Luciana Maria Silva

Synthesis and Biological Evaluation of Novel 3-Alkylpyridine Marine Alkaloid Analogs with Promising Anticancer Activity

Cancer continues to be one of the most important health problems worldwide, and the identification of novel drugs and treatments to address this disease is urgent. During recent years, marine organisms have proven to be a promising source of new compounds with action against tumoral cell lines. Here, we describe the synthesis and anticancer activity of eight new 3-alkylpyridine alkaloid (3-APA) analogs in four steps and with good yields. The key step for the synthesis of these compounds is a Williamson etherification under phase-transfer conditions. We investigated the influence of the length of the alkyl chain attached to position 3 of the pyridine ring on the cytotoxicity of these compounds. Biological assays demonstrated that compounds with an alkyl chain of ten carbon atoms (4c and 5c) were the most active against two tumoral cell lines: RKO-AS-45-1 and HeLa. Micronucleus and TUNEL assays showed that both compounds are mutagenic and induce apoptosis. In addition, Compound 5c altered the cellular actin cytoskeleton in RKO-AS-45-1 cells. The results suggest that Compounds 4c and 5c may be novel prototype anticancer agents.

Synthesis and evaluation of antimalarial activity of oxygenated 3-alkylpyridine marine alkaloid analogues.

A series of new oxygenated analogues of marine 3‐alkylpyridine alkaloids were prepared from 3‐pyridinepropanol in few steps and in good yields. The key step for the synthesis of these compounds was a Williamson etherification under phase‐transfer conditions. All new compounds were evaluated for their antiplasmodial activity and cytotoxicity. A significant reduction in parasitaemia was observed for some of the prepared compounds, and the majority of them exhibited a selectivity index (SI) ranging from 2.78 to 15.58, which suggests that these compounds may be a promising class of substances with antimalarial activity.

Developmental biology of the Brazilian ‘Armed’ spider Phoneutria nigriventer (Keyserling, 1891): Microanatomical and molecular analysis of the embryonic stages

Phoneutria (Ctenidae) is among the most dangerous venomous spiders in Brazil. Its venom is composed of a mixture of pharmacologically active components, some of which have been quite extensively studied due to their potentiality as models for new pharmaceutical drugs. Nevertheless, literature data on the venom-producing glands are very limited. In the present study, we follow the biological development of intra-cocoon stages of Phoneutria nigriventer spiders, mainly regarding the formation of the venomous apparatus and venom production. The results showed that the venom glands of Phoneutria are already present in the early 1st pre-larva stage. The venomous apparatus is completely formed in the larva, a stage that precedes the spider eclosion from the cocoon. At embryo stages, transcripts of a vertebrate-active neurotoxin (PhTx1) were shown to be present, as well as, unidentified venom proteins that were immunolabeled by anti-venom antibodies. It seems that venom toxins play roles in the protection and survival of those early developmental stages of Phoneutria spiders.

Histopathological and immunohistochemical aspects of American cutaneous leishmaniasis before and after different treatments

BACKGROUND The histopathology and immune responses of the healing process of leishmaniasis are still poorly studied. OBJECTIVES This study aimed to examine the histopathological and immunological aspects of lesions of patients with cutaneous leishmaniasis before and after different therapeutic methods. METHODS We studied 23 individuals grouped according to the treatments: Glucantime, Glucantime + Leishvacin and Glucantime + Leishvacin associated with Bacillus Calmette-Guerin. For analysis of the histopathological changes present in the dermis and epidermis, histological sections were stained with hematoxylin and eosin. The samples were immunostained before and after treatment to analyze the expression of interferon (IFN)-γ, interleukin (IL) 12, IL-10 and IL-4. RESULTS Before treatment the presence of intense infiltrates of mononuclear cells was noticed and after treatment, even with a diagnosis of clinical cure, the subjects still showed a moderate inflammatory process. In the immunohistochemical analyses, we noticed a difference between the cytokines, with increased expression of cytokines IFN-γ and IL-12 compared to IL 10 and IL-4, both before and after treatment and, comparatively, the difference in this expression was more intense before treatment. However, the cytokine expression analyzed by treatment group showed no statistically significant difference. CONCLUSION We conclude that a clinical cure does not always coincide with the histopathological one, and that before treatment there is a predominance of Th1 cytokines. In terms of treatment type, there was no difference in the progression of healing for all the three types of treatment, indicating their clinical equivalence.

STAT3 as a promising chemoresistance biomarker associated with the CD44+/high/CD24-/low/ALDH+ BCSCs-like subset of the triple-negative breast cancer (TNBC) cell line

Abstract The cancer stem cell (CSC) concept is currently employed to explain the mechanism of multidrug resistance that is implicated in the reduced efficacy of many chemotherapeutic agents, consequently leading to metastatic spread and disease relapse. We searched for potential predictive markers of doxorubicin (DOX) resistance in breast cancer stem cells (BCSCs) of the BT‐549 human triple‐negative breast cancer (TNBC) cell line classified as a claudin‐low subtype. In this study, we show that BT‐549 presents a BCSCs‐like subset determined by a CD44+/high/CD24‐/low/ALDH1+ phenotype. The CD44+/high/CD24‐/low/ALDH+ BCSCs‐like subset presented the downregulation of a majority of the genes analyzed (64 genes), and only 3 genes were upregulated after DOX treatment. Among the upregulated genes, MAPK3, PRKCZ and STAT3, STAT3 presented a higher level of upregulation in the DOX‐treated CD44+/high/CD24‐/low/ALDH+ BCSCs‐like subset. The identification of biomarkers that predict antitumor responses is at the top of cancer research priorities. STAT3 was highlighted as a molecular signature in the CD44+/high/CD24‐/low/ALDH1+ BCSCs‐like subset obtained from the TNBC BT‐549 cell line related to DOX resistance. A majority of the evaluated genes in the EGF pathway appear to be not associated with DOX resistance, as observed in the CD44+/high/CD24‐/low/ALDH1+ BCSCs‐like subset. Abbreviations: AKT1: AKT serine/threonine kinase 1; AKT2: AKT serine/threonine kinase 1; BC: breast cancer; BCSCs: breast cancer stem cells; CAV2: caveolin 2; CSCs: cancer stem cells; DOX: doxorubicin; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; EMT: epithelial‐mesenchymal transition; HER‐2: human epidermal growth factor receptor 2; IC50: half maximal inhibitory concentration; MAPK3: mitogen‐activated protein kinase 3; MUC1: mucin 1, cell surface associated; PLCG1: phospholipase C gamma 1; PPI: protein‐protein interactions; PRKCZ: protein kinase C zeta; RRAS: related RAS viral (r‐ras) oncogene homolog; RHOA: ras homolog family member A; RHOC: ras homolog family member C; RT‐qPCR: real‐time quantitative reverse transcription; STAT3: signal transducer and activator of transcription 3; TS: tumorspheres; TNBC: triple‐negative breast cancer.

Differential expression of small RNA pathway genes associated with the Biomphalaria glabrata/Schistosoma mansoni interaction

The World Health Organization (WHO) estimates that approximately 240 million people in 78 countries require treatment for schistosomiasis, an endemic disease caused by trematodes of the genus Schistosoma. In Brazil, Schistosoma mansoni is the only species representative of the genus whose passage through an invertebrate host, snails of the genus Biomphalaria, is obligatory before infecting a mammalian host, including humans. The availability of the genome and transcriptome of B. glabrata makes studying the regulation of gene expression, particularly the regulation of miRNA and piRNA processing pathway genes, possible. This might assist in better understanding the biology of B. glabrata as well as its relationship to the parasite S. mansoni. Some aspects of this interaction are still poorly explored, including the participation of non-coding small RNAs, such as miRNAs and piRNAs, with lengths varying from 18 to 30 nucleotides in mature form, which are potent regulators of gene expression. Using bioinformatics tools and quantitative PCR, we characterized and validated the miRNA and piRNA processing pathway genes in B. glabrata. In silico analyses showed that genes involved in miRNA and piRNA pathways were highly conserved in protein domain distribution, catalytic site residue conservation and phylogenetic analysis. Our study showed differential expression of putative Argonaute, Drosha, Piwi, Exportin-5 and Tudor genes at different snail developmental stages and during infection with S. mansoni, suggesting that the machinery is required for miRNA and piRNA processing in B. glabrata at all stages. These data suggested that the silencing pathway mediated by miRNAs and piRNAs can interfere in snail biology throughout the life cycle of the snail, thereby influencing the B. glabrata/S. mansoni interaction. Further studies are needed to confirm the participation of the small RNA processing pathway proteins in the parasite/host relationship, mainly the effective participation of small RNAs in regulating their target genes.

Novel Symmetrical 1,4-Disubstituted-bis-1,2,3-triazoles: Synthesis by Double CuAAC and Cytotoxicity Evaluation

BACKGROUND A series of symmetrical 1,4-disubstituted bis-1,2,3-triazoles was prepared by double copper catalyzed Azide-alkyne Cycloaddition (CuAAC) from aliphatic bis-azides and a tetraethylene glycol bis-azide derivative. The eighteen novel compounds were evaluated in vitro for their cytotoxic activity against two human tumor cell lines: Human breast adenocarcinoma (MDA-MB 231) and ovarian adenocarcinoma (TOV-21G). RESULTS AND CONCLUSION The results of colorimetric MTT assays showed that compounds 4j and 4q exhibited a better selectivity index and cell viability comparable with the standard drug doxorubicin. These compounds induced apoptosis in both tested cell lines, as assessed by BrdU assay. The results suggest that these structurally simple compounds may be promising prototypes for antitumoral agents.

Three-Dimensional Cellular Arrangement in Epithelial Ovarian Cancer Cell Lines TOV-21G and SKOV-3 is Associated with Apoptosis-Related miRNA Expression Modulation

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, and the lack of chemoresistance biomarkers contributes to the poor prognosis. Cancer stem cells (CSC) have been investigated in EOC to understand its relationship with chemoresistance and recurrence. In this context, in vitro cultivation-models are important tools for CSC studies. MicroRNAs (miRNAs) play key roles in cancer, CSC regulation and apoptosis. Thus, this study aims to evaluate the tumorsphere model as CSC-enrichment method in EOC studies and investigate apoptosis-related miRNAs in tumorspheres-derived EOC cell lines. TOV-21G and SKOV-3 were cultured in monolayer and tumorspheres. Genetic profiles of cell lines were obtained using COSMIC database. CD24/CD44/CD146/CD177 and ALDH1 markers were evaluated in cell lines and tumorspheres-derived by flow cytometry. Eleven miRNAs were selected by in silico analysis for qPCR analysis. According to COSMIC, TOV-21G and SKOV-3 have eight and nine cancer-related mutations, respectively. TOV-21G showed a CD44+/high/CD24−/low/CD117−/low/CD146−/low/ALDH1low profile in both culture models; thus, no significant difference between cultivation models was identified. SKOV-3 showed a CD44+/high/CD24+/high/ CD117−/low/CD146−/low/ALDH1low profile in both culture models, although the tumorsphere model showed a significant increase in CD24+/high subpopulation (ovarian CSC-like). Among eleven miRNAs, we observed differences in miRNA expression between culture models. MiR-26a was overexpressed in TOV-21G tumorspheres, albeit downregulated in SKOV-3 tumorspheres. MiR-125b-5p, miR-17-5p and miR-221 was downregulated in tumorsphere model in both cell lines. Given that tumorsphere-derived SKOV-3 had a higher ratio of CD24+/high cells, we suggest that miR-26a, miR-125b-5p, miR-17-5p and miR-221 downregulation could be related to poor EOC prognosis.

New 3‐alkylpyridine marine alkaloid analogues as promising antitumor agents against the CD44+/high/CD24−/low subset of triple‐negative breast cancer cell line

Triple‐negative breast cancer (TNBC) is one of the most aggressive cancers in women. Additionally, presence of residual cancer stem cells (CSC) in TNBC has challenged the efficacy of chemotherapy. Thus, the development of new molecules with potential action against CSC is fundamental. In this study, six synthetic analogues of theonelladin C, a 3‐alkylpyridine marine alkaloid, were tested for cytotoxic activity against human TNBC cell line (BT‐549) and tumorspheres derived from BT‐549. Cytotoxicity assay was performed by sulforhodamine B (SRB). BT‐549 and tumorspheres were examined for CD44+/high/CD24−/low markers, indicative of CSC profile, by flow cytometry. Clonogenic assay was performed to verify inhibiting growth of tumorspheres by the synthetic analogues. Cell death by apoptosis was investigated employing annexin V assay. SRB assay on BT‐549 cells revealed that compounds 1c and 2c were the most active of the series, with IC50 values of 18.66 and 9.8 μm, respectively. Compounds 1c and 2c were able to reduce both CSC‐like population (CD44+/high/CD24−/low) and non‐CSC population (CD44+/high/CD24+/high) in tumorsphere model. Clonogenic and annexin V assays confirmed the ability of 1c and 2c to induce growth inhibition and apoptosis in BT‐549 cells and tumorspheres. These preliminary data indicate that these compounds are a promising class for development of anticancer agents.

Ultrastructural characterization of cells in primary cultures from different adult tissues of Biomphalaria tenagophila TAIM, a strain that is absolutely resistant to Schistosoma mansoni infection.

The etiological agent of schistosomiasis in Brazil, Schistosoma mansoni, requires an obligatory passage through Biomphalaria snails to complete its life cycle. In these intermediate hosts, interaction with the parasite is mediated by humoral factors and hemocytes by mechanisms that are not yet fully understood. Extant studies exploring these processes are usually conducted through experimental infection of Biomphalaria with S. mansoni miracidia. Thus, tissue-derived cultures of Biomphalaria may be useful in increasing the understanding of that interaction at cellular level. However, in the absence of morphological characterization of those cells in culture, the application of such models is delayed. In the present work, we cultured different tissues of B. tenagophila, the second most important host of S. mansoni in Brazil, using a strain that is naturally and absolutely resistant to S. mansoni infection. This decision was driven by the view that this strain might be provided with the most effective response against parasite infection. Primary cultures were successfully established from nine Biomphalaria tissues and the respective cells in culture were ultra structurally described. Attention was particularly devoted to cells derived from mantle cavity and kidney tissues. Although they have been considered important centers for hemocyte production in Biomphalaria, no detailed cell characterization is available in the pertinent literature. Herein, kidney-derived cells partially shared hematoblast characteristics. Moreover, under optical microscopy, kidney cells in culture were very similar to those derived from amebocyte-producing organ (APO) cultures, which have been recently shown to be capable of eliminating S. mansoni sporocysts in vitro. Based on the close resemblance of those cultures and their anatomical proximity inside the mantle cavity, we suggest the effective participation of Biomphalaria kidney cells in hematopoiesis and in host response to S. mansoni infection.