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The extracellular pH (pHe) of solid tumors is often acidic, as a consequence of the Warburg effect, and an altered metabolic state is often associated with malignancy. It has been shown that acidosis can promote tumor progression; thus, many therapeutic strategies have been adopted against tumor metabolism; one of these involves alkalinization therapies to raise tumor pH to inhibit tumor progression, improve immune surveillance, and overcome resistance to chemotherapies. Chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) is a noninvasive technique that can measure pH in vivo using pH-sensitive contrast agents. Iopamidol, an iodinated contrast agent, clinically used for computed tomography (CT), contains amide group protons with pH-dependent exchange rates that can reveal the pHe of the tumor microenvironment. In this study, we optimized intraperitoneal (IP) delivery of iopamidol to facilitate longitudinal assessments of orthotopic pancreatic tumor pHe by CEST-MRI. Following IV-infusion and IP-bolus injections, we compared the two protocols for assessing tumor pH. Time-resolved CT imaging was used to evaluate the uptake of iopamidol in the tumor, revealing that IP-bolus delivered a high amount of contrast agent 40 min postinjection, which was similar to the amounts reached with the IV-infusion protocol. As expected, both IP and IV injection protocols produced comparable measurements of tumor pHe, showing no statistically significant difference between groups ( p = 0.16 ). In addition, we showed the ability to conduct longitudinal monitoring of tumor pHe using CEST-MRI with the IP injection protocol, revealing a statistically significant increase in tumor pHe following bicarbonate administration ( p < 0.001 ). In conclusion, this study shows the capability to measure pHe using an IP delivery of iopamidol into orthotopic pancreatic tumors, which is important to conduct longitudinal studies.

Rationale: Hypoxic regions (habitats) within tumors are heterogeneously distributed and can be widely variant. Hypoxic habitats are generally pan-therapy resistant. For this reason, hypoxia-activated prodrugs (HAPs) have been developed to target these resistant volumes. The HAP evofosfamide (TH-302) has shown promise in preclinical and early clinical trials of sarcoma. However, in a phase III clinical trial of non-resectable soft tissue sarcomas, TH-302 did not improve survival in combination with doxorubicin (Dox), possibly due to a lack of patient stratification based on hypoxic status. Therefore, we used magnetic resonance imaging (MRI) to identify hypoxic habitats and non-invasively follow therapies response in sarcoma mouse models. Methods: We developed deep-learning (DL) models to identify hypoxia, using multiparametric MRI and co-registered histology, and monitored response to TH-302 in a patient-derived xenograft (PDX) of rhabdomyosarcoma and a syngeneic model of fibrosarcoma (radiation-induced fibrosarcoma, RIF-1). Results: A DL convolutional neural network showed strong correlations (>0.76) between the true hypoxia fraction in histology and the predicted hypoxia fraction in multiparametric MRI. TH-302 monotherapy or in combination with Dox delayed tumor growth and increased survival in the hypoxic PDX model (p

Tumor acidosis plays a major role in tumor aggressiveness, invasion, and resistance. Therefore, it is considered an important target for novel anticancer strategies. In this study, we tested the efficacy of a transdermal treatment applied directly to the tumor area to reduce interstitial acidosis. The product involved is a patented transdermal cream formulation (DYV800) produced by Dyve Biosciences that contains 33% sodium bicarbonate and serves as a buffer medicament. C57BL/6 mice bearing B16-F10 melanoma subcutaneous tumors were treated with DYV800, and pH was measured at different time points after cream application. The MRI pH imaging technique used in this study is the Chemical Exchange Saturation Transfer (CEST) method, which involves the injection of an iodinated contrast agent, iopamidol (Isovue, Bracco Imaging), to measure extracellular pH (pHe) in vivo. In the first experiment to examine the time-course effect of DYV800 on tumor acidosis, mice (N=30, 15 untreated and 15 treated) were treated only once with 100 µL of cream. pH acquisitions were done with untreated mice for baseline pH measurements (N=15) and at different time points after cream application in another set of mice (1, 2, 4 and 24 hours; N=15). As revealed by the pH measurements, the highest increase in pH was reported after 2 hours of the cream application (6.71 vs. 6.84, untreated vs. post 2hrs, p-value 0.025 ANOVA with Bonferroni comparison test), and although pH was higher for all the other time points compared to baseline values, no statistical significance was found (post 1hr 6.75; post 4hrs 6.78; post 24hrs 6.75). In a second experiment, mice (N=10) were untreated (N=3) or received prolonged treatment that lasted 5 days, with one group of mice treated once-a-day (N=3) and the second group of mice treated twice-a-day (N=4) with 100 µL of cream for each application. MRI pH imaging was performed the next day after the treatment regimen was completed. Baseline measurements confirmed that B16 tumors have an acidic pH of 6.7 that increased to 6.82 in the once-a-day treated group (p-value 0.037 unpaired t-test vs. baseline) and to 6.77 in the twice-a-day treated group (not significant compared to baseline). To complete the pH evaluations, untreated mice reported a slight reduction in pHe (6.65) after 6 days of the baseline acquisitions. These experiments demonstrate for the first time that the extracellular pH of tumors can be increased upon application of a buffer formulation transdermally delivered to mice. Future experiments are expected to provide insights into the effect of DYV800 cream application on tumor growth and immune cell migration and activation in the tumor microenvironment. Citation Format: Pietro Irrera, Bruna Victorasso Jardim-Perassi, Dominique Abrahams, Thomas Sherlock, Samantha Byrne, Andrew Ojeda, Justin Y. Lau, Nathan Fitzsimmons, Audrene Rice, Chuck Harbert, Renee Arakawa, Arig Ibrahim-Hashim, Ryan Beal, Shari Pilon-Thomas. Transdermal alkalinization treatment induces pH changes in a murine melanoma model as measured with MRI-CEST pH imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2391.

It is well-recognized that solid tumors are genomically, anatomically, and physiologically heterogeneous. In general, more heterogeneous tumors have poorer outcomes, likely due to the increased probability of harboring therapy-resistant cells and regions. It is hypothesized that the genomic and physiologic heterogeneity are related, because physiologically distinct regions will exert variable selection pressures leading to the outgrowth of clones with variable genomic/proteomic profiles. To investigate this, methods must be in place to interrogate and define, at the microscopic scale, the cytotypes that exist within physiologically distinct subregions (“habitats”) that are present at mesoscopic scales. MRI provides a noninvasive approach to interrogate physiologically distinct local environments, due to the biophysical principles that govern MRI signal generation. Here, we interrogate different physiologic parameters, such as perfusion, cell density, and edema, using multiparametric MRI (mpMRI). Signals from six different acquisition schema were combined voxel-by-voxel into four clusters identified using a Gaussian mixture model. These were compared with histologic and IHC characterizations of sections that were coregistered using MRI-guided 3D printed tumor molds. Specifically, we identified a specific set of MRI parameters to classify viable-normoxic, viable-hypoxic, nonviable-hypoxic, and nonviable-normoxic tissue types within orthotopic 4T1 and MDA-MB-231 breast tumors. This is the first coregistered study to show that mpMRI can be used to define physiologically distinct tumor habitats within breast tumor models. Significance: This study demonstrates that noninvasive imaging metrics can be used to distinguish subregions within heterogeneous tumors with histopathologic correlation.

Background:The angiogenesis process is regulated by many factors, such as Hypoxia-Inducible Factor-1 (HIF-1) and Vascular Endothelial Growth Factor (VEGF). Metformin has demonstrated its ability to inhibit cell growth and the LY294002 is the major inhibitor of PI3K/AKT/mTOR pathway that has antiangiogenic properties.Methods:Canine mammary tumor cell lines CMT-U229 and CF41 were treated with metformin and LY294002. Cell viability, protein and gene expression of VEGF and HIF-1 were determined in vitro. For the in vivo study, CF41 cells were inoculated in female athymic nude mice treated with either metformin or LY294002. The microvessel density by immunohistochemistry for CD31 as well as the gene and protein expression of HIF-1 and VEGF were evaluated.Results:The treatment with metformin and LY294002 was able to reduce the cellular viability after 24 hours. The protein and gene expression of HIF-1 and VEGF decreased after treatment with metformin and LY294002. In the in vivo study, there was a decrease in tumor size, protein and gene expression of HIF-1 and VEGFA, in addition to the decreasing of CD31 expression after all treatments.Conclusion:Our results demonstrate the effectiveness of metformin and LY294002 in controlling the angiogenesis process in mammary tumors by VEGF and HIF-1, the most important angiogenic markers.

Hypoxic regions (habitats) within tumors are heterogeneously distributed and can be widely variant. Hypoxic habitats are generally pan-therapy resistant. For this reason, hypoxia-activated prodrugs (HAPs) have been developed to target these resistant volumes. The HAP evofosfamide (TH-302) has shown promise in preclinical and early clinical trials of sarcoma. However, in a phase III clinical trial, TH-302 did not improve survival in combination with doxorubicin (dox), most likely due to a lack of patient stratification based on hypoxic status. Herein, our goal was to develop deep-learning (DL) models to identify hypoxic habitats, using multiparametric (mp) MRI and co-registered histology, and to non-invasively monitor response to TH-302 in a patient-derived xenograft (PDX) of rhabdomyosarcoma and a syngeneic model of fibrosarcoma (RIF-1). A DL convolutional neural network showed strong correlations (>0.81) between the true hypoxic portion in histology and the predicted hypoxic portion in multiparametric MRI. TH-302 monotherapy or in combination with Dox delayed tumor growth and increased survival in the hypoxic PDX model (pOne Sentence SummaryDevelopment of non-invasive MR imaging to assess hypoxia is crucial in determining the effectiveness of TH-302 therapy and to follow response.

Melatonin is a ubiquitous molecule with a broad spectrum of functions including widespread anti-cancer activities. Identifying how melatonin intervenes in complex molecular signaling at the gene level is essential to guide proper therapies. Using meta-analysis approach, herein we examined the role of melatonin in regulating the expression of 46 microRNAs (miRNAs) and their target genes in breast, oral, gastric, colorectal, and prostate cancers, and glioblastoma. The deregulated miRNA-associated target genes revealed their involvement in the regulation of cellular proliferation, differentiation, apoptosis, senescence, and autophagy. Melatonin changes the expression of miRNA-associated genes in breast, gastric, and oral cancers. These genes are associated with cellular senescence, the hedgehog signaling pathway, cell proliferation, p53 signaling, and the hippo signaling pathway. Conversely, colorectal and prostate cancers as well as glioblastoma and oral carcinoma present a clear pattern of less pronounced changes in the expression of miRNA-associated genes. Most notably, colorectal cancer displayed a unique molecular change in response to melatonin. Considering breast cancer network complexity, we compared the genes found during the meta-analysis with RNA-Seq data from breast cancer-bearing mice treated with melatonin. Mechanistically, melatonin upregulated genes associated with immune responses and apoptotic processes, whereas it downregulated genes involved in cellular aggressiveness/metastasis (eg, mitosis, telomerase activity, and angiogenesis). We further characterized the expression profile of our gene subsets with human breast cancer and found eight upregulated genes and 16 downregulated genes that were appositively correlated with melatonin. Our results pose a multi-dimension network of tumor-associated genes regulated by miRNAs potentially targeted by melatonin.

Melatonin is a pleiotropic anti-cancer molecule that controls cancer growth by multiple mechanisms. RNA-Seq can potentially evaluate therapeutic response and its use in xenograft tumor models can differentiate the changes that occur specifically in tumor cells or in the tumor microenvironment (TME). Melatonin actions were evaluated in a xenograft model of triple-negative breast cancer. Balb/c nude mice bearing MDA-MB-231 tumors were treated with melatonin or vehicle. RNA-Seq was performed on the Illumina HiSeq. 2500 and data were mapped against human and mouse genomes separately to differentiate species-specific expression. Differentially expressed (DE) genes were identified and Weighted Gene Co-expression Network Analysis (WGCNA) was used to detect clusters of highly co-expressed genes. Melatonin treatment reduced tumor growth (p

Although cancer often is referred to as "a disease of the genes," it is indisputable that the (epi)genetic properties of individual cancer cells are highly variable, even within the same tumor. Hence, preexisting resistant clones will emerge and proliferate after therapeutic selection that targets sensitive clones. Herein, the authors propose that quantitative image analytics, known as "radiomics," can be used to quantify and characterize this heterogeneity. Virtually every patient with cancer is imaged radiologically. Radiomics is predicated on the beliefs that these images reflect underlying pathophysiologies, and that they can be converted into mineable data for improved diagnosis, prognosis, prediction, and therapy monitoring. In the last decade, the radiomics of cancer has grown from a few laboratories to a worldwide enterprise. During this growth, radiomics has established a convention, wherein a large set of annotated image features (1-2000 features) are extracted from segmented regions of interest and used to build classifier models to separate individual patients into their appropriate class (eg, indolent vs aggressive disease). An extension of this conventional radiomics is the application of "deep learning," wherein convolutional neural networks can be used to detect the most informative regions and features without human intervention. A further extension of radiomics involves automatically segmenting informative subregions ("habitats") within tumors, which can be linked to underlying tumor pathophysiology. The goal of the radiomics enterprise is to provide informed decision support for the practice of precision oncology.

Breast cancer progression is composed of multiple steps that are influenced by tumor cell adaptations to survive under acidic conditions in the tumor microenvironment. Regulation of this cell survival behavior is a promising strategy to avoid cancer development. Melatonin is a natural hormone produced and secreted by the pineal gland capable of modulating different biological pathways in cancer. Although the anti-cancer effects of melatonin are currently widespread, its role in the acid tumor microenvironment remains poorly understood. The aim of the present study was to investigate the effect of low pH (6.7) on human breast cancer cell lines MCF-7 and MDA-MB-231, and the effectiveness of melatonin in acute acidosis survival mechanisms. Cell viability was measured by a MTT assay and the protein expression of glucose transporter (GLUT)-1, Ki-67 and caspase-3 was evaluated by immunocytochemical (ICC) analysis following low pH media and melatonin treatment. In both cell lines the viability was decreased after melatonin treatment (1 mM) under acidosis conditions for 24 h. ICC analysis showed a significant increase in GLUT-1 and Ki-67 expression at pH 6.7, and a decrease after treatment with melatonin for 12 and 24 h. The low pH media decreased the expression of caspase-3, which was increased after melatonin treatment for 12 and 24 h. Overall, the results of the present study revealed melatonin treatment increases apoptosis, as indicated by changes in caspase-3, and decreases proliferation, indicated by changes to Ki-67, and GLUT-1 protein expression under acute acidosis conditions in breast cancer cell lines.

Made available in DSpace on 2018-12-11T16:48:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-08-15 Faculdade de Medicina de São José do Rio Preto Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Mammary tumorigenesis can be modulated by melatonin, which has oncostatic action mediated by multiple mechanisms, including the inhibition of the activity of transcription factors such as NF-κB and modulation of interleukins (ILs) expression. IL-25 is an active cytokine that induces apoptosis in tumor cells due to differential expression of its receptor (IL-17RB). IL-17B competes with IL-25 for binding to IL-17RB in tumor cells, promoting tumorigenesis. This study purpose is to address the possibility of engaging IL-25/IL-17RB signaling to enhance the effect of melatonin on breast cancer cells. Breast cancer cell lines were cultured monolayers and 3D structures and treated with melatonin, IL-25, siIL-17B, each alone or in combination. Cell viability, gene and protein expression of caspase-3, cleaved caspase-3 and VEGF-A were performed by qPCR and immunofluorescence. In addition, an apoptosis membrane array was performed in metastatic cells. Treatments with melatonin and IL-25 significantly reduced tumor cells viability at 1 mM and 1 ng/mL, respectively, but did not alter cell viability of a non-tumorigenic epithelial cell line (MCF-10A). All treatments, alone and combined, significantly increased cleaved caspase-3 in tumor cells grown as monolayers and 3D structures (p

Melatonin is a hormone produced by the pineal gland and has been shown different antitumor effects, as immunomodulatory, antioxidant, pro-apoptotic, anti-proliferative, antimetastatic and antiangiogenic, however, the pathways by which melatonin exerts its action need to be identified. Thus, the aims of this study were to perform the transcriptome analysis to evaluate the pathways of melatonin action in triple-negative breast cancer. Triple-negative breast cancer cells (MDA-MB-231) were injected into the mammary gland of the athymic nude mice (n=10), which were treated with melatonin (40 mg/kg) or vehicle during 21 days. RNA-Seq libraries were created using Truseq RNA-Seq Library Prep Kit v2. The experiment was paired-end with 100nt read length, performed on the Illumina HiSeq2500 sequencer, producing about 30 million reads per library. To differentiate human and mouse expression, the alignment was performed to filter out mouse-like reads before mapping to the human reference and vice versa, and data were mapped against human (GRCh37/hg19) and mouse (NCBI37/mm9) genomes separately, using the TopHat software. The HTSeq was employed for analyses of read counts and DESeq2 was used to identify genes differentially expressed between melatonin treated and control tumors. Differentially expressed genes (DEGs) were identified based on a false discovery rate (FDR) q-value threshold of less than 0.05. Also, we applied Weighted Gene Co-expression Network Analysis (WGCNA) to detect clusters of highly co-expressed genes (modules). Results showed that animals treated with melatonin had smaller tumors volume than controls (p0.05). In mouse cells, which represent the tumor microenvironment, there were 34 DEGs between animals treated with melatonin and controls (adjP0.6), which were functionally enriched for GO terms like lipid metabolic process, response to drug, oxidoreductase activity and PPAR signaling (adjP Citation Format: Jardim-Perassi BV, Sonehara NM, de Paula-Junior R, Chammas R, Coutinho LL, Reis Júnior O, Alexandre PA, Fukumasu H, Zuccari DAPC. Melatonin treatment: A transcriptomic networks in a xenograft model of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-28.

Made available in DSpace on 2020-12-12T02:23:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Mammary gland tumors are a heterogeneous group of neoplastic diseases. Genetic studies make it possible to determine genetic profiles and identify new molecular markers. The aim of the study was to evaluate the gene expression profile of canine mammary carcinomas and identify potential prognostic markers. Twelve mammary cancer samples from bitches were collected for the evaluation of global gene expression. Microarray assays were performed using commercial kits. Statistical analysis of the microarray was done using moderate t-statistic and adjusted using the Benjamini and Hochberg procedure. Differential connectivity analysis was also performed. Enrichment analyses were conducted using WebGestalt. P-values were calculated using hypergeometric statistics and adjusted using the Benjamini and Hochberg procedure. The HYAL-1 gene was validated using quantitative PCR (qPCR). There were 878 upregulated genes and 821 downregulated genes in the neoplasms studied. Enrichment analysis (individual analysis) identified the HYAL-1 gene as a potential marker of tumorigenesis and tumor recurrence. Differential connectivity analysis demonstrated 262 differentially connected genes. Postgraduate Program in Veterinary Surgery UNESP-FCAV, Jaboticabal Department of Molecular Biology FAMERP, São José do Rio Preto Department of Veterinary Medicine FZEA-USP, Pirassununga Postgraduate Program in Veterinary Surgery UNESP-FCAV, Jaboticabal CNPq: 140624/2013-9

Tumors are spatially heterogeneous, stemming from selection pressures on physiologically distinct sub-regions, also called tumor habitats in an analogy to evolutionary ecology. Different habitats may respond differently to therapy, which has a great impact in the clinical outcome. Non-invasive imaging analyses capable of assessing distinct tumor sub-regions rather than the entire tumor, are promising to longitudinally and more realistically monitor the tumor progression and response to therapy. Habitat imaging is a technique that aspires to interrogate these distinct local environments, in which Magnetic Resonance Imaging (MRI) has been explored due to the biophysical principles that govern MRI signal generation. Different functional imaging sequences can be used to segment the tumor into distinct sub-regions. However, biological assumptions from imaging should be made with caution, and a ground truth must be provided by corresponding histological data. This chapter presents studies that have proposed different analyses in specific MRI sequences, as well as multiparametric MRI, to define tumor sub-regions and to assess intratumoral heterogeneity.

Angiogenesis is the process of new blood vessel formation, regulated by a number of pro- and antiangiogenic factors and usually begins in response to hypoxia. Exogenous administration of melatonin has shown numerous anti-tumor effects and appears to inhibit tumor angiogenesis. However, many factors involved in the anti-angiogenic effect of melatonin are still under investigation. Here, we evaluate the effects of melatonin on cell viability and expression of angiogenic factors in MCF-7 and MDA-MB-231 breast cancer cells under hypoxic conditions. Cell viability was investigated by MTT and gene and protein expression of the hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF-A) were verified by qPCR and immunocytochemistry after melatonin treatment (1 mM) under hypoxic conditions. Additionally, a protein array with 20 different cytokines/factors was performed on tumor cell lysates. The results showed that 1 mM of melatonin reduced the viability of MCF-7 and MDA-MB-231 cells (p < .05). This treatment also decreased both gene and protein expression of HIF-1α and VEGF-A under hypoxic conditions (p < .05). Among the proteins evaluated by protein array, melatonin treatment during hypoxia reduced VEGF-C, VEGFR receptors (VEGFR2 and VEGFR3), matrix metalloproteinase 9 (MMP9) and Angiogenin in MCF-7 cells. In MDA-MB-231 cells, a significant decrease was observed in VEGFR2, epidermal growth factor receptor (EGFR) and Angiogenin (p < .05). Taken together, these results showed that melatonin acts in the regulation of angiogenic factors in breast tumor cells and suggests an anti-angiogenic activity, particularly under hypoxic conditions.

Tumor habitats are phenotypically and spatially distinct intratumoral regions. Hypoxic habitats are associated with reduced accessibility of chemotherapeutics, resulting in resistance. Hypoxia targeted prodrugs (HAPs) may overcome this resistance. However, in a phase III clinical trial, the HAP evofosfamide (TH-302) did not improve survival of sarcomas when used in combination with the standard of care doxorubicin (Dox). This may have been due to a lack of patient stratification based on hypoxic status. Thus, identification of hypoxic tumor habitats (Jardim-Perassi et al. 2019) would allow for pre-therapy patient stratification, and monitoring of habitats post-therapy. Our goal was to use multiparametric (mp) MRI and co-registered histology to classify hypoxic habitats in preclinical models of sarcoma, and to use Habitat Imaging to monitor therapy response. A patient-derived xenograft (PDX) of rhabdomyosarcoma (obtained from St. Jude's) and a syngeneic model of fibrosarcoma (RIF-1) were randomized into four groups, receiving saline (control), Dox, TH-302, or the combination (Dox + TH-302). mpMRI was acquired pre and post therapy, and parameter maps were calculated for T2, T2*, diffusion weighted imaging (DWI), and Dynamic contrast-enhanced (DCE). MRI based 3D printed tumor molds were used to guide MRI and histology co-registration. Machine learning was used to classify MRI into viable, hypoxic and non-viable tumor habitats using their co-registered histological counterparts as ground-truth. TH-302 monotherapy or the Dox + TH-302 combination reduced tumor growth and increased overall survival in the PDX model (p Citation Format: Bruna Victorasso Jardim Perassi, Wei Mu, Dominique F. Abrahams, Michal R. Tomaszewski, Jan Poleszczuk, Jack Higgins, Gary V. Martinez, Robert J. Gillies. Targeting hypoxic habitats with hypoxia pro-drug evofosfamide in preclinical models of sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2772.

Made available in DSpace on 2019-10-06T16:32:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Chalcones are valuable structures for drug discovery due to their broad bioactivity spectrum. In this study, we evaluated 20 synthetic chalcones against estrogen-receptor-positive breast cancer cells (MCF-7 line) and triple-negative breast cancer (TNBC) cells (MDA-MB-231 line). Antiproliferative screening by MTT assay resulted in two most active compounds: 2-fluoro-4’-aminochalcone (11) and 3-pyridyl-4’-aminochalcone (17). Their IC50 values ranged from 13.2 to 34.7 µM against both cell lines. Selected chalcones are weak basic compounds and maintained their antiproliferative activity under acidosis conditions (pH 6.7), indicating their resistance to ion-trapping effect. The mode of breast cancer cells death was investigated and chalcones 11 and 17 were able to induce apoptosis rather than necrosis in both lines. Antiproliferative target investigations with MCF-7 cells suggested 11 and 17 upregulated p53 protein expression and did not affect Sp1 protein expression. Future studies on chalcones 11 and 17 can define their in vivo therapeutic potential. Department of Chemistry and Environmental Chemistry Institute of Biosciences Humanities and Exact Sciences (IBILCE) São Paulo State University (UNESP) Department of Molecular Biology Medicine College of São José do Rio Preto (FAMERP) Department of Biology Institute of Biosciences Humanities and Exact Sciences (IBILCE) São Paulo State University (UNESP) Biotechnology Unit University of Ribeirão Preto (UNAERP) Department of Chemistry and Environmental Chemistry Institute of Biosciences Humanities and Exact Sciences (IBILCE) São Paulo State University (UNESP) Department of Biology Institute of Biosciences Humanities and Exact Sciences (IBILCE) São Paulo State University (UNESP)

Mammary tumors are the most common type of tumor in dogs, with approximately half of these tumors being malignant. Hypoxia, characterized by oxygen levels below normal, is a known adverse factor to cancer treatment. The hypoxia-inducible factor 1α (HIF-1α) is a central regulator of the pathophysiological response of mammalian cells to low oxygen levels. HIF-1α activates the transcription of vascular endothelial growth factor (VEGF), which in turn promotes angiogenesis through its ability to stimulate the growth, migration and invasion of endothelial cells to form new blood vessels, contributing to tumor progression. In this study, we evaluated the serum concentration and gene expression of VEGF and HIF-1α linking them with clinicopathological parameters and survival of dogs with mammary tumors in order to infer the possible prognostic value of these factors. We collected blood and tumor fragments of 24 female dogs with malignant mammary tumors (study group) and 26 non-affected female dogs (control group) to verify the gene expression of VEGF and HIF-1α by quantitative real-time PCR (qPCR) and the serum levels by ELISA (enzyme-linked immunosorbent). The results showed high serum levels of VEGF in the study group and its correlation between abundant vascularization, lymph node involvement, metastasis, death rate and low survival (p

Background: NF-kB (nuclear factor kappa B) is a transcription factor composed of two subunits, p50 and p65, which plays a key role in the inflammatory process. Melatonin has oncostatic, antiangiogenic and antimetastatic properties, and some recent studies have indicated an inhibitory effect of melatonin on NF-kB in some types of cancer. This work aims to investigate the effects of melatonin treatment on the expression of NFkB in breast and liver cancer models. Method: The breast cancer xenographic model was performed using female Balb/c nude athymic mice injected with MDA-MB-231 cells. The animals were treated with 40 mg/Kg of melatonin for 21 days. Volume of the tumors was measured with a digital caliper. Hepatocarcinoma model was developed by using the HepG2 cells in vitro, treated with 1 mM melatonin for 24 h. The expression of NF-kB protein was verified by immunohistochemistry and immunocytochemistry and quantified by optical densitometry, in vivo study and in vitro study, respectively. NF-kB gene expression was performed by quantitative RT-PCR. Results: The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size (P=0.0022). There was a decrease in NF-kB protein staining (P=0.0027) and gene expression (P=0.0185) in mice treated with melatonin. The opposite results were observed for the hepatocarcinoma model. HepG2 cells treated with melatonin showed an increase in the NF-kB immunostaining when compared to control cells (P=0.0042). Conclusion: Our results indicated that the treatment with melatonin was able to decrease both gene and protein expressions of NF-kB in breast cancer cells and, conversely, increase the transcription factor protein expression in hepatocarcinoma cells. These data highlighted a double role in the expression of NF-kB, depending on the cell type. Further studies are needed to better elucidate the action of melatonin in NF-kB, since this transcription factor acts on different signaling pathways that are fundamental for carcinogenesis.

Mammary neoplasias are the most common tumors observed in female dogs. Identification of these tumors is valuable in order to identify beneficial therapeutic agents as alternative treatments for this tumor type. Oral administration of melatonin appears to exert an oncostatic effect on mammary neoplasia and may have a possible mechanism of action through its interaction with estrogen receptors on epithelial cells. Hence, we analyzed the potential therapeutic value of melatonin in tumors that are estrogen-dependent or -independent, and established a relationship of its action with the expression of the melatonin receptors MT1 and MT2. Furthermore, we analyzed the rate of cell proliferation and apoptosis after treatment with melatonin. Cell cultures were performed using 10 canine mammary tumor fragments and were divided into estrogen receptor (ER)-positive and ER-negative tumors. The results showed that both ER-positive and ER-negative tumors had decreased cell viability and proliferation after treatment with melatonin (p

Breast cancer is the most common tumor in women and it has high mortality mainly due to the occurrence of tumor metastasis. Both the processes of cell migration and anchorage to the substrate are essential for the development of metastasis. These processes occur by rearrangements of the actin cytoskeleton, regulated by Rho-associated protein kinase 1 (ROCK-1). The degradation of the extracellular matrix, influenced by metalloproteinase 9 (MMP-9) also exerts greater cell invasiveness. The present study evaluated the ROCK-1 and MMP-9 proteins using an immunohistochemical method through the selection of invasive ductal breast carcinoma. The protein expression was correlated to clinicopathological parameters and overall survival of the patients. High expression of the ROCK-1 protein was correlated statistically to the status of lymph nodes ( p = 0.007) and showed variable expression in different clinical stages of the tumor. MMP-9 showed a strong immunostaining in patients with metastasis that had died, whereas there was no marker in normal breast tissues. In addition, 46.6% of patients classified as poor prognosis showed high expression of ROCK-1 and MMP-9 protein and another 40.0% just showed high expression of MMP-9. Thus, the differential expression of ROCK-1 and MMP-9 proteins suggests their potential use as prognostic markers in breast cancer.

Background: Oral cancer is the most common type of head and neck cancer and its high rate of mortality and morbidity is closely related to the processes of angiogenesis and tumor metastasis. The overexpression of the pro-angiogenic genes, HIF-1α and VEGF, and pro-metastatic gene, ROCK-1, are associated with unfavorable prognosis in oral carcinoma. Melatonin has oncostatic, antiangiogenic and antimetastatic properties in several types of neoplasms, although its relationship with oral cancer has been little explored. This study aims to analyze the expression of the genes HIF-1α, VEGF and ROCK-1 in cell lines of squamous cell carcinoma of the tongue, after treatment with melatonin. Methods: SCC9 and SCC25 cells were cultured and cell viability was assessed by MTT assay, after treatment with 100 μM of CoCl 2 to induce hypoxia and with melatonin at different concentrations. The analysis of quantitative RT-PCR and the immunocytochemical analysis were performed to verify the action of melatonin under conditions of normoxia and hypoxia, on gene and protein expression of HIF-1α, VEGF and ROCK-1. Results: The MTT assay showed a decrease in cell viability in both cell lines, after the treatment with melatonin. The analysis of quantitative RT-PCR indicated an inhibition of the expression of the pro-angiogenic genes HIF-1α (P < 0.001) and VEGF (P < 0.001) under hypoxic conditions, and of the pro-metastatic gene ROCK-1 (P < 0.0001) in the cell line SCC9, after treatment with 1 mM of melatonin. In the immunocytochemical analysis, there was a positive correlation with gene expression data, validating the quantitative RT-PCR results for cell line SCC9. Treatment with melatonin did not demonstrate inhibition of the expression of genes HIF-1α, VEGF and ROCK-1 in line SCC25, which has different molecular characteristics and greater degree of malignancy when compared to the line SCC9. Conclusion: Melatonin affects cell viability in the SCC9 and SCC25 lines and inhibits the expression of the genes HIF-1α, VEGF and ROCK-1 in SCC9 line. Additional studies may confirm the potential therapeutic effect of melatonin in some subtypes of oral carcinoma.

The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK-1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti-angiogenic effects and can modulate ROCK-1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK-1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK-1 gene expression and protein expression were determined in vitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5 wk. The metastases were evaluated by X-ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK-1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK-1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of 'hot' spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK-1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer in vitro and in vivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK-1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment.

Introduction: The angiogenesis process is regulated by numerous factors, especially the hypoxia-inducible factor 1alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). The HIF-1 is a central regulator of pathophysiologic response of cells to hypoxia conditions, able to activate transcription of the gene that promotes the induction of VEGF, which in turn promotes angiogenesis through its ability to stimulate growth, migration, and invasion of endothelial cells, leading to the formation of new blood vessels and subsequent tumor growth. Metformin has demonstrated its ability to reduce the incidence of cancer in diabetic patients with positive results, especially in breast cancer, inhibiting cell growth by AMPK/Akt/mTOR signaling pathway. Likewise, LY294002, a PI3K signaling pathway inhibitor, has antiangiogenic features. In this context, the aim of this study was to evaluate the effectiveness of metformin and LY294002 inhibitor treatment in angiogenesis as a therapeutic strategy in mammary tumors in an in vitro and in vivo study. Materials and Methods: In the in vitro study, canine mammary tumor cell line (CF41) was cultured in DMEM high-glucose culture medium, at 37°C in 5% CO2. Cell viability was measured by MTT assay after treatment with different concentrations of metformin and LY294002 inhibitor. Once established the concentration of 5 mM for metformin and 5 µM of LY294002, the protein and gene expression of HIF-1alpha and VEGF were detected by immunocytochemistry and real-time PCR, respectively. For an in vivo study, CF41 were injected in nude athymic female mice and treated with metformin (200 mg/kg i.p.) for 4 weeks and LY294002 (7.5 mg/kg intratumorally) every 3 days for three times. At the end, mice were euthanized and the tumors were collected to determine the protein and gene expression of HIF-1alpha and VEGF and microvessel density by immunohistochemistry for CD31. The immunostaining was quantified by optical densitometry technique, slides were analyzed and photographed in Nikon Eclipse E200 microscope at 40X objective, and proteins were quantified by the ImageJ software analysis. For real-time PCR, the relative expression of the genes of interest was determined by DataAssist v3.0 software, using Ct method. Results: There was a significant decrease of cell viability after treatment with different concentrations of metformin and LY294002 in 24 hours. Both HIF-1alpha and VEGFA protein and gene expression significantly decreased after treatment with metformin and LY294002 or in combination. Furthermore, in animals, there was a decrease in tumor size, protein and gene expression of HIF-1alpha and VEGFA, in addition to a decrease of CD31 expression after all treatments. Conclusion: Our results suggest the potential effectiveness of metformin and LY294002 acting in angiogenesis process of mammary tumors. Financial support: FAPESP. Citation Format: Marina Gobbe Moschetta, Camila Leonel, Larissa Bazela Maschio-Signorini, Thaiz Ferraz Borin, Gabriela Bottaro Gelaleti, Lívia Carvalho Ferreira, Bruna Victorasso Jardim-Perassi, Nathália Martins Sonehara, Debora Aparecida Pires de Campos Zuccari. Meformin and LY294002 treatment regulate angiogenesis process through PI3K-MAPK/AKT/mTOR signaling pathways in mammary tumors [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B25.

Breast cancer shows high intratumoral heterogeneity, which can be visualized as localized sub-populations of tumor cells with different malignancy potential and treatment responsiveness. These regions are like “habitats” that contain different combinations of environmental selection forces, such as hypoxia, acidosis, or nutrient availability. Thus, agents that can perturb these forces may be useful in limiting cancer progression. Studies have shown that melatonin can affect tumor metabolism and we hypothesize that this may be a mechanism by which the disruption of circadian melatonin production by light exposure at night (LAN) promotes breast cancer initiation and progression. The aim of this study was to evaluate the effect of melatonin on intratumoral heterogeneity measured by non-invasive MR imaging in a syngeneic mouse model of breast cancer (4T1). To visualize habitats, clusters were generated from combined MR images generated by different pulse sequences. These were co-registered with histology using tumor specific 3-D printed cradles to classify these tumor habitats. Methods: Mice were exposed to LAN (Control) or LAN plus melatonin. The melatonin metabolite 6-sulfatoxymelatonin (6-SMT) was assessed in urine as a measure of melatonin production. Mice were imaged in a 7T MRI system with: 1) anatomical T2 images, 2) T2*, 3) diffusion-weighted MRI (DWI); and 4) T1 weighted dynamic contrast enhanced (DCE). DCE maps were obtained through semi-quantitative analysis of the pre- and post-contrast agent bolus time-series on a pixel-by-pixel basis. These were combined to the corresponding T2, T2* and Apparent Diffusion Coefficient (ADC) values to generate parameter maps. Parameter maps were used to classify multiple clusters, based on a Gaussian mixture model. Following euthanasia, a 3D printed mold based on the T2 tumor isosurface was created in order to co-register the MRI and histology. Tumors were stained with H&E and for pimonidazole (hypoxia) by immunohistochemistry. Results: Melatonin treatment increased the 6-SMT in urine of mice exposed to LAN and statistically reduced tumor growth by day 15 (p Citation Format: Bruna V. Jardim-Perassi, William Dominguez-Viqueira, Asmaa El-Kenawi, Dominique F. Abrahams, Mikalai Budzevich, Epifanio Ruiz, Suning Huang, Pedro M. Enriquez-Navas, Gary Martinez, Debora Aparecida Pires de Campos Zuccari, Robert J. Gillies. Detection of intratumoral heterogeneity using MR-defined tumor habitats in breast cancer model under melatonin treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 884. doi:10.1158/1538-7445.AM2017-884

The formation of a new blood vessel is stimulated by angiogenic factors. Curcumin, which is the active ingredient of the spice plant Curcuma longa L and is used as food and traditional medicine, has shown anticancer effects against different types of cancers. We evaluated the effects of curcumin on angiogenesis/pro-angiogenic factors in a mouse model of human breast cancer. Cell viability was measured by the MTT assay after curcumin treatment in triple-negative breast cancer cells (MDA-MB-231). For the in vivo study, human breast cancer was induced in athymic mice and treated with 300 mg/kg/day of curcumin administered intraperitoneally. Tumor size was measured weekly, and the animals underwent single photon emission computed tomography (SPECT) scanning with Tc-99m tagged VEGF-c to detect the in vivo expression of VEGFR2/3. In addition, the expression of proangiogenic/ growth factors in the tumor extracts was evaluated by a membrane antibody array. Histological analysis was performed to confirm the effect of curcumin on neovascularization. The MTT assay showed that curcumin significantly reduced the cell viability of MDA-MB-231 cells. In breast cancer xenografts, curcumin treatment led to a decrease in tumor volume and cell proliferation (Ki-67) compared with the vehicle treated group. Tc-99m-HYNIC-VEGF-c-SPECT imaging showed decreased uptake to the tumor, which may indicate a lower expression of VEGFR2/3 in curcumin treated tumors; however, a statistically significant difference was not achieved (p>0.05). Additionally, curcumin treatment showed a significantly low level of expression of pro-angiogenic factors (p

Made available in DSpace on 2014-12-03T13:08:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-01-09Bitstream added on 2014-12-03T13:23:27Z : No. of bitstreams: 1 WOS000329866300068.pdf: 1484144 bytes, checksum: fa772f190a9b2020764b15b21313cc66 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) NIH As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p

Breast cancer is the most common cancer among women and has a high mortality rate. The rapid tumor growth makes difficult the perfusion of O2 mainly in the tumor center, and this hypoxia in tumor microenvironment can exerts selective pressure on the tumor cells, selecting subpopulations with advantage for survival in adverse conditions, characterizing the intratumor heterogeneity. In this context, melatonin, a hormone naturally produced by the pineal gland, has shown antitumor activity, as immunomodulatory, antioxidant, pro-apoptotic, anti-proliferative, antimetastatic, antiangiogenic and with potencial effects in intratumor heterogeneity. The aims of this study was to evaluate the effectiveness of melatonin in a xenograft model of breast cancer, focusing on intratumor heterogeneity verified by PET/CT and by immunohistochemical markers of hypoxia. The tumors were developed by the implantation of 5 million triple-negative breast cancer cells (MDA-MB-231) into the flank of female Balb/c nude mice (n = 14). Treatment with melatonin (n = 7) or vehicle (n = 7) was initiated 10 days after tumor implantation and continued for 14 days. At the end, micro-PET/CT scanning was performed with 18F-FDG, which is an analogue of glucose, and with 18F-FAZA, a specific radiopharmaceutical for hypoxia detection. The radiopharmaceuticals were injected by retro-orbital via and the images were acquired using Albira PET/SPECT/CT Carestream Molecular Imaging. Then, markers of hypoxia were evaluated by immunohistochemistry in mammary tumors. The results showed that tumor growth in animals treated with melatonin was lower than those treated with vehicle (p Citation Format: André L. Mota, Bruna V. Jardim-Perassi, Thaiz F. Borin, Nathália M. Sonehara, Lorena Pozzo, Emerson S. Bernardes, Bruno Cogliati, Debora APC Zuccari. Melatonin action in xenograft model of breast cancer, comparing radiopharmaceuticals in the detection of intratumor heterogeneity by PET/CT confirmed by immunohistochemical markers. [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 2408.

Breast cancer is the most common neoplasia among women and has been frequently associated to high morbidities and mortalities rates. Cancer progression is strongly influenced by conditions of tumor microenvironment. Rapid tumor growth results in decreased of oxygen intake, restricting metabolic demands in specific regions of tumor. This poor perfusion coupled with preferential anaerobic glycolysis leads to acidification of tumor microenvironment. Survival mechanisms of tumor cells under acidic conditions are not well characterized, but has been shown that adverse conditions in tumor microenvironment can select phenotypes that are able to adapted to survival and growth in low pH conditions, hence resulting in tumor progression. Thus, controlling these adverse conditions in tumor microenvironment is a promising tactic in limiting cancer development. Melatonin, a hormone naturally produced and secreted by pineal gland, appears to play multiple roles in cancer and it is proposed as a possible keeper of an extreme environment hindering the survival of these cells. In this study, the benefits of melatonin treatment on cell viability of breast cancer cell lines under acidosis condition were evaluated. Breast cancer cell lines estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) were cultured in Dulbecco's Modified Eagle's medium (DMEM) at 37°C in 5% CO2. Cell viability was measured using an MTT assay. Individual wells of a 96-well plate were seeded with 100 µL of medium containing 0.05 x 106 cells and cells were separated into the following groups: Group 1) Cells cultured in pH 7.4 with DMEM (control); Group 2) Cells cultured in pH 6.7 with DMEM supplemented with 25 mmol/L buffer 2- (N-Morpholino) ethanesulfonic acid (MES) for 24 hours, to mimic an acute acidosis in tumor microenvironment; Group 3) Cells cultured with DMEM supplemented with 25 mmol/L MES and treated with 1 mM of melatonin for 24 hours; Group 4) Cells cultured with DMEM supplemented with 25 mmol/L MES for 12 hours and treated with 1 mM of melatonin for an addition 12 hours. After treatments, MTT solution were added to each well, and the plates were incubated at 37°C for an additional 4 hour. To solubilize the MTT formazan crystals, the cells were incubated with dimethyl sulfoxide (DMSO) for 10 minutes and absorbance was measured at 540 nm. Cell viability (%) was calculated for all groups compared to control samples. The results were compared by ANOVA, followed by the Bonferroni test. The results showed that there was a decrease of both MCF-7 and MDA-MB-231 cells viability under acute acidosis conditions (Group 1 vs Group 2, p < 0.05). MCF-7 cells showed 65.36 ± 1.16 % of cell viability while MDA-MB-231 cells showed 52.98 ± 0.90 of cell viability under acidosis conditions (Group 2). Melatonin treatment for 24 hours was effective in reducing cell viability in acidosis conditions, leading to a decrease in MCF-7 cells (Group 3: 52.41 ± 1.32 %) and MDA-MB-231 cells (Group 3: 47.83 ± 0.67 %) when compared with cells under acidosis without treatment (Group 2 vs Group 3, p < 0.05). Furthermore, in both cell lines there was a decrease in cell viability in Group 4 (MCF-7 = 54.24 ± 1.22 %; MDA-MB-231 = 45.60 ± 0.45 %) compared to cells under acidosis (Group 2 vs Group 4, p < 0.05), and no difference was observed when compared with cells under acidosis and treated with melatonin for 24 hours (Group 3 vs Group 4, p > 0.05). In conclusion, our results show that acute acidosis reduces the viability of breast cancer cells and melatonin treatment is effective, leading to decrease the cell viability under acute acidosis.In addition, studies are being madeto evaluate the effectiveness of melatonin also in chronic acidosis conditions, and to investigate its signaling pathways in the extreme tumor microenvironment, in order to confirm potential effectiveness of melatonin in breast cancer in these conditions. Citation Format: Nathália Martins Sonehara, Bruna Victorasso Jardim-Perassi, André de Lima Mota, Marina Gobbe Moschetta, Debora Aparecida Pires de Campos Zuccari, Rubens de Paula, Jr. Melatonin efficacy in breast cancer microenvironment under acidosis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B38.

Introduction: The PI3K-AMPK / Akt / mTOR signaling pathways are the most important signaling pathways involved in the angiogenesis process, which is regulated by numerous factors, being the most important the hypoxia-inducible factor - 1α (HIF-1α) and vascular endothelial growth factor (VEGF). Metformin, the prescribed drug for patients with metabolic syndrome, has been suggested as a new therapeutic agent with potential anti-neoplastic action capable to inhibit cell growth by AMPK signaling pathway. In the same way, the LY294002, an important inhibitor of PI3K / Akt / mTOR signaling pathway, has anti- angiogenic properties able to decrease the release of growth factors, such as VEGF. In this context, the aim of this study was to evaluate the effectiveness of treatment with metformin and LY294002 in angiogenesis process. Methods: Canine mammary tumor cell line ER-positive (CMT-U229) was cultured in HAM-F12 culture medium, at 37°C in 5% CO2. Cell viability was measured by MTT assay after treatment with varying concentrations of metformin and LY294002. Once stablished the concentration of metformin (1mM) and LY294002 (5µM) treatment, the protein and gene expression of HIF-1α and VEGF were detected by immunocytochemistry and real time PCR, respectively. The immunostaining was quantified by optical densitometry technique, slides were analyzed and photographed in Nikon Eclipse E200 microscope at 40X objective, and proteins were quantified by the ImageJ software analysis. For real time PCR, the relative expression of the genes of interest was determined by DataAssist v3.0 software, using ΔΔCt method. Results: There was a significantly decrease of cell viability after treatment with all concentrations (1mM, 2mM, 3mM and 5mM) of metformin, 5µM and 10µM of LY294002 (p Financial support: FAPESP Citation Format: Marina Gobbe Moschetta, Larissa Bazela Maschio, Bruna Victorasso Jardim-Perassi, Gabriela Bottaro Gelaleti, Camila Leonel, Lívia Carvalho Ferreira, Naiane Nascimento Gonçalves, Debora Aparecida Pires De Campos Zuccari. Simultaneous block of angiogenesis through PI3K-AMPK/AKT/mTOR signaling pathways after treatment with metformin and LY294002 in canine mammary tumor cell line. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A10.

The rapid tumor growth results in hypoxia on tumor microenvironment, leading to a cascade of events that induce angiogenesis and subsequent cancer progression. Thus, the identification of therapeutic agents that can inhibit angiogenesis is essential for the control of tumor progression. Exogenous administration of melatonin, a hormone secreted by the pineal gland, has been shown several oncostatics effects on different types of cancers. The aim of this study was to evaluate the effectiveness of melatonin treatment on angiogenesis in breast cancer, in the in vitro and in vivo studies. In the in vitro study, breast cancer cell lines (MCF-7 and MDA-MB-231) were treated with melatonin under cobalt chloride (CoCl2)-induced hypoxic conditions. Cell viability was measured by MTT assay, the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF-A) was assessed by real-time PCR and immunocytochemistry. Additionally, other proteins involved in angiogenesis were evaluated by the Protein Array. In the in vivo study, the MDA-MB-231 cells were implanted in athymic nude mice, which were treated with melatonin (40 mg/kg) for 21 days. The tumor was measured weekly and evaluation of angiogenesis was performed by single-photon emission computed tomography (SPECT) with Tc-99m-HYNIC-VEGF-c, which is specific for VEGF receptors (VEGFR2/VEGF3). Moreover, VEGFR2, VEGFR3, von Willebrand factor (vWF) and cell proliferation marker (Ki-67) were evaluated in tumor tissue by immunohistochemistry, and other angiogenic proteins by Protein Array. Results from the in vitro study showed that 1 mM of melatonin under hypoxic conditions (200 µM CoCl2) led to decreased cell viability, protein levels of HIF-1α and gene and protein expression of VEGF-A in both cell lines (p < 0.05). Among other proteins evaluated, melatonin treatment under hypoxia resulted in a decrease of VEGF-C, VEGFR2, VEGFR3, matrix metalloproteinase 9 (MMP-9) and angiogenin in MCF-7 cell line (p < 0.05). For MDA-MB-231, a significant reduction was observed for VEGFR2 protein, epidermal growth receptor (EGFR), and angiogenin (p 0.05). The reduction of VEGFR2 in tumors treated with melatonin was confirmed by immunohistochemistry (p Support: FAPESP. Citation Format: Debora C Zuccari, Bruna V Jardim-Perassi, Mateus R Lourenço, Gabriel M Doho, Gabriela B Gelaleti, Lívia C Ferreira, Thaiz F Borin, Marina G Moschetta. Melatonin on angiogenesis in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-04-01.

Background: Angiogenesis plays an important role in the pathogenesis of breast cancer. For growth, tumor requires the formation of new blood vessels, which are stimulated by angiogenic factors to initiate the proliferation of endothelial cells. The major contributors to angiogenesis are the vascular endothelial growth factor (VEGF) and its receptors, which promote cell proliferation, migration, permeability and survival. Controlling tumor-associated angiogenesis and metastasis are promising tactics in limiting cancer progression. Curcumin, a polyphenolic compound derived from the spice plant Curcuma longa, displays multiple actions on solid tumours including anti-angiogenic effects. In this study we evaluated the benefits of curcumin treatment on tumor growth and angiogenesis in breast cancer. Methods: MDA-MB-231 breast cancer cell line was cultured and cell viability was measured by MTT assay after incubation with different concentrations of curcumin (25 uM, 50 uM, 100 uM). We performed an in vivo study implanting cells in athymic nude mice. Mice were treated with 300 mg/kg of curcumin (n = 5) or vehicle (n = 8) daily, starting in the same day as tumor implantation and continued for 21 days. Tumors were measured with a digital caliper on day 7, 14 and 21 and the animals underwent SPECT scanning with Tc-99m tagged VEGF-c to detect in vivo angiogenesis (expression of VEGFR2/3). In addition, the markers of angiogenesis (VEGF-A, VEGF-C, VEGFR2, VEGFR3 and von Willebrand Factor (vWF)) and cell proliferation (Ki-67) were evaluated by immunohistochemistry in mammary tumor tissues. Results: Curcumin treatment in vitro was able to significantly decrease cell viability, after 4 hours of exposure, maintaining its efficacy at higher doses after 24 hours exposure (p0.05). However, decrease of VEGF-A, VEGF-C and VEGFR2 in curcumin treated mice was confirmed by immunohistochemistry (p Citation Format: Lívia Carvalho Ferreira, Ali Syed Arbab, Bruna Victorasso Jardim-Perassi, Thaiz Ferraz Borin, Naiane Nascimento Gonçalves, Ravi Sankara Varma Nadimpalli, Debora Aparecida Pires de Campos Zuccari. Effect of curcumin on the tumor growth and angiogenesis of breast cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A02. doi:10.1158/1538-7445.CHTME14-A02

A selective inhibitor of 20-HETE synthesis, HET0016, has been reported to inhibit angiogenesis. 20-HETE has been known as a second mitogenic messenger of angiogenesis inducing growth factors. HET0016 effects were analyzed on MDA-MB-231 derived breast cancer in mouse and in vitro cell line. MDA-MB-231 tumor cells were implanted in animals’ right flank and randomly assigned to early (1 and 2), starting treatments on day 0, or delayed groups (3 and 4) on day 8 after implantation of tumor. Animals received HET0016 (10 mg/kg) treatment via intraperitoneal injection for 5 days/week for either 3 or 4 weeks. Control group received vehicle treatment. Tumor sizes were measured on days 7, 14, 21, and 28 and the animals were euthanized on day 22 and 29. Proteins were extracted from the whole tumor and from cells treated with 10 µM HET0016 for 4 and 24 hrs. Protein array kits of 20 different cytokines/factors were used. ELISA was performed to observe the HIF-1α and MMP-2 protein expression. Other markers were confirmed by IHC. HET0016 significantly inhibited tumor growth in all treatment groups at all-time points compared to control (p

Background: Breast cancer has a high mortality rate, mainly due to tumor progression and metastatic spread. Once tumor lesion exceeds a few millimeters in diameter, hypoxia triggers a cascade of events to allow neovascularization. The major contributors to angiogenesis are the vascular endothelial growth factor (VEGF) and its receptors, which promote cell proliferation, migration, permeability and survival. Thus, controlling tumor-associated angiogenesis is a promising tactic in limiting cancer development. The melatonin administration, a hormone naturally produced and secreted in the pineal gland, appears to play an important oncostatic role in cancer and have been currently proposed different mechanisms of action. In this study we evaluated the benefits of melatonin treatment on tumor growth and angiogenesis in breast cancer. Methods: MDA-MB-231 breast cancer cell line was cultured and cell viability was measured by MTT assay after incubation with different concentrations of melatonin (5 mM, 10 mM, 20 mM). We performed an in vivo study implanting cells in athymic nude mice. Mice were treated with 1mg of melatonin (n = 5) or vehicle (n = 8) daily, starting in the same day as tumor implantation and continued for 21 days. Tumors were measured with a digital caliper on day 7, 14 and 21 and the animals underwent SPECT scanning with Tc-99m tagged VEGF-c to detect in vivo angiogenesis (expression of VEGFR2/3). In addition, the markers of angiogenesis (VEGFR2, VEGFR3 and von Willebrand Factor (vWF)) and cell proliferation (Ki-67) were evaluated by immunohistochemistry in mammary tumor tissues. Results: Melatonin treatment in vitro was able to significantly decrease cell viability, after 4 hours of exposure, maintaining its efficacy at higher doses after 24 hours exposure (p0.05). However, decrease of VEGFR2 in melatonin treated mice was confirmed by immunohistochemistry (p Conclusion: Taken together, our results showed that melatonin inhibits tumor growth, cell proliferation and blocks tumor angiogenesis in breast cancer, directing the development of a clinical study to determine its therapeutic potential for breast cancer. Citation Format: Bruna Victorasso Jardim-Perassi, Ali S. Arbab, Lívia Carvalho Ferreira, Thaiz Ferraz Borin, Nadimpalli Ravi S. Varma, Adarsh Shankar, Asm Iskander, Meser M. Ali, Debora Aparecida Pires de Campos Zuccari. Effect of melatonin on the tumor growth and angiogenesis of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A009.