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

Abstract

Abstract 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, Marília, São 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, Cecília Patrícia Popolin, Ana Maria Plutin, Edson Luis Maistro, Alzir Azevedo Batista, Márcia 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.