Anticancer Potential of Marine Microorganisms Recovered from Sediments Collected Along the Coast of São Paulo

Dhiego B. Rigato1, Bruna G. Chain1, Bruna V. Souza1, Gabriela C. Falcão1, Helori V. Domingos2, Paola C. Branco2, Letícia V. Costa-Lotufo2, Paula C. Jimenez1*

1Laboratório de Bioprospecção de Organismos Marinhos, Departamento de Ciências do Mar, Universidade Federal de São Paulo, Santos, SP – Brazil; 2Laboratório de Farmacologia Marinha, Instituto de Ciências Biomédicas I, Departamento de Farmacologia, Universidade de São Paulo, São Paulo, SP – Brazil; *[email protected]

Introduction: According to data from the WHO, by the year 2030, over 13 millions deaths should occur as a consequence of cancer. Despite the current therapeutic arsenal, mortality due to this disease remains unacceptably high, so demands for new remedies are ever increasing. Therefore, it is important to search for new molecules that provide more effective and selective anticancer treatments. The oceans are proving to be an immeasurable source of new molecules with pharmacological applications, especially among microorganisms from the diverse Actinobacteria class. Due to their distinct secondary metabolism, they are a traditional terrestrial source of bioactive molecules, many of which have been developed into drugs. Herein, the goal of this study was to bioprospect the anticancer potential of extracts produced by marine actinobacteria recovered from sediments collected on the coast of São Paulo.

Methods: Sediment was collected across 10 points off the coast of São Paulo, including Bertioga, São Sebastião, Ilha Bela, Santos and São Vicente, stored in sterile plastic bags and frozen until processing. Sediment samples were processed by two methods prior to inoculation on agar dishes: M1- desiccation and stamping on agar; or M2- heating and striking on agar. Moreover, three culture media with different nutritional levels (SWA, TMA and A1) were used. Selected individualized colonies were transferred to fresh agar dishes and purified strains were grown in liquid media, under agitation, during 5 to 10 days. An aliquot of this broth was homogenized in glycerol for cryopreservation while the remainder was extracted using ethyl acetate. Extracts were then analyzed for cytotoxicity against HCT-116 cells (human colorectal carcinoma) by the MTT assay at two concentrations (5 g/mL e 50 g/mL). Extracts that inhibited greater than 70% of cell growth in the highest concentration were considered active. Selected active extracts were evaluated in a quantitative approach, by exposing cells to various concentrations (from 0.0032 to 50 g/mL) in order to determine their respective IC50.

Results: A total of 241 strains were isolated from the 10 sediment samples collected. For all locations, SWA was the most competent media, recovering 51% of strains. Of the 225 extracts produced and tested, 83 (37%) were considered positive for cytotoxicity in the qualitative approach. IC50 calculated for selected extracts varied over 1000-fold, from 0.013 to 20.13 g/mL, for BRB-058 and BRB-178, respectively. Some extracts, displayed a steep curve with a high slope (2.63), indicating a classic cytotoxic effect. In contrast, other extracts, showed a mild curve, with a lower slope (0.13) suggesting the presence of cytostatic compounds. For the highly cytotoxic extracts derived from strains BRB-191 (Itaguaré Beach) and BRB-248 (São Sebastião’s Cove), IC50 was not obtained, as it may be below the lowest concentration tested.

Conclusion: These results have shown that the coast of São Paulo has a rich biomedical potential stored in marine microorganisms. Additional biological and chemical studies are in progress, aiming at identifying and isolating the molecules responsible for the bioactivities and elucidating their mechanisms of action.

Financial Support: INCT BioNat (CNPq/INCT Process #465637/2014-0), FAPESP (Process #2015/17177-6), CAPES, ICB – USP and PPG Bioprodutos e Bioprocessos – UNIFESP.