Screening of antibacterial and cytotoxic activities of sponges, collected off Togean Islands, Indonesia, using well agar diffusion and MTT methods, followed by determination of the apoptosis mechanism by Annexin V-FTIC assay have found the high potency of Melophlus sarasinorum and Axinella sp to be antibacterial and anticancer drugs.
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.1 The 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. Among marine organisms, marine sponges (porifera: Demospongiae) have been considered as a largest source of unusual metabolites and bioactive compounds that attributes to various biomedical pharmaceutical importance such as antibacterial, anticancer, antifungal, antiprotozoal and antiviral activities.2-5 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.6
Indonesian 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.7
Previously, our study on soft corals of Sarcophyton trocheliophorum have found numerous novel metabolites possessing antibacterial and antitumor activities.8 In 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.
MATERIALS AND METHODS
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 km2 and contains 66 islands of which Batudaka, Talatakoh, Waleakodi, Waleabahi, Una-una and Togean are the largest.9
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.
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 was performed against two types of bacteria: Staphylococcus aureus and Escherichia coli by using agar well diffusion method.10 Briefly, 0.1 mL of suspended bacterium in sterile medium (1.5 x 108 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 480C and then incubated for 24 h at 370C 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 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.11 Doxorubicin 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 104 cells/well in fresh complete growth medium in 96-well microliter plastic plates at 370C for 24 h under 5% CO2 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 370C in 5% CO2 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 IC50 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 105 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.
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 IC50 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 IC50 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.
a= Chloramphenicol was tested at 1 mg/mL.
ND= Not determined, NT= No toxicity.
The aims of this study are to screen for the most potent antibacterial and cytotoxic activity of sponges collected off Togean Islands, identified as Spheciospongia inconstan, Melophlus sarasironum, Oceanapia amboinensis, Biemna sp and Axinella sp. (Figure 3).
Melophlus sarasinorum (family Ancorinidae) has been reported to contain nine triterpene glycosides, namely sarasinosides A1, A2, A3, B1, B2, B3, C1, C2, and C3.12,13 Interestingly, these sarasinosides compounds exhibited cytotoxic against several cell lines. Schmitz et al.(1988) reported the cytotoxicity of sarasinoside A1 against human lymphocytic leukemia cell line with the ED50 of 2.8 μg/mL.14,15 Lee et al. (2000) proved the cytotoxic activities of sarasinosides A2 and A3 against human leukemia cell line K562 with ED50 of 6.5 and 17.1 μg/mL, respectively.16 Moreover, four new tetramic acid derivates, namely Melophlins P, Q, R and S, have potent cytotoxicity against murine leukemic cell lines with the IC50 of 20.0, 10.5, 0.85 and 5.13 uM, respectively.17 Meanwhile, Axinella sp has been reported to contain polyalkilated cyclopentindoles, herbindoles A, B and C, which are cytotoxic to KB cells.18 These 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.19 The 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%, respectively. Meanwhile, on MCF-7, methanolic extract of Melophlus sarasinorum showed significant percentage of early and late apoptosis of 66.80% (Figure 4). Although some studies revealed the potency of bioactive secondary metabolites from these two sponges, the mechanism of anticancer via apoptosis induction obtained in this study suggested for further isolation and purification for their metabolites.
Melophlus sarasinorum and Axinella sp are the most potential extracts from our biological activity screening that have broad spectrum of antibacterial activity. Moreover, it also found to have potent cytotoxicity and apoptosis induction on HCT-116. This study suggested for further isolation and identification of the bioactive compound from these two sponges.