Total de Trabalhos: 00037

XXXIII Reunião Anual da FeSBE

19.001 Evaluation of synergism and combination effect of sulfasalazine and valproic acid on glioblastoma cells using a software based on Median-Effect Equation and Combination Index Theorem

Autores CARLOS GUSTAVO GARCIA 1, FLAVIA LIMA 2, MARCELO COSSENZA 3

Instituição 1 UNIAN - CENTRO UNIVERSITÁRIO ANHANGUERA DE NITERÓI, 2 ICB/UFRJ -

INSTITUTO DE CIÊNCIAS BIOMÉDICAS, 3 UFF - DEPARTAMENTO DE FISIOLOGIA E

FARMACOLOGIA

Resumo

Introduction: Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor and still lacks effective therapeutic strategies. It has already been shown that old drugs like sulfasalazine (SAS) and valproic acid (VPA) present antitumoral activities in glioma cell lines. A novel study from Garcia and colleagues, 2018 (https://doi.org/10.1007/s12035-018-0895-1) was the first to show the combined effects of both drugs in GBM in vitro models. However, in this work pharmacological interactions based on potency and efficacy between SAS and VPA remained to be explored.

Objective: to verify the presence of synergism and dose-reduction relation using CompuSyn, a software designed to detail those relations using different human (commercial U87MG and a biopsy GBM02) and rat (C6) glioma lineages.

Methodology: Concentration-effect curves were obtained from the previous paper from of group (above mentioned). The same data points were used for data generation using CompuSyn (ComboSyn Development, NY, USA). Data were plotted following software’s user guide instructions. CompuSyn automatically generated, as demanded, a report containing graphs and tables concerning drugs’ concentration alone and in combination.

Results: We chose isobolograms for effect dose 90, 75 and 50% (ED90, 75 and 50 respectivelly), fraction affected-combination index (Fa-CI) plot, fraction affected-dose reduction index (DRI) logarithm [Fa-Log(DRI)] plot as parameters to evaluate. For isobolograms, we observed synergism in the combination of SAS and VPA for all cell lines. Noteworthy, for U87MG and GBM02 cells, combined doses to achieve 90% effect are close to the ones for drugs alone to reach 75%. Regarding Fa-CI plots, we noticed that SAS and VPA exhibited CI< 1 in concentrations above 0.5mM for GBM02 and U87MG cells indicating synergism between drugs. However, C6 lineage did not exhibit the same profile, once CI< 1 values were achieved from 1mM on. Last, we observed that in all cell lines VPA showed higher DRI levels compared to SAS, although positive values were achieved for SAS from 0.5mM on.

Conclusions: SAS and VPA exhibit synergism in GBM in vitro cell lines when in combination regarding loss of cell viability.

Palavras-chaves: Glioma, Glioblastoma, Synergism

19.002 THE CYTOTOXIC EFFECT OF THE MARINE COMPOUND, FISTULARIN 3, IN HUMAN CANCER CELLS

Autores Luíse Araújo de Albuquerque Simões 1, Bruna Braga Dantas 1, Manoel Oliveira de Morais Junior 1,

Barbara Viviana de Oliveira Santos 1, Demetrius Antonio Machado de Araújo 1

Instituição 1 UFPB - Universidade Federal da Paraíba

Resumo

Introduction: According to the World Health Organization 2017, cancer is one of the biggest causes of death by disease in the world, accounting for 8.8 million deaths per year. In this context, it has been conducted surveys with new compound, from a marine animal, to find out the need for more effective molecules against this illness.

Objective: The aim of this study was to evaluate the cytotoxic effect of Fistularin 3 (FIS3), a compound purified from a marine sponge, in some cell cultures, such as acute monocytic leukemia (THP-1), chronic myeloid leukemia (K562), mammary adenocarcinoma (MDA-MB-231) and also in non-cancerous human umbilical vein endothelial cells (HUVEC). Furthermore, it was determined the cell death characteristics in THP-1 cells.

Methodology: It was done the MTT reduction assay and was performed experiments, such as, evaluation of cell cycle by flow cytometry, the visualization of DNA fragmentation in agarose gel and fluorescence experiments, under microscopy. It was also performed the influence of FIS3 on the mitochondrial depolarization. After three independent assays in triplicate, these data were analysed by the software GraphPad Prism 6.0 and expressed with the mean ± standard deviation followed by a analysis of variance (ANOVA) and post-test bonferroni.

Results: It was shown that FIS3 was selective for cancer cells, displayed an IC50 of 135.20 ± 3.24 M in 72 hours in HUVEC cells during 72 hours of treatment. In the cell lines THP-1 and K562, were shown an

IC50 below 100 M in the periods of 24, 48 and 72 hours of incubation. However, in MDA-MB-231 cells, the IC50 was only obtained in 72 hours. Moreover, in THP-1 cells, FIS3 caused an increase in the percentage of DNA during the SubG1 phase and caused a halt in the G1 cell cycle, in the period of 24 hours. We also observed that FIS3 induced an increase of DNA fragmentation in agarose gel in 48 hours of incubation and in the same period was induced 20.24 ± 11.32% of mitochondrial depolarization, which it is almost 2 times greater than was visualized in the control condition, and we cannot observe any change in the fluorescence intensity. FIS3 also caused 1.67 ± 3.32% of reactive oxygen species (ROS) production. Moreover, in comparison to the control, 78.94 ± 3.80% of cells were positive for the presence of acid vesicles organelles (AVOs) and also had a growth in their fluorescence intensity, suggesting an autophagic effect. In addition, we verified a small percentage of early and late apoptosis of 5,83 ± 1,22 % and 8,90 ± 1,15 %, respectively.

Conclusion: We conclude that FIS3 showed a selective cytotoxic effect and the cytotoxicity action in THP-1 cells can be mediated by an autophagic process demonstrating that compound has promising anticancer effects.

Funding: CNPq and CAPES.

Palavras-chaves: Autophagy, Cancer, Cell death, Cytotoxicity, Leukemia

19.003 Effects of BPP-10c on clinical outcomes of neuroblastoma xenograft-injected mice.

Autores Rodrigo Seeger 1, Poliana Martins 1, Luiz Juliano Neto 2, Claudiana Lameu 1

Instituição 1 USP - Universidade de São Paulo, 2 UNIFESP - Universidade Federal de São Paulo

Resumo

Introduction: Nitric oxide (NO) was first discovered and characterized as a secondary messenger that regulates vasodilation signalling in the cardiovascular system. NO is synthesized from L-arginine and oxygen by a family of enzymes termed nitric oxide synthases (NOS). The NO effects on regulation of tumor progression and metastasis establishment have been described for several types of cancer. In addition, few works have analysed NO on cancer stem-like cells, which are a cell population found in tumours, characterized with self-renew and drugs resistance properties therefore crucial for tumor progression and metastasis. BPP-10c, a decapeptide isolated from Bothrops jararaca venom, is well-characterized to induce NO production. This peptide increases the L-arginine levels due to the activation of argininosuccinate synthase (ASS). Some works have reported the correlation between the expression of ASS and the development of metastasis in several cancer types. Hence, this decapeptide became an interesting tool to clarify the role of NO in neuroblastoma progression and metastasis.

Objective: The objective of this work was to investigate the effects of BPP-10c on NO production and analyse its effects on tumor development and the outcomes in mice xenografts derived from neuroblastoma tumorspheres.

Methodology: Firstly, the neuroblastoma cell line (SH-SY5Y) was cultured on low adhesion flasks with suitable medium for cancer stem-like cells (tumorspheres) formation and simultaneously treated for 96h with BPP-10c 10µM, L-NAME 3mM and MDLA 3mM. Nitrite concentration was measured using 2, 3- Diaminonaphthalene (2, 3-DAN). Regarding the animal experiments, the tumorspheres were dissociated and resuspended in Matrigel®, then injected subcutaneously on Balb/c Nude mice (n=14, Certificate CEUA IQ-USP 13/2017). Tumor size and body weight were monitored during 90 days at every 2 days.

Results: The treatment of the cells with BPP-10c 10µM caused a significant increase in nitrate concentration (0.45 µM ± 0.02) when compared to control (0.38 µM ± 0.019). The treatment with L-NAME (3mM) plus BPP-10c 10µM (0.33 µM ± 0.016) caused a decrease when compared to the control. But, when the cells were treated with MDLA (3mM) plus BPP-10c 10µM (0.45 µM ± 0.09) did not shown any significant difference compared to control. The animals injected with the tumorspheres control demonstrated significant weight loss (17.2g ± 4.9) compared to the animals receiving BPP-10c- treated tumorspheres (22.8g ± 3.3) at 90th day. The tumor xenografts size and volume were statistically different between control group (11.7mm ± 1.5) and BPP-10c-treated group (8.4 mm ± 0.6) at 64th day. However, at 90th day no significant difference in the size and volume was observed (control - 16.1mm ± 5.9 and BPP-10c-treated tumorspheres 17.47mm ± 3.9).

Conclusion: Our results are in agreement with previous works that demonstrated that BPP-10c caused a significant increase in NO production (i.e. nitrate). Furthermore, the injection of BPP-10c-treated tumorspheres caused a dramatic protection on mice weight loss when compared when inoculated with control tumorspheres. These effects could be attributed to the allosteric activation of ASS and production of NO. Nevertheless, to clarify the differences in the clinical outcome of the mice, further investigation is required to elucidate the characteristics of the BPP-10c- treated tumorspheres as well mechanisms of the NO action on these cells.

Funding: FAPESP

Palavras-chaves: Bradykinin-potentiating peptide, Neuroblastoma, Nitric Oxide, Tumor Xenografts

19.004 ACTIVATION OF ESTROGEN RECEPTOR REGULATES THE EXPRESSION OF GALECTIN-3 IN ANDROGEN-INDEPENDENT PROSTATE CANCER CELLS.

Autores Deborah Simão de Souza 1, Carolina Meloni Vicente 1, Gustavo José S. Pereira 1, Roger Chammas 2,

Catarina Segreti Porto 1

Instituição 1 EPM-UNIFESP - Escola Paulista de Medicina-UNIFESP, 2 FMUSP-ICESP - Faculdade de

Medicina-USP, Instituto do Câncer do Estado de São Paulo, São Paulo, SP, Brazil

Resumo

Introduction. Galectins play important roles in diverse physiological and pathological processes, including cancer development, progression and metastasis. The mechanisms of regulation of galectin-3 expression are still poorly understood. Our laboratory have shown that androgen-independent prostate cancer cells PC-3 and DU-145 express both estrogen receptors ESR1 (ERα) e ESR2 (ERβ) (Pisolato et al., Steroids 107:74, 2016). It is important to emphasize that the estrogen receptor (ER) dimer binds to target sites on DNA either directly on ERE (estrogen response element) or indirectly through transcription factors, which are required for modulation of gene expression by ERs. The promoter region of the human galectin-3 LGALS3 gene contains several binding sites for transcription factors, including SP1, AP-1, NF-κB and CREB (Dumic et al., Biochim. Biophys. Acta 1760:616, 2006). Aim. This study was performed to investigate whether ERs could regulate galectin-3 expression. Methods. The experimental procedures were approved by the Research Ethical Committee at EPM-UNIFESP (3527220917). Androgen-independent prostate cancer cells DU-145 and PC-3, and human post pubertal prostate normal cells PNT1A were grown in culture medium as previously described. Western blot assays were performed for detection of ESR1, ESR2 and galectin-3 (Pisolato et al., 2016). DU-145 and PC-3 cells, after 24 h of bovine fetal serum removal, were incubated in the absence (control) and presence of 17beta-estradiol (E2) or ESR2-selective agonist (DPN) at concentrations of 0.1; 1; 10 and 100 nM, for 24 h and Western blot assays were performed for detection of galectin-3. Results. ESR1, ESR2 and galectin-3 were detected, respectively, as a single protein band of about 66; 56 and 31 kDa in PNT1A, DU-145 and PC-3 cells. The expression of ESR1 and ESR2 was higher (2-fold, n=4) in DU-145 cells than in PC-3 cells. Low levels of ESR1 and ESR2 were detected in PNT1A cells. On the other hand, PNT1A presented the highest expression of galectin-3, comparing with cancer cells. The expression of galectin-3 was higher (2-fold, n=4) in DU-145 than PC-3 cells, indicating a differential expression of galectin-3 between tumor cells. It is important to emphasize that PC-3 cells derive from bone metastasis and DU-145 cells from brain metastasis, suggesting that the microenvironmental stimuli may play different roles in the regulation of galectin-3. Furthermore, the treatment of PC-3 and DU-145 cells with E2 or DPN increased the expression levels of galectin-3 compared with control (untreated cells). Conclusion. The expression of estrogen receptors is higher in androgen-independent prostate cancer cells, while galectin-3 is more expressed in post pubertal prostate normal cells. In addition, E2-ERs may play a role in galectin-3 expression. It remains to be demonstrated whether the crosstalk between galectin-3 and ER signaling pathways could affect the proliferation, migration and invasion of prostate cancer cells.

Grant support: FAPESP, CNPq, CAPES.

Palavras-chaves: estrogen receptor, galectin-3, prostate cancer cells

19.005 Inhibition of cell migration and in vivo antitumor effect of peptides derived from Brn-2 in murine melanoma

Autores Maria Carolina Mariano Cesar 1, Victória Santos Souza 1, Fernanda Fernandes Miranda da Cunha 1,

Thaysa Paschoalin 2, Luiz R. Travassos 2, Denise Costa Arruda 1

Instituição 1 UMC - Universidade de Mogi das Cruzes, 2 Unifesp - Universidade Federal de São Paulo

Resumo

Introduction: Melanoma is a malignant skin cancer causing 55,000 deaths/year, according to the World Health Organization. Peptides derived from immunoglobulin CDRs and transcription factors have been investigated displaying antitumor activities including anti-melanoma effects. Brn-2 is a transcription factor regulated by MAPK, PI3K/Akt and Wnt/β-catenin cell signaling pathways, which is expressed in melanocytes and overexpressed in melanoma. High Brn-2 leads to tumor proliferation and metastasis. Peptides derived from Brn2 can inhibit tumor cell migration and proliferation. These molecules have low toxicity, penetrate tumor cells by endocytosis and may act by competition with the transcription factor for binding to the gene receptor. They are thus candidates to be developed as specific and effective drugs against the disease.

Objective: The present work aims at determining the inhibitory effects on B16F10-Nex2 melanoma cell migration and cell invasion and the protective effects in vivo of C9K and E24G peptides derived from the transcription factor Brn-2.

Methodology: B16F10-Nex2 cells were cultivated and treated with 0.5 mM of C9K and 1 mM of E24G peptides. The wound-healing method was used to measure migration using four different times. The inhibition of cell invasion was determined using transwell inserts with matrigel and colouring nuclei with DAPI. The cell readings were obtained in a fluorescent microscope, and 1 mM was the concentration of each peptide. To determine the antitumor protective effect in vivo, the melanoma metastatic model was used: cells were inoculated intravenously in C57Bl/6 mice and the treatment consisted in i.p. injection of 300 µg/day of the peptide or PBS, for nine days. Treated and untreated mice were euthanized to remove lungs and count the melanotic nodules. This assay was approved by the Committee of Ethics in Animal Use (CEUA) (Protocol 005/2015). The statistics used Student’s test t and Mann-Whitney.

Results: The C9K and E24G peptides were not cytotoxic to B16F10-Nex2 cells at 1 mM concentration. E24G (1 mM) and C9K (0.5 mM) inhibited tumor cell migration. Both peptides were able to reduce the number of lung melanoma nodules in C57BL/6 syngeneic mice. a) Untreated control mice, mean of 160 nodules; E24G-treated mice, mean of 33 nodules, p<0.05.

Conclusion: The C9K and E24G peptides derived from transcription factor Brn-2 inhibited melanoma cell migration and showed protective effect in vivo against B16F10-Nex2 cells in a syngeneic metastatic model in C57BL/6 mice. These peptides are, in conclusion, promising candidates for cancer treatment.

Funding: CAPES and FAPESP.

Palavras-chaves: Skin cancer, Transcription Factor, Metastases, Melanoma, Peptides

19.006 Determination of cell internalization and localization of R18H peptide derived from transcription factor Brn-2 in murine melanoma cells

Autores Raquel Tamires Soares Santos 1, Fernanda Fernandes Miranda da Cunha 1, Dayane Batista Tada 2,

Renata Arruda Mortara 3, Luiz R. Travassos 3, Denise Costa Arruda 1

Instituição 1 UMC - Universidade de Mogi das Cruzes, 2 UNIFESP - Universidade Federal de São Paulo, São

José dos Campos, SP , 3 UNIFESP - Universidade Federal de São Paulo,

Resumo

Introduction:

Melanoma is formed by malignant transformation of melanocytes, which are present in the basal layer of the epidermis and are melanogenic. The melanoma process involves signaling pathways such as MAPK, PI3K-Akt and Wnt/β-catenin that are directly related to tumor development. In addition, these pathways can activate the transcription factor Brn-2 which is expressed in melanocytes and overexpressed in melanoma. Peptides derived from Brn-2 can inhibit the transcription factor and induce cell death. We have shown that R18H peptide induced significantly antitumor activity in vitro and in vivo. Treated cells showed death by apoptosis. In the present work, cell internalization mechanisms were examined.

Objective:

Determine the in vitro antitumor activity, mechanisms of cell internalization and localization of R18H peptide, derived from transcription factor Brn-2.

Methodology:

B16F10-Nex2 cells were treated with R18H peptide in different concentrations for 2 or 4 h. To determine the mechanism of cell internalization, cells were pre-incubated for 1 h with the inhibitors methyl β-cyclodextrin, nocodazole, amiloride and potassium depletion buffer and treated with peptide for 3 hours. Cell viability was determined with Trypan Blue exclusion dye. For statistic analysis Student’s t test was used. For cell internalization assays, cells were treated with the biotinylated peptide for 4 or 24 h, incubated with immune fluorescent buffer and stained with streptavidin AlexaFluor-488, rhodamine phalloidin and DAPI and imaged in a confocal microscope. Images were processed using ImageJ.

Results:

The R18H peptide showed in vitro cytotoxic activity in B16F10-Nex2 cells after treatment for 2 and 4 h, with EC 50% of 0.663 and 0.589mM, respectively (p<0.0001). Peptide showed antitumor activity in presence of internalization inhibitors such as: caveolae (mehtyl - β-cyclodextrin) (p<0,005), clathrin (potassium depletion buffer) (p<0,001), amiloride (proton pump) (p<0,001) and nocodazole (microtubule polymerization) (p<0,001), suggesting an additional internalization mechanism. Confocal microscopy suggests that the peptide has been internalized since co-localization dots with nucleus and F-actin are demonstrated.

Conclusion:

Our results showed that the peptide has in vitro antitumor effect in melanoma cells. The internalization mechanism is still unknown since the peptide showed antitumor activity in presence of internalization inhibitors. However, confocal microscopy analyzes confirmed the biotinylated peptide internalization and co-localization with the nucleus and F-actin.

FUNDING:

UMC and FAPESP

Palavras-chaves: Melanoma, Internalization , Antitumor activity, Peptide , Localization

19.007 ACTIVATION OF ESTROGEN RECEPTORS INCREASES COLONY FORMATION IN ANDROGEN-INDEPENDENT PROSTATE CANCER CELLS PC-3.

Autores Ana Paola Giometti Lombardi 1, Carolina Meloni Vicente 1, Catarina Segreti Porto 1

Instituição 1 UNIFESP - Universidade Federal de S'ao Paulo

Resumo

Introduction. Our laboratory have shown the expression of estrogen receptors ESR1 (ERα) and ESR2 (ERβ) in androgen-independent prostate cancer cells (PC-3), used as castration-resistant prostate cancer models. Furthermore, estrogen plays a role in PC-3 cell proliferation through a novel pathway, involving ESR2-mediated activation of beta-catenin (Lombardi et al., Mol. Cell. Endocrinol. 430:12, 2016). ESR2 also participates in PC-3 cell migration and invasion through SRC, PI3K/AKT and β-catenin. In addition, ESR1 is also involved in these processes (Lombardi et al., Next Frontiers to Cure Cancer Congress, 2018). Aim. This study was performed to investigate the effects of estrogen receptors activation on colony formation in androgen-independent prostate cancer cells PC-3. Methods. All experimental procedures were approved by the Research Ethical Committee at EPM-UNIFESP (#4330100615). To further analyze the tumorigenic potential of estrogen receptors in PC-3 cells, we performed the colony formation, anchorage-independent growth assay. PC-3 cells were grown in culture medium (Lombardi et al., 2016) embedded in soft agar for 21 days. Cells were incubated in the absence (control) and presence of 17beta-estradiol (E2, 10 nM), ESR2-selective agonists (DPN 10 nM) every other day, after medium renewal. The cells were also untreated or pretreated with ESR2-selective antagonist (PHTPP, 10 nM), ESR1-selective antagonist (MPP, 10 nM) for 30 minutes. Afterwards, the cells are stimulated with agonists of ERs. The morphology of colonies was observed under microscope and the images were captured using a Floid Cell Imaging Station and analyzed by Image J. The numbers and size of colonies were determined. Results. E2, PPT and DPN, respectively, increased in 3-, 3- and 2.5-fold the number of colonies formed by PC-3 cells (rounds spheroids structures) compared with control (absence of agonists). E2, PPT and DPN, respectively, increased in 2-, 2.5- and 2.5-fold the size of colonies. Furthermore, E2, PPT and DPN treatment also resulted in stellate or invasive structures. These effects were blocked by MPP and PHTTP, indicating that ESR1 and ESR2 are upstream receptors regulating these processes. Conclusion. 17beta-estradiol and estrogen receptors play an important role in prostate cancer tumorigenesis. The signaling pathways involved in colony formation remain to be explored.

Grant support: FAPESP, CNPq, CAPES

Palavras-chaves: colony formation, estrogen, estrogen receptors, prostate cancer

19.008 THE ROLE OF POLYUNSATURATED FATTY ACIDS IN MULTIDRUG RESISTANCE IN GLIOBLASTOMA

Autores Janaína Alessandra Silva 1,1, Alison Colquhoun 1,1

Instituição 1 ICB - Instituto de Ciências Biomédicas

Resumo

Introduction: Current therapeutic strategies for glioblastoma are surgery, chemotherapy and radiotherapy. However, such treatments fail due to the tumor’s acquired resistance mechanisms, such as increased activity of multidrug resistance proteins (MRPs). Polyunsaturated fatty acids (PUFAs) are described as modulators of the expression of these proteins.

Objective: To evaluate the role of ω-3 and ω-6 in drug resistance of human glioblastoma cells, verifying their effects upon tumor growth and chemotherapeutic sensibility of Vincristine (VCR) and Temozolomide (TMZ).

Methodology: Drug resistant U87MG and T98G cell lines were produced with VCR and TMZ in different concentrations (0.4 nM and 25 μM in U87MG) (5 nM and 112.5 µM in T98G). MRP activity was assessed through a Rhodamine 123 efflux activity assay in U87MG and TMZ-resistant (TMZ-R) and VCR-resistant (VCR-R) U87MG-derived cells. Cell growth assays with PUFAs treatments were performed using GLA (ω-6) and EPA (ω-3) in U87MG, TMZ-R and VCR-R. For statistical analysis, ANOVA with Bonferrini posttest were used to evaluate the statistical relevance in cell growth assay (when n≥ 2).

Results: The preliminary results showed that TMZ-R cells number decreased when treated with GLA [50, 100 and 150 µM] compared with Albumin (BSA) (p<0.05; 72 h; n=2, in triplicate). TMZ + EPA [50 µM] decreased cell growth after 72 h when compared with EPA (p<0.05; n=2, in triplicate). In U87MG the decrease of cell growth was more evident with VCR + GLA [50 µM; 48 h] [100 µM and 150 µM; 72 h] than VCR (p<0.05 and p<0.001, respectively; n=2, in triplicate). In U87MG, EPA [100 µM and 150 µM] decreased cell growth after 72 h compared with BSA (p<0.01 and p<0.001, respectively; n=2, in triplicate). In VCR-R, VCR + EPA [100 µM] decreased cell growth compared to VCR after 72h (p<0.001; n=2, in triplicate). The efflux activity of MRPs appears to be decreased in the presence of PUFAs and TMZ in TMZ-R more than U87MG after 100 min (n=1, in duplicate).

Conclusion: Co-treatment with PUFAs and chemotherapies reveals that resistant cells respond differently compared with non-resistant cells when we analyze the cell growth assay and efflux activity. GLA and VCR might be more effective in non-resistant cells however EPA and TMZ or VCR might be more effective in resistant cells. In efflux activity, TMZ-R may have a lower efflux when treated with TMZ and fatty acids, suggesting these cells’ MRPs have a lower activity. In U87MG this activity is not different in presence of drugs and PUFAs. Further analysis must be done to understand the association between the resistance phenotype and response to these treatments.

Funding: FAPESP

Palavras-chaves: Fattys Acids, Glioblastoma, Resistance, MRPs, Cancer

19.009 STUDY OF ANTICANCER ACTIVITY OF Extracts from Apodanthera congestiflora

Autores Sâmela Jordão Sâmela Jordão Pinto 1

Instituição 1 ufpe - universidade federal de pernambuco

Resumo

STUDY OF ANTICANCER ACTIVITY OF EXTRACTS FROM Apodanthera congestiflora

Sâmela Jordão1, Larissa Cardoso2, Matheus Barros3, Thiago Napoleão4, Teresinha Silva5, Patrícia Paiva6

1,2,3,4,6 Department of Biochemistry, 5 Department of Antibiotics, UFPE, Recife/ PE

Indroduction: Apodanthera congestiflora (Curcubitaceae) is popularly known as “batata de teiú” and its root is used in folk medicine in northeastern Brazil. However, there are few studies on the biological activities and chemical composition of this plant.

Objective: This work investigated the cytotoxicity and morphological changes promoted by the treatment with the extracts of A. congestiflora on cancer cells, as well as the cytotoxicity on human peripheral blood mononuclear cells.

Methodology: Extracts were obtained with the solvents hexane, ethyl acetate and methanol, in this order, through two methods: hot and cold extraction. Cytotoxicity of extracts (50 µg/mL) was evaluated by MTT method using the cancer cell lines NCI-H292, HT-29, HEP-2, HL-60, K562, MOLT-4 at concentrations of 105 cell/mL or 0.3x106 cell/mL. Extracts that reduced cell viability above 70% were tested against normal and cancer cells serial concentrations (1.5625 to 50 μg/mL). PBMC test was performed with about 6 mL of blood of volunteer and added in 3 mL of PBS (Ethics Committee 60107916.8.0000.5208). Lymphocytes and monocytes were obtained with addition of 3 ml of ficoll followed by a centrifugation of 1500 rpm for 3 min. The cell pellet was resuspended in RPMI 1640 with 20% fetal bovine serum. Phytohemagglutinin (3%) was added. Cells were plated and incubated for 24h at 37 °C. The extracts were added (1.5625 to 50 μg/mL) and incubated for 72h at 37 °C. 25 μl of MTT (0.5 mg/mL), was added and after incubation at 37 °C for 3 h, 100 μL of dimethylsulfoxide (DMSO). The optical density of the wells was performed and the IC50 was performed using Graphpad prism version 5.0 software.The morphological analysis of the NCI-H292 cell line was performed with the most active extracts at concentrations of 24 μg/mL, 8.48 μg/mL and 17 μg/mL (hexane extract obtained by soxhlet) and 4.62 μg/mL, 9.26 μg/mL and 18.52 μg/mL (ethyl acetate extract obtained by maceration). After 48 h, an aliquot of the cell suspension (50 μL) was collected to prepare the slides. The cells were fixed with methanol, stained with May-Grünwald-Giemsa and analyzed under an optical microscope.

Results: Methanolic extract was not active against the cancer cells tested in the single concentration screening. Hexane extract obtained by the soxhlet method showed IC50 ranging from 3.75 μg/mL to 15.53 μg/mL, while the ethyl acetate extract had IC50 ranging from 18.02 μg/mL to 20.95 μg/mL. The hexane and ethyl acetate extracts obtained by maceration showed IC50 ranging from 7.33 μg/mL to 21.23 μg/mL and from 5.52 μg/mL to 24.74 μg/mL, respectively. The extracts showed low cytotoxicity against normal cells (IC50 ranging from 13.1 to 32.1 μg/mL[L4] ).Morphological analysis of NCI-H292 cells by light microscopy revealed that the hexane extract promoted apoptosis (presence of apoptotic bodies) whereas the ethyl acetate extract promoted necrosis (increased cell volume and disintegration of the cytoplasm).

Conclusion: The data show that the hexane extract obtained by soxhlet and the ethyl acetate obtained by maceration are the most promising since they contains cytotoxic substances for cancer cells and little cytotoxicity for normal cells.

Funding: Capes, CNPq

Palavras-chaves: anticancer, curcubitaceae, Cytotoxicity, extracts, root

19.010 Antitumor potential of antipsychotic drugs against neuroblastoma

Autores Talita Gomes dos Santos 1, Gemina S. B. Vanderley 1, Wagner A. S. Júdice 2, Tiago Rodrigues 1

Instituição 1 UFABC - Universidade Federal do ABC, 2 UMC - Universidade de Mogi das Cruzes

Resumo

Introduction: Neuroblastoma is a malignant disease characterized by the transformation of neuroblasts in cancer cells in the adrenal gland, neck, chest, or spinal cord. It is the most common extracranial solid tumor in childhood. Main symptoms are bone pain and a lump in the abdomen, neck, or chest and it is diagnosed by biopsy. Chemotherapy, radiotherapy, and surgery in combination are used to treat neuroblastoma, depending on the stage of the disease. The chemotherapy consists of carboplatin, cyclophosphamide, doxorubicin, and etoposide, although several side effects and

drug resistance were already described. Thus, in this work we evaluated the cytotoxic effects and underlying mechanisms of cell death of tricyclic antipsychotic drugs on neuroblastoma cells. Material and Methods: The viability of Neuro 2a neuroblastoma cells was estimated by using the MTT reduction assay and LDH release was measured by kinetic method of pyruvate reduction. The mitochondrial transmembrane potential, ROS production, and GSH levels were evaluated spectrofluorimetrically by using rhodamine 123, DCFDA, and OPT. Results: Chlorpromazine (CPZ) and thioridazine (TR) presented a concentration-dependent cytotoxicity against Neuro 2a cells after 24 h incubation associated to increased ROS generation, GSH oxidation, and mitochondrial transmembrane potential dissipation. It was observed an apoptotic cell death profile and TR effect was more potent than CPZ, with an EC50 about 27 and 68 µM, respectively. Conclusions: The phenothiazines TR and CPZ exhibited potent cytotoxic effect against neuroblastoma cells and, considering that these drugs are clinically used for other purposes, they have a pharmacological potential to be used in the antitumor chemotherapy.

Palavras-chaves: antipsychotics, apoptosis, cancer, mitochondria, neuroblastoma

19.011 PHARMACOLOGICAL INHIBITION OF AUTOPHAGIA AND POSSIBLE INCREASE IN ANTILEUKEMIC EFFECTS OF CYTARABINE AND IDARUBICIN IN MYELOID LEUKEMIA

Autores Larissa Gonçalves Bueno 1,2, Caroline Palmeira dos Santos 1, Marina Yukari Kubota 1, Soraya Soubhi

Smaili 1, Gustavo José da Silva Pereira 1, Claudia Bincoletto Trindade 1

Instituição 1 UNIFESP - Universidade Federal de São Paulo, 2 CUSC - Centro Universitário São Camilo

Resumo

Introduction: Leukemia is the general term for hematological cancers characterized by uncontrolled cell proliferation. Even using standard treatment in AML therapy, many individuals recur in a short time, which often results in low percentages of cure. Within this context, research involving strategies to improve the response of leukemic patients to current therapy should be encouraged not only to decrease systemic toxicity but also to improve the rate of complete remission over a longer period of time. Recently, studies indicate that autophagy is dysregulated in leukemias, as it promotes the survival of leukemic cells, suggesting that their inhibition may become an interesting pharmacological target. Objective: To evaluate the possible increase in the antileukemic effects of the combination cytarabine–idarubicin (Ara-c–IDA) in the presence of the autophagy inhibitor chloroquine (CQ). Methods: Flow cytometry assays were performed to evaluate cell death by labeling fragmented DNA with propidium iodide, in line U937 E HL60, analyzing Ara-C (1, 5 and 10μM) and IDA (0.2, 0.5 and 1μM) effects and comparing with CQ (25 and 50μM) per 24h. Statistical analysis was performed with ANOVA or Student's T-test, followed by Tukey or Dunnet test when necessary (* p <0.05 in relation to the control group; #p <0.05 compared to Ara-C/IDA alone treatment). While the autophagic flow study by determination of the proteins involved in autophagy (LC3-II and pg62) was performed using the Western Blotting (WB) technique on cells treated with Ara-10 (μM) for 1, 2 and 4 hours isolated and in the presence of NH4Cl (autophagy inhibitor). Results: Data obtained to the moment have shown that the combination of CQ and cytarabine results in a decrease in the cytotoxic effects of this drug on U937 cells. Induction of autophagy appears to participate in this event because cytarabine, in principle, increases the protein levels of LC3II in these cells. Concerning HL60, Initial WB data showed that the treatment of HL60 cells with Ara-C alone and along with NH4Cl did not lead to any additional accumulation of LC3I, suggesting that cytarabine, principle acts as an inhibitor of autophagy and that its combination with CQ leads to increased effects of Ara-C. Conclusions: With the data obtained so far, we suggest that at first we are faced with different responses regarding the two lines studied, where inhibition of autophagy by CQ seems to decrease cell death in U937 cells and increase HL60 cells, if confirmed, we will be faced with important data that point to the need for individual autophagy measures so that its pharmacological modulation is a reality in the treatment of leukemias. Therefore, complementary studies on cell death and differentiation are underway.

Funding: FAPESP; CNPq; CAPES.

Palavras-chaves: Autophagy, Cell death, Cytarabine, Idarubicin, Leukemia

19.012 Prostaglandin signaling pathway alters Fascin-1 and Cofilin-1 gene expression in human glioblastoma cell lines T98G and U87MG

Autores Lucas Rossetti 1, Alison Colquhoun 1

Instituição 1 ICB-USP - Instituto de Ciências Biomédicas da Universidade de São Paulo

Resumo

Introduction: Glioblastoma (GBM) is the most common malignant brain tumor and there is no efficient option for therapeutic management. GBM cells migrate and invade the surrounding healthy tissue, making it more difficult to properly treat patients. Prostaglandin signaling pathways can interfere in migration. However the mechanism remains unclear. Some studies have shown that the actin-binding proteins Cofilin-1 (CFL1) and fascin-1 (FSCN1) could be the link between prostaglandins and migration.

Objective: To investigate the influence of prostaglandin signaling pathways on CFL1 and FSCN1 gene expression.

Methodology: T98G and U87MG glioblastoma cell lines were treated with: 5uM PGE2; 5uM PGF2-alpha; 50uM Ibuprofen; 40uM mPGES1 inhibitor CAY10526, for 12 hours and 24 hours. Pellets were collected and RT-qPCR was performed to evaluate CFL1 and FSCN1 expression.

Results: After 12 hour of treatment with PGE2, PGF2-alpha and CAY10526, CFL1 expression was reduced in U87MG cells. In T98G cells, Ibuprofen increased CFL1 expression after 12 hours. CFL1 expression levels were not altered after 24 hours of any treatment. FSCN1 expression was reduced after PGE2, PGF2-alpha and CAY10526 treatments for 12 hours in U87MG cells. PGE2 and PGF2-alpha increased FSCN1 expression in T98G cells, while Ibuprofen and CAY10526 decreased after 12 hours. After 24 hour, only CAY10526 treatment reduced FSCN1 expression in U87MG cells.

Conclusion: These preliminary results suggest that interfering with the prostaglandin signaling pathway alters CFL1 and FSCN1 expression after 12 and 24 hours of treatment. Further experiments will investigate the effect of these treatments in migration assays.

Funding: FAPESP and CNPq.

Palavras-chaves: Glioblastoma, Prostaglandin, Migration, Fascin-1, Cofilin-1

19.013 Astaxanthin induces cytotoxic effects and morphological alterations in vitro in B16F10-Nex2 melanoma cells

Autores Fernanda Fernandes Miranda da Cunha 1, Angela Pedroso Tonon 2, Denise Costa Arruda 1

Instituição 1 UMC - Universidade de Mogi das Cruzes, 2 USP - Universidade de São Paulo

Resumo

Introduction: There are some chemotherapy-resistant by melanoma cells used in therapeutics, demonstrating the importance to study new drugs for this kind of skin cancer. Astaxanthin is a carotenoid that belongs to a xanthophylls family can be found in microorganisms such as, bacteria, yeast and

microalgae. In addition, astaxanthin presents greater biological activity compared to other carotenoids. The astaxanthin inhibitory effect in growth of different tumor cells including colon, fibrosarcoma, breast, prostate cancer cells and fibroblasts has been reported in the scientific literature. Astaxanthin has several possibilities and promising applications in the cancer research, but there are not reports in the literature of their effect on melanoma cells yet.

Objective: Determination of the cytotoxic activity in vitro and evaluation morphologic changes induced by astaxanthin in melanoma cells.

Methodology: Astaxanthin was purified from Haematococcus pluvialis. B16F10-Nex2 cells were incubated with natural astaxanthin and viability was determined by MTT assay. In addition, was evaluated cytotoxic effect in the presence of necroptosis inhibitor (necrostatin), caspases inhibitor (Z-VAD) and ROS inhibitor (N-acetyl L-cysteine). Moreover, was observed morphologic changes in treated and untreated cells by transmission electronic microscopy.

Results: Astaxanthin demonstrated cytotoxic effects in B16F10-Nex2 cells in vitro (p<0.005). Cells were treated with increasing concentrations ranging from 5.0 to 80.0 µM, for 12, 24 and 48 hours. The EC 50% was determined for 12, 24 and 48h, being 55.00 ± 0.94 µM, 38.05 ± 0.98 µM and 29.26 ± 1.22 µM, respectively. The cytotoxic effect astaxanthin was not inhibited when B16F10-Nex2 cells were incubated in presence of necrostatin and Z-VAD. However the cytotoxic effets of astaxanthin was partially inhibited in presence of N-acetyl L-cysteine. Melanoma cells treated with astaxanthin showed significant alterations morphologic as appearance of large numbers of vacuoles and chromatin condensation.

Conclusion: We have shown that astaxanthin display antitumor effects in vitro and the cytotoxic effects was not inhibited in presence of necroptosis inhibitor and caspases inhibitor. The cytotoxic effect of astaxanthin was partially inhibited in presence of N-acetyl L-cysteine suggesting a mechanism of death related to the production of superoxide anion. In addition, astaxanthin induces morphological alterations in B16F10-Nex2 cell line which may be related to death cell by apoptosis and autophagy.

Funding: CAPES and FAPESP

Palavras-chaves: Astaxanthin, Haematococcus pluvialis, Melanoma

19.014 Evaluation of the effect of an aqueous fraction from Synadenium grantii latex on breast tumor and normal epithelial cells

Autores Julia Salles Oliveira 1, Maria Marta Martins 2, Maria Thereza Gamberini 1, Wagner Ricardo Montor 1

Instituição 1 Department of Physiological Sciences, FCMSCSP - Department of Physiological Sciences of Santa

Casa de São Paulo School of Medical Sciences, 2 Department of Gynecology and Obstetrics, ISCMSP

- Department of Gynecology and Obstetrics of Santa Casa de São Paulo Hospital

Resumo

Introduction: Synadenium grantii is a shrub popularly known as "leiterinha”. In Brazil people use it empirically, as an oral solution prepared from 18 drops of its latex in a liter of water, for cancer treatment.

Objective: Analyze the antiproliferative properties of the aqueous fraction (AF) obtained from the latex in the MCF-7 and MDA-MB231 breast cancer cells and HUVEC, a non-tumor epithelial cell line. The phytochemical composition of this fraction was also analyzed by HPLC.

Methods: The latex was lyophilized after being collected. To the lyophilized material MilliQ H2O was added for the extraction of polar the compounds. The antiproliferative activity of AF (5; 10 and 50μg/mL) was analyzed by the determination of its effects on the growth curve of the aforementioned cells, plus MTT assays. ROS formation assays were also performed. All the experiments were performed in six replicates, except for the detection of ROS, which was done in quadriplicates. Statistical analysis was performed with one- and two-way ANOVA and Bonferroni post-test for significance represented by p<0.05. For HPLC analysis, AF (250ug/mL) was injected to a C18 column

(Luna Phenomenex, 5µm; 250x10mm; 100Å), detection was done at =240 nm, using the solvent systems A: 0.1% TFA/H2O; and B: 90% acetonitrile/0.1% TFA/H2O with a gradient of 0-90% B in 150min, at a flow rate of 1mL/min.

Results: In the growth curve, MCF-7 was shown to be more sensitive, since it presented only 5,3% of viable cells on the 7th day after treatment with 50ug/mL (p<0,0001); while MDA-MB-231 showed to be more resistant, with inhibition of cell proliferation by the 5th day, making remain only 11,5% of viable cells on the 7th day with 50ug/mL (p<0,0001); HUVEC did not show cell number decrease in the growth curve but grew less than the control, remaining 41,1% of viable cells on the 7th day with 50ug/mL (p<0,0001). In the MTT assay, 5 and 10ug/mL promoted up to 10% reduction in metabolic activity in 24 and 48h in HUVEC, while 50μg/mL promoted a reduction to 38,5% in 24h and 42,4% in 48h (p<0,0001); in MCF-7, 5 and 10ug/ml promoted reduction in approximately 40% in 24h and 48h, and 50ug/ml promoted greater reduction in 48h, with 38% (p<0,0001); in MDA-MB-231, 5 and 10 μg/mL reduced in up to 30% in 24h and 40% in 48h, while 50ug/ml approximately 55% at 24 and 48h (p<0,0001). The AF showed to decrease ROS formation in the non-tumor cells, indicating that it decreased the oxidative stress of these cells.

Conclusion: The AF has antiproliferative effects on the breast tumor cell lines, according to the results of the cell growth curve assays. It has also been shown not to kill non-tumoral epithelial cells and does not present toxicity (MTT) to the cells except for 50ug/mL, and decreases the formation of basal ROS, indicating a possible protective effect for them when exposed to stressing agents. The HPLC analysis shows the presence of a major compound(s) with high polarity. Additional analyzes by LC-MS and NMR are being carried out for chemical identification of the bioactive(s) compound(s).

Funding – FAPESP

Palavras-chaves: cancer, cell biology, cell proliferation, Chemoprevention, Synadenium grantii

19.015 MODULATION OF THE ACTIVITY OF CYSTEINE CATHEPSIN PROTEASES BY METALLIC PALLADIUM COMPOUNDS

Autores Larissa Joana Vitorino 1, Aline Aparecida de Souza 1, Adelino Vieira de Godoy Netto 2, Renan Lira

de Farias 2, Wagner Alves de Souza Júdice 1

Instituição 1 UMC - Universidade de Mogi das Cruzes , 2 UNESP - Instituto de Química - IQ - Unesp -

Araraquara

Resumo

Introduction: Cysteine lysosomal proteases, among them cathepsin B and L, participate in tumor progression, metastasis, viral capsid processing, bacterial toxin processing, in facilitating the invasion of parasitic and fungi microorganisms, are also involved in the apoptotic program in tumor cells, programmed cell death and senescence, and in the control of cell proliferation and differentiation. By their deleterious actions, they are interesting targets for the development of new drugs. Objectives: To study the effects of metallic palladium compounds on the activity of cathepsins B and L as possible enzymatic modulators. Methodology: The enzymatic activity was monitored using specific buffer in spectrofluorimeter Shimadzu RF6000 at λEx = 360nm and λEm = 480nm using as fluorogenic probe Z-FR-MCA in the absence and presence of the compounds. We performed a screening and determination of the inhibition mechanism by time course and calculated Ki, k-3 and k3 using the program Grafit 5. Results: In the screening of the metallic palladium compounds at 25 μM, we observed that the inhibition of cathepsin B ranged from 0 to 38%, and for cathepsin L we observed inhibition between 52% and 72%. Interestingly, the compound [PdCl(TSC-H,PH)]2 did not promote inhibition of cathepsin B at 25μM, but it reduced the activity of cathepsin L by 72%, therefore, showing selectivity to this cathepsin. The other compounds [PdCl(TSC-H,H)]2; [PdCl(TSC-H, M)]2; [PdCl(TSC-H,E)]2 did not show significant differences in inhibition of cathepsin-L, being around average of 46%. In the evaluation of the mechanism by time course we observed that the compounds presented competitive reversible inhibition of the slow binding type. The compound [PdCl(TSC-H,E)]2 showed k-3=6.10-5±4.10-6s-1, k3=4.9.10-5±1.10-61µM-1.s-1 and Ki=1,23±0,07µM (Ki=k-3/k3) for the cathepsin L. In the inhibition of cathepsin B, were determined the values of k-3=6.10-5±3.10-6s-1, k3=1.9.10-5±1.10-6µM-1-s-1 and Ki=1,23±0,7µM. The compound [PdCl(TSC-H,H)]2 showed k-3=1.2.10-4±2.10-5s-1, k3=1.5.10-4±4.10-5µM-1.s-1 and Ki=0.83±0,02µM for cathepsin B. In relation to cathepsin L we found k-3=2.5.10-5±1.10-5s-1, k3=3.2.10-4±2.10-5µM-1.s-1 and Ki=0,077±0,004µM. For the compound [PdCl(TSC-H,M)]2 we observed k-3=4.6.10-5±1.7.10-5s-1, k3=1.1.10-4±2.10-5µM-1.s-1 and Ki=0,41±0,06µM for cathepsin B. In relation to cathepsin L, we found k-3=3.8.10-7±1.9.10-7s-1, k3=3.1.10-4±1.10-5µM-1.s-1 and Ki=0,0012±0,0001µM. For the compound [PdCl(TSC-H,PH)]2 we observed k-3=5.6.10-5±1.5.10-5s-1, k3=4.7.10-5±6.10-6µM-1.s-1 and Ki=1,2±0,4µM for cathepsin B. In relation to cathepsin L, we found k-3=8.2.10-5±1.10-5s-1, k3=2.2.10-4±2.10-5µM-1.s-1 and Ki=0,37±0,03µM. Conclusion: The evaluated compounds presented a competitive mechanism of inhibition and low Ki values characterizing high inhibitory effectiveness, however the microscopic constants k-3 and k3 presented very low values establishing a slow binding mechanism.

Supported by: FAPESP, FAEP, OMEC, CAPES, CNPQ.

Palavras-chaves: Cathepsin, inhibitor, Palladium, Proteases, Time course

19.016 Evaluation of Lactate as a Modulator of Tumorigenesis

Autores Alan Clavelland Ochioni 1, Thainá Magalhães Demaria 1, Mauro Sola-Penna 1, Patrícia Zancan 1

Instituição 1 UFRJ - Universidade Federal do Rio de Janeiro

Resumo

INTRODUCTION There are many elements that contribute for the establishment and progression of the tumor, one of them is related to changes in the metabolism of tumor cells. The Warburg Effect or aerobic glycolysis drives the glycolytic flux to the lactate production and contributes to the development of tumor resistance. Moreover, recent studies have demonstrated that higher lactate production could be related to the polarization of Tumor Associated Macrophages (TAM) towards M2 (alternative) instead of M1 (inflammatory), thus increasing tumor progression and survival. OBJECTIVES The objective of this project is to evaluate whether the inhibition of aerobic glycolysis and, consequently, of the lactate production will promote a shift of macrophage polarization (M1>M2). We are evaluating the effects of clotrimazole (CTZ), a drug that acts as PI3K inhibitor and glycolytic inhibitor, thus decreasing lactate production by the tumor cell. MATERIALS AND METHODS Our in vitro experiments were conducted with B16-F10 (human melanoma cells). For the polarization studies, we used J774 (mouse macrophages) cells. We evaluate cell viability through MTT assay and lactate production after the treatment with CTZ. The polarization of the macrophages was evaluated by qPCR. DISCUSSION AND RESULTS We observed that B16-F10 cells are less susceptible to the treatment with CTZ when we compare with other cell lines

evaluated in previous studies. B16-F10 cells viability starts to decrease after 24h of treatment with 50 M CTZ. At the concentration of 40 μM CTZ, we did not observe viability loss but a reduction in lactate production under normoxia and hypoxia conditions. This particular condition will allow us to analyze the effects of lactate reduction on the polarization of macrophages linked to tumor cells. Regarding the effects of lactate on macrophage polarization, preliminary results showed that it could be modulated by different concentrations of lactate, suggesting the effect of this metabolite on macrophages polarization.

CONCLUSION

Our results suggest that CTZ could interfere with the lactate production of tumor cells without loss of viability, which may assist in the study of the interaction of lactate production by the tumor cell and the polarization of macrophages. In preliminary analyzes we observed that lactate can modulate the polarization of macrophages, suggesting that it has an important role in the differentiation of these cells.

FUNDING :CAPES and FAPERJ

Palavras-chaves: Clotrimazol, Lactato, Macrófagos , Polarização

19.017 BJcuL, a snake venom lectin is cytotoxic to in differentiation neuroblastoma cells.

Autores Gabriela Mondini 1, Karlla Beatriz Vital Monti 1, Sheron Cogo 2, Jéssica Goetten 2, Luciana de F

Chaves de Mello Zischler 1, Selene Elifio Esposito 2

Instituição 1 ECV/PUCPR - School of life sciences, Pontificia Universidade Católica do Paraná, 2 PPGCS -

Programa de Pós-Graduação em Ciências da Saúde

Resumo

BJcuL, a snake venom lectin is cytotoxic to in differentiation neuroblastoma cells

Introduction: Neuroblastoma (NB) is the most common extra cranial solid pediatric tumor. The prevalence of this tumor is preferentially in young children with a median age at diagnosis of 18 months, and accounts for 15% of pediatric cancer deaths. It arrives from neural crest cells, and remains distinct from other solid tumors due to its biological heterogeneity, as NB tumors vary within a spectrum from spontaneous regression to a highly-aggressive metastatic disease. NB cell lines can represent tumor cells characteristics with different levels of malignancy: more undifferentiated cells are more aggressive but they are also more susceptible to chemo and radiotherapy. On the other hand in-differentiation cells seem to be less aggressive while can be more refractory to treatment. Snake venoms are mixtures of biological active compounds, mainly enzymatic and non-enzymatic proteins. BJcuL is a D-galactose binding C-type lectin isolated from the venom of Bothrops jararacussu which has previously demonstrated cytotoxic action over different tumor cell types. However, there is no data about the investigation of its effect in neuroblastoma cells.

Objective: The goal of this study was to evaluate the viability of neuroblastoma cells treated with BJcuL.

Methodology: Three NB cell lines were used in this project aiming to surrogate the heterogeneity of NB tumors. SK-N-SH cells present a highly heterogeneous phenotype, capable of originating differentiated NB populations (N-type and S-type), due to the presence of cells classified as I-type. SH-SY5Y presents mainly N-type cells, and CHLA-20 cell line, established from a metastatic tumor post chemotherapy treatment, though more resistant to chemotherapy compounds. Viability was accessed by the MTT or methylene blue methods after cellular incubation with BJcuL (0.1 to 5 µM) for 24h. Cells were also evaluated by clonogenic assay (incubation with 0.3 µM BJcuL followed by incubation with fresh medium for 3 weeks) and by the scratch wound healing method, to analyze BJcuL effect on cell migration. The fibroblast cell line HFF-1 was used as a non-tumoral control.

Results: BJcuL (5 µM) had cytotoxic effect over the more differentiated cell lines, reducing cell viability of SH-SY5Y and CHLA-20 cells in about 30%, but had no effect in the viability or migration of SK-N-SH cells. The capacity of colony formation of CHLA cells was reduced to 40% after BJcuL incubation when compared with non-treated cells. This effect was abolished when the lectin was previously incubated with D-galactose, indicating that it should act through its carbohydrate recognition domain. BJcuL was also cytotoxic to fibroblasts at the highest concentrations (2.5 and 5.0 µM).

Conclusions: BJcuL was cytotoxic to the more differentiated and more adherent NB cells. This action was not specific as the lectin also reduced the viability of non-tumoral fibroblasts.

Funding: Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Estado do Paraná.

Palavras-chaves: BJcuL, Cancer, Neuroblastoma, Pediatric Cancer, Solid pedriatic tumor

19.018 A NOVEL 1,2,3-TRIAZOL DERIVATIVE WITH SELECTIVE ACTIVITY AGAINST HUMAN BREAST CANCER CELLS

Autores

AMANDA OLIVEIRA 1, JESSICA R. BRANCO 1, VANESSA G. OLIVEIRA 2, INGRID C.

CHIPOLINE 2, MIRIAM F. O. LIMA 2, FERNANDA C. S. BOECHAT 2, FERNANDO C. DA

SILVA 2, VÍTOR F. FERREIRA 2, MAURO SOLA-PENNA 3, MARIA C. B. V. DE SOUZA 2,

PATRICIA ZANCAN 1

Instituição 1 LabOMol - Laboratório de Oncobiologia Molecular, 2 UFF - Departamento de Química Orgânica,

Programa de Pós-Graduação em Química, 3 LabECom - Laboratório de Enzimologia e Controle do

Metabolismo

Resumo

INTRODUCTION: Breast cancer is a major cause of death among women worldwide. Although several successful approaches to control human breast cancer, it is still a target for the scrutinization of novel drugs due to its ability to become resistant and, most of all, the large number of side-effects of the current medicine. The side-effects are most due to the lack of selectiveness of the used drugs that affect both, cancer and non-cancer cells. Thus, there is the constant search for a more selective drug that affect cancer cells with minimal effects on non-cancer cells. Recently, we have identified an azolic drug with selective effects against breast cancer. However, the effects are obtained at relatively high concentrations (50 µM). Therefore, we synthesized novel 1,2,3-triazolic derivatives targeting the amelioration of the antitumor effects.OBJECTIVE:To test the antitumor properties of novel 1,2,3-triazole derivatives using tumor and non-tumor human breast cells (MCF-7 and MCF10A, respectively). METHODOLOGY:We evaluated cell viability, ROS production, apoptosis induction, mitochondrial potential and glucose metabolism in the cells treated with the drugs.RESULTS:Initially, 5 novel 1,2,3-triazol oxoquinolines conjugated with naphtoquinones were screened for the ability to selectively interfere with cancer cells viability. All the compounds were toxic for the cells, presenting different potencies, but only one, named VI03, was able to decrease breast cancer cell (MCF-7) viability (IC50 = 5 µM) without interfering with non-cancer breast cell, MCF10A. Therefore, VI03 was used in the further experiments to characterize its mechanism of action. Selectively, VI03 induced apoptosis in MCF-7 cells, with concomitant increment in ROS production by these cells. This effect is not related to impaired mitochondrial activity, since the drug does not affect mitochondrial potential in these cells. On the other hand, glucose consumption, lactate production and intracellular ATP levels were negatively impacted by the treatment with the drug. All of these effects were exclusively observed in MCF-7 cells, being the non-tumor cell line MCF10A insensitive to the drug.Conclusion: Our results are indicative that VI03 selectively interfere with breast cancer cells by promoting a ROS-mediated apoptosis in these cells. ROS might be being produced through the impaired glycolytic flux, which also facilitate the induction of apoptosis.

Palavras-chaves: 1,2,3-triazol, breast cancer, naphtoquinones

19.019 ANTITUMOR ACTIVITY OF AN ACRIDINE DERIVATIVE ON EHRLICH ASCITES CARCINOMA MODEL

Autores

Vivianne Mendes Mangueira 1, Tatyanna Kelvia Gomes de Sousa 1, Tatianne Mota Batista 1, Ryldene

Marques Duarte da Cruz 1, Renata Albuquerque de Abrantes , Francisco de Assis Silveira Oliveira 1,

Ana Ligia da Costa Pereira 2, Vanessa de Lima Serafim 2, Ricardo Olímpio de Moura 2, Marianna

Vieira Sobral 1

Instituição 1 UFPB - Universidade Federal da Paraiba, 2 UEPB - Universidade Estadual da Paraiba

Resumo

Introduction: Acridine derivatives have antitumor activity, however, side effects, development of resistance and their low bioavailability have limited their use. N0-(2-

chloro-6-methoxy-acridin-9-yl)-2-cyano-3-(4-dimethylaminophenyl) acrilohidrazida (ACS-AZ10) is an acridine derivative with no reports in literature.

Objectives: To evaluate the in vivo antitumor activity and mechanisms of action (cell cycle profile and antiangiogenic effect) of ACS-AZ10 on Ehrlich ascites carcinoma (EAC) model.

Methodology: Procedures were approved by the Ethics Committee on the Use of Animals (CEUA)/UFPB, no 0801/14. EAC cells were implanted in the female mice’s peritoneal cavity (n=6/group). After 24 h, ACS-AZ10 (7.5, 15 or 30 mg/kg) was administered intraperitoneally (i.p.), for nine days. 5-fluorouracil (5-FU) (25 mg/kg, i.p.) was used as a positive control and Tween 80 at 12% (v/v) in saline as negative control. The animals were euthanized and ascitic fluid was collected for the assessment of tumor weight and volume, and tumor cell total count. For cell cycle analyses, cells from ascitic fluid (groups: tumor control, 25 mg/kg 5-FU and 7.5 or 15 mg/kg ACS-AZ10) were stained with propidium iodide. BD FACS CANTO IITM (USA) was used, acquiring 10.000 events/sample. To evaluate peritoneal angiogenesis, the peritoneum was cut open and the inner lining of the peritoneal cavity was examined for angiogenesis in tumor control, 25 mg/kg 5-FU and ACS-AZ10 (7.5 or 15 mg/kg) groups, and photographed. Microvessel density was determined by selecting the blood vessel area per field in selected vascularized areas divided by the whole area, using AVSOFT® software. The results are expressed as the mean±standard error of mean, and the differences were compared by analysis of variance (ANOVA), followed by Tukey’s test (p<0.05).

Results: ACS-AZ10 induced a significant decrease on all the analyzed parameters: tumor weight 15 mg/kg (1.95 ± 0.11 g) or 30 mg/kg (2.80 ± 0.24 g); volume 15 mg/kg (0.04 ± 0.01 mL) or 30 mg/kg (0.04 ± 0.01 mL); and tumor cell total count 15 mg/kg (0.04 ± 0.03x 107

cells) or 30 mg/kg (0.03 ± 0.02 x 107 cells), when compared to tumor control group (13.50 ± 0.78 g; 12.07 ± 0.48 mL; 134.50 ± 24 x 107 cells). Similar results were observed for the standard drug (5-FU – 25 mg/kg). Significant change in cells distribution on the different cell cycle phases was observed. After the treatment with 7.5 mg/kg ACS-AZ10, it was observed an increase in sub-G1 peak to 9.2%, associated with decrease in G0/G1 (34.2%) phase, when compared to the tumor control group (1.9%; 47.7%, respectively). ACS-AZ10 15 mg/kg induced decrease in G0/G1 (37.7%), accompanied by increase on G2/M (23.5%) phases when compared to the tumor control group (47.7%; 17.7%, respectively). ACS-AZ10 (7.5 and 15 mg/kg) induced a significant decrease of the microvessel density (19.3 ± 6.8% and 19.4 ± 6.2%, respectively) when compared to the tumor control group (75.3 ± 13.9%).

Conclusion: ACS-AZ10 has in vivo antitumor activity by induce cell cycle arrest in G2/M phase and antiangiogenic action.

Funding: CNPq and Capes

Palavras-chaves: Acridine derivatives, cell cycle, Ehrlich ascites carcinoma, in vivo antitumor activity, peritoneal angiogenesis

19.020 INFLUENCE OF BMI IN THE SURVIVOR OF PATIENTS WITH HEAD AND NECK CANCER

Autores Lucas André Silva Bonela 1, João Victor da Silva Coutinho 1, Karine Gadioli de Oliveira 1, Sonia

Alves Gouvea 1

Instituição 1 UFES - Universidade Federal do Espírito Santo

Resumo

Introduction: The variation in nutritional status of patients is one of the most common intercurrence in oncology, including head and neck cancer. The higher risk in these patients can be justified by their previous eating habits and by the location of the tumor. Therefore, evaluate the nutrition status of the patient trough the body mass index seem to be an interesting option that can be justified by the simplicity and non invasiveness. All these facilities contributes to the use of this method on

the nutritional monitoring of the oncological patient during antineoplastic therapy and before its starts. The monitoring before the antineoplastic treatment helps to determinate a possible intervention in the diet, in order to improve the survival rate. Object: Evaluate the influence from body mass index on survival rate of head and neck patients and relate to tobacco and alcohol consumption, tumor size (T), lymph node involvement (N), staging and systolic arterial pressure. Methods: These study was approved by CEP/UFES (n° 99.242/2012). Were included patients with histopathology result of squamous cells carcinoma from head and neck. The corrections were made by Spearman an surveiliescence were made by Kaplan-Meier analysis and Long-Rank were used to compare both curves made. Were considered significant p<0,05. Results: The survival rate from the group with BMI<18,5 Kg/m² were significant smaller than the group with BMI 8,5-25 Kg/m², 40% and 65,6% respectively. The group with BMI >25 Kg/m² had the survival rate of 77,8%, witch is the higher between the groups. We still observe that was diference between those who uses tobacco and those who don’t (22,74 ± 3,926 and 25,51 ± 3,31 in case of p=0,0057). No relation between BMI and alcohol consumption were identified. Those who uses both tobacco and alcohol had a smaller BMI then the ones that don’t uses neither (23,11 ± 4,094 Kg/m2 vs 27,72 ± 2,189 Kg/m2, P=0,0023). The factor T (0,003), N (0,007) and stage (0,001) had an inverse correlation with BMI. Conclusion: Patients with head and neck cancer have difficulty in alimentation that can became worse during the antineoplastic treatment. The body mass index take charge, in a good way, in patients survival rate. The use of alcohol and tobacco impact in the loss of body mass reducing the BMI and relating with a poor clinic prognostic. Because of all seen we conclude that BMI is a relevant factor in evaluation of prognostic and survival rate of patients with head and neck cancer.

Palavras-chaves: Alcohol consumption, BMI, head and neck cancer, survival rate, smoking

19.021 EFFECT OF BOTRYOSPHAERAN, β-(1→3)(1→6)-D-GLUCAN, ON TUMOR DEVELOPMENT IN HIGH-FAT HIGH-SUGAR DIET-INDUCED OBESE RATS.

Autores

Mariana Costa Ribeiro 1, Patrícia Karina Comiran 1, Amadeu Zattoni da Silva 1, John Hebert Gomes

da Silva 1, Ana Emília Finamor Chiaradia 1, Izabella Santos 1, Kamila Ortega Martins 1, Morenna

Alana Giordani 1, Robert Frans Huibert Dekker 2, Aneli de Melo Barbosa Dekker 3, Pâmela Alegranci 1, Eveline Aparecida Isquierdo Fonseca de Queiroz 1

Instituição 1 UFMT - Universidade Federal de Mato Grosso- Campus Sinop , 2 UTFPR - Universidade

Tecnológica Federal do Paraná , 3 UEL - Universidade Estadual de Londrina

Resumo

Introduction: Cancer is a multifactorial disease characterized by an uncontrolled growth of cells that can invade other tissues causing metastasis. Studies have demonstrated that obesity increases the development of several types of cancer, like breast cancer, and that it is associated with insulin resistance, hyperinsulinemia and low chronic inflammation, frequently present in obesity. Fungal β-D-glucans of the (1→3)-type are known to exhibit direct antitumor effects, and can also indirectly decrease tumor proliferation through immunomodulatory responses. Botryosphaeran, a β-(1→3)(1→6)-D-glucan, produced by the fungus Botryosphaeria rhodina MAMB-05 have been demonstrated to present antimutagenic, hypoglycaemic, hypocholesterolaemic, antiproliferative and pro-apoptotic activities. However, its effects on tumor development are not well understood. Objective: To evaluate the effects of botryosphaeran on tumor development in high-fat and high sugar diet-induced obese rats, as well as analyze its mechanism of action. Methodology: Male wistar rats (~30 days) were divided randomly in two groups, control and obese. Obese rats received high-fat diet (49.7% energy from fat) and water with sucrose (300g/L) for 8 weeks. Control received standard diet and water without sugar. On 8th week, 1x107

Walker-256 tumor cells were subcutaneously injected in the right flank of the rats and concomitantly the treatment with botryosphaeran (12 mg/kg/15 days, via gavage) started. Thus, the rats were subdivided in four groups: control tumor (CT), control tumor botryosphaeran (CTB), obese tumor (OT) and obese tumor botryosphaeran (OTB). On 10th week, the tumor development and botryosphaeran effect were

analyzed. Obesity was characterized; feed intake, glucose and lipid profiles, glucose tolerance, insulin resistance and hemogram were analyzed. Tumor weight, percentage of rats that developed tumor and presence of metastasis were evaluated, and protein expression was determined by Western blotting. Data was analyzed by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. The project was approved by the Ethics Committee for Animal Use and Experimentation at UFMT (Protocol n. 23108.722108/2017-19). Results: Tumor development was higher in OT rats compared to CT rats, and botryosphaeran was effective in reducing this development (g/100g b.w.; CT=2.44±1.13, CTB=4.78±3.98, OT=8.06±4.89* and OTB=5.79±3.68). OT rats showed significant increases in retroperitoneal, periepididymal and mesenteric adipose tissue, reduced muscle, presented glucose intolerance, insulin resistance and hyperglycemia, as well as anemia and leukocytosis. Botryosphaeran improved glucose tolerance, insulin resistance and reduced the hyperglycemia. In addition, botryosphaeran did not correct the anemia, but increased the total leukocyte count (x103/mm3; CT=15.1±3.9, CTB=19.3±0.2, OT=33,2±4,7 and OTB=45,7±29,9*). CT and CTB rats presented granulocitosis and monocitosis, suggesting an immediate immune response. OT and OTB rats presented granulocytosis, lymphocytosis and monocytosis, suggesting an adaptive immune response. No difference was observed in Bax, Bcl-2, caspase-3 and p27 protein expression. Conclusion: Our data allows us to suggest that botryosphaeran, reducing the stimulatory effect of obesity on tumor development and increasing the immune response, has a potential role in the management of cancers. * p<0,05 vs CT. n=6-8 Funding: UFMT.

Palavras-chaves: Botryosphaeran, Cancer, Immune response, Insulin resistance, Obesity

19.022 ANTITUMOR ACTIVITY OF A NEW PIPERINE ANALOGUE

Autores

Tatyanna Kélvia Gomes de Sousa Tatyanna Kélvia Gomes de Sousa 1,1,1,1, Vivianne Mendes

Mangueira Vivianne Mendes Mangueira 1,1,1,1, Tatianne Mota Batista Tatianne Mota Batista 1,1,1,1,

Ryldene Marques Duarte da Cruz Ryldene Marques Duarte da Cruz 1,1,1,1, Renata Albuquerque de

Abrantes Renata Albuquerque de Abrantes 1,1,1,1, Camyla Caroliny Neves de Andrade Camyla

Caroliny Neves de Andrade 1,1,1,1, Robson Cavalcante Veras Robson Cavalcante Veras 1,1,1,1,

Helivaldo Diógenes da Silva Souza Helivaldo Diógenes da Silva Souza 1,1,1,1, Bruno Freitas Lira

Bruno Freitas Lira 1,1,1,1, Petrônio Filgueiras de Athayde-Filho Petrônio Filgueiras de Athayde-Filho 1,1,1,1, Marianna Vieira Sobral Marianna Vieira Sobral 1,1,1,1

Instituição 1 UFPB - Universidade Federal da Paraíba

Resumo

Introduction: N-(p-ethylphenyl)acetamide piperinoate (HE-03) is a new piperine analogue, an amide alkaloid with antitumor effect.

Objectives: To evaluate the in vivo antitumor activity and mechanisms of action (cell cycle profile and antiangiogenic effect) of HE-03 in Ehrlich ascites carcinoma (EAC) model.

Methodology: Procedures were approved by the Ethics Committee on the Use of Animals (CEUA)/UFPB, no 0901/14. EAC cells were implanted in the female mice’s peritoneal cavity (n=6/group). After 24 hours and for 9 days, HE-03 (6.25, 12.5 or 25 mg/kg) was administered intraperitoneally (i.p.). 5-flurouracil (5-FU) (25 mg/kg, i.p.) was used as positive control and Tween 80 at

12% (v/v) in saline as negative control. After euthanasia, ascitic fluid was collected for the assessment of cellular viability, tumor volume and weight, and tumor cell total count. For cell cycle analysis, cells (groups: tumor control, 25 mg/kg 5-FU and 12.5 mg/kg HE-03) were stained with Propidium Iodide (PI). BD FACS CANTO IITM (USA) was used, acquiring 10.000 events/sample. To evaluate peritoneal angiogenesis, the peritoneum was cut open and the inner lining of the peritoneal cavity was examined in tumor control, 25 mg/kg 5-FU and 12.5 mg/kg HE-03 groups, and then they were photographed. Microvessel density was determined by selecting the blood vessel area per field in selected vascularized areas divided by the whole area, using AVSOFT® software. The results are expressed as the mean ± standard error of mean, and the differences were compared by analysis of variance (ANOVA), followed by Tukey’s test (p<0.05).

Results: Treatments with HE-03 (12.5 or 25 mg/kg) and 5-FU, respectively, induced a significant decrease on all the analyzed parameters - tumor weight (4.4 ± 1.3; 3.5 ± 0.8; 1.8 ± 0.2 g), tumor volume (3.5 ± 1.4; 1.1 ± 0.2; 0.09 ± 0.03 mL), cell viability (8.5 ± 1.9; 6.6 ± 2.5; 0.3 ± 0.07 x106 cells/mL), and tumor cell total count (3.9 ± 2.0; 0.8 ± 0.2; 0.02 ± 0.01 x107 cells), while 6.25 mg/kg HE-03 reduced the cell viability (9.5 ± 6.7 x106 cells/mL) and tumor cell total (68.4 ± 8.0 x107 cells), when compared to tumor control group (tumor weight: 13.5 ± 0.8 g; volume: 10.4 ± 1.0 mL; cell viability: 123.5 ± 13.0 x106 cells/mL; tumor cell total: 150.0 ± 25.7 x107 cells). After the treatment with 12.5 mg/kg HE-03, it was observed an increase in sub-G1 peak to 24.8%, associated with decrease in G0/G1 (31.0%) phase, when compared to the tumor control group (2.2%; 47.7%, respectively). 5-FU induced increase in sub-G1 peak (76.1%), accompanied by reduction on G0/G1 (2.3%) and G2/M (2.8%) phases. HE-03 (12.5 mg/kg) and 5-FU (25 mg/kg) induced a significant decrease of the microvessel density (11.5 ± 1.5% and 8.0 ± 2.1%; respectively) when compared to the tumor control group (75.3 ± 13.9%).

Conclusion: HE-03 has in vivo antitumor activity by interfering in the cell cycle progression and presenting antiangiogenic effect.

Funding: CNPq and Capes

Palavras-chaves: Antitumor, Ehrlich ascites carcinoma , Piperine

19.023 Mechanism of antitumor action of a piperine analogue

Autores

tatianne mota batista 1, jephesson alex floriano santos 1, monalisa taveira brito 1, vivianne mendes

mangueira 1, tatyanna kelvia gomes de sousa , renata albuquerque abrantes 1, ana paula gomes de

moura 1, rafael carlos ferreira 1, Ryldene Marques Duarte da Cruz 1, bruno farias lira 1, helivaldo

diogenes da silva souza 1, petronio figueiras de athayde-filho 1, marianna vieira sobral 1

Instituição 1 UFPB - universidade federal da paraíba

Resumo

Title: Mechanism of antitumor action of a piperine analogue

1Batista, T.M., 1Santos, J. A. F., 1 Brito, M.T., 1Mangueira, V.M., 1Sousa, T.K.G., 1Abrantes, R.A., 1Moura, A. P., 1Ferreira, R.C., 1Cruz, R.M.D, 2Lira, B.F., 2Souza, H.D.S, 2Filho, P.F.A, 1Sobral, M.V.

1Departamento de Ciências Farmacêuticas, UFPB, João Pessoa/PB, 2Departamento de Química, UFPB, Paraíba/PB.

Introduction: Natural products still have an important part as prototypes to synthesis of new drug candidates. Piperine is an alkaloid amide, obtained from Piper species, which shows antitumor activity along to significant toxicity. Several piperine analogues are being studied, among them the 2-oxo-2-(4-nitrophenylamine) ethyl piperinoate (HE-02). Our previous data have shown significant in vivo antitumor activity for HE-02 on Ehrlich ascitic carcinoma (EAC) model.

Objective: To evaluate mechanisms of antitumor action of HE-02 (cell cycle profile, antiangiogenic action and nitric oxide dosage).

Methodology: Procedures were approved by the Ethics Committee on the Use of Animals (CEUA)/UFPB, n. 005/2016. EAC cells were implanted in the female mice’s peritoneal cavity (n=6/group). After 24 hours and for 9 days, HE-02 (12.5 mg/kg) was administered intraperitoneally (i.p.). Tween 80 at 12% (v/v) in saline was used as negative control and 5-FU (25 mg/kg, i.p.) as positive control. For cell cycle analysis, the animals were euthanized and ascitic fluid cells were collected, and stained with propidium iodide. BD FACS CANTO IITM (USA) was used, acquiring 10.000 events/sample. To evaluate peritoneal angiogenesis, the peritoneum was cut open and the inner lining of the peritoneal cavity was examined for angiogenesis, and then they were photographed. Microvessel density was determined by selecting the blood vessel area per field in selected vascularized areas divided by the whole area, using AVSOFT® software. Nitrite levels in the peritoneal fluid of animals of all treatment groups were quantified using Griess reagent, consisting of 0.1% N-1-naphthyl-ethylenediamine, 1% sulfanilamide in 2.5% phosphoric acid solution, using NaNO2 as standard. Concentrations of nitrite were calculated from a standard curve previously established with known molar concentrations of sodium nitrite. The tests were done in quadruplicate and the values expressed in μmolar. Results are expressed as the mean±standard error of mean, and the differences were compared by analysis of variance (ANOVA), followed by Tukey’s test (p<0.05).

Results: In cell cycle analysis, HE-02 induced only an increase of 10% in the sub-G1 peak compared to the tumor control group, suggesting that changes on cell cycle profile do not represent an important mechanism for the HE-02 antitumor effect. 5-FU induced increase in sub-G1 peak (93.43%), accompanied by reduction on G0/G1 (1.36%) and G2/M (0.69%) phases. The treatment with HE-02 and 5-FU reduced the peritumoral microvessel density (0,24 ± 0,02%; 0,16 ± 0,02%, respectively) when compared to the tumor control group (0,39% ± 0,03%), indicating antiangiogenic action. 5-FU did not induce significant changes in nitrite concentration. However, for the group treated with HE-02, a significant increase in nitrite concentration (703 ± 85.44 μM) was observed in comparison to the tumor control group (410.4 ± 38.66 μM).

Conclusions: These results together suggest that HE-02 has antitumor activity by induce antiangiogenic action and cellular damage associated with NO production.

Funding: CNPq and Capes

Palavras-chaves: antiangiogenic action , Mechanism of antitumor action , nitric oxide, Natural products , Piperine analogue

19.024 THIOSEMICARBAZONES AS ANTICANCER PRODRUGS AND RADIOSENSITISING AGENTS

Autores Barbara Guedes Louzada 1, Nayara Santana Peixoto 1, Alexandre Almeida Oliveira 2,1, Heloisa

Beraldo 2, RAQUEL GOUVEA SANTOS 1

Instituição 1 CDTN - Centro de Desenvolvimento da Tecnologia Nuclear, 2 UFMG - Universidade Federal de

MInas Gerais

Resumo

Indroduction: Radiotherapy and chemotherapy are extensively used in the treatment of carcinomas. However several tumors have specific molecular factors that contribute to tumor resistance and a possible recurrence. Considerable efforts have been made to maximize drug efficiency and minimize side effects. Our group have developed many compounds based on Schiff base as antitumor agents in order to improve tumor therapy outcomes. In this study, new compounds derived from thiosemicarbazones were studied as pro-drugs and radiosensitizing agents for cancer therapy.

Objective: To identify the cytotoxic potential of compounds derived from thiosemicarbazone and imidazol against human glioblastoma cell line(U87) and characterize their potential as prodrug and radiosensitising agents.

Methodology: Glioma cells were treated with increasing concentrations (10-10 to 10-4 M) of the compounds for 48h. The effect of radiotherapy was performed by cell irradiation with Co-60 gamma cell (0-3Gy). The effect of drugs and ionising radiation on metabolic viability and cell survival were evaluated respectively by MTT assay and clonogenic assay. In order to evaluate radiosensitising potential, cells were treated with chemotherapy followed by irradiation. Cellular morphology was qualitatively evaluated by contrast microscopy. Experiments were done in triplicates and repeated at least three times in order to confirm reproducibility. Data were considered significant when P-value<0.05.

Results: Drugs induced morphological alterations such as rounding, reduction of cytoplasmic volume, appearance of vesicles and “blebs suggesting cell death. IC50 values (concentration of the compound that produces 50% of cell death) were determined after normalization and non linear regression fit of dose response curve plot (log (inhibitor) vs. response) using the Graphpad Prism program. The IC50 of HBL 106 and HBL 128 were 10.2 and 40.0 µM, respectively. (drug monotherapy). After drug combination with radiotherapy (3Gy) cell survival decreased about 50% (+7%) and 87% (+7%) respectively suggesting radiosensitisation of the glioma cells.

Conclusion: All compounds tested showed cytotoxic effect at micromolar range. The combined therapy with gamma radiation and thiosemicarbazones derived from imidazol increased the induction of morphological alterations suggesting cell death as well as decreased cell proliferation. The radiosensitising potential of these compounds suggests that they can serve as a promising platform to develop radiosensitising molecules against malignant glioma.

Funding: FAPEMIG, CNPq and CDTN/CNEN.

Palavras-chaves: Thiosemicarbazones, anticancer, prodrugs, radiosensitisation

19.025 Antitumor potential of the hydantoin derivative 3,5-diphenyl-imidazolidine-2,4-dione.

Autores

Renata Albuquerque de Abrantes Renata Albuquerque de Abrantes 1,1, Daiene Martins Beltrão

Daiene Martins Beltrão 1, Tatyanna Kelvia Gomes Sousa Tatyanna Kelvia Gomes Sousa 1, Vivianne

Mendes Mangueira Vivianne Mendes Mangueira 1, Tatianne Mota Batista Tatianne Mota Batista 1,

Ryldene Marques Duarte da Cruz Ryldene Marques Duarte da Cruz 1, Thais Bezerra Mongeon

Honorato Thais Bezerra Mongeon Honorato 1, Severino Araujo de Souza Severino Araujo de Souza 1,

Petrônio Filgueiras de Athayde-Filho Petrônio Filgueiras de Athayde-Filho 1, Marianna Vieira Sobral

Marianna Vieira Sobral

Instituição 1 UFPB - Universidad Federal da Paraiba

Resumo

Title: Antitumor potential of the hydantoin derivative 3,5-diphenyl-imidazolidine-2,4-dione. 1Abrantes, R.A.*, 1Beltrão, D.M., 1Sousa, T.K.G., 1Mangueira, V.M, 1Batista, T.M., 1Cruz, R.M.D., 1Honorato, T.B.M., 2Souza, S.A., 2Athayde-Filho, P.F., 1Sobral, M.V., 1Departamento de Ciências Farmacêuticas, 2Departamento de Química, UFPB, Paraíba/PB

Introduction: Cancer is a group of heterogeneous genetic diseases characterized by uncontrolled cell proliferation. Problems in regard to effectiveness, safety as well as development of resistance to treatment foment the need for researches on new molecules with antitumor potential. The synthesis of hydantoin derivatives has been widely used because of the reactivity of its ring system and its biological properties, such as, anticonvulsant, antimicrobial and antitumor.

Objectives: To evaluate the in vivo antiangiogenic and antitumor activity of the hydantoin derivative 3,5-difenil-imidazolidina-2,4-diona (IM-15) on Ehrlich ascites carcinoma (EAC) model.

Methodology: Procedures were approved by the Ethics Committee on the Use of Animals (CEUA)/UFPB, n. 0511/11. For in vivo antitumor activity, EAC cells were implanted in the female mice’s peritoneal cavity (n=6/group). After 24 hours and for 9 days, IM-15 (12.5, 25 or 50 mg/kg) was administered (intraperitoneally, i.p.). 5-Fluorouracil (5-FU) (25 mg/kg, i.p.) was used as positive control and Tween 80 at 12% (v/v) in saline as negative control. The animals were euthanized and ascitic fluid was collected for the assessment of cellular viability (x106/mL), and tumor volume (mL) and weight (g). To evaluate peritoneal angiogenesis, the peritoneum was cut open and the inner lining of the peritoneal cavity was examined for angiogenesis in tumor control, 25 mg/kg 5-FU and HE-03 (25 and 50 mg/kg) groups, and then they were photographed. Microvessel density was determined by selecting the blood vessel area per field in selected vascularized areas divided by the whole area, using AVSOFT® software. All results are expressed as the mean±standard error of mean, and the differences were compared by analysis of variance (ANOVA), followed by Tukey’s test (p<0.05).

Results: IM-15 (25 or 50 mg/kg) and 5-FU, respectively, induced a significant decrease on all the analyzed parameters - tumor weight (6,74 ± 2,09; 5,29 ± 1,52; 0,78 ± 0,22 g), tumor volume (6.00 ±

1.22; 4.65 ± 1.09; 0.01 ± 0.01 mL) and cell viability (77.44 ± 13.11; 64.44 ± 10.21; 64.60 ± 10.11 x 106 cells/mL), while 12.5 mg/kg IM-15 reduced only the cell viability (112.00 ± 9.10 x 106 cells/mL), when compared to tumor control group (tumor weight: 13,33 ± 0,46 g; volume: 10,88 ± 0,39 mL; cell viability: 177,40 ± 12.42 x 106 cells/mL). In addition, IM-15 (25 and 50 mg/kg) and 5-FU (25 mg/kg) induced a significant decrease of the microvessel density (33.8 ± 7.7% and 28,8 ± 7.4%; 27.6 ± 2.5%, respectively) when compared to the tumor control group (77.8 ± 7,8%).

Conclusion: IM-15 has significant in vivo antitumor activity by inducing antiangiogenic action.

Funding: CNPq

Palavras-chaves: Hidantoína, ATIVIDADE ANTIANGIOGENICA , Carcinoma ascítico de Ehrlich

19.026 ROLE OF KININ RECEPTORS IN LUNG CANCER CELLS AND TUMOR-ASSOCIATED-MACROPHAGES

Autores Isabella Ogusuku 1, Henning Ulrich 1, Isis Nascimento 1

Instituição 1 IQUSP - Instituto de Química da Universidade de São Paulo

Resumo

Title: ROLE OF KININ RECEPTORS IN LUNG CANCER CELLS AND TUMOR-ASSOCIATED-MACROPHAGES

1 Ogusuku, I. E. Y., 1 Ulrich, H., 1 Nascimento, I. C.; 1 Department of Biochemistry, IQUSP, São Paulo/SP

Introduction: Inflammation, a hallmark of cancer, supplies tumor cells with factors that are important for their progression. Inflammatory cells, especially tumor-associated macrophages (TAMs), are recruited by tumor cells and infiltrate tissues, thus promoting the tumor development by angiogenesis and suppression of the adaptive immunity.Macrophages in tumor microenvironment have been characterized as classically (M1) or alternatively activated (M2) based on surface receptors, gene signatures and secretion of inflammatory mediators. Previous studies showed that M1 and M2 subtypes have anti- and pro- tumoral effects, respectively. Kinins are generated within inflammatory tissue microenvironments and promote cell proliferation, cell migration and leukocyte activation. Kinin receptors (B1R and B2R) are also related to cancer progression, however, whether or not they are significant to macrophage-tumor cell interactions is still unknown.

Objective: To investigate the effects of lung cancer conditioned-medium (CM) on macrophage polarization and the role of B1R and B2R in the interactions between macrophages and A549 cells.

Methodology: Lung cancer A549 cell line and macrophages obtained from monocytic cells THP-1 were used as models of study. IFN-y and IL-4 were used as M1 and M2 control treatments for polarization, respectively. Macrophages were also treated with CM collected from five different A549 groups: previously treated with agonists BK or LDBK; antagonists HOE or R715; and non treated. To characterize the macrophage subtypes and analyze the expression of kinin receptors in all cells, we used Flow Cytometry (FC) and Immunocytochemistry (IC).

Results: FC and IC confirmed B1R and B2R expression in A549 cells and in all macrophages subtypes. FC anlysis showed that the expression of CCR7 was almost exclusively expressed in M1. CD80 was also up-regulated in M1, although the basal expression was high in M2. CD206 and CD23 were more highly expressed in M2. After the treatment with A549 CM, the macrophages presented high expressions of CD14 and M1 receptors (CD80 and CCR7). The treatment with CM obtained from A549 previously treated with BK, LDBK, HOE or R715 resulted in up-regulated expressions of M2 (CD206 and CD23) and down-regulated expressions of M1 characteristic receptors (CCR7 and CD80).

Conclusion: A549 secretome induce macrophage polarization to M1 subtype. A549 Kinin receptors modulation may promote polarization to M2 subtype.

Funding: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP#2017/00386-7)

Palavras-chaves: Inflammation, Kinin Receptors, Lung Cancer, Macrophages

19.027 Mechanisms of antitumor action of the hydantoin derivative 3,5-diphenyl-imidazolidine-2,4-dione

Autores

Daiene Martins Beltrão 1,2, Ana Paula Gomes Moura 1,1, Monalisa Tavares Brito 1, Camyla Andrade 1,

Tateanne Mota Batista 1, Tatyanna Kelvia Gomes Sousa 1, Vivianne Mendes Mangueira 1, Renata

Albuquerque de Abrantes 1, Sâmia Sousa Duarte 1, Severino Araújo de Souza 1, Bruno Freitas Lira 1,

Petrônio Filgueiras de Athayde-Filho 1, Marianna Vieira Sobral 1

Instituição 1 UFPB - Universidade Federal da Paraíba, 2 FACENE - Faculdade Nova Esperança

Resumo

Introduction: The 3,5-diphenyl-imidazolidine-2,4-dione (IM-15) is a hydantoin derivative with few reports in the literature. Nevertheless, hydantoin derivatives are heterocyclic compounds which show several biological activities because of the reactivity of its ring system, including, antimicrobial, anticonvulsant and antitumor activities.

Objectives: To evaluate the IM-15 effect on cell cycle profile, as well as to investigate the participation of cytokines in the mechanism of antitumor action of IM-15 on Ehrlich ascites carcinoma (EAC) model.

Methodology: All procedures were approved by the Ethics Committee on the Use of Animals of Federal University of Paraíba (nº. 0511/11). EAC cells were implanted into female mice peritoneum (n=6/group) and, after 24 hours, IM-15 was administered (intraperitoneally, i.p.) at 25 or 50 mg/kg doses. 5-Fluorouracil (5-FU), 25 mg/kg, was the standard drug and Tween 80 at 12% (v/v) in saline was used as negative control. After 9 days of treatment, animals were euthanized, and the ascitic fluid was collected from the peritoneal cavity for the cytokines (IL-1β, IL-4, IL-10, IL-12, CCL2, TNF-α, IFN-γ) dosage by ELISA, following the manufacturer's instructions. In addition, for cell cycle analysis, cells from ascitic fluid of all treatment groups were stained with Propidium Iodide (PI). BD FACS CANTO IITM (USA) was used, acquiring 10.000 events/sample. The results are expressed as the mean±standard error of mean, and the differences were compared by analysis of variance (ANOVA), followed by Tukey’s test (p<0.05).

Results: IM-15 induced a significant decrease on IL-4 (1.36 ± 0.76 pg/mL), IFN-γ (12.32 ± 5.36 pg/mL), TNF-α (81.16 ± 12.4 pg/mL) and CCL2 (818.7± 121.0 pg/mL) levels in animals treated with 50 mg/kg IM-15, when compared to tumor control group (IL-4: 7.2 ± 0.9 pg/mL; IFN-γ: 1278 ± 226.4 pg/mL; TNF-α: 194.7 ± 28.88 pg/mL and CCL2: 3102 ± 43.5 pg/mL). For 5-FU, it was observed an increase on IL-12 (45.8 ± 5.6 pg/mL) level, in comparison to tumor control group (16.46 ± 3.7 pg/mL), as well as, a decrease on CCL2, IFN-γ and TNF-α levels (140.2 ± 9.19 pg/mL; 78.80 ± 12.17 pg/mL and 32.72 ± 4.32 pg/mL, respectively). Regarding to cell cycle analysis, IM-15 did not induce any change on cell cycle profile, however, 5-FU induced a significant increase of sub-G1 peak to 91.7%, comparing to tumor control group (4.8%).

Conclusions: IM-15 reduced the cytokines IL-4, TNF-α, IFN-γ, and chemokine CCL-2 levels, suggesting that the antitumor potential of IM-15 involves the modulation of the immune response related to the control of cell proliferation, apoptosis and angiogenesis.

Funding: CNPq and CAPES.

Palavras-chaves: Hydantoin, Antitumor activity, Ehrlich ascites carcinoma, Anti-inflammatory activity, Toxicity

19.028 Antiproliferative activity of annonacione, a annonaceous acetogenins from Annona muricata seeds

Autores

Gabriel Gusmão Grisi Rocha 1, Renan da Silva Santos 1, Maria Francilene Santos Silva 1, Andrea

Felinto Moura 1, Lucas Moreira Brito 1, Claudio Costa dos Santos 2, Claudia do Ó Pessoa 1,3, Manoel

Odorico de Moraes Filho 1

Instituição 1 UFC - Universidade Federal do Ceará, 2 UFERSA - Universidade Federal Rural do Semiárido , 3

FIOCRUZ - Fundação Oswaldo Cruz

Resumo

Introduction: Annonaceous acetogenins (ACGs) are a class of most predominant bioactive compounds of Annonaceous family, growing in tropical and

subtropical regions. They are natural polyketides and have shown anti-cancer properties. The ACGs have been reported to inhibit the activity of NADH-

ubiquinone oxide-reductase complex I of the mitochondrial electron transport system. Objective: In this context, the present study aimed to evaluate the

cytotoxic potential of annonacinone in human tumour cell lines from bioguided fractionation of the acetonic extract of A. muricata seeds. Methods: The

fractionation of acetone extract of A. muricata seeds (AMSA) was obtained by column chromatography and the purification by high performance liquid

chromatography. Nuclear magnetic resonance, infrared and mass spectroscopy were used for identification and structure determination of the compound.

To evaluate the cytotoxic potential of annonacinone MTT assay was perfomed. The compound was tested against nine cancer cell lines: HCT-116 (colon),

HEPG2 (liver), HL-60 (leukemia), NCI-H460 (lung), PC-3 (prostate), PC-9 (lung), SF-295 (brain), SNB-19 (brain), SW620 (colon) and one non-tumoral

cell line, L-929 (murine fibroblast). Results: The annonacinone shown IC50 values ranged from 3.7 to 0.19 µM in SW620 and SF-295, respectively and

no cytotoxic effect in L929 at the maximum concentration tested (5 µM). Conclusion: These results showed relevant in vitro cytotoxic of annonacinone

against different human tumour lines, with greater effect in SF-295 cells. Annonaceous acetogenins phytochemical and pharmacological studies have

further revealed a novel therapeutic role as an anticancer agent for these natural products. Funding: CNPq, PRONEX, CAPES, and FUNCAP.

Palavras-chaves: Annonacinone, Acetogenins, Anticancer, Cytotoxicity, Annona muricata L.

19.029 ANALYSIS OF THE EFFECT OF 1-METHYL-D-TRYPTOPHANE ON THE SIGNALING ROUTES MEASURED BY INDOLEAMINE 2,3-DIOXYGENASE IN BLADDER CARCINOMA: POSSIBLE INVOLVEMENT IN THE DETERMINATION OF INVASIVE PHENOTYPE

Autores

Stephanie Vanin Dalmazzo 1,1,1,1, Hellen Joyce de Souza Pereira dos Santos 1, Luiz Henrique Gomes

Matheus 1, Marina Baptista Floriani 1,1,1,1, Lucas Alves Pereira 1, Sabrina Thalita dos Reis Faria 1,

José Pontes Junior 1, Humberto Delle 1

Instituição 1 UNINOVE - Universidade Nove de Julho

Resumo

ANALYSIS OF THE EFFECT OF 1-METHYL-D-TRYPTOPHANE ON THE SIGNALING ROUTES MEASURED BY INDOLEAMINE 2,3-DIOXYGENASE IN BLADDER CARCINOMA: POSSIBLE INVOLVEMENT IN THE DETERMINATION

OF INVASIVE PHENOTYPE

DALMAZZO, S.V.1, SANTOS, H.J.S.P.2, MATHEUS, L.H.G.3, FLORIANI, M.B.4, PEREIRA, L.A.5, FARIA, S.T.R.6, PONTES JUNIOR, J..7, DELLE, H.8.

Pós-graduação em Medicina1, Universidade Nove de Julho (UNINOVE)2, São Paulo, Brasil.

INTRODUCTION: Bladder cancer (CB) is among the most common of the urinary system and although the most frequent non-muscle-invasive form, approximately 10% progress to this form after surgical resection. The mechanisms by which cells acquire muscle-invasive phenotype are still unclear. Our group has studied the enzyme indoleamine 2,3-dioxygenase (IDO), classically recognized as immunomodulatory, to demonstrate effects on CB independent of immune mechanisms, including the induction of invasive phenotypes.

OBJECTIVE: To evaluate the expression of IDO in two non-muscle-invasive (RT4) and one muscle-invasive (T24) lines of CB, also to evaluate the difference between expressions when cells are treated with 1-Methyl-D -Triptophane (MT) and verify the activation of the GCN2 and aryl hydrocarbon receptor (AHR) pathways, classic IDO pathways in cell control.

METHODS: Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics in 75 cm2 bottles. After subculture in 6 wells plates and upon reaching confluence of 70-80%, total RNA was extracted. Real-time PCR was performed for the analysis of IDO, CHOP (GCN2 pathway marker), AHR and CYP1A1 (AHR pathway marker).

RESULTS: Both strains had IDO constitutive expression, however, T24 cells presented a greater 500X expression than RT4 cells (relative expression of 1.0 ± 0.2 in RT4 and 498.7 ± 95.6 in T24; P

CONCLUSION: Expression of IDO is greater in T24 cells compared to RT4 and during incubation of MT cells did not influence the expression of IDO and CHOP, but significantly increased the expression of CYP1A1. Of the two classic IDO pathways analyzed, only GCN2 was increased in T24, indicating a possible role of IDO in CB via GCN2.

FUNDING: 2017/19668-2

Palavras-chaves: Indoleamine 2,3-Dioxygenase, Bladder cancer, 1-Methyl-D-Tryptophan, Invasive Muscle, Non-Invasive Muscle

19.030 Determination of the antitumor activity of the L21R peptide derived from the Brn-2 transcription factor in murine melanoma cells B16F10-Nex2

Autores Caroline Ramos da Silva Siqueira 1, Luiz Rodolpho Raja Gabaglia Travassos 2, Denise Costa Arruda 1

Instituição 1 UMC - Universidade Mogi das Cruzes, 2 Unifesp - Universidade Federal de São Paulo

Resumo

Determination of the antitumor activity of the L21R peptide derived from the Brn-2 transcription factor in murine melanoma cells B16F10-Nex2

Siqueira, C.R.S ¹; Travassos, L.R.²; Arruda D.C.¹

¹Laboratório de Biologia Experimental do Câncer, Universidade Mogi das Cruzes, SP, Brasil.²Unidade de Oncologia Experimental, Universidade Federal de São Paulo, SP, Brasil.

INTRODUCTION: The Brn-2 protein is related with the normal development of melanocytes. When this protein is super-expressed it can activate cell proliferation and invasion as in malignant melanoma. The transcription factor Brn-2 is regulated by three signaling pathways, Wnt/β-catenin, MAPK and PI3K/AKT. The use of specific inhibitors of Brn-2, such as peptides derived from the DNA-binding domain of the transcription factor, may represent a strategy for melanoma growth and metastasis inhibition. Some studies showed that peptides competing with transcription factors induce cell death. The L21R peptide, derived from the Brn-2 transcription factor could interfere in both the mechanisms of cell death and tumor growth factors.

OBJECTIVE: To study the in vitro and in vivo antitumor activity of L21R peptide, derived from the Brn-2 transcription factor, as well as, determine the cell death mechanism induced by this peptide.

METHODS: Cytotoxicity of the peptide was determined in B16F10-Nex2 (1.104) cells plated in 96-well plates and incubated with L21R peptide at different concentrations. The EC50% was determined in a dose-response curve. A time susceptibility curve was determined in cells treated with the L21R peptide for 30 minutes, 1, 2, 4, 8, 12 and 24 h. To determine if the L21R cytotoxicity depended on ROS, the effect was tested in presence of reducing N-acetylcisteine. Cell viability was defined, in all tests, by cell counting in the presence of Trypan Blue exclusion dye. Statistics was based on the GraphPad Prism program using the t-test. To determine the antitumor effect in C57Bl/6 mice, cells were inoculated intravenously and mice were treated intraperitoneally with injections of 300 µg/day of the peptide, for three alternating days.This assay was approved by the Committee of Ethics in Animal Use (CEUA) (Protocol 018/2017).

RESULTS: L21R exhibited cytotoxicity in B16F10-Nex2 cells, the EC50% approximately of 0.07 mM. Furthermore, a time-activity and dose-dependent, decreasing cytotoxicity by ROS was noticed with N-acetylcisteine. In vivo assay was inconclusive because L21R was toxic to mice at 300 μg/ mouse / day.

CONCLUSIONS: L21R peptide displays antitumor effects and induces cell death in B16F10-Nex2 cell line. The mechanism involved cell stress and increased reactive oxygen species. Further in vivo studies will be performed using lower concentrations of the peptide. Peptide L21R from Brn-2 transcription factor is a promising anti-melanoma cytotoxic agent.

FUNDING: FAPESP.

Palavras-chaves: melanoma, Brn-2, cell death

19.031 RIPK3 AND MLKL AS PROGNOSTIC MARKERS IN RECTAL CANCER

Autores Guilherme Vergara 1, Ricardo Weinlich 1

Instituição 1 HIAE - Hospital Israelita Albert Einstein

Resumo

Introduction: Necroptosis is a novel cell death pathway characterized by plasma membrane rupture and cytoplasmic content leakage, which leads to a pro-inflammatory phenotype. The core of this machinery is composed by RIPK3 and MLKL. Reduced RIPK3 expression levels have been reported in different cancer types, associated with diminished sensitivity to necroptotic stimuli. RIPK3 and MLKL down-modulation have also been suggested as biomarkers for poor prognosis in breast and cervical cancer, respectively. In many cell lines, the ablation of those molecules could be reverted by treatment with a demethylating agent, whereas in others, such as colon cancer cells, there was no gain in expression. Colorectal cancer is the third most diagnosed cancer and the third most lethal. Usually, rectal cancers are the most aggressive subtype, despite the high heterogeneity regarding treatment responses, ranging from complete response to no response at all. Interestingly, there is no information regarding MLKL and RIPK3 expression levels in rectal cancer patients. Objective: Our aim is to investigate whether RIPK3 and MLKL can be used as a prognostic marker for overall survival, response to treatment or chance of relapse in rectal cancer patients. Methodology: In silico z-score-standardized genomic expression data from a retrospective cohort of 231 patients was used to test the correlation between RIPK3 or MLKL expression and several clinical outcomes, using Kruskall-Wallis test with Dunn’s post-hoc and Kaplan-Meier survival analysis. The normality of our sample was tested by Shapiro-Wilk test. As next steps, we aim to assess predictive value of our variables with Cox’s proportional hazard regression models, both in univariate and multivariate approaches. Results: Our data suggests that RIPK3 expression is different comparing tumor-free and relapsed patients, being higher in the latter group. The same could not be seen for MLKL expression. The survival data showed no difference between expression profiles of both RIPK3 and MLKL. Conclusion: Differently to what it was found to other cancer types, RIPK3 and MLKL expression levels are not good prognostic markers in cancer patients regarding Overall Survival. As next step, we will check the correlation between cell death genes expression levels and therapeutic response of patients with rectal cancer. Financial Support: FAPESP. Disclosure: The authors declare that they have no conflicts of interest

Palavras-chaves: Biomarker, RIPK3, MLKL, Cancer, Prognostic

19.032 Circulating cortisol levels and its association with worst prognosis parameters in overweight and obese breast cancer women

Autores Aedra Carla Bufalo Kawassaki 1,2, Elaine Minatti Dias 2, Thalita B Scandolara 2, Daniel Rech 2,3,

Carolina Panis 2, Wander Rogério Pavanelli 1

Instituição 1 UEL - Universidade Estadual de Londrina, 2 UNIOESTE - Universidade Estadual do Oeste do

Paraná, 3 CEONC - Hospital do Câncer Francisco Beltrão

Resumo

Introduction: Obesity is an emerging risk factor associated with the development and evolution of breast cancer due to the continued formation of inflammatory compounds. Excessive body fat is also related with changes in the hypothalamic-pituitary-adrenal axis, with increased synthesis of stress hormones such cortisol.

Objective: The aim of this study was to investigate the correlation among circulating levels of cortisol and clinicopathological parameters in patients diagnosed with breast cancer.

Methodology: This study included 135 women diagnosed with breast cancer attended at the Francisco Beltrão Cancer Hospital in the period from 2015 to 2017. This research was approved by the National Committe for Ethics in research (CONEP) under the number CAAE 35524814.4.0000.0107. Samples were taken from patients outside the morning cortisol peak of morning, between 14 and 17h, by peripheral venous puncture (10 mL), prior to the start of chemotherapy, and immediately frozen after separation of the plasma for further analysis. Cortisol levels were measured in plasma by a commercial kit (AccuBind ELISA kit, USA). Plasma cortisol levels were correlated with clinical and pathological data collected from patients' records. The results were analyzed using statistical software GraphPadPrism 5.0 and SPSS Estatistic 20, with a significance level of 95%.

Results: Circulating levels of cortisol in breat cancer patients ranged from 0.92 to 23.68 ug / dL, with an average of 11. 28 ± 0.5 μg / dL. Statistical differences were observed in circulating levels of cortisol when patients were categorized according to their body mass index (BMI) in eutrophic patients (10.02 ± 0.89 μg / dL), overweight (12 ± 0.83 μg / dL) and obese (12.46 ± 0.90 ug / dL), in the comparison between obese and obese women (p = 0.049). Patients with ompromised marginsn had significant reduction in circulating cortisol levels (12.3 ± 0.7 μg / dL in those with negative margins and 10.23 ± 0.71 μg / dL in those with positive margins, p = 0.0427). The other clinicopathological parameters (age, molecular subtypes, histological grade, tumor size, angiolympjatic clots and menopausal status) were not significant. It was observed that in obese patients there was a negative correlation between serum cortisol levels and the histological grade of tumors (p = 0.028, R = - 0.366) and a

positive correlation with angiolymphatic clots (p = 0.035, R = 0.368). In the eutrophic patients, a negative association was observed between circulating cortisol levels and the presence of lymph node metastases.

Conclusion: The data obtained allow concluding that excessive body fat negatively affects the prognosis of breast cancer, and that cortisol levels are associated with the parameters that determine the worse prognosis of this disease.

Funding: Research Program for SUS (PPSUS), State University of Western Paraná.

Palavras-chaves: breast cancer, cortisol, prognosis, overweight, obese

19.033 Preclinical evaluation of the losartan reversing hypoxia in treated murine breast tumors.

Autores Evelyn Martins 1,2

Instituição 1 (LIM-43) FMUSP - Laboratório de Medicina Nuclear , 2 CTO-ICESP (LIM24) - Centro de

Investigação Translacional em Oncologia do Instituto do Câncer do Estado de São Paulo

Resumo

Introduction: AGTR1, encoding gene for angiotensin II type I receptor, was found to be one of the most highly overexpressed genes in 10-20 % of breast cancers in independent microarray studies. Receptor blockage impairs ECM components like collagen and hyaluronan by reducing CAFs density in stroma, ending in narrow interstitial pressure and improved tumour perfusion. Reversing tumour hypoxia has potential clinical importance due to its hole as major resistance factor to radio and chemotherapy. Several radiotracers, including 99mTc-HL91, have been used to detect and measure changes in hypoxia status in tissues.

Objective: The study aimed to evaluate the efficacy of losartan decreasing hypoxia areas, using 99mTc-HL91 uptake and immunohistochemical analysis to detect changes in oxygen supply in the tumor.

Methodology: The project was approved by ethical committee process number (colocar o número do comitê de ética). Tumour model and treatment: Murine breast tumour 4T1 cell line (5x104 cells/0.1 mL) were orthotopically injected into the mammary fat pad of young female Balb/c mice (8 wks, 15-21 g). Two groups (n = 4/group) were stablished and treated with aqueous solution of losartan (1mg/animal, oral gavage) or vehicle. Radiolabelling, in vivo biodistribution: HL91 freeze-dried kits were radiolabelled with 740 MBq of Na99mTcO4 (25°C, 15 min). Radiochemical purity (RP) was evaluated by planar chromatography. Mice were injected in tail vain with 37 MBq of 99mTc-HL91, 2 h before image acquisition. The pimonidazole, a “gold standard” for IHC hypoxic probe, was injected intraperitoneally (60 mg/kg) one hour before animal euthanasia. SPECT/CT images were acquired in small animal imaging equipment Triumph™ Trimodality, and semiquantitative analysis was done using AMIDE (Free Software – UCLA/SU). Statistical analyses were performed using Student’s T test. Ex vivo analysis: Tumour was excised from euthanized animal, and 40 µm sections, obtained from cryostat cut, were immediately exposed to a storage phosphor screen for autoradiographic images. Adjacent tissue was paraffin embedded and submitted to H&E staining, CD31 and pimonidazole immunohistochemical (IHC). Slices were imaged in EVOS® FL Auto Imaging System.

Results: 99mTc-HL91 was obtained with RP ≥ 90%. In vivo determined Tumour/Normal tissue ratio (T/N) uptake was 4.71±0.41 for treated group (n=3), and 5.22±1.07, for control group (n=4). Student’s T test showed no significant difference between groups (p=0.354). Histologic assessment revealed regions consistent with necrotic patterns in agreement with hypocaptant areas seen by autoradiography. Autoradiography and losartan IHC image gave similar regions for both hypoxia detecting compounds, but not for CD31 staining, confirming that the activity belongs to intracellular uptake and not tumour blood pool.

Conclusion: Similarity between the radiotracer and pimonidazole staining localization in tumor tissues confirms 99mTc-HL91 as specificity and suitability for hypoxia tracer. The observance for non-difference in tumor perfusion, both losartan treated and not treated groups, suggest the necessity to reevaluate experimental conditions, including losartan dose, treatment time, and SPECT resolution for small change oxygen supply in treated tumor.

Funding: Fellowship from Capes and grant from FAPESP.

Palavras-chaves: Hypoxia, HL91, radiotracers, Vascular normalization, breast cancer

19.034 The combination of coffee compounds attenuates preneoplastic lesion development in the liver by altering proliferation/apoptosis balance

Autores Sara Viana Mattioli 1, Gabriel Bacil Prata 1, Guilherme Ribeiro Romualdo 2, Luís Fernando Barbisan 1

Instituição 1 IBB - UNESP - Instituto de Biociências - Universidade Estadual Paulista, 2 FMB - UNESP -

Faculdade de Medicina - Universidade Estadual Paulista

Resumo

Introduction: Literature shows that daily coffee consumption has a significant impact on the decrease of hepatic fibrosis, cirrhosis and hepatocellular carcinoma, while decaffeinated coffee does not (1,2). A single cup of common or espresso coffee contains hundreds of molecules (3), which include caffeine (CAF), trigonelline (TRI) and chlorogenic acid (CGA). These compounds individually showed beneficial effects over experimental steateatohepatits, fibrosis or hepatocarcinogenesis (3,4), however, there are no studies on the effects of coffee compound combinations.

Objective: Thus, the protective effects of CAF individually or combined with TRI and/or CGA over fibrosis-associated hepatocarcinogenesis were assessed.

Methodology: In order to induce fibrosis-associated hepatocarcinogenesis process in male C3H/HeJ mice (n=10/group), animals received a single dose of diethylnitrosamine [DEN, 10 mg/Kg body weight (b.wt.), intraperitoneally (i.p.), in 0.9% saline] at week 2 and multiple and increasing carbon tetrachloride doses (CCl4, 0.25 to 1.50 µL/g b.wt., i.p., 3x/week, in 10% corn oil) from weeks 8 to 16. Furthermore, mice were treated with CAF alone (50 mg/Kg b.wt., gavage, 5x/week, in distilled water) or combined to TRI and/or CGA (25 mg/Kg b.wt., both, same regimen) from weeks 7 to 17. The CAF dose is based on human high filtered coffee consumption (3-4 cups of coffee/day, ~300 mg CAF/day) while TRI and CGA doses are based on TRI or CGA/CAF proportion found in filtered coffee (5,6). Mice were euthanized at week 17. Liver was collected for histological analysis (H&E and immunohistochemistry). Incidence data were analyzed by Fisher’s Exact test. Other data were analyzed by ANOVA or Kruskall-Wallis and post hoc Tukey test (p<0.05). Data are presented as mean±standard deviation or the proportion of affected animals (%). CEUA 2017/994.

Results: Rather than CAF alone, CAF+TRI and CAF+CGA treatments, only the combination of CAF+TRI+CGA reduced the number of preneoplastic foci/liver area compared to DEN/CCl4 group (3.07±1.92 vs. 6.55±3.33, p=0.035). This treatment diminished the incidence preneoplastic clear cell foci as well [0/10 (0%) vs. 6/10 (60%), p=0.011]. In accordance, only CAF+TRI+CGA treatment reduced cell proliferation (Ki-67) within preneoplastic lesions (221.48±142.73) when compared to DEN/CCl4 (441.89±289.14) and CAF alone (608.67±392.51) (p=0.006). Despite of not altering apoptosis (cleaved caspase-3) in these lesions, the CAF+TRI+ACG treatment increased the apoptosis (117.01±26.27) in surrounding hepatic tissue (avoiding lesions) compared to DEN/CCl4 (88.80±15.30) and CAF (69.53±15.00) (p<0.001) groups.

Conclusion: The findings indicate that the combination of three coffee compounds, rather than CAF alone or combined to TRI or CGA, attenuates preneoplastic lesion development, reducing cell proliferation inside these lesions. Further mechanisms will be evaluated.

References: 1. Plos One. 10:11, 2011. 2. Int. J. Cancer. 136:8, 2015. 3. Genes Nutr. 10:6, 2015. 4. Asian Pac. J. Trop. Dis. 8:8, 2015. 5. Food Chem. Toxicol. 64, 2014. 6. FASEB J. 22, 2008.

Funding: FAPESP (2016/14420-0, 2016/12015-0).

Palavras-chaves: Hepatocarcinogenesis, Liver Fibrosis, Caffeine, Trigonelline, Chlorogenic acid

19.035 Evaluation of Cu(SDMX)(phen) on the viability of breast cancer cells

Autores Ramon Handerson Gomes Teles 1, Douglas Hideki Nakahata 2, Angélica Ellen Graminha 1, Pedro

Paulo Corbi 2, Márcia Regina Cominetti 1

Instituição 1 UFSCar - Universidade Federal de São Carlos, 2 Unicamp - Universidade de Campinas

Resumo

Introduction: Breast cancer is the second cause of mortality in women, only exceeded by lung cancer. Chemotherapy consists in a systemic treatment that can be administered either intravenously or orally. However, the main drugs used for breast cancer chemotherapy, such as doxorubicin and cisplatin, present severe side effects. Thus, the search for new more specific and effective drugs is necessary. Objective: To investigate the cytotoxic activity of a copper-compound in different breast cancer cell lines. Methods: The Cu(SDMX)(phen) (where SDMX: sulfadimethoxine, phen: 1,10-phenantroline) compound and the following tumor cell lines were used: SKBR3, MCF-7, MDA-MB-231. As a control, the non-tumor breast cell line MCF-10A was used. To evaluated the cytotoxicity activity of the compound, MTT assay was performed. For this, 1x104cells/100μl/well were seeded in appropriated medium into 96-well plates, which were maintained in an incubator with an environment containing 5% CO2 and 37ºC for 24h. After, cells were treated with different concentrations of the Cu(SDMX)(phen) dissolved in specific culture medium. After 24 and 48h of incubation, the supernatant was removed and a solution containing MTT (0.5 mg/ml) was added. After 3h of incubation, crystals were solubilized in DMSO and the plates were read (λ=540nm). The data were analyzed using Hill’s equation. To study the ability of the compound to inhibit colony formation, 1x103cells/well were seeded into sterile 6-well plates. After 24h cells were treated with different concentrations of the compound Cu(SDMX)(phen) for 5h and the medium was changed for a new medium without treatment. After 10 days the plates were stained (crystal violet 0.5%) and the number and size of colonies were analyzed. To evaluate the effects of the compound on cell migration, wound healing assays were performed. For that, 5x105cells/well were seeded into sterile 12-well plates, after 24h a cell-free area was created in a confluent monolayer by mechanical damage. The cell migration into the gap was measured in the wells where cells were treated with different concentrations of the compound after 36h and images were captured. The data were normalized in percentage compared to untreated control. Data was statistically analyzed using one or two-way ANOVA, depending on the experimental design, and by Dunnett’s test. Results: The compound showed IC50 values under 10μM in 48h for all cell lines studied (MDA-MB-231: 2.23μM, SBKR3: 4.07μM, MCF-7: 5.11μM, MCF-10A: 3.47μM). In the MDA-MB-231 cell line colonies number and size decreased in 51% and 53%, respectively, upon treatment with a concentration of the compound corresponding to its IC50 value for this cell line. In the wound healing assay there was an inhibition of about 36% in MDA-MB-231 cell migration. Conclusion: The copper compound is promising against breast cancer in vitro, affecting tumor cell viability and migration. However, more studies should be performed to understand how this compound acts in breast cancer.

Palavras-chaves: breast cancer, copper-compound, oncology, viability cell, proliferation cell

19.036 PD-L1 antibody expressing chimeric antigen receptor (CAR) T cells for anhydrase carbonic IX positive renal cell carcinoma – construction with CD28 and CD3-ζ signaling domains

Autores Eloah Rabello Suarez 1,2,3, Wayne Anthony Marasco 2,3

Instituição 1 UFABC - Universidade Federal do ABC, 2 DFCI - Dana-Farber Cancer Institute , 3 HMS - Harvard

Medical School

Resumo

Introduction: A new generation of immmunotherapy able to armoring T cells against cancer using chimeric antigen receptors has revolutionized the treatment of hematological tumors, however their efficacy against solid tumors is still a challenge due to several factors including their functional impairment in the immunosuppressive

microenvironment of the solid tumors. The immunosuppression of T cells is favored by the expression of immune checkpoint molecules by the tumors, including the programmed cell death ligand-1 (PD-L1). PD-L1 is able to interact with the programmed cell death receptor-1 (PD-1) on T cells, resulting in an exhausted phenotype of T cells that cannot restrain tumor progression. The immune checkpoint blockade with antibodies has led to improved progression-free survival in several types of cancer. The current therapies used to treat metastatic clear cell renal cell carcinoma (ccRCC) are toxic, rarely achieve durable long-term complete responses and are not curative.

Objectives: Construction of a new combination immunotherapy that consists of human anti-carbonic anhydrase IX (CAIX)-targeted chimeric antigen receptor (CAR) T cells engineered to secrete human anti-programmed death ligand 1 (PD-L1) antibodies at the tumor site to treat ccRCC.

Methodology: A single bicistronic lentiviral vector containing anti-CAIX (G36) scFv linked to CD28 and CD3-ζ signaling domains (G36-CD28z CAR) in the first cassette was used to clone anti-PD-L1 IgG1 and IgG4 antibodies in its second expression cassette. The functional characterization of these CAR T cells was performed, including the evaluation of their effector activity and their effects on T cell exhaustion in vitro and in an orthotopic mouse model of human RCC in vivo. IRB protocol approval 11-104.

Results: The local antibody delivery led to marked immune checkpoint blockade. Tumor growth diminished 5 times and tumor weight reduced 50–80% when compared with the anti-CAIX CAR T cells alone in a humanized mice model of ccRCC (N= 6 mice per group). The expression of PD-L1 and Ki67 in the tumors decreased and an increase in granzyme B levels was found in CAR T cells. The anti-PD-L1 IgG1 isotype, which is capable of mediating ADCC, was also able to recruit human NK cells to the tumor site in vivo.

Conclusion: These armed second-generation CAR T cells empowered to secrete human anti-PD-L1 antibodies in the ccRCC milieu to combat T cell exhaustion is an innovation in this field that should provide renewed potential for CAR T cell immunotherapy of solid tumors where limited efficacy is currently seen.

Funding: This research was supported by the National Foundation for Cancer Research (NFCR, USA) and Sao Paulo Research Foundation (FAPESP, Brazil).

Palavras-chaves: CAR T cells, immune checkpoint blockade, PD-1, PD-L1, Renal cell carcinoma

19.037 Jamelão (S. cumini) alleviates liver fibrosis while Jaboticaba [Myrciaria jaboticaba (Vell.) O.Berg] attenuates preneoplastic lesion development

Autores Lucas Vilhegas de Souza 1, Isadora Penedo de Souza 1, Guilherme Ribeiro Romualdo 2, Luis

Fernando Barbisan 1

Instituição 1 IBB - UNESP - Department of Morphology, Biosciences Institute, 2 FMB - UNESP - Department of

Pathology, Botucatu Medical School – UNESP

Resumo

Introduction: Epidemiological studies indicate that frequent vegetable consumption reduces the risk of hepatocellular carcinoma [1], which is considered the second most deadly cancer worldwide and frequently associated with the setting of liver fibrosis/cirrhosis [2]. In this way, the consumption of fruits and their food derivatives are potentially useful for the prevention and/or treatment of chronic liver

diseases. Especially, the fruits of Myrtaceae family are rich in anthocyanins in their peels, polyphenols with antioxidant and anti-inflammatory properties [3].

Objective: We assessed the protective effects of jaboticaba, jamelão or jambo peels on liver fibrosis and carcinogenesis in mice.

Methodology: The fruit peels were submitted to the determination of anthocyanin profile and levels by high performance liquid chromatography in duplicate. Female mice of the C3H/HeJ strain were randomly allocated into 5 experimental groups (n=13 animals/group). The animals were submitted to a liver fibrosis/carcinogenesis model by receiving diethylnitrosamine (DEN)/carbon tetrachloride (CCl4) (G2-G5), or vehicle (G1). For ten weeks, mice received basal diet (G1 and G2) or basal diet enriched (2%) with jaboticaba [Myrciaria jaboticaba (Vell.) O.Berg] (G3), jamelão (Syzygium cumini) (G4) and jambo (Syzygium malaccense) (G5) dehydrated peels.

The animals were euthanized at the end of the 17th week (CEUA IBB-UNESP 2018/1035) and liver samples for histological and biochemical analysis. Data were analyzed by ANOVA or Kruskal-Wallis followed by Tukey's post hoc test (p<0.05). The incidence data were analyzed by Fisher's exact test (p<0.05). Data are expressed as mean±standard deviation or the proportion of affected animals (%).

Results: Jaboticaba peels displayed the highest amount of anthocyanins (802.89mg/100g) compared to jamelão (575.19mg/100g) and jambo (156.05mg/100g), but jamelão showed greatest variability of anthocyanins (delphinidin-3,5-diglucoside, cyanidin 3,5-diglucoside, petunidin 3,5-diglucoside and malvidin 3,5-diglucoside) in comparison to jaboticaba (cyanidin 3-glucoside and delphinidin 3-glucoside) and jambo (cyanidin 3-glucoside and 3,5-diglucoside). All Myrtaceae fruit peel interventions (G3-G5) reduced liver oxidative stress (lipid hydroperoxide levels) compared to DEN/CCl4 group (G2) (271.9±28.15, 284.2±21.93, 269.1±53.23 vs. 341.6±34.55, respectively, p=0.002). Jamelão (G4) and jambo (G5) diets increased catalase activity compared to DEN/CCl4 group (G2) (31.38±6.88, 31.60±7.24 vs. 19.05±4.51, respectively, p<0.001) while jaboticaba (G3) enhanced total glutathione compared to G1 (31.66±0.65 vs. 24.94±0.23, p=0.009). Moreover, jamelão dietary intervention (G4) reduced liver collagen levels (Sirius red) compared to the positive control (G2) (1.90±0.50 vs. 2.59±0.58, p<0.001) while jaboticaba-treated group (G3) exhibited a lower incidence of liver preneoplastic foci than the positive control group (G2) [5/13 (38%) vs. 12/13 (92%), p=0.011).

Conclusion: The results suggest that consumption of jaboticaba attenuates liver preneoplastic lesion development and jamelão alleviates liver fibrosis. These different effects can be attibutted to different anthocyanin profile and levels.

References: 1. Br J Cancer. 112:7, 2015; 2. Globocan, 1:0, 2012; 3. Food Nutr Res. 61:1, 2017.

Palavras-chaves: jaboticaba, jamelão, jambo, liver fibrosis, liver carcinogenesis