Amanda Blanque Becceneri

Purification and differential biological effects of ginger-derived substances on normal and tumor cell lines

This study describes an optimization of [6]-, [8]- and [10]-gingerol isolation and purification in semi-preparative HPLC scale and their anti-proliferative activity. The gingerols purification was carried out in HPLC system using a Luna-C₁₈ and the best mobile phase evaluated was MeOH/H₂O (75:25, v/v). This new methodology for the gingerols isolation was very effective, since considerable amounts (in the range of milligrams) with a good purity degree (∼98%) were achieved in 30 min of chromatographic run. [6]-, [8]- and [10]-Gingerol purified by this methodology inhibited the proliferation of MDA-MB-231 tumor cell line with IC₅₀ of 666.2±134.6 μM, 135.6±22.6 μM and 12.1±0.3 μM, respectively. These substances also inhibited human fibroblasts (HF) cell proliferation, however in concentrations starting from 500 μM. In conclusion, our results demonstrate an optimization of gingerols isolation and their specific anti-proliferative activities against tumor cells, suggesting their use as important models for drug design in an attempt to develop new compounds with fewer side effects when compared to conventional chemotherapy.

(6)-gingerol as a Cancer Chemopreventive Agent: A Review of Its Activity on Different Steps of the Metastatic Process

For many years, ginger or ginger root, the rhizome of the plant Zingiber officinale, has been consumed as a delicacy, medicine, or spice. Several studies have been conducted on the medicinal properties of ginger against various disorders, including cancer. Cancer is the second leading cause of death, and chemoprevention is defined as the use of natural or synthetic substances to prevent cancer initiation or progression. Evidence that ginger-derived compounds have inhibitory effects on various cancer cell types is increasingly being reported in the scientific literature. In this review we focused on the cancer chemopreventive effects of [6]-gingerol, the major pungent component of ginger, and its impact on different steps of the metastatic process.

Characterization of metabolic profile of intact non-tumor and tumor breast cells by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy

(1)H high-resolution magic angle spinning nuclear magnetic resonance ((1)H HR-MAS NMR) spectroscopy was used to analyze the metabolic profile of an intact non-tumor breast cell line (MCF-10A) and intact breast tumor cell lines (MCF-7 and MDA-MB-231). In the spectra of MCF-10A cells, six metabolites were assigned, with glucose and ethanol in higher concentrations. Fifteen metabolites were assigned in MCF-7 and MDA-MB-231 (1)H HR-MAS NMR spectra. They did not show glucose and ethanol, and the major component in both tumor cells was phosphocholine (higher in MDA-MB-231 than in MCF-7), which can be considered as a tumor biomarker of breast cancer malignant transformation. These tumor cells also show acetone signal that was higher in MDA-MB-231 cells than in MCF-7 cells. The high acetone level may be an indication of high demand for energy in MDA-MB-231 to maintain cell proliferation. The higher acetone and phosphocholine levels in MDA-MB-231 cells indicate the higher malignance of the cell line. Therefore, HR-MAS is a rapid reproducible method to study the metabolic profile of intact breast cells, with minimal sample preparation and contamination, which are critical in the analyses of slow-growth cells.

Copper (II) and 2,2′-Bipyridine Complexation Improves Chemopreventive Effects of Naringenin against Breast Tumor Cells

Cancer is the second leading cause of death worldwide and there is epidemiological evidence that demonstrates this tendency is emerging. Naringenin (NGEN) is a trihydroxyflavanone that shows various biological effects such as antioxidant, anticancer, anti-inflammatory, and antiviral activities. It belongs to flavanone class, which represents flavonoids with a C6-C3-C6 skeleton. Flavonoids do not exhibit sufficient activity to be used for chemotherapy, however they can be chemically modified by complexation with metals such as copper (Cu) (II) for instance, in order to be applied for adjuvant therapy. This study investigated the effects of Cu(II) and 2,2′-bipyridine complexation with naringenin on MDA-MB-231 cells. We demonstrated that naringenin complexed with Cu(II) and 2,2′-bipyridine (NGENCuB) was more efficient inhibiting colony formation, proliferation and migration of MDA-MB-231 tumor cells, than naringenin (NGEN) itself. Furthermore, we verified that NGENCuB was more effective than NGEN inhibiting pro-MMP9 activity by zymography assays. Finally, through flow cytometry, we showed that NGENCuB is more efficient than NGEN inducing apoptosis in MDA-MB-231 cells. These results were confirmed by gene expression analysis in real time PCR. We observed that NGENCuB upregulated the expression of pro-apoptotic gene caspase-9, but did not change the expression of caspase-8 or anti-apoptotic gene Bcl-2. There are only few works investigating the effects of Cu(II) complexation with naringenin on tumor cells. To the best of our knowledge, this is the first work describing the effects of Cu(II) complexation of a flavonoid on MDA-MB-231 breast tumor cells.

[10]-gingerol induces apoptosis and inhibits metastatic dissemination of triple negative breast cancer in vivo

There is increasing interest in the use of non-toxic natural products for the treatment of various pathologies, including cancer. In particular, biologically active constituents of the ginger oleoresin (Zingiber officinale Roscoe) have been shown to mediate anti-tumour activity and to contribute to the anti-inflammatory, antioxidant, antimicrobial, and antiemetic properties of ginger. Here we report on the inhibitory properties of [10]-gingerol against metastatic triple negative breast cancer (TNBC) in vitro and in vivo. We show that [10]-gingerol concentration-dependently induces apoptotic death in mouse and human TNBC cell lines in vitro. In addition, [10]-gingerol is well tolerated in vivo, induces a marked increase in caspase-3 activation and inhibits orthotopic tumour growth in a syngeneic mouse model of spontaneous breast cancer metastasis. Importantly, using both spontaneous and experimental metastasis assays, we show for the first time that [10]-gingerol significantly inhibits metastasis to multiple organs including lung, bone and brain. Remarkably, inhibition of brain metastasis was observed even when treatment was initiated after surgical removal of the primary tumour. Taken together, these results indicate that [10]-gingerol may be a safe and useful complementary therapy for the treatment of metastatic breast cancer and warrant further investigation of its efficacy, either alone or in combination with standard systemic therapies, in pre-clinical models of metastatic breast cancer and in patients.

Cytotoxicity and anti-tumor effects of new ruthenium complexes on triple negative breast cancer cells

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype. The high rate of metastasis associated to the fact that these cells frequently display multidrug resistance, make the treatment of metastatic disease difficult. Development of antitumor metal-based drugs was started with the discovery of cisplatin, however, the severe side effects represent a limitation for its clinical use. Ruthenium (Ru) complexes with different ligands have been successfully studied as prospective antitumor drugs. In this work, we demonstrated the activity of a series of biphosphine bipyridine Ru complexes (1) [Ru(SO4)(dppb)(bipy)], (2) [Ru(CO3)(dppb)(bipy)], (3) [Ru(C2O4)(dppb)(bipy)] and (4) [Ru(CH3CO2)(dppb)(bipy)]PF6 [where dppb = 1,4-bis(diphenylphosphino)butane and bipy = 2,2’-bipyridine], on proliferation of TNBC (MDA-MB-231), estrogen-dependent breast tumor cells (MCF-7) and a non-tumor breast cell line (MCF-10A). Complex (4) was most effective among the complexes and was selected to be further investigated on effects on tumor cell adhesion, migration, invasion and in apoptosis. Moreover, DNA and HSA binding properties of this complex were also investigated. Results show that complex (4) was more efficient inhibiting proliferation of MDA-MB-231 cells over non-tumor cells. In addition, complex (4) was able to inhibit MDA-MB231 cells adhesion, migration and invasion and to induce apoptosis and inhibit MMP-9 secretion in TNBC cells. Complex (4) should be further investigated in vivo in order to stablish its potential to improve breast cancer treatment.

Effects of Limonoid Cedrelone on MDA-MB-231 Breast Tumor Cells in vitro

Cancer is the second leading cause of death, preceded only by cardiovascular diseases, and there is epidemiological evidence that demonstrate this tendency is emerging worldwide. Brazil has an extensive vegetal biodiversity with more than 55,000 species listed. Such biodiversity collaborates with the finding of compounds which could be the basis for the design of new anti-tumor drugs, with fewer side effects than the conventional chemotherapy used currently. Cedrelone is a limonoid isolated from Trichilia catigua (Meliaceae) which is a native Brazilian plant. This study demonstrates that cedrelone inhibits proliferation, adhesion, migration and invasion of breast tumor cells from the line MDA-MB-231. The effects of cell migration and invasion on MDA-MB-231 cell may be explained, at least in part, by the ability of cedrelone to inhibit MMP activity. We also demonstrate that cedrelone is able to induce apoptosis in MDA-MB-231 cells. There are only a few works investigating the effect of limonoids in cellular processes closely related to tumor progression such as adhesion, migration and invasion. To the best of our knowledge, this is the first work describing the effects of a limonoid on tumor and non-tumor cell adhesion process.

Abstract A16: Evaluation of the genotoxicity of ruthenium complex, trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6, by comet assay in vitro on breast cells and in vivo

Cancer remains a major public health problem, accounting for one in six deaths worldwide. One of the most common types of cancer in women is breast cancer, which is extremely heterogeneous and composed of different subtypes. The majority of drugs currently used in cancer treatment show no selectivity for tumor cells, provoking undesired side effects. Cisplatin, discovered more than 40 years ago, and successive generations of platinum-based antitumor drugs have demonstrated that metal complexes may play an important role in the cancer treatment. Metals have unique characteristics that can be exploited by the chemistry coordination for the design of new drugs with fewer side effects. In the last two decades, ruthenium complexes have been widely studied and gained prominence for cancer treatment due to their unique characteristics, such as high rate of ligand exchange, accessible oxidation states, and the ability to mimic the iron to bind biologic molecules. This study aimed to evaluate the genotoxicity effects of the ruthenium complex trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6 in vitro and in vivo by the comet assay, which can be used to measure DNA damage in individual eukaryotic cells. Briefly, for in vitro assays, MDA-MB-231 malignant breast cells and MCF-10A nonmalignant breast cells were seeded (1x105/1000µL) into sterile 12-well plates. Cells were allowed to grow at 37°C in 5% CO2 for 24h and then treated or not (control) with different concentrations (2, 4 or 8µM) of the ruthenium complex or positive control cisplatin (8µM) for 1h. After this period, cells were harvested, centrifuged and resuspended in 500μL of culture medium. For in vivo assays, the animals, swiss mice, were kept in a climate-controlled environment (22±2°C) with a 12/12h light/dark cycle and food and water ad libitum. The Animal Bioethics Committee (CEUA/UNESP FAMEMA, Marilia, Sao Paulo, Brazil) approved this study (protocol no. 828/2016). Mice were divided into five groups with five animals in each group. The ruthenium complex (12.5, 25 or 50mg/kg), positive control doxorubicin (20mg/kg), or negative control (vehicle - DMSO) were administered intraperitoneally for 3 days at 24h interval. On the third day, peripheral blood from the tail vein was collected after 4h after the administration of the last treatment. Then both, cell suspension or blood, were mixed with 120µL of 0.5% low-melting point agarose and dropped on slides precoated with 1.5% normal melting point agarose and taken to a lysis solution for 1h. After denaturation (20min) and alkaline electrophoresis (25V, 300mA, 20min) the slides were neutralized and fixed. The slide staining was performed with GelRed® (Uniscience) and comet analysis of at least 150 random cells per group was done visually according to comet tail size using a fluorescence microscope (Olympus BX61-TRF5). In vitro results of genotoxic evaluation of trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6 demonstrate that this complex was able to induce significant DNA damage in MDA-MB-231 cells at the highest concentrations (4 and 8μM), as well as the positive control, cisplatin (8μM). However, the complex did not cause DNA damage in MCF-10A cells; only cisplatin induced damage to these cells. The results of genotoxic evaluation of in vivo assays indicated that only the group of animals treated with the highest dose of the complex (50mg/kg) and the animals treated with doxorubicin presented DNA damage. These results demonstrate that this complex causes DNA damages in vitro with more specificity for tumor cells and that only in the highest dose it causes DNA damages in vivo. Therefore, more studies should be performed to evaluate its potential for breast cancer treatment. Citation Format: Amanda Blanque Becceneri, Cecilia Patricia Popolin, Ana Maria Plutin, Edson Luis Maistro, Alzir Azevedo Batista, Marcia Regina Cominetti. Evaluation of the genotoxicity of ruthenium complex, trans-[Ru(ThySMet)(PPh3)2(bipy)]PF6, by comet assay in vitro on breast cells and in vivo [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A16.

ÓLEOS ESSENCIAIS DOS FRUTOS E FOLHAS DE Kielmeyera coriacea: ATIVIDADE ANTITUMORAL E ESTUDO QUÍMICO

Kielmeyera coriacea e uma planta tipica do Cerrado conhecida como pau-santo sendo muito utilizada pela medicina popular no tratamento de diversas doencas. O presente trabalho teve como objetivo caracterizar quimicamente os oleos essenciais dos frutos e das folhas de K. coriacea e avaliar sua atividade antitumoral frente as linhagens de celulas tumorais humanas MDA-MB-231(tumor mama) e DU-145 (tumor de prostata) na busca de novos compostos bioativos de origem natural. Os oleos essenciais foram extraidos por hidrodestilacao em aparato clevenger. O rendimento dos oleos essenciais das folhas e frutos foi de 0,38 % e 0,0041 %, respectivamente. Os compostos majoritarios presentes no oleo essencial das folhas de K. coriacea , foram: 2- Acetato de (2 E )-tridecenol ( 9,06 % ), Acido 1,2-benzenodicarboxilico ( 8,28 % ) e E - nerolidol ( 7,70 % ). E no oleo essencial dos frutos os constituintes majoritarios identificados foram: D-germacreno (18,94 %), neo-intermedeol (12,18 %) e biciclogermacreno (12,00 %). Este e o primeiro relato da composicao quimica do oleo essencial dos frutos de Kielmeyera coriacea . O oleo essencial dos frutos inibiu a ploriferacao celular das linhagens tumorais DU-145 e MDA-MB-231 e das celulas nao tumorais (FH), enquanto que o oleo essencial das folhas nao exibiu atividade frente a nenhuma linhagem tumoral testada. Destaca-se ainda que ambos os oleos essenciais analisados foram citotoxicos frente as celulas normais (FH).

Abstract B40: [10]-gingerol as a pro-apoptotic molecule in triple negative breast cancer

The aim of this work was to study the pro-apoptotic effects of [10]-gingerol and connect them to anti-tumor and anti-metastatic activities in triple negative breast cancer (TNBC) cell lines and in an spontaneous triple negative breast cancer model in vivo. Breast cancer has the highest incidence among women and metastasis is the major cause of death in cancer. TNBC affects 20% of women with breast cancer. This cancer type is very aggressive and has a high propensity to form lung and brain metastases. Nowadays, there is no effective treatment for TNBC and therefore, new therapies are required. Studies to discovery new molecules to be used in more efficient treatments for this disease are essential. Molecules that can interfere with cell proliferation, migration, invasion, apoptosis and cell cycle can be used as a model or tools to develop new anti-metastatic drugs. Epidemiologic studies have shown that Asian populations have lower incidence of breast cancer comparing to western populations. This has been attributed in part to differences in dietary habits. In particular, gingerols, natural compounds extracted from ginger rhizomes, have been shown to present anti-cancer properties against various tumor cell types in vitro and in vivo. MTT assay was performed to verify the inhibitory effects of [10]-gingerol on cell proliferation. To investigate [10]-gingerol mechanisms of action, apoptosis, cell cycle assays and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) were performed. For the in vivo data an spontaneous model of breast cancer metastasis was used. Results : [10]-gingerol was able to inhibit cell proliferation in different cell lines. In the murine cell line (4T1Br4), [10]-gingerol induced apoptosis, cell cycle arrest at sub-G0 phase and also induced DNA damage demonstrated by TUNEL assay, therefore interfering with DNA and provoking its damage. [10]-gingerol inhibited lung, bone and brain metastases in an intra-mammary fat pad spontaneous breast cancer model. Conclusion : [10]-gingerol showed to be a non-toxic compound with anti-proliferative and DNA damaging effects, inducing cell apoptosis in vitro and also inhibiting lung, bone and brain metastases in a spontaneous TNBC model in vivo. Patent deposit: BR 10 2015 024093 7. Approved by ethical committee – E507 and 3224-1. Citation Format: Ana Carolina B. M. Martin, Rebeka Tomasin, Amanda Blanque Becceneri, Angelina Fuzer, Paulo Cezar Vieira, Marcia Regina Cominetti. [10]-gingerol as a pro-apoptotic molecule in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B40.