Marine and Soil Fungi Extracts with Antiproliferative Activity Induce Morphological Alterations in Breast Cancer Cells Alice Abreu Ramos 1 , Fernanda Malhão 1,2 , Ana Ferreira 2 , Ângela Alves 1,2 , Bruno Castro-Carvalho 1,2 , Maria Prata- Sena 1,2 , Daniela Gargiulo 1,3 , Tida Dethoup 4 , Sudaret Buttachon 1,2 , Alexandre Lobo-da-Cunha 1,2 , Anake Kijjoa 1,2 , Eduardo Rocha 1,2 1. Interdisciplinary Center for Marine and Environmental Research (CIIMAR), CIMAR Associated Laboratory (CIMAR LA), University of Porto (U.Porto), Porto, Portugal. 2. Microscopy Department, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto (U.Porto), Porto, Portugal. 3. UNIBH - University of Minas Gerais, Brazil. 4. Faculty of Agriculture, Kasetsart University, Bangkok, Thailand. Cancer is one leading cause of death in world today, with high human and social costs [1]. Research of new anticancer drugs with a higher efficiency and specificity for cancer cells has been a hard work. In the last decades, bioprospection of marine environments has brought to light some new compounds with a promising anticancer activity [2]. Some of them are acting by inhibition of cell proliferation and/or induction of cell death mechanisms such as apoptosis [3]. In this vein, the aim of this study was to assess morphological alterations induced by marine-derived fungi extracts, that previously showed antiproliferative activity on a breast cancer cell line (MCF7) evaluated by both light and electron microscopy. The crude ethyl acetate extracts tested were obtained from a marine fungus Neosartorya laciniosa KUFC 7896 (E7) and a soil fungus Neosartorya fischeri KUFC 6344 (E2). MCF7 cells were incubated with the IC50 (determined by MTT assay) of E2 and E7 for 48h. Doxorubicin (an antitumor drug) was used as a positive control. Effects on cell morphology were first observed on phase contrast microscopy. Then cells were collected, the pellet fixed and semithin sections (1 μm thick) were performed and stained with methylene blue/azure II and Sudan black. For ultrastructural morphology analysis, ultrathin sections ( 90 nm thick) were observed and photographed in a JEOL 100CXII electron microscope. MCF7 cell cultures treated with E2 and E7 extracts and doxorubicin showed a decrease of cell density (antiproliferative effect), and morphological alterations, such as rounded and detached cells (indicative of cell death), were observed with a phase contrast microscope (Fig. 1). Another observation was the presence of dot-like cytoplasmic inclusions, mainly when cells were treated with E2 and E7 extracts. These structures were not observed in control cells. The analysis of semithin sections confirmed the appearance of (light and dark) inclusions in cells treated with extracts. Most of these inclusions are lipid droplets, as confirmed by the positive staining with black Sudan. Ultrastructurally, cells treated with E2 and E7 showed a moderate and abundant lipid accumulation, respectively (Fig. 2). With both extracts, degenerated mitochondria were observed. Cells treated with doxorubicin also showed disrupted mitochondria, besides chromatin condensation and vacuoles with debris, presumably of autophagic nature. By the nuclear condensation assay we observe that E2, E7 and doxorubicin induce cell death in MCF7 cells. Our results, for which electron microscopy was found quite useful, suggest that in line with doxorubicin, despite in lower degree, both extracts show an apoptotic activity. However, the exact pathways by which cell death are induced seem to be different and further studies should be done to clarify them [4]. Microsc. Microanal. 21 (Suppl 5), 2015 © Microscopy Society of America 2015 83 doi:10.1017/S1431927615014221