M5 and M25 cell lines. M5 cell line have 404 and 394 up- and downregulated genes in comparison to M25 cell line, respectively. Gene ontology of DE genes showed the more malignant cell (M25) significantly enriched for pathways related to drug metabolism, ECM-receptor interaction, cell cycle, DNA replication, focal adhesion and cell differentiation. In conclusion, M25 cell line is more malignant than the M5 which is supported by their transcriptome. Thus, it is possible to use these cells as a comparative translational model for drug development especially if one wishes to work with molecular targets as described above. Citation Format: Heidge Fukumasu, Yonara G. Cordeiro, Pedro L. Xavier, Arina L. Rochetti, Pamela A. Alexandre, Claudia C. Mori, Silvio H. Freitas, Ricardo F. Strefezzi. Transcriptomic profiling of two canine mammary cancer cell lines for translational oncology studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 819. doi:10.1158/1538-7445.AM2017-819

Caffeine increases Nr1i3 expression and potentiates the effects of its ligand, TCPOBOP, in mice liver

A cafeina e uma das substâncias mais consumidas mundialmente, estando presente no cafe, cha-verde e guarana, entre outros. O receptor sensor de xenobioticos Receptor Nuclear subfamilia 1, grupo I, membro 3 (Nr1i3, mais conhecido como Androstano Consititutivo - Car) e um regulador chave da biotransformacao e excrecao de substâncias e nenhuma descricao consistente dos efeitos da cafeina sobre este receptor foi feita. Entao, para avaliar os efeitos da cafeina sobre este receptor, realizamos experimentos em camundongos. Primeiramente, camundongos C57/Bl/6 foram tratados diariamente com cafeina (50 mg/kg) por 15 dias e apresentaram um leve, mas significativo, aumento na expressao do Car e do seu gene alvo Cyp2b10. Assim, um segundo experimento foi realizado para verificar os efeitos da cafeina sobre o TCPOBOP (1,4-bis-[2-(3,5-dicloropiridiloxi)]benzeno,3,3′,5,5′-tetracloro-1,4-bis(piridiloxi)benzeno), o mais potente agonista do Nr1i3 de camundongos conhecido. Interessantemente, a cafeina potencializou os efeitos pleiotropicos do TCPOBOP no figado dos camundongos, como hepatomegalia, hepatotoxicidade, proliferacao celular e perda da comunicacao intercelular por juncoes do tipo gap. Os camundongos tratados com cafeina e TCPOBOP apresentaram maior expressao genica de Nr1i3 e Cyp2b10, quando comparados aos camundongos tratados apenas com cafeina ou TCPOBOP. Juntos, nossos resultados indicam que a cafeina aumenta a expressao do receptor CAR em figados de camundongos C57/Bl/6, porem nesta etapa ainda nao e possivel afirmar se estes efeitos sao direta ou indiretamente mediados pelo Nr1i3.

Lafoensia pacari extract induces apoptosis mediated by caspase-3 and inhibition of growth in human lung cancer cells

Background Lafoensia pacari is a native species from South America which has been used in traditional medicine as antiulcerogenic and anti-inflammatory for several diseases, but the antineoplastic potential still have not been elucidated so far, though its etnopharmacological indication. The aim of this study was to evaluate the anti-neoplastic effect of L. pacari ethanolic extract in three human lung neoplastic cell lines.