This short note reports results from a pilot study to investigate new anticancer agents from deepsea corals in which colonizing sponges were encountered. The pure white stylasterid coral fans of Errina dabneyi (Pourtalès, 1871) are a conspicuous feature on the upper bathyal slopes in Azorean waters and can be found in depths from 215 to more than 500 m (Wisshak et al. 2009; Braga-Henriques et al. 2013). From the 26 species of Errina known worldwide (most from deeper waters) (Cairns 1983), E. dabneyi seems to be endemic to the Azores Archipelago and the adjacent Mid-Atlantic Ridge region (Zibrowius & Cairns 1992; Braga-Henriques et al. 2013).
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Colaço, Ana

Lino, Sílvia P. P.

Santos, Ricardo S.

Xavier, Joana

Repositório da Universidade dos Açores

           Arquipelago  -­‐‑  Life  and  Marine  Sciences        ISSN:  0873-­‐‑4704   SHORT  COMMUNICATION  Sponge  perforating  lace  coral  with  anticancer  activity  SILVIA  LINO,  J.R.  XAVIER,  R.S.  SANTOS  &  A.  COLAÇO Lino, S., J.R. Xavier, R.S. Santos & A. Colaço 2015. Sponge perforating lace   coral with anticancer activity. Arquipelago. Life and Marine Sciences 32. Silvia Lino (e-mail: silvialino@uac.pt), Ricardo S. Santos & Ana Colaço, MARE – Marine and Environmental Sciences Centre, and Department of Oceanography and Fisheries, Uni-versity of the Azores, PT-9901-862 Horta, Portugal; Joana R. Xavier, Centre for Geobiolo-gy and Department of Biology, University of Bergen, Thor Møhlensgate 53A/B, NO-5020, Bergen, Norway. This short note reports results from a pilot study to investigate new anticancer agents from deep-sea corals in which colonizing sponges were en-countered. The pure white stylasterid coral fans of Errina dabneyi (Pourtalès, 1871) are a conspicu-ous feature on the upper bathyal slopes in Azorean waters and can be found in depths from 215 to more than 500 m (Wisshak et al. 2009; Braga-Henriques et al. 2013). From the 26 spe-cies of Errina known worldwide (most from deeper waters) (Cairns 1983), E. dabneyi seems to be endemic to the Azores Archipelago and the adjacent Mid-Atlantic Ridge region (Zibrowius & Cairns 1992; Braga-Henriques et al. 2013).     The anticancer activity of the lipid extract from the lace coral Errina dabneyi (Figure 1A) was being investigated as part of a broader ongoing bio prospecting research on deep-sea inverte-brates from the Azores area. Deep-sea fisheries in the Azores use longline gears at depths where cold-water corals are common (Braga-Henriques et al. 2011; Pham et al. 2014). The specimens studied were collected accidently (bycatch) at Princess Alice Seamount (near the Azorean cen-tral group of islands) at 200 m in April 2012 by a local commercial fishery fleet. Observers onboard froze samples immediately after collection and kept them at -20ºC until being extracted. A sub-sample was taken and preserved for the biological reference collection at University of the Azores (DOP/UAc COLETA), with ID number - DOP 9044. When processing Errina dabneyi, we noted that part of the coral (from the base to the middle of the primary branches) had been bored by what appeared to be a sponge in the interior of the skeleton (Figure 1B). Since it was impossible to fully separate the sponge from the coral, the part of the coral that was colonized was treated as an independent sample (CS; Figure 1c). For identifi-cation purposes, a small fragment of sponge tis-sue was excised, digested in sodium hypochlorite, and washed in a water-ethanol series. The ob-tained spicules were then mounted in Canada balsam, observed and measured under a micro-scope. The sponge was identified as Thoosa ar-mata Topsent, 1888 (Demospongiae, Astrophori-da), a species previously known to occur associ-ated with deep-sea corals in the Azores (Topsent 1904). Thoosa is one of several demosponge gen-era known for their ability to excavate calcareous substrates (see review in Cairns 1983), a genus of demosponge in the family Thoosidae. This genus is known for boring holes in corals (Schoenberg 2008) as can be seen in Figure 1B.  Total lipids were extracted using a modified Bligh and Dyer method as described in Lino et al. (2013). Cyto-toxicity essays were performed on HCT-116 hu-man colon adenocarcinoma (tumorous) cell line. Briefly, cells were inoculated into plates at 2,5×104 cells/ml (150 µl/well), and incubated overnight 37ºC and 5% CO2 before treatment.  Series of dilutions in DMSO of each of the              Lino  et  al.    Fig. 1. a) Errina dabneyi specimen deposited in the reference collection at the University of the Azores DOP/UAç COLETA); b) Errina dabneyi specimen where it is possible to see the sponge inside (pointed by the black arrow); c) Specimen of coral E. dabneyi plus sponge (CS) investigated for their anticancer potential in this study - the white arrows indicate the holes done by the Thoosa sponge in the coral skeleton in order to connect with the exteri-or and which indicates  the presence of the sponge within the coral.  extracts (Errina dabneyi (C) and Errina plus sponge (CS)), were added to the cells followed by further incubation at 37ºC for 72 h. Each extract was essayed in duplicate. Cell viability was de-termined by a colorimetric [3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphen-yl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay. After the exposure time, 20 µL of MTS/ PMS (phena-zine methosulfate - electron coupling agent) solu-tion was added to each well. Plates were incubat-ed for 3 h before absorbance was measured at 490 nm using an EMax® Endpoint ELISA Plate Reader. Cells in DMSO were used as negative control, and cells treated with Etoposide (VP-16) were used as positive control. Relative cellular survival was determined by using the measured optical density (OD) and was calculated as fol-lows: (% Survival for sample) = [(OD of sam-ple)/(Average of negative control)] x100. Con-centration that killed 50% of the cancer cells            Sponge  with  anticancer  activity     (IC50 values) was automatically calculated by SoftMax Pro software. Results for anticancer ac-tivity showed that the extract from Errina dab-neyi did not show any activity but the extract from the coral sample harbouring the sponge was active presenting IC50 values of 25.29 and 32.72 µg/mL for the two essays. To our knowledge this is the first time that anticancer activity is reported in a coral perforating sponge.     Considering the interesting preliminary results on the anticancer activity for the sponge and tak-ing in consideration the high bio eroding ratio observed in Errina dabneyi specimens deposited in the collection at University of the Azores, more consistent studies should be made to address this matter. ACKNOWLEDGMENTS The authors would like to thank the observer onboard Daniel Pereira for bringing the samples in good preservation conditions. We are grateful to Professor William Fenical at Center for Marine Biotechnology and Biomedicine, Scripps Institu-tion of Oceanography, University of California San Diego, who kindly accepted  Silvia Lino as collaborator at his lab to investigate  the natural marine products of our  samples and to Lauren Paul for performing the cytotoxicity essays. We thank Valentina Matos and Andreia Braga-Henriques for assisting with sampling and coral taxonomic identification. Silvia Lino was sup-ported by PhD grant from Fundação para a Ciên-cia e Tecnologia, (FCT - Ministry of Science and Technology of Portugal), with reference SFRH/BD/72154/2010.   REFERENCES Braga-Henriques, A., M. Carreiro-Silva, F. Porteiro, V. Matos, Í. Sampaio, O. Ocaña, & S.P. Ávila 2011. The association between a deep-sea gastropod Pedicularia sicula (Caenogastropoda: Pedicularii-dae) and its coral host Errina dabneyi (Hydrozoa; Stylasteridae) in the Azores. ICES Journal of Marine Sciences 68: 399–407. Braga-Henriques, A., F. M Porteiro, P.A. Ribeiro, V. de Matos, I. Sampaio, O. Ocaña, & R.S. Santos 2013. Diversity, distribution and spatial structure of the cold-water coral fauna of the Azores (NE Atlantic).  Biogeosciences 10: 4009–4036. Cairns, S.D. 1983. A generic revision of the Stylasterina (Coelenterata: Hydrozoa). Part 1. Description of the genera. 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Skeletal reorganisation affects geochemical signals, exemplified in the stylasterid hydrocoral Errina dabneyi (Azores Archipelago). Marine Ecology Progress Series 397: 197-208. Zibrowius, H. & S.D. Cairns 1992. Revision of the northeast Atlantic and Mediterranean Stylasteridae (Cnidaria: Hydrozoa). Mémoires du muséum national d'histoire naturelle A 153:1–136.  Received 12 Dez 2014. Accepted 06 Feb 2015,  Published online 13 February 2015.. 

Sponge perforating lace coral with anticancer activity

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