Antibacterial and Cytotoxic Activities of Sponges Collected off the Coast of Togean Islands , Indonesia

Context: Marine sponges (Porifera: Demospongia) have astonishing structural diversity and broad biological activities. Aims: To evaluate the antibacterial and cytotoxic activities of five sponges collected off the coast of Togean Islands, Indonesia, identified as Spheciospongia inconstan, Melophlus sarasironum, Oceanapia amboinensis, Biemna sp and Axinella sp. Methods and Material: All dried sponges materials were extracted by maceration method using methanol and then evaporated by the rotary evaporator to obtain viscous extracts. The determination of antibacterial activity was performed by well agar diffusion method against Staphylococcus aureus and Escherichia coli while the cytotoxic activity was determined by MTT methods on human breast adenocarcinoma (MCF-7) and human colon colorectal carcinoma (HCT-116), followed by determination of the apoptosis mechanism by Annexin V-FTIC assay. Results: M. sarasinorum and Axinella sp showed strong inhibition against S.aureus and E.coli with the diameter of inhibition of 14.21 ± 0.92 mm and 14.36 ± 0.92 mm, and 10.01 ± 2.65 mm and 12.07 ± 1.54 mm, respectively. Moreover, they also exhibited potent cytotoxicity on HCT-116 with IC50 values of 0.002 and 8.518 μg/mL, respectively. Meanwhile, on MCF-7, only M. sarasinorum showed moderate inhibition with an IC50 value of 87.35 μg/mL. Annexin V-FTIC assay clearly showed that the cytotoxic mechanism of M. sarasinorum and Axinella sp on HCT-116 and MCF-7 was via apoptosis induction. Conclusion: The sponges of M. Sarasinorum and Axinella sp are undergoing further analysis to identify the active constituents which could be developed as potential antibacterial and anticancer agents.


INTRODUCTION
The increasing case of infection diseases caused by bacteria, as well as the growth of antibiotics and anticancer drugs resistance in the worldwide have encourage scientist to search the new source of antibiotic and anticancer drugs, particularly from marine bioactive compounds. 1The exploration of marine environment have progressively improved in the past 50 year that possessing around thousands of unique chemical structure of new bioactive compounds from marine.][4][5] Some of them are halichondrin B from Halichondria okadai which is under preclinical anticancer agent, aurantosides from Siliquariaspongia japonica and Homophynia conferta and spongistatin 1 from Hyrtio serecta which are commercially available. 6ndonesian coast is one of the richest biodiversity for marine organisms in the world.Literature studies and informal database record-based review confirmed the richness of sponges along the Indonesian's shoreline.However, the published knowledge based of Indonesian sponge's organisms is sorely incomplete.Much of the sponge's materials are still needing to be described for species identification and many sponge's locations still need to be explored. 7reviously, our study on soft corals of Sarcophyton trocheliophorum have found numerous novel metabolites possessing antibacterial and antitumor activities. 8n continuing our focus research on marine organism to identify compounds with medicinal prospect, particularly from Indonesian sea, our study now starting on some sponges collected off Togean Islands, Indonesia.Here, we report the screening assay for antibacterial and cytotoxic activities of sponges methanolic extracts on two pathogenic bacteria Staphylococcus aureus and Escherichia coli, two human carcinoma cells MCF-7 and HCT-116 and two human normal cells CCD and NHDF followed by apoptosis assay for the most cytotoxic extracts.

Study area
Togean islands is in the north, eastward-projecting peninsula of central Sulawesi (Figure 1 and 2).It occupies the central portion of Tomini Bay, stretching over about 90 km.The land area of the Togean Group covers about 755 km 2 and contains 66 islands of which Batudaka, Talatakoh, Waleakodi, Waleabahi, Una-una and Togean are the largest. 9

Materials
All samples of sponges were collected in January 2017 on Togean Islands, Tojo Una-una, Indonesia at a depth of 5-10 m and identified by Tri Aryono Hadi (co-author) at Research Center for Oceanography-Indonesian Institute of Science, Jakarta.A voucher sample was deposited at Laboratory of Phytochemistry, Department of Pharmacy, Tadulako University.

Extraction
Material of sponges were minced and repeatedly extracted for 3-5 x 24 h by maceration method with Me-OH as a solvent at room temperature.Then each obtained extract was evaporated by using rotary evaporator to reach a viscous extract.Each of extracts was subjected to antibacterial and cytotoxic activity tests.

Antibacterial Screening
Antibacterial screening was performed against two types of bacteria: Staphylococcus aureus and Escherichia coli by using agar well diffusion method. 10Briefly, 0.1 mL of suspended bacterium in sterile medium (1.5 x 10 8 CFU/mL) was spread on nutrien agar media.Then 50 μL of each sample (1000, 500 and 250 mg/mL) was poured into the wells (6-mm diameter).All plates were left for 1 h at 48 0 C and then incubated for 24 h at 37 0 C for bacteria.Inhibition zone diameters formed around the well were measured and the mean diameter of three replicates was calculated.DMSO was used as a negative control and chloramphenicol as a positive control.

Cytotoxic activity
Cytotoxic activity was applied on human breast adenocarcinoma (MCF-7), human colon colorectal carcinoma (HCT-116) and two normal cell lines NHDF and CCD-118 by MTT method as described in our previous study. 11Doxorubicin and fluorouracil were used as a positive standard anticancer drug.The stock samples were diluted with RPMI-1640 medium to desired concentrations of 62.5, 125, 250, 500 and 1000 μg/mL.The final concentration of dimethyl sulfoxide (DMSO) in each sample was 1 % v/v.The cancer cells were batch cultured for 10 d, then seeded in 96 well plates of 1 x 10 4 cells/well in fresh complete growth medium in 96-well microliter plastic plates at 37 0 C for 24 h under 5% CO 2 using a water jacketed carbon dioxide incubator (CelCulture, Esco Medical ApS, Denmark).The medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of sample.After 48 h of incubation, cells were added with 10 μl/well of MTT (5 mg/mL) and incubated for 4 h in incubator at 37 0 C in 5% CO 2 humidified atmosphere.The reaction was stopped by 100 μl dimethylsulfoxide (DMSO).The plate was then incubated for 15 min.The absorbance of each well was read at 550 nm wavelength in Elisa Reader (Infinite M200 pro Nano Quant, Tecan, Switzerland), using wells without cells as blanks.All experiments were performed in triplicate.The effect of compounds on proliferation of cancer cells was expressed as the % cytoviability, using the following formula: The IC 50 calculation was done statistically by probit analysis using SPSS 17.0 (SPSS.Inc, Chicago IL, USA), in which the series of dose-response data and the percentage of cytoviability were plotted together.

Annexin V-FITC Apoptosis Assay
Annexin V-FITC Apoptosis Assay on cancer cells (MCF-7 and HCT-116) were seeded as described above and then incubated with different treatments for 24 h.Cells were harvested, washed twice with PBS and centrifuged.In brief, 1 x 10 5 of cells were treated with annexin V-FITC and propidium iodide (PI) using the apoptosis detection kit (BD Biosciences, San Jose, CA) according to the manufacturer's protocol.Annexin V-FITC and PI binding were analysed by flow cytometry on FACScanto II (BD Biosciences, San Jose, CA) without gating restrictions using 10,000 cells.Data were collected using logarithmic amplification of both the FL1 (FITC-A) and the FL2 (PI-A) channels.Quadrant analysis of coordinate dot plots was performed with Cell Quest software.Unstained cells were used to adjust the photomultiplier voltage and for compensation setting adjustment to eliminate spectral overlap between the FL1 and the FL2 signals.

RESULTS
Antibacterial test was performed by well agar diffusion method.Staphylococcus aureus and Escherichia coli were chosen as tested bacteria as the representative of gram positive and gram-negative bacteria.The inhibition of both bacteria used to be said that the extract has broad spectrum type of inhibition.As it can be seen in Table 1, Only Melophlus sarasinorum and Axinella sp have inhibition on Staphylococcus aureus and Escherichia coli, with diameter of inhibition zone of 14.21 ± 0.92 mm and 14.36 ± 0.92 mm, and 10.01 ± 2.65 mm and 12.07 ± 1.54 mm, respectively.Therefore, it can be suggested that the extract of Melophlus sarasinorum and Axinella sp have a broad spectrum of antibacterial activities.Further cytotoxic activity against human colon carcinoma (HCT-116) of the all extracts of sponges, found that the cytotoxic effects were dose and time dependent.It showed that Melophlus sarasinorum and Axinella sp methanolic extract have time dependent potent cytotoxicity with the IC 50 of 0.002 and 8.518 μg/mL, respectively after 48 h incubation (Table 2).Meanwhile, only Melophlus sarasinorum showed cytotoxicity on MCF-7 with the IC 50 of 87.35 μg/mL after 48 h incubation.Axinella sp showed high selectivity on cell growth inhibition where it is found to be not toxic on both CCD and NHDF normal cells.20.0, 10.5, 0.85 and 5.13 uM, respectively. 17Meanwhile, Axinella sp has been reported to contain polyalkilated cyclopentindoles, herbindoles A, B and C, which are cytotoxic to KB cells. 18These reported data supported our result.However, there is still a lack in the mechanism of cytotoxicity reported.Therefore, we further identify the possible apoptosis mechanism of the potential extracts (Melophlus sarasinorum and Axinella sp) by Annexin V-FITC assay.Apoptosis is the programmed cell death process.
It plays an important role in the regulation of tissue development and homeostatis.Therefore, the induction of apoptosis will suppress the tumor progression. 19The result showed that methanolic extract of Melophlus sarasinorum and Axinella sp have significant percentage of early and late apoptosis on HCT-116 cell lines with the value of 89.70% and 34.00%,

Table 1 : Antibacterial activity of Togean island sponges.
a= Chloramphenicol was tested at 1 mg/mL.