Murilo F. Cerri

Evaluation of PvuII and XbaI polymorphisms in the estrogen receptor alpha gene (ESR1) in relation to menstrual cycle timing and reproductive parameters in post-menopausal women

OBJECTIVE To evaluate the association of -397T>C and -351A>G single nucleotide polymorphisms (SNPs) - also called PvuII and XbaI, respectively - located on estrogen receptor alpha (ERS1) gene with age at menarche, menopause onset, fertility and miscarriage in a population of post-menopausal women. STUDY DESIGN Cross-sectional study with 273 healthy, high miscegenated, post-menopausal women (mean age of 63.1±9.7 years old). Subjects were genotyped for PvuII and XbaI SNPs by PCR-RFLP and confirmed by automatic sequencing. Reproduction informations (age at menarche, age at menopause, number of pregnancies, fertility rate and miscarriages) were obtained by retrospective study using a questionnaire. RESULT(S) Age at menarche, menopause onset, number of pregnancies, total fertility rate, and parity did not seem to be influenced by any of the studied genotypes (chi-square, p>0.05). However, women carrying the xx genotype showed a 44% higher chance of miscarriage, whereas this value did not trespass 16% for any other genotype analyzed. It has been also observed a higher occurrence of miscarriage in association with combined xxpp genotype of ERS1 gene (chi-square, p<0.01). CONCLUSION(S) The present data indicate that the studied SNPs on ERS1 gene do not influence the menstrual cycle timing and parity but there is a strong relationship between the xx ERS1 SNP genotype and the incidence of miscarriage in the post-menopausal population analyzed.

Synthesis, Antitumor Activity and Docking of 2,3-(Substituted)-1,4-Naphthoquinone Derivatives Containing Nitrogen, Oxygen and Sulfur

Eleven 2,3-(substituted)-1,4-naphthoquinone derivatives were synthesized in yields ranging from 52-89%. These derivatives were evaluated for their cytotoxic effects on human lungs (H460), triple-negative breast (MDA-MB-231) and ovarian (A2780) cancer cell lines. Compounds 5f and 8 showed IC50values of 3.048 × 10-5 mol L-1 and 4.24 × 10-6 mol L-1 for H460; 5c and 8showed IC50 values of 2.16 × 10-5 mol L-1 and 1.60 × 10-5 mol L-1 for MDA-MB-231, and 5gand 8 showed IC50 values of 2.68 × 10-6 mol L-1 and 3.89 × 10-6 mol L-1 for A2780. Additionally, we conducted a docking study with the four most active compounds and the therapeutic targets PI3K and topoisomerase II showing the pharmacophoric conformation of these compounds.

Abstract 2626: Novel naphtoquinone PIC21 targets PI3K in triple negative breast cancer cell line

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Triple negative breast cancer (TNBC) is a heterogeneous subgroup (ER-, PR-, and HER2-) of aggressive breast cancer, which poor prognosis is partially due to chemoresistance to available drugs. We have synthesized and screened 43 novel naphtoquinone-derived drugs (patent-protected), rationally designed to act through multiple pathways to avoid tumor chemoresistance, in MDA-MB231 by cellular metabolic viability and IC50 calculation. Of these, the most promising drugs PIC20 and PIC21 showed significantly higher AE than cisplatin, doxorubicin and paclitaxel in the lineage. Methods: Based on the crystalline structure of protein deposited on Protein Data Bank ( 1E7U, PI3K), and PIC20 and PIC21 tridimensional structures, computational molecular dock studies were conducted to investigate the molecules PIC20 e PIC21 tridimensional conformation and bounding energy to PI3K (Autodock Vina software). In vitro PI3K inhibition by PIC21 in MDA-MB231 was assessed using the Flowcellect PI3K/Mapk Dual Pathway Activation and Cancer Marker Detection. For PI3K/Akt and MAPK signaling pathways, cells were pre treated with PIC21 (0,1mM, 2h) or Wortmannin (Wort) (0,1μM, 30 min). Cells were then treated with insulin (0,6μM, 5 min) for stimulation of PI3K/Akt and MAPK pathways and incubated with Anti-phospho-Akt1/PKBα Alexa Fluor® 488 Conjugated Antibody and Anti-phospho-Erk1/2- R- Phycoerythrin conjugated antibody. A FACSCanto II cytometer was used for the flow cytometric analysis. FSC and SSC were used to establish size gates and exclude cellular debris from the analysis. In each measurement, a minimum of 30000 events were analyzed. Data were acquired and analyzed using BD FACSDiva and DeNovo FCS software. Findings: PIC21 targeting of PI3K was confirmed by docking studies, which data demonstrated that the interaction energy (E) for PI3K and PIC21, and PI3K inhibitors, LY294002 and Wort, was: PIC20 (E=-8.9), PIC21(E=-8.2), LY294002 (E=-9.5), Wort (E=-8.8). Cell treatment with PIC21 and Wort decreased the pAkt+ cell population (control=23.02%, PIC21 treated=6.63%, Wort treated=4.20%), whereas the pERK+ cell population increased (control=0,58%, PIC21 treated=6,41%, Wort treated=10,14%. This is due to the pathways crosstalk, by which pAkt inhibits the MAPK/ERK pathway. Conclusions: Our data strongly points to PIC21 as a novel PI3K inhibitor. A high prevalence of PI3K mutation, leading to its constitutive activation, has been described in TNBC, favoring tumor cells growth, proliferation, metabolism, and survival. Therefore, PIC21 opens a new avenue to overcome TNBC dramatic epidemiological scenario, bringing hope to improve patients overall outcome and quality of life. Citation Format: Renata D. Daltoe, Klesia P. Madeira, Murilo F. Cerri, Joao F. Allochio Filho, Maicon Delarmelina, Marcella L. Porto, Silvana S. Meirelles, Silvana S. Meirelles, Ian V. Silva, Sandro J. Greco, Leticia B A Rangel. Novel naphtoquinone PIC21 targets PI3K in triple negative breast cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2626. doi:10.1158/1538-7445.AM2014-2626

Abstract 3343: Anomalous expression of claudin 16 in ovarian cancer: Role of PKC, PI3K and estrogen

Background: We have demonstrated that 63% of primary epithelial ovarian cancer (EOC) anomalously overexpressed CLDN16 in the cell cytoplasm, regardless of tumor histological type or grade, thus suggesting that this is an early event in EOC development, and might serve as an EOC marker. The present work aimed to enlighten cellular mechanisms that modulate CLDN16 expression in EOC. Methods: PKA, PKC, and PI3K pathways affect CLDNs cellular localization and function, cancer development and progression. We investigated the effect of the pathways, and of intra-tumoral estrogen (0.5, 5, 50 and 500 nM), on CLDN16 expression in the serous and platinum-resistant EOC model, the OVCAR3 cell line. Immunofluorescence experiments were carried out to determine the cellular localization of CLDN16 in OVCAR3. CLDN16 expression was assessed by real-time RT-PCR, in the presence or absence of the pathway modulators cAMP (PKA activator, 10-6M), KT5720 (PKA inhibitor, 10-7M), PMA (PKC activator, 10-8M), and Wortmannin (WORT: PI3K activator, 10-7M). CLDN16 relative expression analyses followed the CT method of Pffalf, using the REST program (Version 2.0.13, 2009). Data, expressed as mean ± SE, were analyzed by Bonferroni and Dunnet multiple comparisons tests. Findings: OVCAR3 expressed CLDN16 exclusively in the cytoplasm compartment, resembling primary tumors, so being a reliable in vitro model to our expression study. While the PKA pathway did not affect CLDN16 expression in OVCAR3, the PKC pathway lead to an increase of the transcript by1.95-fold (PMA; p Conclusions: Our data strongly suggest that the expression of CLDN16 is stimulated by the PKC and PI3K, and intra-tumoral aromatase-produced estrogen in EOC. Whereas the anomalous cytoplasm overexpression of CLDN16 still puzzles us, we speculate that fragments of the molecule could be possibly secreted. If this is the case, screening for circulatory portions of the protein could serve as an EOC screening molecule, considering that it is overexpressed early in disease course. Despite the fact that more studies are necessary to elighten the mechanisms that control the expression and function of CLDN16 in EOC, our study opens a new avenue to overcome EOC dramatic epidemiological scenario, bringing hope to improve patients overall outcome and quality of life. Citation Format: Nayara G. Tessarollo, Marcela Paes, Murilo Cerri, Alice Herlinger, Klesia Madeira, Renata Daltoe, Ian Silva, Leticia Rangel. Anomalous expression of claudin 16 in ovarian cancer: Role of PKC, PI3K and estrogen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3343. doi:10.1158/1538-7445.AM2014-3343

Abstract 463: Evaluation of relative expression of SLC34A2/NaPi-IIb in lung cancer cell lines treated with estrogen and PKC and PKA pathway modulators

Background: Lung cancer (LC) represents a major health challenge worldwide, being the leading cause of cancer-related deaths in both men and women. SLC34A2 is a member of the solute carrier family and encodes the type IIb sodium phosphate cotransporter (NaPi-IIb). SLC34A2 expression is reduced by ten-fold in LC samples compared to normal lung. Estrogen is a known risk factor for LC in women, and a relationship between its action and SLC34A2/NaPi-IIb super-expression in certain cell lines has been proposed. Furthermore, based on the central role of PKC isoenzymes in carcinogenesis, and on studies showing that administration of cAMP stimulates the NaPi-IIb mRNA expression in adult rats alveolar type II cells, this work aimed to evaluate the correlation between PKA (cAMP mediator), PKC, estrogen signaling pathways and SLC34A2/NaPi-IIb expression levels in human LC cells. Methods: For both LC cell lines studied, A549 and H460, the effects of PKC and PKA activating and inhibitory reagents (PMA, calphostin C, dB cAMP and KT 5720) were tested in relation to SLC34A2/NaPi-IIb expression. Additionally, a dose-response curve of 17-beta-estradiol was performed for the expression of SLC34A2/NaPi-IIb in A549 cell line. SLC34A2/NaPi-IIb gene expression was analyzed by qRT-PCR, using SYBR Green PCR Master Mix as the detection system. Relative quantification of gene expression was performed by calculating the delta-delta Ct using two housekeeping genes: GAPDH and Beta-Actin. Findings: In our analysis we found that PKC and PKA did not influence the expression of SLC34A2/NaPi-IIb in LC cell line evaluated. There was, however, a considerable increase in the expression of this gene when treated with calphostin C, probably by a mechanism independent of its classic role in the PKC inhibition. There was also a trend of decreased SLC34A2/NaPi-IIb gene expression in LC cell line following the treatment with 17 beta-estradiol, and SLC34A2/NaPi-IIb - which expression is already reduced in LC - undergoes a greater reduction in its relative expression. Interpretation: We believe that maintaining the reduced expression of SLC34A2/NaPi-IIb should provide benefits to LC cells. Calphostin C induces apoptosis in several tumor cell lines by mechanisms not yet fully elucidated. Thus, we suggest a possible involvement of SLC34A2/NaPi-IIb in this pro-apoptotic pathway, which corroborates the fact that LC tumors present a reduced SLC34A2/NaPi-IIb expression compared to normal lung. Likewise, based on our findings, we believe that the inclusion of selective Estrogen receptor modulators and aromatase inhibitors in the routine clinical practice of LC might help to control the disease that is still the leading cause of cancer-related deaths worldwide. Citation Format: Murilo F. Cerri, Lucas C d Rezende, Marcela F. Paes, Klesia P. Madeira, Renata D. Daltoe, Nayara G. Tessarollo, Ian V. Silva, Leticia B a Rangel. Evaluation of relative expression of SLC34A2/NaPi-IIb in lung cancer cell lines treated with estrogen and PKC and PKA pathway modulators. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 463. doi:10.1158/1538-7445.AM2014-463

In vitro antineoplastic activity in triple-negative breast cancer cell line and in vivo

Background Triple negative breast cancer (TNBC) is a heterogeneous subgroup (ER-, PR-, and HER2-) of invasive breast cancer, associated to poor prognosis, partially due to its resistance to available drugs. Therefore, it is imperative to discover new treatment options for the disease. In this context, we have synthesized and screened novel naphtoquinone-derived drugs (patent-protected), rationally designed to act through multiple pathways to avoid tumor chemoresistance.

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

Abstract 3812: Retrospective analysis of Claudin-16 (CLDN16) overexpression in ovarian cancer (OVCA): Evaluation of a biomarker candidate and a novel potential therapeutic target

Our group has previously documented the overexpression of CLDN16 in OVCA; nonetheless the role of the phenomenon in cancer development and progression remains enlightened, even thought it points to cell permeability modification and tumor increased invasion potential. The present study aims to establish the clinical relevance of CLDN16 overexpression in OVCA, as well as to elucidate its role in ovarian carcinogenesis in order to future develop OVCA CLDN16-based diagnosis and/or therapeutic strategies. We had generated an OVCA database including patients clinical follow up. CLDN16 expression in tissue array platforms has been accessed by immunohistochemistry. The studied population profile corroborates to the worldwide OVCA statistics, with a prevalence of serous adenocarcinoma (serous: 42.5%, endometrioid: 17%, mucinous: 16%, clear cell: 11.3%), in menopausal women (mean age at diagnosis, 55.9 years), at advanced staged (FIGO stage I: 7.5%, II: 8.5%, III: 41.5%, VI: 27.4%; poor differentiated: 37.7%, moderately differentiated : 22.7%, well differentiated: 2.8%). Surprisingly, patients median survival rate was 12%, considerably lower than that estimated by WHO (30%). Investigation of CLDN16 expression profile have revealed, so far, that 67.74% of the cases overexpress the protein in cytoplasm. It has been also observed that CLDN16 expression is an OVCA-specific feature, as previously observed by our group. Despite the cellular localization of CLDN16 in OVCA cells, it has been postulated by others that cellular trafficking of CLDNs in cancer cells might derive from post-translational events, and might be related to vesicle trafficking or cellular matrix modification. CLDN16 is overexpressed in all OVCA histological types. To this point, we have found no differential CLDN16 expression in OVCA regarding the tumor degree of diferentiation, tumor platin responsiveness, patients survival rate or stages. Altogether, our data suggest that CLDN cytoplasm overexpression in OVCA cells might be an early event in ovarian malignant transformation, therefore playing a central role in ovarian tumorigenesis. Considering that CLDN16 silencing has been associated exclusively to decreases in magnesium serum levels, which can be exogenously replaced, it is a potential novel OVCA therapeutic target. Even though we still have to confirm CLDN16 cytoplasm localization in OVCA cells, we aim to develop a therapeutic strategy against OVCA. One possibility is encapsulate CLDN16 monoclonal antibody in lipid IgG-NaPi-Iib-immunoconjugated formulations. Ultimately, we believe that CLDN16 is an interesting and novel target to fight OVCA, which is the leading cause of gynecological cancers-related deaths among women. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3812. doi:10.1158/1538-7445.AM2011-3812