Leticia Ba Rangel

Abstract 2571: Metformin inhibits proliferation and acts synergistically with paclitaxel and doxorubicin in triple negative breast cancer cell lines

Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer (BC) characterized by its unique molecular profile, aggressive behavior, distinct patterns of metastasis and unavailability of effective target therapy. Indeed, despite good initial response to chemotherapy, even platinum- or anthracyclines-based, TNBC relapse and mortality rates are high even in early-stage disease. Metformin, one of the most commonly used medications for treatment of type 2 diabetes mellitus, has emerged as a potential anticancer agent. This study aimed to evaluate the combination treatment of metformin, at lower concentrations (10uM), with conventional chemotherapeutic agents on TNBC cell lines. We investigated the combination effect of metformin with doxorubicin (DOX), cisplatin (CDDP) and paclitaxel (PTX). Methods: Three TNBC cell lines (MDAMB-231, HCC-70, HCC-1937) and one luminal BC cell line (MCF-7) were used. Cell proliferation was determined by MTT assay. Clonogenic assay was conducted and apoptosis was analyzed by Annexin V-FITC assay. Western immunoblotting was performed to determine the expression of the downstream targets of PI3K, MAPK and AMPK pathways. Findings: Metformin potently inhibited the proliferation of all BC cell lines in a dose-dependent manner. Likewise, metformin caused a significant dose-dependent reduction in clonogenicity of TNBC cell lines (P Citation Format: Isabella S. Guimaraes, Nayara G. Tessarollo, Laura FRL Oliveira, Roger C. Zampier, Ian V. Silva, Cinthya Sternberg, Leticia BA Rangel. Metformin inhibits proliferation and acts synergistically with paclitaxel and doxorubicin in triple negative breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2571. doi:10.1158/1538-7445.AM2015-2571

Abstract 1441: Effect of chemotherapy on cytokine production by solid tumor cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tumor microenvironment strongly influences cancer progression, regulating several cellular processes and cancer immune surveillance. Albeit the influence of cytokines from immune cells on tumor progression has already been described, little is known on the role of cancer cells on the production of these cytokines, especially in response to chemotherapy. To address this aspect of tumor biology, we exposed the serous epithelial ovarian cancer (EOC) cell line A2780, the clear cell EOC cell line TOV21G, the endometrioid EOC cell line MDAH-2774 (further referred as 2774), the breast cancer (BC) cell line MDA-MB 231, and the non-small cell lung carcinoma (NSCLC) cell line A549 to cisplatin (CI), cyclophosphamide (CY), doxorubicin (DO), and paclitaxel (PA) for 5 days, at their IC50 range, prior to cytokine measurement by ELISA in cells conditioned media (CM). Cytokines (IL-17, IFN-γ, CXCL-1, and TGF-β) were measured in the pool of CM from 3 well after 3 and 5 days of exposure to each drug or untreated cells (UT). Results were normalized by the mean protein content of wells and are expressed in pg/mL/μg of protein. Cytokines’ level varied among UT cell lines, probably reflecting tumor heterogeneity. IL-17 secretion was higher in 2774, MDA-MB 213 and A549 (4.6, 8.9, and 10.6, respectively). UT TOV21G cells did not secrete IL-17. In response to CI, IL-17 levels decreased; whereas response to the other drugs greatly varied. INF-γ levels showed the lesser variation among cell lines (0.01 - 0.2). Upon exposure to the drugs, IFN-γ secretion tend to increase by all drugs in A2780 and 2774 lines, and in TOV21G exposed to CY; however it was strongly decreased by PA in this line and in A549. EOC cells A2780 and 2774 were the only lines secreting CXCL-1 in UT condition (∼0.015 on D5). CXCL-1 secretion in most conditions either was unaltered or decreased. However, CI was the only drug that induced its secretion in A2780, TOV21G and MDA-MB 231. As this chemokine has already been described as a factor promoting tumor cell migration and invasion, the relation between CI and CXCL-1 is worth of further analysis. Worth noticing, TGF-β was highly secreted by NSCLC cells (42.7 on D5); all other cells’ level was < 0.96 on D5. The most striking change was in A540 where all drugs decreased its levels, especially CI (0.73 on D5). Similar data were observed in A2780 cells, which is not in concordance with our previous data. This finding highlights the importance of precisely define drug dose in cancer therapy and its impact on patients’ clinical outcome, as the assays were conducted under distinct CI concentration. Overall, our data corroborated the great heterogeneity among different cancers and its response to chemotherapy, also highlighting the importance to consider the effect of different drugs on tumor immune surveillance, therefore introducing a novel aspect in predicting tumor responsiveness to conventional antineoplastic drugs, and ultimately to control cancer progression. Citation Format: Alice L. Herlinger, Iuri C. Valadao, Mariana C. de Souza, Maria das Gracas MO Heniques, Celso Caruso-Neves, Leticia BA Rangel. Effect of chemotherapy on cytokine production by solid tumor cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1441. doi:10.1158/1538-7445.AM2013-1441

Abstract A96: Cisplatin induces CSC phenotype and modulates cytokine production in ovarian cancer cells.

Cancer stem cells (CSC) are considered major contributor to tumor progression. Furthermore, it has been reported that standard chemotherapy regimens, as the cisplatin (CIS)-based schemes, might induce CSC phenotype in epithelial ovarian cancer (EOC) cells. Nonetheless, the influences of CSC on tumor microenvironment, as well as its role on cancer immunosurveillance remain inconclusive. Herein, we present data derived from the exposure of the CIS-sensitive EOC cell line A2780 to CIS at IC50 750 nM and half IC50 375 nM, for 5 days; after which the expression of CD44, CD24, OCT-4, citokeratin-18 (CK-18), vimentin, epithelial specific antigen (ESA), nestin and nanog was evaluated by qPCR, applying the ΔΔCt method. In parallel, protein expression of nestin and nanog was assessed by Western blot. Data were analyzed by one way ANOVA and Newmann-Keuls post-test, always using beta actin as the reference molecule. CSC markers transcripts OCT-4 (P Citation Format: Alice L. Herlinger, Mariana C. de Souza, Maria das Gracas MO Henriques, Celso Caruso-Neves, Leticia BA Rangel. Cisplatin induces CSC phenotype and modulates cytokine production in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A96.

Abstract 924: In vitro study of the potential antineoplastic effect of synthetic naphthoquinones in cisplatin-resistant ovarian cancer lineage

Background: Cancer development has been associated with alterations in polyamine biosynthesis and metabolism, which induce cell proliferation, angiogenesis, expression of genes related to tumor invasion and metastasis; whereas inhibit apoptosis. Based on the strong rational to develop novel polyamine depleting molecules, and adding the strategy to have substances that can control cancer through different cellular pathways aiming to bypass the acquisition of drug resistant phenotype by cancer cells; this work aimed to screen, in an ovarian cancer (OVCA) line, 40 novel rationally developed potential anti-cancer compounds, following rapid, high efficient, and low cost synthetic methodologies, then confirmed by spectroscopic techniques. OVCA is the most lethal gynecological malignancy, with high rates of chemoresistance and disease relapse; therefore, supporting the urge to generate novel anti-OVCA agents. Methods: Novel naphthoquinone-derived compounds were rationally designed to act through multiple cellular pathways aiming the avoidance of drug resistant phenotype acquisition by cancer cells, and were synthesized by rapid, efficient and low cost synthetic method. Drugs antineoplastic efficacy (AE) was accessed in OVCAR3, through the evaluation of cellular metabolic viability (CMV) (MTT method). Drugs structures are protected by patent. Cells were cultured in RPMI media supplemented with 10% (v/v) FBS, antibiotics and antifungics, in 5% CO 2 , until subconfluence; then, 1.5x10 5 cells/well were subcultured for 72h prior to treatment with drugs in different concentrations (10 −4 , 10 −5 , 10 −6 , 10 −7 , and 10 −8 M). After 24h, CMV was assessed. Experiments in which the lineage was treated with cisplatin, doxorubicin or paclitaxel were run in parallel. The mean and standard-deviation of the absorbancies were used to calculate CMV and drugs IC 50 (PrismaGraphPad version 5.1). Findings: We have screened the AE of 40 novel naphtoquinone-derived drugs in OVCAR3; five have decreased its CMV by, at least, 70%, namely: M8 (IC 50 1.64x10 −5 M; CMV decrease of 90%); M10 (IC 50 1.37x10 −5 M; CMV decrease of 96%); M14 (IC 50 1.45x10 −5 M; CMV decrease of 85%); PIC10 (IC 50 9.13x10 −6 M; CMV decrease of 95%); PIC20 (IC 50 5.31x10 −5 M; CMV decrease of 70%). The IC 50 for cisplatin, the gold therapy against OVCA was 3.87x10 −5 M and CMV decrease was 85%. Interpretation: We herein present novel drugs to treat OVCA; whereas PIC 10 is more potent that cisplatin, M8, M10, M14, PIC10 and PIC20 seem to have similar or higher antineoplastic efficacy in treating cisplatin-resistant OVCA. We strongly believe that the present pre-clinical research project is innovative, as it introduces novel anti-OVCA drugs, economically viable and socially important, as it might bring hope to put OVCA treatment in a perspective in which the disease control is a real possibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 924. doi:1538-7445.AM2012-924