Anticancer Potential in Actionobacteria Recovered from Sediments Collected at Itaguaré Beach, São Paulo, Brazil

Introduction: Marine microbes from the Actinobacteria Phylum are an important source of bioactive molecules of pharmacological relevance. Due to a diversified secondary metabolism, these bacteria have shown to be especially promising when considering anticancer compounds. In this context, the aim of the present study was to search, in sediment samples from Itaguaré Beach (SP, Brazil) for actinobacteria producers of molecules with antiproliferative activity against cancer cell lines. Methods: Approximately 20 g of a sediment sample was collected at 23°46'49.51"S, 45°58'7.78"O using a van-veen catcher dropped to 0.5 m, stored in a sterile plastic bag and frozen at -20 oC and until processing. Sediment was subjected to two stressors prior to inoculation onto agar containing dishes: 1) M1, which included desiccation and stamping on agar; or 2) M2, accounted by heating at 55 oC and striking on agar). Moreover, three culture media with increasing nutritional levels (SWA, TMA and A1) were used for bacterial isolation. Selected individualized colonies were transferred to fresh agar dishes, purified by sequential streaking onto fresh agar then cultured in liquid A1 media (5 to 10 days; room temperature; agitation of 150 rpm). An aliquot of this broth was homogenized in glycerol for cryopreservation while the remainder was extracted with ethyl acetate. After solvent removal, the obtained crude extracts were initially analyzed for cytotoxicity against one cell line, HCT-116 (human colorectal carcinoma), at two concentrations (5 and 50 µg/mL) by the MTT assay. Extracts that inhibited over 75% cell growth at the highest concentration were considered active. In addition, those that were active at both concentrations were selected for further evaluation of cytotoxicity against an expanded human cell line panel, added by a breast carcinoma (MCF-7), a melanoma (MM200) a non-tumor model (RPE), through the MTT assay in a qualitative manner (concentrations ranged over 0.0032 to 50 µg/mL), aiming at determining their respective IC50 for each cell. An aliquot (2 mg) of each crude extract was dissolved in methanol (2 mL), submitted to cleanup on C-18 SPE cartridges and analyzed on Agilent HPLC-DAD Infinity 1260 with a C-18 ACE-5 (250 x 4.6 mm, 5 µm) column (0.8 mL/min flow rate, 20 µL injection volume, 30°C temperature). The solvents used were water (A) and acetonitrile (B) in a gradient: 0-5 min (15 % B), 5-40 min (15-80% B) 40-41 min (80-100%B), 41-45 min (100%B) and chromatograms were recorded at 254 and 360 nm. Selected strains were further subjected to taxonomical identification through a molecular approach. Results: 22 strains were isolated from the sediment sample. The minimum media (SWA) was responsible for the largest number of recovered strains (~59%), while M1 was the most effective stressor, representing about 68% of the strains. Nine extracts were considered active against HCT-116 cells at the highest concentration. Among those, 6 extracts, most of which yielded by members of the Streptomyces genus, showed to be active in both concentrations, and were further studied for their bioactivity. BRB-094 and BRB-191 produced the generally more cytotoxic extracts, with IC50 ranging from 0.16 to 0.91 µg/mL (BRB-094), and 0.01 to 1.04 µg/mL (BRB-191). Still, the extract from BRB-191 showed promising selectivity for tumor cell lines, with selectivity index (SI) above 100 towards the breast cancer and the melanoma cell lines. Moreover, BRB-090 and BRB-201 showed interesting SI towards the colorectal carcinoma cells and, both above 10 times. Chromatographic profile analysis showed distinct profiles among the 22 samples. Five samples presented both an interesting chemical profile and compelling biological activity, and are promising candidates for isolation of bioactive substances: BRB-090, BRB-094, BRB-104, BRB-191 and BRB-201. Conclusion: Additional chemical and biological studies are crucial to identify the molecules of interest and to elucidate their mechanisms of action. However, the results disclosed herein show that the marine actinobacteria present in sediments of Itaguaré Beach have a relevant biomedical potential. Financial Support: INCT BioNat (CNPq #465637/2014-0), FAPESP (#2015/17177-6), CAPES.