Abstract B10: Effects of limonoid cedrelone on MDA-MB-231 breast tumor cells in vitro

Abstract

Abstract Introduction: Cancer is the second leading cause of death, preceded only by cardiovascular diseases, and epidemiological evidence demonstrates that this tendency is emerging worldwide. The number of cancer-related deaths is expected to increase 45% between 2007 and 2030. Excluding skin cancer, breast cancer is the most common malignancy among women, accounting for nearly 1 in 3 cancers diagnosed in the United States. Brazilian data confirm that breast cancer is the leading type of cancer in women and that, over the past 30 years, mortality has increased. 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), a native Brazilian plant popularly known as “catuaba” or “catigua”. Material and Methods: Cedrelone was isolated from the fruits of Trichilia catigua collected at Escola Superior de Agricultura Luiz de Queiroz (ESALQ), Piracicaba, Brazil. The aryls were reduced to powder and extracted with dichloromethane and methanol at room temperature. Dichloromethane fraction was chromatographed on a silica gel, similar fractions were grouped and fraction eight was recristalized and produced 323.4 mg of pure cedrelone. MDA-MB-231 human breast tumor and HF cell lines, obtained from ATCC, were maintained at 37°C in 5% CO2 in DMEM medium (Vitrocell) containing FBS 10%, penicillin (100 UI/ml), streptomycin (100 mg/ml) and L-glutamine (2 mM). For proliferation assays, the MTT method was used (Mosmann et al., 1983). Wound-healing migration assay was based on the repopulation of wounded cultures. Adhesion assays were performed in 96 well plates. Different concentrations of cedrelone were incubated with MDA-MB-231 cells before being plated on the wells, which were previously coated with a solution of collagen type I. Unbound cells were washed and adhered cells were stained with crystal violet solution. Absorbance reading was performed at 595nm. The effect of cedrelone on MDA-MB-231 tumor cell invasiveness was determined by the ability to transmigrate through a layer of matrigel in a transwell chamber (BD Biosciences). Zymography experiments were performed with supernatants of the wound healing assay which were collected and tested for MMP activity as previously described (Leber and Negus, 2001). Finally, the apoptotic activity of cedrelone on MDA-MB-231 cells was analyzed by flow cytometry with the FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Results: 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. Furthermore, after nimbolide, this is the second work to demonstrate the effects of a limonoid on cell migration and invasion, in addition to the effect on MMP activity, and the first work performed with MDA-MB-231 breast tumor cell line. Conclusion: Our results show that cedrelone could be the basis for the design of a new anti-tumor drug to be used in chemotherapy. Citation Format: Amanda Blanque Becceneri, Angelina Maria Fuzer, Julio Cesar Conceição Filho, Paulo Cezar Vieira, João Batista Fernandes, Cristiane Cazal, Márcia Regina Cominetti. Effects of limonoid cedrelone on MDA-MB-231 breast tumor cells in vitro. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B10.