Screening for Anti-Cancer Compounds in Marine Organisms in Oman

OBJECTIVES
Marine organisms are a rich source of bioactive molecules with potential applications in medicine, biotechnology and industry; however, few bioactive compounds have been isolated from organisms inhabiting the Arabian Gulf and the Gulf of Oman. This study aimed to isolate and screen the anti-cancer activity of compounds and extracts from 40 natural products of marine organisms collected from the Gulf of Oman.


METHODS
This study was carried out between January 2012 and December 2014 at the Sultan Qaboos University, Muscat, Oman. Fungi, bacteria, sponges, algae, soft corals, tunicates, bryozoans, mangrove tree samples and sea cucumbers were collected from seawater at Marina Bandar Al-Rowdha and Bandar Al-Khayran in Oman. Bacteria and fungi were isolated using a marine broth and organisms were extracted with methanol and ethyl acetate. Compounds were identified from spectroscopic data. The anti-cancer activity of the compounds and extracts was tested in a Michigan Cancer Foundation (MCF)-7 cell line breast adenocarcinoma model.


RESULTS
Eight pure compounds and 32 extracts were investigated. Of these, 22.5% showed strong or medium anti-cancer activity, with malformin A, kuanoniamine D, hymenialdisine and gallic acid showing the greatest activity, as well as the soft coral Sarcophyton sp. extract. Treatment of MCF-7 cells at different concentrations of Sarcophyton sp. extracts indicated the induction of concentration-dependent cell death. Ultrastructural analysis highlighted the presence of nuclear fragmentation, membrane protrusion, blebbing and chromatic segregation at the nuclear membrane, which are typical characteristics of cell death by apoptosis induction.


CONCLUSION
Some Omani marine organisms showed high anti-cancer potential. The efficacy, specificity and molecular mechanisms of anti-cancer compounds from Omani marine organisms on various cancer models should be investigated in future in vitro and in vivo studies.

5][6] This is due to the fact that sessile organisms often accumulate toxic and repellent compounds in their body, not only to compensate for their lack of mechanical defences and protective structures, but also to protect themselves from predators, pathogens and the accumulation of unwanted material on their surfaces. 7[10][11] Cancer remains one of the major causes of mortality worldwide; unsurprisingly, many research groups are currently focusing on finding novel anticancer drugs to enhance chemotherapy treatment and increase survival rates. 123][14] Bryostatin-1 is a macrolide compound produced by endosymbiotic bacteria of the bryozoan Bugula neritina which induces apoptosis at nanomolar concentrations in cancer cells and is enhanced by protein kinase C overexpression; it is currently in phase II clinical trials. 13Another product, didemnin B, was isolated from the tunicate Trididemnum solidum and revealed high activity against myelomas and breast, ovarian, cervical and lung cancers; unfortunately, it was excluded from any further consideration as an anti-cancer agent after it was found to be highly toxic. 7,13ata are limited regarding natural products from marine organisms and their application as traditional medications in the Arabian Gulf area, particularly Oman.While the activity of pure anti-cancer com-pounds isolated from marine organisms has been reported previously, it is possible that new anti-cancer compounds can be isolated from Omani organisms.Several novel natural products have been isolated from Spatoglossum variabile and Dictyota dichotoma algae gathered from the coast of the Arabian Sea in Karachi, Pakistan, but their pharmaceutical activity has not yet been investigated. 15,16An antifungal phenolic compound with aromatic unsaturation was produced from the bacterium Pseudomonas aeruginosa CMG1055, also isolated from the Arabian Sea coast of Pakistan. 17Gelliodes spp.and Spheciospongia spp.1 and spp.2 sponges from the Persian Gulf coast in Bushehr, Iran, were identified as the most active against a panel of bacterial pathogens such as Bacillus subtilis, Staphylococcus aureus, P. aeruginosa and Escherichia coli; unfortunately, no bioactive compounds were identified. 18In a previous study in Oman, researchers screened the anti-microbial, antidiatom and antilarval properties of Holothuria atra and H. edulis sea cucumbers collected from the Bandar Al-Khayran region; their findings suggested the presence of cytotoxic compounds. 19To the best of the authors' knowledge, there is no information in the literature about anti-cancer compounds derived from marine organisms in Oman.Therefore, the aim of this study was to screen the anti-cancer activity of natural products and extracts of marine organisms collected from the Gulf of Oman.

Methods
This study was carried out between January 2012 and December 2014 at the Sultan Qaboos University, Muscat, Oman.A total of 40 natural products were obtained from different species of fungi, bacteria, marine sponges, algae, soft corals, tunicates, bryozoa, mangrove tree samples and sea cucumbers collected from seawater at Marina Bandar Al-Rowdha (23 º 34.55' North, 58 º 36.27'East) and Bandar Al-Khayran (23 º 75' North, 58 º 75' East) in Oman. 20cteria and fungi were isolated from the seawater using a marine broth (Oxoid Ltd., Basingstoke, UK) according to previously described methods. 21All macroorganisms were freeze-dried and extracted with methanol and ethyl acetate solvents (Sigma-Aldrich Corp., St. Louis, Missouri, USA).For microbes,

-Ultrastructural analysis and staining of the Michigan Cancer Foundation-7 cell line indicated the role of the apoptotic pathway in
triggering cell death.

Application to Patient Care -The results of this study indicate possibilities for the development of new treatments for breast adenocarcinomas and other cancers.
cultures were centrifuged at 5,000 g and the bacterial cell pellets and fungi mycelium were extracted using the methanol and ethyl acetate solvents.All extracts were filtered using filtration paper (Whatman ® grade 1 filtration paper, Sigma-Aldrich Corp.) and the solvents were removed by evaporation under reduced pressure using a rotary evaporator (Büchi Labortechnik AG, Flawil, Switzerland).The extracts were separated and purified using open-air silica for non-polar extracts or C18 columns for polar extracts.Highperformance liquid chromatography was performed using a Shimadzu system (Shimadzu Corp., Kyoto, Japan) in order to further purify the fractions.Finally, the structure of pure secondary metabolites was elucidated on the basis of spectroscopic data, including infrared and ultraviolet radiation, high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.The novelty of the isolated compounds was assessed using the Royal Society of Chemistry MarinLit ® database of marine natural products. 22he dry extracts and pure compounds were then redissolved either in dimethyl sulfoxyde (DMSO; Sigma-Aldrich Corp.) or methanol.
A breast adenocarcinoma cell line, Michigan Cancer Foundation (MCF)-7 (ATCC ® HTB-22 ™ , American Type Culture Collection, Manassas, Virginia, USA), and a control line of human fibroblasts were cultured in Dulbecco's Modified Eagle medium (DMEM; Gibco ® , Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).This was supplemented with 10% fetal bovine serum (FBS) and a 1% antibiotic antimycotic cocktail (Gibco ® , Thermo Fisher Scientific Inc.) containing 10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin and 25 µg/mL of amphotericin B. The cells were maintained in a humidified incubator at 37 º C with a 5% carbon dioxide atmosphere.Extracts or solutions of pure compounds (1 µL) were applied to each well of a 96-well plate (Nunc ™ MicroWell ™ plate, Thermo Fisher Scientific Inc.).The MCF-7 cells or human fibroblasts were then seeded in the 96-well plate at a density of 1,500 cells per well in 100 μL of DMEM supplemented with 10% FBS before being incubated for 24 hours.At the end of the experiment, the cells were observed with an inverted microscope and the status of the cells was determined.The experiment was repeated three times.
Due to its activity and availability, the crude extract of Sarcophyton sp. was selected for further analysis to determine the mode of its anti-cancer action.The MCF-7 cells were treated for 24 hours with different concentrations (12.5, 25.0, 50.0, 100.0, 150.0, 200.0, 300.0, 350.0 and 400.0 μg/mL) of the crude extract prepared in DMEM, without phenol red solution or FBS.Cells treated with DMSO were included as negative controls for each concentration.Cells were seeded in a six-well plate and treated with the determined inhibitory concentration 50% (IC 50 ) of Sarcophyton sp.extract on attainment of 70-80% confluence.The cells were subsequently stained with Hoechst dye in order to identify the mode of cell death induced by the bioactive extracts of Sarcophyton sp. in the MCF-7 breast cancer model.Treated cells were harvested, suspended in 40 μL of Hoechst-formalin solution (ratio: 1:50) and incubated overnight in the dark at 4 º C. Hoechst-stained cells were then mounted on a glass slide and examined under the microscope using the 4' ,6-diamidino-2-phenylindole filter (excitation: 350 nm; emission: 461 nm).Cells treated with DMSO were included as a negative control.Following the cell viability assay, electronic microscopy analysis of the treated cells was used to elucidate the mechanism by which the extracts destroyed the cells.The MCF-7 cell lines were visualised using a transmission electron microscope (TEM; JEOL, Peabody, Massachusetts, USA). 23Briefly, the cell samples were fixed, dehydrated using an alcohol series and embedded in epoxy resin.Ultra-thin sections were obtained using an ultramicrotome and stained with uranyl acetate and Reynolds' lead citrate.

Results
A total of eight pure compounds and 32 extracts of marine organisms in Oman were investigated, with 22.5% showing strong or medium anti-cancer activity against the MCF-7 cells, including 62.5% of the compounds and 12.5% of the extracts [Table 1].The greatest anti-cancer activity was observed for malformin A, kuanoniamine D, hymenialdisine and gallic acid compounds, as well as the soft coral Sarcophyton sp.extract.Medium activity was observed for the aaptamin compound and the bryozoan Schizoporella unicornis, gorgonian coral Acanthogorgia sp. and sponge Mycale sp.extracts.No quantifiable activity on the control human fibroblast cells was detected.
Treatment of MCF-7 cells with different concentrations of Sarcophyton sp.extract indicated a gradual increase in anti-cancer bioactivity as observed from escalating levels of cell death with increasing treatment concentration.The IC 50 was 97 µg/mL; concentrations of cell death induction were most potent at doubled IC 50 .The presence of fragmented nuclei was observed in treated cells in comparison to the negative control cells [Figure 1].This highlighted the role of the apoptotic pathway in triggering cell death.Furthermore, the TEM ultrastructural analy-   A and C isolated from sponges has been found to inhibit the growth of tumour and non-tumour cell lines, as well as an oestrogen-dependent breast cancer cell line. 28Smith et al. revealed that hymenialdisine has anti-cancer properties against human colorectal carcinomas. 29[32] In the current study, the tested compounds had no measurable effects on human fibroblast cells.This may suggest that these compounds are non-toxic, although alternatively this could indicate that fibroblasts are very resistant compared to the MCF-7 breast cancer cell line.This observation is in agreement with earlier findings regarding the resistance of fibroblasts to several toxic compounds. 32Moreover, MCF-10A cells are not considered a good control model since they have a transformed phenotype and are abnormal epithelial cells. 33New epithelial non-transformed cells are commercially available; however, they are difficult to handle and propagate. 33As such, there is a need for future in vivo studies to be carried out in order to assess the toxicity of marine natural products using immunosuppressed mice.
Analysis of the anti-cancer activity of Sarcophyton sp.extracts in the present study indicated the potency of this extract in inducing cell death in the MCF-7 breast cancer model at the IC 50 of 97 µg/mL.Moreover, TEM microscopic analysis and Hoechst staining indicated the presence of fragmented nuclei within the treated cells.Typical characteristics of cell death induction by apoptosis were noted, such as nuclear fragmentation, membrane blebbing and increased vacuolisation.The anti-cancer activity of soft coral extracts has been previously reported. 34,35For example, diterpenes from the soft coral Xenia elongata were found to induce apoptosis in a genetically engineered mouse cell line which was D3-deficient in the BAK1 and BAX genes. 35he preliminary results of the current study therefore indicate the potential benefit of Sarcophyton sp.extracts in cancer treatment.An in-depth analysis of the molecular effects, specificity and efficacy of the extract for breast, ovarian, colon and prostate cancers is required through further in vitro and in vivo studies; this may potentially lead to the development of new treatments for breast adenocarcinomas and other cancers.

Conclusion
The results of this study highlight the anti-cancer potential of various marine organisms in Oman.The active anti-cancer compound extracted from Sarcophyton sp. is of particular interest and should be isolated in future experiments.The compound should be tested on in vitro models of various cancers to determine the specificity of its anti-cancer activity.Finally, the molecular mechanism and pathways activated in response to treatment with this compound should be investigated.Such an investigation will provide possibilities for the development of new cancer treatments.
Figure 1A & B: Hoechst stains at x40 magnification of Michigan Cancer Foundation-7 breast cancer cells treated with (A) 97 µg/mL of Sarcophyton sp.extract for 24 hours and (B) dimethyl sulfoxide as a negative control.Note the presence of nuclear fragmentation in the cells treated with the Sarcophyton sp.extract (arrows).

Figure
Figure 2A-D: Transmission electron microscopy ultrastructural analysis at x40 magnification of (A-C) Michigan Cancer Foundation (MCF)-7 breast cancer cells treated with 97 µg/mL of Sarcophyton sp.extract for 24 hours and (D) control cells treated with solvents.Intensive blebbing, vacuolisation and chromatic segregation (arrows) due to apoptosis were observed in the MCF-7 cells.N = nucleus.

Table 1 :
Pure compounds* and extracts isolated from Omani marine organisms and their activity against the breast cancer Michigan Cancer Foundation-7 cell line ACA = anti-cancer activity; DMSO = dimethyl sulfoxide; MeOH = methanol.*All compounds were dissolved either in MeOH or DMSO prior to the experiments and MeOH solutions were also evaporated beforehand.Correspondent MeOH and DMSO controls were included.† Anti-cancer activity was classified as either none (no activity), weak (<1,000 µg/mL), medium (100-1,000 µg/mL) or strong (>100 µg/mL).