Search

In this study, eight naphthoquinone derivatives were synthesized in yields ranging from 52 to 96% using easy, fast, and low-cost methodologies. All naphthoquinone derivatives were screened for their in vitro anti-proliferative activities against OVCA A2780 cancer cell line. Amongst all analysed compounds, derivatives 3 - 5 presented the most prominent cytotoxic potential. Naphthoquinones 3 and 4, bearing sulfur-containing groups, were identified as having high potential for ROS production, in particular the superoxide anion. Furthermore, 3 and 4 compounds caused a decrease in the cell population in G0/G1 and induced more than 90% of the cell population to apoptosis. Compound 5 did not act in any of these processes. Finally, compounds 3 - 5 were tested for their inhibitory ability against PI3K and MAPK. Compounds 3 and 4 do not inhibit the PI3K enzyme. On the other hand, the naphthoquinone-polyphenol 5 was only able to inhibit reduced the percentage of cells expressing pERK.

cDNA microarray data conducted by our group revealed overexpression of CXCL2 and CXCL8 in ovarian cancer (OC) microenvironment. Herein, we have proven that the chemokine receptor, CXCR2, is a pivotal molecule in re-sensitizing OC to cisplatin, and its inhibition decreases cell proliferation, viability, tumor size in cisplatin-resistant cells, as well as reversed the overexpression of mesenchymal epithelium transition markers. Altogether, our study indicates a central effect of CXCR2 in preventing tumor progression, due to acquisition of cisplatin chemoresistant phenotype by tumor cells, and patients’ high lethality rate. We found that the overexpression of CXCR2 by OC cells is persistent and anomalously confined to the cellular nuclei, thus pointing to an urge in developing highly lipophilic molecules that promptly permeate cells, bind to and inhibit nuclear CXCR2 to fight OC, instead of relying on the high-cost genetic engineered cells.

We developed a novel method for the synthesis of bis-naphthoquinones (BNQ), which are hybrids of lawsone (2-hydroxy-1,4-naphthoquinone) and 3-hydroxy-juglone (3,5-dihydroxy-1,4-naphthoquinone). The anticancer activity of three synthesized compounds, named 4 (RC10), 5 (RCDFC), and 6 (RCDOH) was evaluated in vitro against two metastatic prostate cancer (PCa) cell lines, DU145 and PC3, using MTT assays. We found that 4 (RC10) and 5 (RCDFC) induced cytotoxicity against DU145 and PC3 cells. Flow cytometry analysis revealed that these two compounds promoted cell cycle arrest in G1/S and G2/M phases, increased Sub-G1 peak and induced inhibition in cell viability. We also showed that these effects are cell-type context dependent and more selective for these tested PCa cells than for HUVEC non-tumor cells. The two BNQ compounds 4 (RC10) and 5 (RCDFC) displayed promising anticancer activity against the two tested metastatic PCa cell lines, DU145 and PC3. Their effects are mainly associated with inhibition of cell viability, possibly through apoptotic cell death, besides altering the SubG1, G1/S and G2/M phases of cell cycle. 5 (RCDFC) compound was found to be more selective than 4 (RC10), when comparing their cytotoxic effects in relation to HUVEC non-tumoral cells. Future work should also test these compounds in combination with other chemotherapeutic drugs to evaluate their effects on further sensitizing drug-resistant metastatic PCa cells.

Osteopontin (OPN) is upregulated in several types of tumor and has been associated with chemoresistance. However, the contribution of OPN splicing isoforms (OPN‑SIs) to chemoresistance requires further investigation. The present study aimed to evaluate the expression patterns of each tested OPN‑SI in cisplatin (CDDP)‑resistant ovarian carcinoma cell lines, focusing on the role of the OPN‑c isoform (OPNc) in drug resistance. ACRP ovarian cancer cells resistant to CDDP, as well as their parental cell line A2780, were used. Analyses of the transcriptional expression of OPN‑SIs, epithelial‑mesenchymal transition (EMT) markers and EMT‑related cytokines were performed using reverse transcription‑quantitative PCR. OPNc was silenced in ACRP cells using anti‑OPNc DNA oligomers and stably overexpressed by transfecting A2780 cells with a mammalian expression vector containing the full length OPNc cDNA. Functional assays were performed to determine cell proliferation, viability and colony formation. The results demonstrated that among the three tested OPN‑SIs, OPNc was the most upregulated transcript in the ACRP cells compared with the parental A2780 cells. In addition, the expression levels of P‑glycoprotein multidrug transporter were upregulated in CDDP‑resistant ACRP cells compared with those in A2780 cells. OPNc knockdown sensitized ACRP cells to CDDP treatment and downregulated P‑gp expression levels compared with those in the negative control group. Additionally, silencing of OPNc impaired cell proliferative and colony formation abilities, as well as reversed the expression levels of EMT markers and EMT‑related cytokines compared with those in the negative control cells. Notably, although stable OPNc overexpression resulted in increased A2780 cell proliferation, it notably increased CDDP sensitivity compared with that in the cells transfected with a control vector. These results suggested that OPNc silencing may represent a putative approach to sensitize resistant ovarian cancer cells to chemotherapeutic agents.

The standard treatment for locally advanced cervical cancer (CC) is chemoradiotherapy. Once the bladder receives part of the radiation, a typical inflammatory condition that configures radiation-induced cystitis may develop. Chronic radiation-induced cystitis is commonly characterized by the bladder new submucosal vascularization, which is typically fragile and favors hematuria. The current study aims to investigate if Hypoxia-Induced Factor (HIF-1α) and its transcriptional target Vascular Endothelial Growth Factor A (VEGF-A) could be a primary pathway leading to increased submucosal vascularization. HIF-1α and VEGF-A mRNA levels in bladder core biopsies from CC patients treated with radiotherapy versus untreated (non-irradiated) patients were analyzed using a droplet digital polymerase chain reaction technology. Gene expression results showed that HIF-1α and VEGF-A had no significant differences between bladder samples from patients previously irradiated and untreated patient samples. However, a direct relationship between the degree of late morbidity and the expression of HIF-1α and VEGF-A has been demonstrated. Despite the lack of statistical significance precludes a definitive conclusion, the data presented herein suggests that further studies investigating the role of HIF-1α in bladder neovascularization in radiation-induced cystitis are highly recommended.

We developed a novel method for the synthesis of bis-naphthoquinones (BNQ), which are hybrids of lawsone (2-hydroxy-1,4-naphthoquinone) and 3-hydroxy-juglone (3,5-dihydroxy-1,4-naphthoquinone). The anticancer activity of three synthesized compounds, named 4 (RC10), 5 (RCDFC), and 6 (RCDOH) was evaluated in vitro against two metastatic prostate cancer (PCa) cell lines, DU145 and PC3, using MTT assays. We found that 4 (RC10) and 5 (RCDFC) induced cytotoxicity against DU145 and PC3 cells. Flow cytometry analysis revealed that these two compounds promoted cell cycle arrest in G1/S and G2/M phases, increased Sub-G1 peak and induced inhibition in cell viability. We also showed that these effects are cell-type context dependent and more selective for these tested PCa cells than for HUVEC non-tumor cells. The two BNQ compounds 4 (RC10) and 5 (RCDFC) displayed promising anticancer activity against the two tested metastatic PCa cell lines, DU145 and PC3. Their effects are mainly associated with inhibition of cell viability, possibly through apoptotic cell death, besides altering the SubG1, G1/S and G2/M phases of cell cycle. 5 (RCDFC) compound was found to be more selective than 4 (RC10), when comparing their cytotoxic effects in relation to HUVEC non-tumoral cells. Future work should also test these compounds in combination with other chemotherapeutic drugs to evaluate their effects on further sensitizing drug-resistant metastatic PCa cells.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase widely expressed in cervical tumors, being correlated with adverse clinical outcomes. EGFR may be activated by a diversity of mechanisms, including transactivation by G-protein coupled receptors (GPCRs). Studies have also shown that platelet-activating factor (PAF), a pro-inflammatory phospholipid mediator, plays an important role in the cancer progression either by modulating the cancer cells or the tumor microenvironment. Most of the PAF effects seem to be mediated by the interaction with its receptor (PAFR), a member of the GPCRs family. PAFR- and EGFR-evoked signaling pathways contribute to tumor biology; however, the interplay between them remains uninvestigated in cervical cancer. In this study, we employed The Cancer Genome Atlas (TCGA) and cancer cell lines to evaluate possible cooperation between EGFR, PAFR, and lysophosphatidylcholine acyltransferases (LPCATs), enzymes involved in the PAF biosynthesis, in the context of cervical cancer. It was observed a strong positive correlation between the expression of EGFR × PAFR and EGFR × LPCAT2 in 306 cervical cancer samples. The increased expression of LPCAT2 was significantly correlated with poor overall survival. Activation of EGFR upregulated the expression of PAFR and LPCAT2 in a MAPK-dependent fashion. At the same time, PAF showed the ability to transactivate EGFR leading to ERK/MAPK activation, cyclooxygenase-2 (COX-2) induction, and cell migration. The positive crosstalk between the PAF-PAFR axis and EGFR demonstrates a relevant linkage between inflammatory and growth factor signaling in cervical cancer cells. Finally, combined PAFR and EGFR targeting treatment impaired clonogenic capacity and viability of aggressive cervical cancer cells more strongly than each treatment separately. Collectively, we proposed that EGFR, LPCAT2, and PAFR emerge as novel targets for cervical cancer therapy.

Melanoma is considered the most lethal skin cancer and its incidence has increased during the past decades. About 10 % of cases are classified as hereditary melanoma. Genetic predisposition usually manifests itself clinically as early onset and multiple cutaneous melanomas. Several genes have been identified as involved to melanoma susceptibility, some of them still with unknown clinical relevance. Beyond melanoma, the affected families are also more prone to develop other malignancies, such as pancreatic cancer. The identification of risk families and involved genes is of great importance, since different forms of oncological surveillance are recommended. However, well established guidelines to standardize both the selection of individuals and the genetic panel to be requested are still lacking. Given the importance of the genetic counseling and testing in the context of clinical suspicion of hereditary melanoma, this paper aims to review the literature regarding genetic panel indications worldwide.

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.

This abstract was not presented at the conference. Citation Format: Guimarães I, Gimba E, Castelo-Branco M, Sternberg C, Silva I, Rangel L. Not presented [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-08-01.

Objectives This study aim was to review cases of acinic cell carcinoma (the main differential diagnosis of secretory carcinoma) that were diagnosed and treated at the National Cancer Institute of Brazil (INCA) between 1996 and 2016. The primary objective was to identify underdiagnosed cases of secretory carcinoma via a clinical, immunopathological and molecular reassessment. Materials and methods This is a cross sectional study, with retrospective data collection from medical records and histological specimen review, with staining for periodic acid-Schiff (PAS) and PAS with diastase, immunohistochemistry for S-100, mammaglobin, and DOG-1, and droplet digital RT-PCR for ETV6-NTRK3. The Research Ethics Committee approved this study, and the patients allowed their participation through informed consent. Results Eighty-three cases of acinic cell carcinoma were diagnosed and treated in the specified period at INCA, of which, seven had their diagnosis changed to secretory carcinoma. Conclusion The present study adds seven cases of secretory carcinoma to the literature, contributing to a better understanding of the epidemiological, histological, immunohistochemical and molecular characteristics of this recently described tumor. Also, the use of a comprehensive diagnostic approach, including immunohistochemical and molecular methods, along with classical morphological studies, allowed the reclassification of acinic cell carcinoma to secretory carcinoma.

Cisplatin-based chemoradiation is the standard treatment for cervical cancer, but chemosensitizing strategies are needed to improve patient survival. EGFR (Epidermal Growth Factor Receptor) is an oncogene overexpressed in cervical cancer that is involved in chemoresistance. Recent studies showed that EGFR upregulates multiple elements of the coagulation cascade, including tissue factor (TF) and the protease-activated receptors (PAR) 1 and 2. Moreover, many G protein-coupled receptors, including PARs, have been implicated in EGFR transactivation. However, the role of coagulation proteins in the progression of cervical cancer has been poorly investigated. Herein we employed cervical cancer cell lines and The Cancer Genome Atlas (TCGA) database to evaluate the role of EGFR, TF and PAR2 in chemoresistance. The SLIGKL-NH2 peptide (PAR2-AP) and coagulation factor VIIa (FVIIa) were used as PAR2 agonists, while cetuximab was used to inhibit EGFR. The more aggressive cell line CASKI showed higher expression levels of EGFR, TF and PAR2 than that of C33A. PAR2 transactivated EGFR, which further upregulated cyclooxygenase-2 (COX2) expression. PAR2-AP decreased cisplatin-induced apoptosis through an EGFR- and COX2-dependent mechanism. Furthermore, treatment of CASKI cells with EGF upregulated TF expression, while treatment with cetuximab decreased the TF protein levels. The RNA-seq data from 309 TCGA samples showed a strong positive correlation between EGFR and TF expression (P = 0.0003). In addition, the increased expression of EGFR, PAR2 or COX2 in cervical cancer patients was significantly correlated with poor overall survival. Taken together, our results suggest that EGFR and COX2 are effectors of the TF/FVIIa/PAR2 signaling pathway, promoting chemoresistance.

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. Indeed, EOC epidemiological scenario is partially due to its late diagnosis, when most of tumor cells have acquired metastatic and chemoresistant phenotype. Despite the initial satisfactory response of EOC cells to platinum/taxanes-based therapy, chemoresistance emerges in an uncontrolled pace, specifically when the patient carries a high-grade serous adenocarcinoma (HGS-AC). Therefore, EOC clinic urges for novel treatment possibilities, aiming the reversal of chemoresistance, the improvement of patients’ quality of life, and their survival rates. cDNA microarray data from our group demonstrated that chemokines secreted to the tumor microenvironment, as CXCL2, IL-6, and IL-8 likely play a crucial role in EOC cells chemoresistance. Thereafter, the present study focused on the role of CXCL2, which binds to its receptor CXCR2 in the membrane of target cells. Nonetheless its function in HGS-AC is unclear. Our study model was based in two HGS-AC lineages: chemosensitive A2780, and its pan-resistant derived ACRP (IC50 to cisplatin 7.3uM vs. 26,56uM, p Citation Format: Taciane Henriques, Diandra dos Santos, Isabella Guimaraes, Nayara Tessarollo, Paulo Lyra, Patricia Mesquisa, Diana Padua, Ana Luisa Amaral, Luisa Pereira, Bruno Cavadas, Ian Silva, Raquel Almeida, Leticia Rangel. Role of CXCR2 in the acquisition of pan-resistant phenotype in high grade serous ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A133. doi:10.1158/1535-7163.TARG-19-A133

Breast cancer (BC) is the second most common kind of cancer in the world and the most common among women. Its frequency rate is becoming alarming, being this way a major challenge to global health. It is worth mentioning that the lack of effective therapeutic options, in particular for certain subtypes, such as triple-negative tumor, still present a challenge for clinicians who often have to resort to highly unspecific cytotoxic therapies. Unfortunately, most patients relapse after a period of remission and eventually tumors becomes refractory to frontline therapy. This makes it desirable to identify drugs which synergize with chemotherapeutics and potentially reduce the dose of them that is necessary to treat patients. In the current financial and economical context, drug repositioning is gaining increasing interest as an alternative strategy for drug development. Cancer cells frequently exhibit specific alterations in their metabolic activity. Thus, targeting lipid metabolism in cancer cells could have therapeutic benefits and it does appear as promising auxiliary strategies in the fight against cancer. The present work aimed to investigate the effect of atorvastatin (ATV) on paclitaxel (PTX), cisplatin (CDDP) and olaparib (OLAP) cytotoxic on human BC cell lines MDA-MB-231 and MCF-7, and the mechanisms by which this interaction occurs. ATV decreased cellular metabolic viability (CMV; MTT assay) in a dose-time-dependent manner, especially in MDA-MB-231 being the maximum effect observed in with ATV 100µM (- 90%; p Note: This abstract was not presented at the meeting. Citation Format: Diandra Zipinotti dos Santos, Isabella dos Santos Guimarães, Nayara Gusmão Tessarollo, Taciane Barbosa Henriques Barbosa Henriques, Paulo Cilas Lyra Junior, Marcele L L. de Souza, Maria C. Gomes, Ian Victor Silva, Leticia B. A. Rangel. Enhancement of cisplatin activity against triple negative breast cancer cells by atorvastatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5276.

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.

Cancer is one of the leading causes of death worldwide. Every year 8.2 million people die from the disease. In this context, breast and ovarian cancer are the most incidental among women. Elucidation of cell growth pathways and the observation that these pathways are altered in human cancer have encouraged the search for specific inhibitors. The phosphatidylinositol-3cinase (PI3K)/Protein kinase b (AKT)/Mammalian Target of Rapamycin (mTOR) is an important pathway involved in cell growth, tumorigenesis, cell invasion, and resistance to therapies. This pathway is often activated in breast and ovarian cancers and the deregulation of its signaling can contribute to tumor growth, angiogenesis and metastasis. Metformin is one of the most commonly prescribed antidiabetic drugs in the world whose anticancer effects, mediated by reduced mTOR signaling, have become notable. Therefore, this review provides an overview of signaling pathway PI3K/AKT/mTOR in the ovarian and breast cancers as well as for target therapies of mTOR signaling, with an emphasis on its mechanisms, clinical applicability and future perspectives.

Background: Breast cancer (BC) comprises multiple diseases harboring different genetic alterations, which subtypes respond differently to treatment, and are associated to distinct clinical outcomes. Likewise, triple negative breast cancer (TNBCs) are a heterogeneous subgroup of BC, immunophenotypically negative for estrogen and progesterone receptor, and HER2, that account for 10–15% of all invasive BC, affecting mostly African-American and Hispanic pre-menopausal women. Considering that TNBC have poor prognosis, and cannot be effectively treated with current targeted therapies, the discovery of new treatment options is imperative. Methods: Novel naphtoquinone-derived drugs were rationally designed to act through multiple pathways aiming the avoidance of drug-resistant phenotype acquisition by tumor cells, and were synthesized following high efficiency and low cost method. Drugs antineoplastic efficacy (AE) was accessed in claudin-low TNBC cell line, MDA-MB-231, 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 and antibiotics until subconfluence; then, 1.5×105 cells/well were subcultured for 72h prior to treatment with drugs (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 IC50 (PrismaGraphPad version 5.1). Findings: We screened the AE of 43 novel naftoquinones-derived drugs in MDA-MB-231 lineage; seven have decreased its CMV by, at least, 50%, as: PIC1 (IC50 1.15×10−4M; CMV decrease of 50%); PIC6 (IC50 4.24×10−5M; CMV decrease of 70%); PIC10 (IC50 5.07×10−5M; CMV decrease of 70%); PIC 20 (IC50 1.38×10−5M; CMV decrease of 90%); PIC21 (IC50 5.00×10−5M; CMV decrease of 70%); S5 (IC50 7.26×10−5M; CMV decrease of 60%); M20 (IC50 7.94×10−5M; CMV decrease of 60%). Their AE was significantly higher than that of cisplatin (IC50 1.56×10−4M; CMV decrease < 10%), doxorubicin (IC50 1.76×10−4M; CMV decrease of 38%), and paclitaxel (IC50 5.05×10−7M; CMV decrease of 20%). Interpretation: Altogether, our results present potential novel antineoplastic drugs to treat TNBC from a panel of in house rationally designed naftoquinones-derived compounds, developing an innovative and economically viable project. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-11-13.

Osteopontin (OPN) has been described as an important gene product mediating resistance to chemotherapeutic drugs. However, the specific roles of each OPN splice variant in mediating chemoresistance should be further investigated. This work aimed to evaluate OPNc expression patterns and its correlations with resistance to doxorubicin (DOX) and cisplatin (CIS) in breast and ovarian tumor cell lines, respectively. We used one breast cancer cell line resistant to DOX (MCF-7 DoxR), and one ovarian cancer cell line resistant to CIS (ACRP) and their corresponding parental cell lines (MCF-7 and A2780). Knockdown of OPNc expression levels has been achieved by transfecting cell lines with specific anti-OPNc phosphothiotate modified DNA oligomers. Cytotoxicity assays have been carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), crystal violet and clonogenic assays. Gene expression has been assessed by quantitative real time PCR. Our results showed that drug-resistant cells overexpress OPNc and P-glycoprotein (P-gp), when compared to their age matched parental cell lines. After transfecting both MCF-7 DoxR and ACRP resistant cell lines with the anti-OPNc oligomers, we found a significant knockdown of OPNc isoform expression, as well as the expression of P-gp. Moreover, these cells exhibited a significantly increased sensitivity to DOX and CIS, and inhibited cell growth and colony formation (p Citation Format: Mariana C. Brum, Isabella S. Guimaräes, Luciana Bueno Ferreira, Letícia B. Rangel, Raquel C. Maia, Gabriela N. de Moraes, Etel R. Gimba. Osteopontin-c mediated drug resistance in breast and ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4453.

Ovarian cancer (OC) is the leading cause of gynecologic cancer-related deaths due to late diagnosis and chemoresistance. RNA-seq data, conducted in collaboration with our group, compared the expression of transcription levels between normal Fallopian tube and high-grade serous ovarian carcinoma samples and revealed phosphodiesterase 7-A as a novel potential therapeutic target against the disease. Metabolic cell viability assays (MCV) were conducted following OC cells treatment with selective PDE7 inhibitor (BRL50481) in monotherapy or in association with paclitaxel (PTX). Mechanism of action of the drug was evaluated by cell cycle analysis, anexina V/PI assay, immunoblotting, real-time PCR and scanning/transmission electron microscopy. Data showed that the use of the BRL50481 in monotherapy reduced A2780 cells MCV by ~60% in a dose-dependent manner after 48h treatment, but nothing significant was observed in OVCAR3. On the other hand, the association of BRL50481 and PTX promoted inhibition of MCV in both cell lines analyzed. An increase in the potency of PTX was also observed, an aspect verified with the reduction of its IC50 in relation to monotherapy by 30% (p Citation Format: Nayara G. Tessarollo, Isabella S. Guimarães, Diandra Z. dos Santos, Taciane B. Henriques, Marcele LM Souza, Laura Maciel, João Carlos A. Almeida, Alan Branco, Ian V. Silva, Leticia BA Rangel. Phosphodiesterase 7-A is a novel potential therapeutic target against ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5883.

Resistance to chemotherapeutic drugs corresponds to main causes of treatment failure. Total osteopontin (OPN) has been described as a gene product involved on chemoresistance, besides activating several aspects of tumor progression. OPN regulates the expression of P-glycoprotein (P-gp) in prostate cancer (PCa) cells, besides promoting apoptosis evasion induced by chemotherapy using paclitaxel and estramustin. These data point OPN as a potential therapeutic target to approaches that could reduce chemotherapy tumor resistance. However, most of this data are related to total OPN. Once OPN suffers alternative splicing, producing 3 isoforms, further investigation should demostrate their specific involvement on chemoresistance. Recent data from our group indicated that OPNb and OPNc splicing isoforms activate PCa tumor progression features. Besides, PCa cells that overexpress these splice variants are more resistant to apoptosis induced by docetaxel (DXT). The current work aims to investigate the expression pattern of OPN splicing isoforms (OPN-SIs) and their relationships in DXT-PCa resistant cells. Total RNA has been extracted from PCa cell lines resistant to DXT (PC3-D8 and PC3-D12) and PC3 control cells with similar cell passages (PC3-Ag). Then, cDNa has been synthesized. OPN-SI expression levels were analyzed by quantitative real time PCR (qRT-PCR) using SYBR Green and isoform specific oligonucleotides. Relative expression levels were calculated using ΔΔCT method. Among the 3 OPN-SIs, we observed that OPNb and OPNc variants are overexpressed in relation to OPNa in DXT-PCa resistant cells PC3-D8 and PC3-D12, when compared to PC3-Ag. PC3-D12 cell line, resistant to higher DXT concentrations, expresses higher levels of OPNc, when compared to the other cell lines. Overall, our data further evidence that OPNb and OPNc overexpression in DXT-PCa resistant cells could be involved on resistance to DXT. Functional assays will be performed in order to investigate the molecular mechanisms by which these OPN-SIs mediate resistance to this chemotherapeutic drug. Citation Format: Mariana Concentino Menezes, Amanda O’Neil, Isabella Guimaraes, Letícia Rangel, Etel Rodrigues Pereira Gimba. Expression of osteopontin splicing isoforms in prostate cancer cells resistant to docetaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2937.

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 (Spearman's 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 Spearman's 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

In ovarian carcinoma (OC), invasive cell properties are supported by epithelial mesenchymal transition (EMT). We previously demonstrated that OPNc, one of the glycophosphoprotein osteopontin splicing isoforms (OPN-SI), activates OC progression. However , the involvement of these OPN-SI in EMT in OC cells has not previously been addressed. We aimed to evaluate the expression of OPN-SI in OC cells induced to EMT by TGFβ. ES2 and A2780 OC cell lines were treated with 10ng/ml of TGFβ in order to induce EMT. The expression of OPN-SI and EMT epithelial (E) (Claudin-3, Claudin-4 and E-cadherin) and mesenchymal (M) (slug, vimentin and N-cadherin) markers was tested by quantitative real time PCR. In both cell lines, OPNa is overexpressed in relation to OPNb and OPNc. Upon treatment with TGFβ, in A2780 cells we observed an upregulation of OPNc. Conversely, OPNa and OPNb were downregulated. In ES2 cells, TGFβ also induced OPNc upregulation, but OPNb expression is activated as well, while OPNa expression is inhibited. In order to validate EMT induction by TGFβ, we tested the expression of E and M markers. In A2780 cells, TGFβ induce the downregulation of E-cadherin, but Claudin-3 and Claudin-4 are upregulated. In ES2 cells, Claudin-3 expression is downregulated in this condition. After TGFβ treatment, in A2780 cells all M markers are downregulated, while in ES2 cells, only N-cadherin expression has been inhibited. Our data indicate that the expression of OPN-SI is modulated by EMT induced by TGFβ and that mostly the oncogenic OPNc is upregulated in this condition. Our data evidence that the expression of OPN-SI is modulated by a partial EMT phenotype induced by TGFβ, which is cell-type specific, and that OPN-SI, specially OPNc, could contribute to this key step on tumor progression in OC cells. Citation Format: Ingridy Celestino, Isabella S. Guimarães, Leticia B. A. Rangel, Etel R. P. Gimba. Osteopontin splicing isoforms expression is modulated by partial epithelial mesenchymal transition in ovarian carcinoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A28.

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

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

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% CO2, until subconfluence; then, 1.5x105 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 IC50 (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 (IC50 1.64x10−5M; CMV decrease of 90%); M10 (IC50 1.37x10−5M; CMV decrease of 96%); M14 (IC50 1.45x10−5M; CMV decrease of 85%); PIC10 (IC50 9.13x10−6M; CMV decrease of 95%); PIC20 (IC50 5.31x10−5M; CMV decrease of 70%). The IC50 for cisplatin, the gold therapy against OVCA was 3.87x10−5M 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