Publications by authors named "Debora Aparecida Pires de Campos Zuccari"

31 Publications

Part-time cancers and role of melatonin in determining their metabolic phenotype.

Life Sci 2021 Aug 8;278:119597. Epub 2021 May 8.

Departamento de Biologia Estrutural e Funcional, Instituto de Biociencias, Botucatu, Sao Poalo 18618-689, Brazil.

This brief review describes the association of the endogenous pineal melatonin rhythm with the metabolic flux of solid tumors, particularly breast cancer. It also summarizes new information on the potential mechanisms by which endogenously-produced or exogenously-administered melatonin impacts the metabolic phenotype of cancer cells. The evidence indicates that solid tumors may redirect their metabolic phenotype from the pathological Warburg-type metabolism during the day to the healthier mitochondrial oxidative phosphorylation on a nightly basis. Thus, they function as cancer cells only during the day and as healthier cells at night, that is, they are only part-time cancerous. This switch to oxidative phosphorylation at night causes cancer cells to exhibit a reduced tumor phenotype and less likely to rapidly proliferate or to become invasive or metastatic. Also discussed is the likelihood that some solid tumors are especially aggressive during the day and much less so at night due to the nocturnal rise in melatonin which determines their metabolic state. We further propose that when melatonin is used/tested in clinical trials, a specific treatment paradigm be used that is consistent with the temporal metabolic changes in tumor metabolism. Finally, it seems likely that the concurrent use of melatonin in combination with conventional chemotherapies also would improve cancer treatment outcomes.
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http://dx.doi.org/10.1016/j.lfs.2021.119597DOI Listing
August 2021

Oncolytic effect of Newcastle disease virus is attributed to interferon regulation in canine mammary cancer cell lines.

Vet Comp Oncol 2021 Apr 19. Epub 2021 Apr 19.

Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga, Sao Paulo, Brazil.

Canine mammary carcinoma (CMC) is one of the major health threats in dogs. The oncolytic virotherapy is a promising strategy to treat canine as well as human cancer patients with non-pathogenic replicating viruses. Here, we evaluated the antitumor activity of one lentogenic, non-lytic Newcastle disease virus (NDV) LaSota strain expressing GFP (NDV-GFP) on five different CMCs and one non-tumorigenic cell line, regarding cell viability, cell death, selectivity index, morphology, global and target gene expression analysis. As evidenced by the selectivity index, all CMC cell lines were more susceptible to NDV-GFP in comparison with the non-tumorigenic cells (~3.1× to ~78.7×). In addition, the oncolytic effect of NDV-GFP was more evident in more malignant CMC cells. Also, we observed an inverse association of the IFN pathway expression and the susceptibility to NDV. The downregulated genes in NDV-GFP-sensitive cells were functionally enriched for antiviral mechanisms by interferon and immune system pathways, demonstrating that these mechanisms are the most prominent for oncolysis by NDV. To our knowledge, this is the first description of oncolysis by an NDV strain in canine mammary cancer cells. We also demonstrated specific molecular pathways related to NDV susceptibility in these cancer cells, opening the possibility to use NDV as a therapeutic-targeted option for more malignant CMCs. Therefore, these results urge for more studies using oncolytic NDVs, especially considering genetic editing to improve efficacy in dogs.
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http://dx.doi.org/10.1111/vco.12699DOI Listing
April 2021

Melatonin synthesis in and uptake by mitochondria: implications for diseased cells with dysfunctional mitochondria.

Future Med Chem 2021 Feb 5;13(4):335-339. Epub 2021 Jan 5.

Departamento de Morfología y Biología Celular, Facultad de Medicina, c/JulianClaveria, 6, Oviedo 33006, Spain.

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http://dx.doi.org/10.4155/fmc-2020-0326DOI Listing
February 2021

A meta-analysis of microRNA networks regulated by melatonin in cancer: Portrait of potential candidates for breast cancer treatment.

J Pineal Res 2020 Nov 19;69(4):e12693. Epub 2020 Sep 19.

Department of Cell Systems and Anatomy, UT Health, San Antonio, TX, USA.

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.
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http://dx.doi.org/10.1111/jpi.12693DOI Listing
November 2020

Prognostic phenotypic classification for canine mammary tumors.

Oncol Lett 2019 Dec 5;18(6):6545-6553. Epub 2019 Nov 5.

Laboratory of Molecular Investigation of Cancer, Department of Molecular Biology, Faculty of Medicine of São José do Rio Preto, São José do Rio Preto, São Paulo 15090-000, Brazil.

Mammary neoplasms are a heterogeneous form of disease, and in order to determine its course and biological features with more accuracy, investigations based on tumor phenotypes are required. The aim of the present study was to propose and validate a phenotypic classification for canine mammary tumors and to assess any association between clinicopathological characteristics, survival and prognosis. For the immunohistochemistry analysis, the primary antibodies against estrogen receptor-α, progesterone receptor, human epidermal growth factor receptor 2 (HER-2)/neu and E-cadherin were used. A total of 110 canine mammary tumors were investigated; 42 tumors were classified as luminal A, 41 as luminal B, 17 as triple-negative and 10 as HER-2-positive. The luminal A and B phenotypes were associated with improved prognosis, whereas HER-2positive and triple-negative tumors were more aggressive, and exhibited a significant association with the occurrence of metastasis, a worse Tumor-Node-Metastasis classification and shorter survival time (P<0.05). In addition, there were different levels of E-cadherin expression intensity observed among the four tumor profiles investigated. Luminal A and B phenotypes presented an upregulation of E-cadherin compared with the HER-2 positive and triple-negative phenotypes (P<0.05). From the results of the present study, the proposed immunohistochemical panel and phenotypic classification techniques could be useful diagnostic tools with a good technical applicability in veterinary oncology. The analysis of E-cadherin expression in the panel of tumor markers allowed a more accurate classification for determining the biological features of the mammary tumor.
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http://dx.doi.org/10.3892/ol.2019.11052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876320PMC
December 2019

Antiproliferative activity and p53 upregulation effects of chalcones on human breast cancer cells.

J Enzyme Inhib Med Chem 2019 Dec;34(1):1093-1099

a Department of Chemistry and Environmental Chemistry, Institute of Biosciences, Humanities and Exact Sciences (IBILCE) , São Paulo State University (UNESP) , São Paulo , Brazil.

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 () and 3-pyridyl-4'-aminochalcone (). Their IC 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 and were able to induce apoptosis rather than necrosis in both lines. Antiproliferative target investigations with MCF-7 cells suggested and upregulated p53 protein expression and did not affect Sp1 protein expression. Future studies on chalcones and can define their therapeutic potential.
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http://dx.doi.org/10.1080/14756366.2019.1615485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534249PMC
December 2019

Therapeutic Potential of Melatonin in the Regulation of MiR-148a-3p and Angiogenic Factors in Breast Cancer.

Microrna 2019 ;8(3):237-247

Sao Paulo State University (Unesp), Institute of Biosciences, Humanities and Exact Sciences (Ibilce), Sao Jose do Rio Preto (SP), Brazil.

Background: The high mortality rate of breast cancer is related to the occurrence of metastasis, a process that is promoted by tumor angiogenesis. MicroRNAs are small molecules of noncoding mRNA that play a key role in gene regulation and are directly involved in the progression and angiogenesis of various tumor types, including breast cancer. Several miRNAs have been described as promoters or suppressors angiogenesis and may be associated with tumor growth and metastasis. Melatonin is an oncostatic agent with a capacity of modifying the expression of innumerable genes and miRNAs related to cancer.

Objective: The aim of this study was to evaluate the role of melatonin and the tumor suppressor miR- 148a-3p on angiogenesis of breast cancer.

Method: MDA-MB-231 cells were treated with melatonin and modified with the overexpression of miR-148a-3p. The relative quantification in real-time of miR-148a-3p, IGF-IR and VEGF was performed by real-time PCR. The protein expression of these targets was performed by immunocytochemistry and immunohistochemistry. Survival, migration and invasion rates of tumor cells were evaluated. Finally, the xenograft model of breast cancer was performed to confirm the role of melatonin in the tumor.

Results: The melatonin was able to increase the gene level of miR-148a-3p and decreased the gene and protein expression of IGF-1R and VEGF, both in vitro and in vivo. In addition, it also had an inhibitory effect on the survival, migration and invasion of breast tumor cells.

Conclusion: Our results confirm the role of melatonin in the regulation of miR-148a-3p and decrease of angiogenic factors.
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http://dx.doi.org/10.2174/2211536608666190219095426DOI Listing
July 2020

RNA-Seq transcriptome analysis shows anti-tumor actions of melatonin in a breast cancer xenograft model.

Sci Rep 2019 01 30;9(1):966. Epub 2019 Jan 30.

Faculdade de Medicina de São José do Rio Preto, Av. Brigadeiro Faria Lima, 5419, São José do Rio Preto, SP, 15090-000, Brazil.

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 < 0.01). 57 DE genes were identified in murine cells, which represented the TME, and were mainly involved in immune response. The WGCNA detected co-expressed genes in tumor cells and TME, which were related to the immune system among other biological processes. The upregulation of two genes (Tnfaip8l2 and Il1f6) by melatonin was validated in the TME, these genes play important roles in the immune system. Taken together, the transcriptomic data suggests that melatonin anti-tumor actions occur through modulation of TME in this xenograft tumor model.
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http://dx.doi.org/10.1038/s41598-018-37413-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353949PMC
January 2019

Melatonin regulates tumor aggressiveness under acidosis condition in breast cancer cell lines.

Oncol Lett 2019 Feb 26;17(2):1635-1645. Epub 2018 Nov 26.

Faculty of Medicine of São José do Rio Preto (FAMERP), Department of Molecular Biology, São José do Rio Preto, SP 15090-000, Brazil.

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.
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http://dx.doi.org/10.3892/ol.2018.9758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341749PMC
February 2019

Evaluation of melatonin and AFMK levels in women with breast cancer.

Endocrine 2018 10 24;62(1):242-249. Epub 2018 May 24.

São Paulo State University - UNESP, Cristóvão Colombo, 2265, São José do Rio Preto, 15054-000, São Paulo, Brazil.

Purpose: Changes in the circadian rhythm may contribute to the development of cancer and are correlated with the high risk of breast cancer (BC) in night workers. Melatonin is a hormone synthesized by the pineal gland at night in the absence of light. Levels of melatonin and the metabolite of oxidative metabolism AFMK (acetyl-N-formyl-5-methoxykynurenamine), are suggested as potential biomarkers of BC risk. The aims of this study were to evaluate levels of melatonin and AFMK in women recently diagnosed with BC, women under adjuvant chemotherapy, and night-shift nurses, and compare them with healthy women to evaluate the relation of these compounds with BC risk.

Methods: Blood samples were collected from 47 women with BC, 9 healthy women, 10 healthy night shift nurses, and 6 patients under adjuvant chemotherapy. Compound levels were measured by mass spectrometry.

Results And Conclusions: Our results showed that women with BC had lower levels of melatonin compared to control group women, and even lower in night-shift nurses and in patients under adjuvant chemotherapy. There was no significant difference of AFMK levels between the groups. In addition to this, high levels of melatonin and AFMK were related to patients with metastasis, and high levels of AFMK were related to the presence of lymph node-positive, tumor > 20 mm and patients who sleep with light at night. Our results showed a reduction of melatonin levels in BC patients, suggesting a relation with the disease, and in addition, point to the importance of melatonin supplementation in women that work at night to reduce the BC risk.
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http://dx.doi.org/10.1007/s12020-018-1624-2DOI Listing
October 2018

Efficacy of melatonin, IL-25 and siIL-17B in tumorigenesis-associated properties of breast cancer cell lines.

Life Sci 2017 Aug 15;183:98-109. Epub 2017 Jun 15.

Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP/IBILCE), Programa de Pós-Graduação em Genética, São José do Rio Preto, SP, Brazil; Faculdade de Medicina de São José do Rio Preto (FAMERP). Laboratório de Investigação Molecular do Câncer (LIMC), São José do Rio Preto, SP, Brazil. Electronic address:

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 1mM and 1ng/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<0.05). Semi-quantitative analysis of apoptosis pathway proteins showed an increase of CYTO-C, DR6, IGFBP-3, IGFBP-5, IGFPB-6, IGF-1, IGF-1R, Livin, P21, P53, TNFRII, XIAP and hTRA proteins and reduction of caspase-3 (p<0.05) after melatonin treatment. All treatments reduced VEGF-A protein expression in tumor cells (p<0.05). Our results suggest therapeutic potential, with oncostatic effectiveness, pro-apoptotic and anti-angiogenic properties for melatonin and IL-25-driven signaling in breast cancer cells.
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http://dx.doi.org/10.1016/j.lfs.2017.06.013DOI Listing
August 2017

Inhibition of Epithelial-Mesenchymal Transition and Metastasis by Combined TGFbeta Knockdown and Metformin Treatment in a Canine Mammary Cancer Xenograft Model.

J Mammary Gland Biol Neoplasia 2017 03 11;22(1):27-41. Epub 2017 Jan 11.

Universidade Estadual Paulista "Julio de Mesquita Filho" (UNESP/IBILCE), PostGraduate Program in Genetics, Cristovao Colombo Street, 2265, Jardim Nazareth, Sao Jose do Rio Preto, SP, Brazil.

Epithelial mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties, generating metastases. Transforming growth factor beta (TGF-β) is associated with this malignancy by having the ability to induce EMT. Metformin, has been shown to inhibit EMT in breast cancer cells. Based on this evidence we hypothesize that treatment with metformin and the silencing of TGF-β, inhibits the EMT in cancer cells. Canine metastatic mammary tumor cell line CF41 was stably transduced with a shRNA-lentivirus, reducing expression level of TGF-β1. This was combined with metformin treatment, to look at effects on cell migration and the expression of EMT markers. For in vivo study, unmodified or TGF-β1sh cells were injected in the inguinal region of nude athymic female mice followed by metformin treatment. The mice's lungs were collected and metastatic nodules were subsequently assessed for EMT markers expression. The migration rate was lower in TGF-β1sh cells and when combined with metformin treatment. Metformin treatment reduced N-cadherin and increased E-cadherin expression in both CF41 and TGF-β1sh cells. Was demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-β1sh tumors. This paralleled a decreased N-cadherin and vimentin expression, and increased E-cadherin and claudin-7 expression in lung metastases. This study confirms the benefits of TGF-β1 silencing in addition to metformin as potential therapeutic agents for breast cancer patients, by blocking EMT process. To the best of our knowledge, we are the first to report metformin treatment in cells with TGF-β1 silencing and their effect on EMT.
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http://dx.doi.org/10.1007/s10911-016-9370-7DOI Listing
March 2017

Inhibition of Epithelial-mesenchymal Transition in Response to Treatment with Metformin and Y27632 in Breast Cancer Cell Lines.

Anticancer Agents Med Chem 2017 ;17(8):1113-1125

Laboratory of Molecular Investigation of Cancer (LIMC), FAMERP - Faculdade de Medicina de Sao Jose do Rio Preto, Av. Brigadeiro Faria Lima 5416, 15090.000 Sao Jose do Rio Preto, Sao Paulo, Brazil, Brazil.

Background: ROCK-1 expression is associated with the malignant character of tumors, while inhibiting this molecule results in a significant suppression of tumor metastasis. Likewise, transforming growth factor beta (TGF-β) is associated with this malignancy by having the ability to induce epithelial-mesenchymal transition (EMT). Metformin, a drug used in the treatment of diabetes, has previously been shown to inhibit EMT in breast cancer cells.

Objective: The aim of this study is to evaluate the TGF-β1 action model for induction of EMT and the action of metformin and ROCK-1 inhibitor (Y27632) in EMT process in breast cancer cell lines.

Method: MCF-7 and MDA-MB-231 cell lines were treated with metformin and Y27632, after induction of EMT by TGF-β1, to examine the effects on cell migration as well as the protein expression of the ROCK-1 markers, vimentin, E-cadherin, CD44 and CD24 by immunocitochemistry.

Results: There was a lower protein expression of ROCK-1, vimentin, CD44 and CD24 in both cell lines after treatment with metformin and Y27632. In MDA-MB-231 cells, E-cadherin expression was increased in all treatment groups. Treatment of MDA-MB-231 cell line with metformin and Y27632 significantly reduced the invasion of these cells.

Conclusion: This study confirms the benefits of metformin and Y27632 as potential therapeutic agents in mammary tumors, by blocking EMT process and metastatic potential.
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http://dx.doi.org/10.2174/1871520617666170102153954DOI Listing
September 2017

Evaluation of Melatonin Effect on Human Breast Cancer Stem Cells Using a Threedimensional Growth Method of Mammospheres.

Anticancer Agents Med Chem 2017 ;17(7):961-965

Laboratory of Molecular Research in Cancer (LIMC), Faculty of Medicine of São José do Rio Preto (FAMERP), Brigadeiro Faria Lima, 5416, Vila São Pedro, São José do Rio Preto (SP). Brazil.

Background: The high rates of women&#039;s death from breast cancer occur due to acquired resistance by patients to certain treatments, enabling the recurrence and/or tumor growth, invasion and metastasis. It has been demonstrated that the presence of cancer stem cells in human tumors, as responsible for recurrence and resistance to therapy. Studies have identified OCT4 as responsible for self-renewal and maintenance of pluripotency of stem cells. Thus, it is interesting to study potential drugs that target this specific population in breast cancer. Melatonin, appears to have oncostatic effects on cancer cells, however, little is known about its therapeutic effect on cancer stem cells.

Objective: Evaluate the viability and the expression of OCT4 in breast cancer stem cells, MCF-7 and MDA-MB- 231, after melatonin treatment.

Method: The cells were grown in a 3-dimensional model of mammospheres, representing the breast cancer stem cell population and treated or not with melatonin. The cell viability of mammospheres were evaluated by MTT assay and the OCT4 expression, a cancer stem cells marker, was verified by immunocitochemistry.

Results: Our results demonstrated that the melatonin treatment decreased the cell viability of MCF-7 and MDAMB- 231 mammospheres. Furthermore, it was observed that in both cell lines, the expression of OCT4 was decreased in melatonin-treated cells compared to the control group.

Conclusions: This fact suggests that melatonin is effective against breast cancer stem cells inhibiting the cell viability via OCT 4. Based on that, we believe that melatonin has a high potential to be used as an alternative treatment for breast cancer.
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http://dx.doi.org/10.2174/1871520616666160923093229DOI Listing
August 2017

Melatonin Regulates Angiogenic and Inflammatory Proteins in MDA-MB-231 Cell Line and in Co-culture with Cancer-associated Fibroblasts.

Anticancer Agents Med Chem 2016 ;16(11):1474-1484

Laboratory of Molecular Research in Cancer (LIMC), Faculty of Medicine of São José do Rio Preto (FAMERP), Brigadeiro Faria Lima, 5416, Vila São Pedro, São José do Rio Preto (SP), Brazil.

Background: Cancer-associated fibroblast (CAFs) are the most abundant cells in the tumor microenvironment, able to secrete growth factors and act on tumor progression. Melatonin is associated with several mechanisms of action with oncostatics and oncoprotectors effects, and also participate in the reduction of synthesis of surrounding fibroblasts and endothelial cells in breast cancer.

Objective: The objectives of this study were to determine the effectiveness of melatonin in cell viability and expression of proteins involved in angiogenesis and inflammation in triplenegative mammary tumor cell line (MDA-MB-231) and in co-culture with CAFs.

Method: Cell viability was measured by MTT assay and the protein expression was evaluated by Membrane Antibody Array after melatonin treatment.

Results: Melatonin treatment (1 mM) for 48 hours reduced the cell viability of MDA-MB-231, CAFs and co-culture (p < 0.05). The semi-quantitative protein analysis showed that when monoculture of tumor cells were compared with co-culture of CAFs, there was a regulation of angiogenic and inflammatory proteins (p < 0.05). Melatonin treatment also leads a differential expression of angiogenic and inflammatory proteins in both monoculture and co-culture of tumor cells and CAFs (p < 0.05).

Conclusion: The influence of CAFs under the tumor microenvironment was confirmed, increasing the malignancy of the tumor. In addition, melatonin is effective in both monoculture and co-culture, regulating angiogenic and inflammatory proteins that contribute to tumor progression. This study show an overview of melatonin ability in regulating angiogenic and inflammatory proteins, and opens the way for exploration of each individual protein in further studies.
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http://dx.doi.org/10.2174/1871520616666160422105920DOI Listing
June 2017

Effect of Melatonin in Epithelial Mesenchymal Transition Markers and Invasive Properties of Breast Cancer Stem Cells of Canine and Human Cell Lines.

PLoS One 2016 2;11(3):e0150407. Epub 2016 Mar 2.

Department of Molecular Biology, Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, SP, Brazil.

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44(+)/CD24(low/-) marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44(+)/CD24(low/-) positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150407PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4774906PMC
July 2016

Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression.

J Pineal Res 2016 Jan 20;60(1):3-15. Epub 2015 Oct 20.

Laboratory of Molecular Investigation of Cancer - LIMC, Department of Molecular Biology, Faculdade de Medicina de Sao Jose do Rio Preto - FAMERP, Sao Jose do Rio Preto, SP, Brazil.

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.
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http://dx.doi.org/10.1111/jpi.12270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996347PMC
January 2016

Differential Expression of ADAM23, CDKN2A (P16), MMP14 and VIM Associated with Giant Cell Tumor of Bone.

J Cancer 2015 23;6(7):593-603. Epub 2015 May 23.

1. Laboratory of Genomics Studies, UNESP, São José do Rio Preto, Brazil.

Though benign, giant cell tumor of bone (GCTB) can become aggressive and can exhibit a high mitotic rate, necrosis and rarely vascular invasion and metastasis. GCTB has unique histologic characteristics, a high rate of multinucleated cells, a variable and unpredictable growth potential and uncertain biological behavior. In this study, we sought to identify genes differentially expressed in GCTB, thus building a molecular profile of this tumor. We performed quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry and analyses of methylation to identify genes that are putatively associated with GCTB. The expression of the ADAM23 and CDKN2A genes was decreased in GCTB samples compared to normal bone tissue, measured by qPCR. Additionally, a high hypermethylation frequency of the promoter regions of ADAM23 and CDKN2A in GCTB was observed. The expression of the MAP2K3, MMP14, TIMP2 and VIM genes was significantly higher in GCTB than in normal bone tissue, a fact that was confirmed by qPCR and immunohistochemistry. The set of genes identified here furthers our understanding of the molecular basis of GCTB.
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http://dx.doi.org/10.7150/jca.11238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4466407PMC
June 2015

Effect of Curcumin on Pro-angiogenic Factors in the Xenograft Model of Breast Cancer.

Anticancer Agents Med Chem 2015 ;15(10):1285-96

Laboratorio de Investigacao Molecular no Cancer (LIMC), Faculdade de Medicina de Sao Jose do Rio Preto (FAMERP), Avenida Brigadeiro Faria Lima, 5416, Vila Sao Pedro, CEP 15090-000, Sao Jose do Rio Preto, SP, Brasil.

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<0.05) and a decrease in micro-vessel density (vWF) in animals compared with that of vehicle treated tumors. In conclusion, curcumin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.
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http://dx.doi.org/10.2174/1871520615666150520093644DOI Listing
August 2016

Melatonin Regulates Angiogenic Factors under Hypoxia in Breast Cancer Cell Lines.

Anticancer Agents Med Chem 2016 ;16(3):347-58

Laboratório de Investigação Molecular no Câncer (LIMC), Faculdade de Medicina de São José do Rio Preto/FAMERP. Avenida Brigadeiro Faria Lima, 5416, Vila São Pedro, 15090-000 São José do Rio Preto, SP, Brazil.

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.
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http://dx.doi.org/10.2174/1871520615666150511094201DOI Listing
November 2016

Prognostic value of vascular endothelial growth factor and hypoxia-inducible factor 1α in canine malignant mammary tumors.

Oncol Rep 2015 May 16;33(5):2345-53. Epub 2015 Mar 16.

Laboratory of Cancer Molecular Investigation (LIMC), Faculty of Medicine of Sao Jose do Rio Preto/FAMERP, São José do Rio Preto, SP, Brazil.

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<0.05). The serum percentage of HIF-1α in female dogs with mammary neoplasia was lower than that in the control group and higher in female dogs with tumor metastasis and history of tumor recurrence (p<0.05). Regarding gene expression, there was a gene overexpression of VEGFA in female dogs with poor outcome, in contrast to the gene underexpression of HIF-1A. Taken together, these results suggested that VEGF is important in tumor progression and can be used as a potential prognostic marker in the clinic and may be useful in predicting tumor progression in dogs with mammary neoplasia.
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http://dx.doi.org/10.3892/or.2015.3856DOI Listing
May 2015

Evaluation of melatonin treatment in primary culture of canine mammary tumors.

Oncol Rep 2015 Jan 11;33(1):311-9. Epub 2014 Nov 11.

Laboratory of Molecular Research in Cancer (LIMC), São José do Rio Preto, SP, Brazil.

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<0.05), although treatment was more effective in the ER-positive tumors. Analysis of the relative expression of the MT1 and MT2 genes by quantitative PCR was performed and the data were compared with the expression of ER in 24 canine mammary tumors and the cellular response to melatonin in 10 samples. MT1 was overexpressed in ER-positive tumors (p<0.05), whereas MT2 was not expressed. Furthermore, melatonin treatment in ER-positive tumors showed an efficient oncostatic effect by inhibiting cell viability and proliferation and inducing apoptosis. These results suggest that melatonin decreased neoplastic mammary cell proliferation and viability and induced apoptosis, with greater efficacy in ER-positive tumors that have a high expression of melatonin receptor MT1. This is a strong evidence for the use of melatonin as a therapeutic agent for estrogen-dependent canine mammary tumors.
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http://dx.doi.org/10.3892/or.2014.3596DOI Listing
January 2015

Molecular markers of angiogenesis and metastasis in lines of oral carcinoma after treatment with melatonin.

Anticancer Agents Med Chem 2014 ;14(9):1302-11

Laboratorio de Investigacao Molecular no Cancer (LIMC), Faculdade de Medicina de Sao Jose do Rio Preto/FAMERP. Avenida Brigadeiro Faria Lima, 5416, Vila Sao Pedro, 15090-000 Sao Jose do Rio Preto, SP, Brazil.

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 CoCl2 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.
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http://dx.doi.org/10.2174/1871520614666140812110246DOI Listing
August 2015

Immunoexpression of ROCK-1 and MMP-9 as prognostic markers in breast cancer.

Acta Histochem 2014 Oct 11;116(8):1367-73. Epub 2014 Sep 11.

Faculdade de Medicina de São José do Rio Preto (FAMERP), Laboratório de Investigação Molecular do Câncer (LIMC), Sao Jose do Rio Preto, Sao Paulo 15090-000, Brazil; Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP/IBILCE), Program of Post-Graduate in Genetics, Sao Jose do Rio Preto, Sao Paulo 15054-000, Brazil. Electronic address:

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.
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http://dx.doi.org/10.1016/j.acthis.2014.08.009DOI Listing
October 2014

Comparison of the solution of histidine-tryptophan-alfacetoglutarate with histidine-tryptophan-glutamate as cardioplegic agents in isolated rat hearts: an immunohistochemical study.

Rev Bras Cir Cardiovasc 2014 Jan-Mar;29(1):83-8

Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, SP, Brasil.

Introduction: Cardiac arrest during heart surgery is a common procedure and allows the surgeon to perform surgical procedures in an environment free of blood and movement. Using a model of isolated rat heart, the authors compare a new cardioplegic solution containing histidine-tryptophan-glutamate (group 2) with the histidine-tryptophan-alphacetoglutarate (group 1) routinely used by some cardiac surgeons.

Objective: To assess caspase, IL-8 and KI-67 in isolated rat hearts using immunohistochemistry.

Methods: 20 Wistar male rats were anesthetized and heparinized. The chest was opened, cardioctomy was performed and 40 ml/kg of the appropriate cardioplegic solution was infused. The hearts were kept for 2 hours at 4ºC in the same solution, and thereafter, placed in the Langendorff apparatus for 30 minutes with Ringer-Locke solution. Immunohistochemistry analysis of caspase, IL-8, and KI-67 were performed.

Results: The concentration of caspase was lower in group 2 and Ki-67 was higher in group 2, both P<0.05. There was no statistical difference between the values of IL-8 between the groups.

Conclusion: Histidine-tryptophan-glutamate solution was better than histidine-tryptophan-alphacetoglutarate solution because it reduced caspase (apoptosis), increased KI-67 (cell proliferation), and showed no difference in IL-8 levels compared to group 1. This suggests that the histidine-tryptophan-glutamate solution was more efficient than the histidine-tryptophan-alphacetoglutarate for the preservation of hearts of rat cardiomyocytes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4389473PMC
http://dx.doi.org/10.5935/1678-9741.20140015DOI Listing
December 2014

Effect of melatonin on tumor growth and angiogenesis in xenograft model of breast cancer.

PLoS One 2014 9;9(1):e85311. Epub 2014 Jan 9.

Laboratório de Investigação Molecular no Câncer, Department of Molecular Biology, Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, São Paulo, Brazil ; Department of Molecular Biology, Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, São Paulo, Brazil.

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 (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p<0.05) but the changes were not significant on SPECT (p>0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085311PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887041PMC
September 2014

Pollution-induced metabolic responses in hypoxia-tolerant freshwater turtles.

Ecotoxicol Environ Saf 2013 Nov 29;97:1-9. Epub 2013 Aug 29.

Department of Biology, Centro de Estudo de Quelônios (CEQ) and Laboratório de Hemoglobinas e Genética das Doenças Hematológicas (LHGDH), IBILCE, UNESP - Sao Paulo State University, Sao Jose do Rio Preto, SP 15054-000, Brazil. Electronic address:

The physiological control to support the absence of O2 for long periods of diving, and oxidative damage impact caused by the whole process of hypoxia/reperfusion in freshwater turtles is well known. However, effects of contaminants may act as co-varying stressors and cause biological damage, disrupting the hypoxia/reperfusion oxidative damage control. In order to investigate the action of environmental stressors present in domestic or industrial wastewater effluent, we performed a biochemical analysis of biotransformation enzymes, oxidative stress, as well as neuromuscular, physiological and morphological parameters in Phrynops geoffroanus, an hypoxic-tolerant freshwater turtle endemic of South America, using animals sampled in urban area, contaminated by sewage and industrial effluents and animals sampled in control area. Here we demonstrate the physiological and biochemical impact caused by pollution, and the effect that these changes cause in antioxidant activity. Animals from the urban area exhibited higher EROD (ethoxyresorufin-O-deethylase, CYP1A1), GST (glutathione S-transferase), G6PDH (glucose-6-phosphate deshydrogenase), AChE (acetilcholinesterase) activities and also TEAC (trolox-equivalent antioxidant capacity) and TBARS (thiobarbituric acid reactive substances) values. We examined whether two morphometric indices (K - condition factor and HIS - hepatosomatic index) which help in assessing the general condition and possible liver disease, respectively, were modified. The K of the urban animals was significantly decreased compared to the control animals, but the HIS value was increased in animals from the urban area, supporting the idea of an impact in physiology and life quality in the urban freshwater turtles. We propose that this freshwater turtle specie have the ability to enhance its antioxidants defenses in order to protect from tissue damage caused by hypoxia and reperfusion, but also that caused by environmental contamination and that the oxidative damage control in hypoxic conditions has resulted in an adaptive condition in hypoxic-tolerant freshwater turtle species, in order to better tolerate the release of contaminated effluents resulting from human activity.
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http://dx.doi.org/10.1016/j.ecoenv.2013.06.035DOI Listing
November 2013

Immunohistochemical investigation of the angiogenic proteins VEGF, HIF-1α and CD34 in invasive ductal carcinoma of the breast.

Acta Histochem 2014 Jan 27;116(1):148-57. Epub 2013 Jul 27.

Laboratory of Cancer Molecular Investigation (LIMC), Faculdade de Medicina de São José do Rio Preto/FAMERP, Brigadeiro Faria Lima, 5416, 15090-000 São José do Rio Preto, SP, Brazil; Department of Molecular Biology, FAMERP, Brazil. Electronic address:

The expression of prognostic markers in cancer has become important in diagnostic routine and research. A high mitotic rate compromises the individual cell access to oxygen and nutrients, due to reduced blood supply. Cells undertake adaptive measures such as vascular endothelial growth factor (VEGF), expressed under the control of hypoxia-inducible factor-1α (HIF-1α). CD34 is an endothelial marker which can show the presence and distribution of blood vessels. This study evaluated the presence and relative expression of VEGF, HIF-1α and CD34 using immunohistochemistry of 60 breast tumors and double staining, correlating the findings with clinical and pathological variables. High VEGF expression was correlated with low cell proliferation, lymph node-negative, smaller tumor size and patients not receiving hormone therapy. High HIF-1α expression predominated in younger (<50-year) patients, subjected to neo-adjuvant therapy and in p53-negative tumors. Absence of metastasis, radiotherapy or hormone treatment, and estrogen receptor (ER)-positive tumors showed high CD34 immunoreactivity. We suggest that the angiogenic factors VEGF, HIF-1α and CD34 are important in breast cancer progression and their abundance in breast tumors has prognostic and predictive value.
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http://dx.doi.org/10.1016/j.acthis.2013.06.005DOI Listing
January 2014

An immunohistochemical study of interleukin-8 (IL-8) in breast cancer.

Acta Histochem 2012 Oct 11;114(6):571-6. Epub 2012 Jan 11.

Department of Molecular Biology, Faculdade de Medicina de São José do Rio Preto, FAMERP, SP, Brazil.

The use of prognostic markers for breast cancer is important for routine diagnosis and research. Interleukin-8 is a chemotactic cytokine produced by several cell types in response to inflammation, however, its expression, regulation and function are poorly understood. Recent studies have associated angiogenesis and inflammatory processes with tumor malignancy. The present study investigated the correlation between interleukin-8 expression and breast cancer prognosis. Interleukin-8 expression was assessed in 72 women with mammary neoplasia by immunohistochemistry and the results were statistically correlated with clinical-pathological findings. There was an inverse correlation between interleukin-8 expression and metastasis (p=0.03) and/or local recurrence (p=0.02). In the patient group that received post-surgery chemotherapy and radiotherapy, a lower interleukin-8 expression was found in those women that showed local recurrence (p=0.01). Multivariate logistic regression showed estrogen receptor negativity, progesterone positivity and metastasis with increased risk of death (p<0.05). The data reflect the complexity of the role of interleukin-8 in tumor microenvironment and support its classification as a possible prognostic marker, although more studies are necessary for its inclusion in clinical practice.
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http://dx.doi.org/10.1016/j.acthis.2011.10.007DOI Listing
October 2012

Differentially expressed genes in giant cell tumor of bone.

Virchows Arch 2011 Apr 9;458(4):467-76. Epub 2011 Feb 9.

Laboratory of Genomics Studies, São Paulo State University - UNESP, Cristóvão Colombo, 2265, 15054-000, São José do Rio Preto, SP, Brazil.

Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.
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http://dx.doi.org/10.1007/s00428-011-1047-4DOI Listing
April 2011