Leticia Batista Azevedo Rangel

Regulatory T Cell as a Target for Cancer Therapy

Advances in our understanding of CD4+CD25+Foxp3+ regulatory T cells (TRegs) enabled the characterization of their activities in maintaining peripheral tolerance, preventing autoimmune diseases, and limiting chronic inflammatory diseases. Ironically, an effective action of these cells during tumor development can limit beneficial responses by suppressing immunity and limiting antitumor resistance, whereas one of the main functions of the immune system is to eliminate malignant cells. During the last years, the immunological role, mechanism of action, and clinical importance of these cells were profoundly characterized and the relationship between this subset of lymphocytes and cancerous cells arises as a key factor that influences tumor development. Recent insights obtained from clinical studies and experimental mouse models expand our perception of the potential role of TRegs in cancer treatment. In this review we describe the basic mechanisms of TReg origin and differentiation, their potential role in cancer, as well as the future perspectives concerning the modulation of these cells as a potential approach for anticancer strategies.

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.

Metformin synergistically enhances antiproliferative effects of cisplatin and etoposide in NCI-H460 human lung cancer cells.

OBJECTIVE: To test the effectiveness of combining conventional antineoplastic drugs (cisplatin and etoposide) with metformin in the treatment of non-small cell lung cancer in the NCI-H460 cell line, in order to develop new therapeutic options with high efficacy and low toxicity. METHODS: We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and calculated the combination index for the drugs studied. RESULTS: We found that the use of metformin as monotherapy reduced the metabolic viability of the cell line studied. Combining metformin with cisplatin or etoposide produced a synergistic effect and was more effective than was the use of cisplatin or etoposide as monotherapy. CONCLUSIONS: Metformin, due to its independent effects on liver kinase B1, had antiproliferative effects on the NCI-H460 cell line. When metformin was combined with cisplatin or etoposide, the cell death rate was even higher.

Estrogen receptor alpha (ERS1) SNPs c454-397T>C (PvuII) and c454-351A>G (XbaI) are risk biomarkers for breast cancer development

There are several risk factors related to Breast Cancer (BC) risks and response to chemotherapy with SERMs. Recently some single nucleotide polymorphisms (SNPs) on ESR1 gene have been associated to this disease. However, data are still inconclusive. The present study aimed to investigate the association of SNPs c454-397T>C (also called PvuII) and c454-351A>G (so called XbaI) to incidence of sporadic BC; ERα expression in BC; tamoxifen hormonetherapy (HT-TMX) responsiveness. To do so, a cohort of BC patients was analyzed through retrospective data collection, immunohistochemistry to ERα protein, and genotyping for PvuII and XbaI SNPs by PCR–RFLP, confirmed by sequencing. Significant difference in PvuII alleles frequencies were found BC patients when compared to control samples. Patients with P allele have a 5.14-fold increased BC risk. We found higher P and X alleles frequencies in ERα positive BC and the pp and xx genotypes were observed exclusively in patients with HT-TMX-responsive BC. Taken together, data indicates that P allele as a novel sporadic BC biomarker whereas p and x alleles enhanced chemotherapy responsiveness.

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.

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.

Comparison of immunohistochemical analysis with estrogen receptor SP1 and 1D5 monoclonal antibodies in breast cancer

In the present study, we aimed to evaluate estrogen receptor ER-alpha status in 61 breast cancer cases using Sp1 and 1D5 monoclonal antibodies. Tissue array platforms were generated containing samples of breast cancer and positive controls that were assayed by immunohistochemistry applying monoclonal primary antibodies anti-ER alpha, SP1 and 1D5. We noted a high concordance rate (96.7%) between the referred antibodies. Moreover, we calculated the Kappa factor (0.921), indicating that 1D5 and SP1 provided overlapping ERα expression results. Indeed, we observed controversial results only in 2 samples studied, which were ER-negative when stained with 1D5 and ER-positive when assessed with SP1. Total concordance of PS was obtained (Pearson and intraclass CF, 0.7351 and 0.6193, respectively). However, concordance between the antibodies seems to be more accurate in higher PS values. An excellent IS correlation between antibodies was observed throughout the population (Spearmans CF, ρ=0.9150). Following the Allred score, 17 out of 42 positive BC samples diverged, with 1D5 always pointing to weaker staining than SP1. When calculating Spearmans CF of Total Score (TS) within the population, an excellent correlation between both the antibodies (ρ=0.9238) was noted. Nonetheless, the results were less concordant among the BC-positive cases (ρ=0.7743). Indeed, 20 samples were differentially classified using the antibodies (only 3 had higher TS with 1D5). Considering the mean TS of all samples or of invasive ductal carcinoma, SP1 provided higher scores than 1D5 (p<0.05). We recommend the use of the anti-ER RMAb SP1 due to the high probability that the BC ERα status can be determined accurately as the reagent provides higher IS. Therefore, the A-score was higher than the MMAb 1D5. Ultimately, higher IS and A-score decrease the possibility of ERα status misinterpretation and, consequently, inappropriate BC treatment that would compromise the patients quality of life and overall survival. We recommend the use of anti-ER RMAb SP1 due to the high probability that the BC ER status can be determined accurately as the reagent provides higher IS, therefore higher A-score, than the MMAb 1D5.

Association of PvuII and XbaI polymorphisms on estrogen receptor alpha (ESR1) gene to changes into serum lipid profile of post-menopausal women: Effects of aging, body mass index and breast cancer incidence.

Estrogen is a steroidal hormone involved in several physiological functions in the female body including regulation of serum lipid metabolism and breast cancer (BC). Estrogen actions on serum lipids mostly occur through its binding to intracellular Estrogen Receptor alpha (ERalpha) isoform, expressed in most of tissues. This gene (ESR1) exhibit many polymorphic sites (SNPs) located either on translated and non-translated regions that regulate ERalpha protein expression and function. This paper aimed to investigate the association of two intronic SNPs of ESR1 gene, namely c454-397T>C (PvuII) and c454-351A>G (XbaI) to alterations in serum levels of total cholesterol (T-chol), total lipid (TL), low density lipoprotein cholesterol (LDL), high density lipoprotein (HDL), and triglycerides (TG) in a cohort of post-menopausal women. In addition, we aimed to associate presence of these SNPs to development of BC along 5 years period. To do so, a group of healthy 499, highly miscigenated, post-menopausal Brazilian women, were carried using PCR-FRLP technique and further confirmed by automatic sequence analysis as well followed through 5 years for BC incidence. Measurements of serum lipid profile by standard commercial methods were carried individually whereas Dual Energy X-ray Absorciometry (DXA) measured Body Mass Indexes (BMI), Fat Mass (FM), Lean Body Mass (LBM), and Body Water Content (BWC). No effects of PvuII SNP on ESR1 gene were observed on patient´s serum T-chol, TL, LDL, HDL, and TG. However, c454-397T>C PvuII SNP is associated to lower body fat and higher levels of lean mass and body water and lower incidence of BC. On the other hand, statistically significant effect of XbaI c454-351A>G SNP on serum TG and TL levels. Patients homozygous for X allele were followed up from 2010–2015. They showed higher incidence of breast cancer (BC) when compared to either heterozygous and any P allele combination. Moreover, the increasing of TG and TL serum concentrations associated to SNP XbaI c454-351A>G on ESR1 gene is enhanced in both obese (higher BMI) and elder women. Taken together, these results suggested that XbaI c454-351A>G SNP is associated to changes in lipid profile, particularly in serum TG and TL, in this cohort of post-menopausal woman. Also, this paper shows another link between obesity and BC corroborating the current thesis that both diseases are interlinked.

Abstract 2548: Metformin and everolimus act synergistically with paclitaxel against ovarian cancer

Background: Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, mostly due to the diagnosis of the disease at advanced stages and to chemoresistance to platinum- and taxane-based standard therapy. EOC comprises a heterogeneous group of diseases classified based on morphologic and molecular-genetic features. High grade serous ovarian cancer is the most prevalent EOC subtype, being followed by the clear cell subtype. Both EOC subtypes are vastly aggressive and frequently acquire the chemoresistant phenotype, thus challenging researchers and clinicians. Notably, hyperactivation of PI3K and MAPK pathways are frequently related to resistance to standard therapies in EOC, thus, metformin (MET) and everolimus (EVE) have emerged as novel therapeutic options against EOC. The present work aimed to investigate the action of MET and EVE, in vitro, in ES-2 (clear cell) and A2780 (serous) cell lines. Methods: Metabolic cell viability (MCV) assays were conducted after the treatment of the cells with MET or EVE in monotherapy and in association with paclitaxel (PAC) by the MTT method. Cell cycle progression was evaluated by flow cytometry/propidium iodide. Apoptosis was analyzed by the annexin V-FITC assay. Western blot was performed to investigate MEK/ERK and PI3K/AKT pathways. Findings: MCV of EOC lineages was reduced by MET and EVE in different concentrations. The combination of MET, at low and safe concentration (10uM), with PAC 100nM reduced the MCV in 56.55% for ES-2, and 71.38% for A2780. Similarly, EVE 0.06 nM and PAC 100 nM decreased the VCM in 66.4% and 73.38% for ES-2 and A2780, respectively. MET 10μm and EVE 0.06nM associated with PAC 12.5nM kept the MCV at levels comparable to PAC 100nM. These findings are of extreme clinical relevance as they reveal that the synergism between MET and EVE with PAC likely maintains the drug citotoxicity at lower concentrations than those associated to its severe and limiting side effects, as neurotoxicity. Annexin-FITC assay was conducted in ES-2 lineage treated with PAC alone and combined with MET or EVE. Low percentage of apoptotic/necrotic cells was observed, which suggests the occurrence of additional processes, such as autophagic cell death. EVE induced ES-2 cell cycle arrest; however, MET, at low concentration, did not cause the same effect. There were no significant cell cycle changes due to the combined treatment of ES-2 with PAC and MET or EVE. Western blot analysis demonstrated that the combination of EVE and PAC increased, whereas MET alone or in combination with PAC decreased the phosphorylation of ERK1/2 and AKT in ES2 cells. Conclusions: Our data suggest that MET or EVE exerts synergistic effect with PAC against EOC, thus opening an avenue to explore novel strategies against this highly lethal disease. Ongoing experiments are sought to elucidate the cellular pathways modulated by MET and EVE alone or in combination with PAC in EOC lineages, as well as the elicited cellular events and mechanisms. Citation Format: Nayara G. Tessarollo, Isabella S. Guimaraes, Taciane Ladislau, Ian Victor Silva, Leticia B. A. Rangel. Metformin and everolimus act synergistically with paclitaxel against ovarian cancer. [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 2548. doi:10.1158/1538-7445.AM2015-2548

Chemosensitizing effects of metformin on cisplatin- and paclitaxel-resistant ovarian cancer cell lines

BACKGROUND Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. Primary cytoreductive surgery with adjuvant taxane-platinum chemotherapy is the standard treatment to fight ovarian cancer, however, their side effects are severe, and chemoresistance emerges at high rates. Therefore, EOC clinic urges for novel treatment strategies to reverse chemoresistance and to improve the survival rates. Metformin has been shown to act in synergy with certain anti-cancer agents, overcoming chemoresistance in various types of tumors. This paper aims to investigate the use of metformin as a new treatment option for cisplatin- and paclitaxel-resistant ovarian cancer. METHODS The effects of metformin alone or in combination with conventional drugs on resistant EOC cell lines were investigated using the MTT assay for cell proliferation; Flow Cytometry analysis for cell cycle and the mRNA expression was analyzed using the real-time PCR technique. RESULTS We found that metformin exhibited antiproliferative effects in paclitaxel-resistant A2780-PR, and in cisplatin-resistant ACRP cell lines. The combined therapy containing conventional drugs and metformin improved the effect of the treatment in cell proliferation rate, especially in the resistant cells. We found that metformin, in clinical relevant doses, could significantly reduce the mRNA expression of inflammatory cytokines and NF-κB signaling pathway. CONCLUSIONS Taken together, our observations suggest that metformin inhibits the inflammatory pathway induced by paclitaxel and cisplatin treatment. Furthermore, metformin in combination with paclitaxel or cisplatin improved the sensitivity in drug-resistant ovarian cancer cells. Therefore, metformin may be beneficial treatment strategy, particularly in patients with tumors refractory to platinum and taxanes.