Marcela Ferreira Paes

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.

Prevalence of estrogen receptor alpha PvuII (c454-397T>C) and XbaI (c454A>G) polymorphisms in a population of Brazilian women

The aim of this work was to study the estrogen receptor alpha (ERS1) PvuII and XbaI gene polymorphisms prevalence in randomly selected women population from Rio de Janeiro and Espirito Santo states in Brazil by polymerase chain reaction restriction fragment lengths polymorphism (PCR_RFLP) methodology. It was shown that Rio de Janeiro women exhibited a significantly different prevalence of XbaI polymorphism comparing to Espirito Santo women. Nonetheless, similar prevalence of PvuII polymorphism was found in both women´s populations. Moreover, a strong linkage disequilibrium was observed between these SNPs reinforcing the hypothesis of differential pattern of inheritance observed on such populations.

Noninvasive genetic sampling of endangered muriqui (Primates, Atelidae): efficiency of fecal DNA extraction

The muriqui (Brachyteles) is one of the most endangered primates in the world, however little is known about the viability of the remaining populations. We evaluated the technique of extracting DNA from wild muriqui feces for PCR applications. In order to determine the effect of the DNA in subsequent amplifications, we analyzed five different extracts. The importance of the recommended BSA and the HotStarTaq DNA polymerase was tested. The minimal conditions to successfully amplify highly degraded fecal DNA were determined, showing that the recommended reagents are not required. We envision that this method may be useful in further conservation management studies.

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

Abstract 161: The role of mTOR in the cisplatin resistant phenotype in ovarian cancer lineage

Background Ovarian cancer (OVCA) is the second cause of gynecological-cancer deaths in Brazil, being frequently associated to chemotherapy resistance, a characteristic usually associated to PI3K/Akt/mTOR. We herein present data concerning the role of mTORC2 in cisplatin-resistant phenotype acquisition by OVCA cells. Methods Antineoplastic efficacy of cisplatin, doxorubicin, paclitaxel, and rapamycin was accessed, in different combinations, in the OVCA lineage OVCAR3 (cisplatin-resistant, advanced serous OVCA), through cellular metabolic viability (CMV) (MTT method). Cells were cultured in RPMI media supplemented with 10% (v/v) FBS and antibiotics until subconfluence. To characterize the role of mTORC1 or mTORC2 in OVCAR3 MCV, 1.5x105 cells/well were treated according to: EA1) Inhibition of mTORC1: Drugs at IC50 for 24h; EA2) Inhibition of mTORC1 and mTORC2: Rapamycin 100nM for 72h, followed by drugs at IC50 for 24h. Modulation of MTOR expression by PKA, PKC, and PI3K was investigated by real time RT-PCR. Statistical analysis was performed using PrismaGraphPad version 5.1. Findings OVCAR3 CMV was not significantly changed by cisplatin (IC50 0,08mM) nor paclitaxel (IC50 0,06nM) or rapamycin (IC50 0,02nM) (CMV 95%, 100%, 100%, respectively), but by doxorubicin (IC50 0,02nM) (CMV 60%), especially in combination with cisplatin (CMV 31%), following EA1. Considering the irreversible doxorubicin-induced cardiotoxicity, we pursued with EA2. Intriguingly, OVCAR3 cisplatin sensitivity was, at least partially, recovered (CMV 65%). No addictive effect was observed with cisplatin/doxorubicin following EA2 (CMV 60%), suggesting that OVCA-resistant patient might benefit from less toxic rapamycin-based chemotherapy, leaving doxorubicin for palliative final-staged disease management. Finally, neither PKA nor PI3K modulate MTOR expression; however, PKC suppresses its transcription by 4.42-fold. Interpretation Altogether, our data suggest that mTORC2, but not mTORC1, might influence cisplatin sensitivity in OVCA. Moreover, PKC inhibits the expression of MTORC in cisplatin-resistant OVCA. We propose that the inhibition of mTORC2 followed by cisplatin treatment might lead to a conjunction of autophagic cell death and apoptosis in OVCA. Of remarkable clinical interest, we point to the urge of better design clinical trials, in which not only novel drugs are tested, but also the chronological order of drugs administration to the patient is seen as crucial for treatment efficacy. 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 161. doi:1538-7445.AM2012-161

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