1Department of Marine Science & Fisheries, College of Agricultural & Marine Sciences, Sultan Qaboos University; 2Centre of Excellence in Marine Biotechnology, Sultan Qaboos University; Departments of 3Biochemistry and 4Pathology, College of Medicine & Health Sciences, Sultan Qaboos University; 5Department of Medicine, Sultan Qaboos University Hospital, Muscat, Oman *Corresponding Author e-mail: sergey@squ.edu.om الكشف عن املكونات البيولوجية اليت حتتوي عليها الكائنات البحرية واليت تتسم بأنشطة مضادة للسرطان يف عمان �رسجي دوبريت�سوف، يحيي التاميمي، حممد الكندي، اأكرام بريين abstract: 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. Keywords: Anticancer Agents; Cancer Screening; Breast Cancer; Marine Organisms; Biological Products; Apoptosis; Oman. الطب يف تطبيقات لها تكون قد التي النا�سطة واجلزيئات احليوية باملكونات غني م�سدر البحرية تعتربالكائنات اأهداف: امللخ�ص: والتكنولوجيا احليوية وال�سناعة. وقد مت ا�ستخراج عدد قليل فقط من املركبات واملكونات احليوية النا�سطة. من كائنات بحرية تعي�س يف منطقة اخلليج العربي وبحر عمان. يهد ف هذا البحث إىل عزل وا�ستخال�س مركبات طبيعية والتحري عن الن�ساط امل�ساد لل�رسطان فيها يف 40 من املنتجات الطبيعية التي مت جمعها من بحر عمان منهجية: اأجريت هذه الدرا�سة بني يناير 2012 و دي�سمرب 2014 يف جامعة ال�سلطان قابو�س يف م�سقط/�سلطنة عمان. مت جمع الفطريات والبكترييا والإ�سفنج واملرجان البحري الناعم و اخليار البحري من منطقة مارينا بندر الرو�سة ومنطقة بند اخلريان ب�سلطنة عمان. عملية عزل البكترييا والفطريات متت با�ستخدام مرق البحر. اأما املكونات احليوية متت املطيافية. بالتحاليل عليها احل�سول مت التي البيانات اأ�سا�س على حتديدها ومت اأ�سيتيت والإيثيل بامليثانول ا�ستخال�سها مت فقد .MCF-7 جتربة الأن�سطة امل�سادة لل�رسطان املتواجدة يف الكائنات البحرية على خاليا �رسطانية من�سقة من الثدي واملعروفة بالنموذج نتائج: عدد 8 مركبات نقية و 32 م�ستخل�سات من الكائنات البحرية قد مت درا�ستها. اأظهرت %22.5 من جميع العينات التي مت فح�سها Sarcophyton بكميات خمتلفة من م�ستخل�سات كائنات ال�ساركوفايتون MCF-7 نتائج ايجابية م�سادة لل�رسطان. التجربة علي خاليا ت�سرياإىل اأن موت اخلاليا ال�رسطانية خا�سع اىل الكمية امل�ستعملة من املواد اخلا�سعة للتجربة. ي�سري الفح�س الدقيق للخاليا اخلا�سعة للتجربة ايل وجود جتزئة يف نواة اخللية، مع بروز على الغ�ساء وتكوين فقاعات اىل جانب انعزال الكروماتني عند الغ�ساء النووي والتي تعترب من ال�سمات املميزة يف حالة موت اخللية املربمج. خامتة: حتتوي بع�س الكائنات البحرية العمانية على مواد م�سادة لل�رسطان عالية الفعالية. الدرا�سات امل�ستقبلية ينبغي اأن ت�سلط ال�سوء على فعالية هذه املواد، وخ�سو�سياتها، والطالع علي اآليات التحكم اجلزيئية للمواد م�سادة لل�رسطان من الكائنات البحرية العمانية وذلك با�ستخدام مناذج �رسطانية خمتلفة يف جتارب معملية وعلى فئران التجارب. كلمات مفتاحية: مواد مكافحه لل�رسطان؛ فح�س ال�رسطان؛ �رسطان الثدي؛ الكائنات البحرية؛ املنتجات البيولوجية؛ املوت اخللوي؛ عمان. Screening for Anti-Cancer Compounds in Marine Organisms in Oman *Sergey Dobretsov,1,2 Yahya Tamimi,3 Mohamed A. Al-Kindi,4 Ikram Burney5 clinical & basic research Sultan Qaboos University Med J, May 2016, Vol. 16, Iss. 2, pp. 168–174, Epub. 15 May 16 Submitted 8 Sep 15 Revision Req. 19 Nov 15; Revision Recd. 2 Dec 15 Accepted 3 Jan 16 doi: 10.18295/squmj.2016.16.02.006 Advances in Knowledge - This is the first study from Oman showing the anti-cancer potential of Omani marine organisms. Very few studies of this kind have been conducted in the Gulf Cooperative Council region. - The greatest activity against the breast adenocarcinoma model was observed with the malformin A, kuanoniamine D, hymenialdisine and gallic acid compounds as well as the soft coral Sarcophyton sp. extract. Sergey Dobretsov, Yahya Tamimi, Mohamed A. Al-Kindi and Ikram Burney Clinical and Basic Research | e169 The marine ecosystem covers two-thirds of the Earth’s surface and has diverse environmental conditions, facilitating the specialisation and diversity of marine organisms; as such, these organisms are a rich source of as-yet- untapped bioactive molecules.1‒3 Soft-body sessile marine organisms—such as algae, sponges, soft corals and tunicates—are important sources of bioactive compounds.4‒6 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 Few secondary metabolites have yet been isolated from marine organisms and transformed into pharmaceuticals, but it is expected that novel drugs will be isolated from marine organisms in the future.4,8–11 Cancer remains one of the major causes of mortality worldwide; unsurprisingly, many research groups are currently focusing on finding novel anti-cancer drugs to enhance chemotherapy treatment and increase survival rates.12 A significant number of anti-cancer compounds have been isolated from marine organisms but only a few have been approved for treatment, for example the chemotherapy drugs cytosine arabinoside (isolated from the sponge Cryptotethya crypta) and eribulin (halichondrin B isolated from the sponge Halichondria okadai) and the anti-tumour drug trabectedin (ecteinascidin isolated from the tunicate Ecteinascidia turbinata).12–14 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.13 Another 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.7,13 Data 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.15,16 An antifungal phenolic compound with aromatic unsaturation was produced from the bacterium Pseudomonas aeruginosa CMG1055, also isolated from the Arabian Sea coast of Pakistan.17 Gelliodes 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.18 In 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.19 To 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, bryozoans, mangrove tree samples and sea cucumbers collected from seawater at Marina Bandar Al-Rowdha (23º 34.55' North, 58º 36.27' East) and Bandar Al- Khayran (23º 75' North, 58º 75' East) in Oman.20 Bacteria and fungi were isolated from the seawater using a marine broth (Oxoid Ltd., Basingstoke, UK) according to previously described methods.21 All 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. Screening for Anti-Cancer Compounds in Marine Organisms in Oman e170 | SQU Medical Journal, May 2016, Volume 16, Issue 2 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. High- performance 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.22 The dry extracts and pure compounds were then re- dissolved either in dimethyl sulfoxide (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 supplemen- ted with 10% fetal bovine serum (FBS) and a 1% anti- biotic 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 ºC 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% (IC50) 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 ºC. Hoechst-stained cells were then mounted on a glass slide and examined under the microscope using the 4’,6-diamidino-2-phenylindole filter (exci- tation: 350 nm; emission: 461 nm). Cells treated with DMSO were included as a negative control. Following the cell viability assay, electron 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).23 Briefly, 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 IC50 was 97 µg/mL; concentrations of cell death induction were most potent at doubled IC50. 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 Sergey Dobretsov, Yahya Tamimi, Mohamed A. Al-Kindi and Ikram Burney Clinical and Basic Research | e171 Table 1: Pure compounds* and extracts isolated from Omani marine organisms and their activity against the breast cancer Michigan Cancer Foundation-7 cell line Isolate Organism Species Phylum Solvent ACA† C om po un d Aaptamin Sponge Hemiasterella sp. Porifera DMSO Medium Hymenidin Sponge Halichondria sp. Porifera DMSO Weak 2-Bromoaldisine Sponge Thethya sp. Porifera DMSO None Hymenialdisine Sponge Hemiasterella sp. Porifera DMSO Strong Malformin A Fungus Aspergillus niger Ascomycota DMSO Strong Kojic acid Fungus Aspergillus sp. Ascomycota MeOH None Kuanoniamine D Tunicate Didemnum sp. Chordata DMSO Strong Gallic acid Mangrove tree Avicennia marina Tracheophyta MeOH Strong Ex tr ac t Methanol Soft coral Sarcophyton sp. Cnidaria DMSO None Ethyl acetate Soft coral Sarcophyton sp. Cnidaria DMSO Strong Methanol Soft coral Sinularia sp. Cnidaria DMSO None Ethyl acetate Soft coral Sinularia sp. Cnidaria DMSO None Methanol Soft coral Cladiella sp. Cnidaria DMSO Weak Ethyl acetate Soft coral Cladiella sp. Cnidaria DMSO Weak Methanol Soft coral Scleronephthya sp. Cnidaria DMSO None Ethyl acetate Soft coral Scleronephthya sp. Cnidaria DMSO Weak Methanol Soft coral Dendronephthya sp. Cnidaria DMSO None Ethyl acetate Soft coral Dendronephthya sp. Cnidaria DMSO None Methanol Tunicate Phallusia nigra Chordata DMSO None Ethyl acetate Tunicate Phallusia nigra Chordata DMSO None Methanol Sponge Chondrosia sp. Porifera DMSO None Ethyl acetate Sponge Chondrosia sp. Porifera DMSO None Ethyl acetate Fungus Penicillium sp. Ascomycota DMSO None Methanol Fungus Penicillium sp. Ascomycota DMSO None Methanol Sea cucumber Holothuria edulis Echinodermata DMSO Weak Ethyl acetate Sea cucumber Holothuria atra Echinodermata DMSO Weak Methanol Gorgonian coral Acanthogorgia sp. Cnidaria DMSO Weak Ethyl acetate Gorgonian coral Acanthogorgia sp. Cnidaria DMSO Medium Methanol Bryozoan Schizoporella unicornis Bryozoa DMSO None Ethyl acetate Bryozoan Schizoporella unicornis Bryozoa DMSO Medium Methanol Sponge Mycale sp. Porifera DMSO Medium Ethyl acetate Sponge Mycale sp. Porifera DMSO None Methanol Bacterium Halomonas sp. Proteobacteria DMSO None Ethyl acetate Bacterium Halomonas sp. Proteobacteria DMSO None Methanol Bacterium Marinobacter sp. Proteobacteria DMSO None Ethyl acetate Bacterium Marinobacter sp. Proteobacteria DMSO None Methanol Green alga Ulva sp. Chlorophyta MeOH None Ethyl acetate Green alga Ulva sp Chlorophyta MeOH None Ethyl acetate Bryozoan Bugula sp. Bryozoa MeOH None Methanol Bryozoan Bugula sp. Bryozoa MeOH Weak Control - - - DMSO and MeOH None 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). Screening for Anti-Cancer Compounds in Marine Organisms in Oman e172 | SQU Medical Journal, May 2016, Volume 16, Issue 2 death. Furthermore, the TEM ultrastructural analy- sis of the Sarcophyton sp.-treated cells indicated the presence of cell membrane blebbing, intense vacuo- lisation and chromatic segregation at the periphery of the nucleus [Figure 2]. These changes confirmed the activation of apoptosis in the treated MCF-7 cells. Discussion Numerous studies have indicated the benefits of marine flora and fauna extracts in various fields, including improvement in the prognosis of several diseases such as cancer.24–26 The complexity, poor prognosis and patient-, type- and stage-specificity of cancer requires the investigation and identification of novel compounds with effective clinical utility. In this study, the aim was to investigate the anti-cancer activity of extracts and compounds isolated from marine organisms in Oman. Almost a quarter of the investigated extracts and compounds showed medium or strong anti-cancer activity. The strongest activity against the MCF-7 breast adenocarcinoma model was observed for the malformin A, kuanoniamine D, hymenialdisine and gallic acid compounds and the soft coral Sarcophyton sp. extract. Hagimori et al. observed that malformin can disrupt the cell cycle at the G2 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 2A–D: Transmission electron microscopy ultrastructural analysis at x4,000 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. Sergey Dobretsov, Yahya Tamimi, Mohamed A. Al-Kindi and Ikram Burney Clinical and Basic Research | e173 checkpoint in cancer cells.27 Similarly, kuanoniamine 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.28 Smith et al. revealed that hymenialdisine has anti-cancer properties against human colorectal carcinomas.29 Furthermore, gallic acid has been shown as a potent compound with antimicrobial, anti- oxidant, quorum-sensing inhibitory and anti-cancer properties.30–32 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.32 Moreover, MCF-10A cells are not considered a good control model since they have a transformed phenotype and are abnormal epithelial cells.33 New epithelial non-transformed cells are commercially available; however, they are difficult to handle and propagate.33 As 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 IC50 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.34,35 For 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.35 The 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. a c k n o w l e d g e m e n t s This study was funded by an internal grant from the Sultan Qaboos University (#IG/AGR/FISH/12/01). The authors would like to acknowledge the support given to them by Sultan Qaboos University. c o n f l i c t o f i n t e r e s t The authors declare no conflicts of interest. References 1. Fenical W, Jensen PR. Developing a new resource for drug discovery: Marine actinomycete bacteria. Nat Chem Biol 2006; 2:666–73. doi: 10.1038/nchembio841. 2. Cooper EL, Yao D. Diving for drugs: Tunicate anticancer compounds. Drug Discov Today 2012; 17:636–48. doi: 10.1016 /j.drudis.2012.02.006. 3. Blunt JW, Copp BR, Keyzers RA, Munro MH, Prinsep MR. Marine natural products. Nat Prod Rep 2013; 30:237–323. doi: 10.1039/c2np20112g. 4. Proksch P, Edrada RA, Ebel R. Drugs from the seas: Current status and microbiological implications. Appl Microbiol Biotechnol 2002; 59:125–34. doi: 10.1007/s00253-002-1006-8. 5. Thoms C, Schupp P. Biotechnological potential of marine sponges and their associated bacteria as producers of new pharmaceuticals (part I). J Inter Biotech Law 2005; 2:217–20. doi: 10.1515/jibl.2005.2.5.217. 6. Penesyan A, Kjelleberg S, Egan S. Development of novel drugs from marine surface associated microorganisms. Mar Drugs 2010; 8:438–59. doi: 10.3390/md8030438. 7. Paul VJ, Arthur KE, Ritson-Williams R, Ross C, Sharp K. Chemical defenses: From compounds to communities. Biol Bull 2007; 213:226–51. doi: 10.2307/25066642. 8. Schwartsmann G, Ratain MJ, Cragg GM, Wong JE, Saijo N, Parkinson DR, et al. Anticancer drug discovery and development throughout the world. J Clin Oncol 2002; 20:47S–59S. doi: 10.1200/JCO.2002.07.122. 9. Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007; 70:461–77. doi: 10.1021/np068054v. 10. Abraham I, El Sayed K, Chen ZS, Guo H. Current status on marine products with reversal effect on cancer multidrug resistance. Mar Drugs 2012; 10:2312–21. doi: 10.3390/ md10102312. 11. Terlau H, Olivera BM. Conus venoms: A rich source of novel ion channel-targeted peptides. Physiol Rev 2004; 84:41–68. doi: 10.1152/physrev.00020.2003. http://dx.doi.org/10.1038/nchembio841 http://dx.doi.org/10.1016/j.drudis.2012.02.006 http://dx.doi.org/10.1016/j.drudis.2012.02.006 http://dx.doi.org/10.1039/c2np20112g http://dx.doi.org/10.1007/s00253-002-1006-8 http://dx.doi.org/10.1515/jibl.2005.2.5.217 http://dx.doi.org/10.3390/md8030438 http://dx.doi.org/10.2307/25066642 http://dx.doi.org/10.1200/JCO.2002.07.122 http://dx.doi.org/10.1021/np068054v http://dx.doi.org/10.3390/md10102312 http://dx.doi.org/10.3390/md10102312 http://dx.doi.org/10.1152/physrev.00020.2003 Screening for Anti-Cancer Compounds in Marine Organisms in Oman e174 | SQU Medical Journal, May 2016, Volume 16, Issue 2 12. Sawadogo WR, Schumacher M, Teiten MH, Cerella C, Dicato M, Diederich M. A survey of marine natural compounds and their derivatives with anti-cancer activity reported in 2011. Molecules 2013; 18:3641–73. doi: 10.3390/molecules18043641. 13. Simmons TL, Andrianasolo E, McPhail K, Flatt P, Gerwick WH. Marine natural products as anticancer drugs. Mol Cancer Ther 2005; 4:333–42. 14. Hussain SM, Fareed S, Ansari S, Khan MS. Marine natural products: A lead for anti-cancer. Indian J Geomarine Sci 2012; 41:27–39. 15. Hayat S, Atta-ur-Rahman, Chouldhary MI, Khan KM, Abbaskhan A. Two new cinnamic acid esters from marine brown alga Spatoglossum variabile. Chem Pharm Bull (Tokyo) 2002; 50:1297–9. doi: 10.1248/cpb.50.1297. 16. Ali MS, Pervez MK, Ahmed F, Saleem M. Dichotenol-A, B and C: The C-16 oxidized seco-dolastanes from the marine brown alga Dictyota dichotoma (Huds.) lamour. Nat Prod Res 2004; 18:543–9. doi: 10.1080/14786410310001622059. 17. Uzair B, Ahmed N, Mohammad FV, Ahmad VU. Detection, isolation and partial characterization of antifungal compound(s) produced by Pseudomonas aeruginosa CMG1055. J Chem Soc Pak 2008; 30:649–53. 18. Safaeian S, Hosseini H, Asadolah AA, Farmohamadi S. Antimicrobial activity of marine sponge extracts of offshore zone from Nay Band Bay, Iran. J Mycol Med 2009; 19:11–16. doi: 10.1016/j.mycmed.2008.11.003. 19. Dobretsov S, Al-Mammari IM, Soussi B. Bioactive compounds from Omani sea cucumbers. J Agricultural Mar Sci 2009; 14:49–53. 20. Riguera R. Isolating bioactive compounds from marine organisms. J Mar Biotech 1997; 5:187–93. 21. Dobretsov SV, Qian PY. Effect of bacteria associated with the green alga ulva reticulata on marine micro- and macrofouling. Biofouling 2002; 18:217–28. doi: 10.1080/08927010290013026. 22. Royal Society of Chemistry. MarinLit: A database of the marine natural products literature. From: pubs.rsc.org/marinlit/ Accessed: Jan 2016. 23. Skalli O, Boykins LG, Coons L. Electron microscopy. In: Dobretsov S, Williams DN, Thomason JC, Eds. Biofouling Methods. London, UK: Wiley-Blackwell, 2014. Pp. 26–43. doi: 10.1002/9781118336144. 24. Lee JC, Hou MF, Huang HW, Chang FR, Yeh CC, Tang JY, et al. Marine algal products with anti-oxidative, anti-inflammatory, and anti-cancer properties. Cancer Cell Int 2013; 13:55. doi: 10.1186/1475-2867-13-55. 25. Sithranga Boopathy N, Kathiresan K. Anticancer drugs from marine flora: An overview. J Oncol 2010; 2010:214186. doi: 10.1155/2010/214186. 26. Cooper EL, Albert R. Tunicates: A vertebrate ancestral source of antitumor compounds. In: Kim SK, Ed. Handbook of Anticancer Drugs from Marine Origin. Geneva, Switzerland: Springer, 2015. Pp. 383–96. doi: 10.1007/978-3-319-07145-9_18. 27. Hagimori K, Fukuda T, Hasegawa Y, Omura S, Tomoda H. Fungal malformins inhibit bleomycin-induced G2 checkpoint in Jurkat cells. Biol Pharm Bull 2007; 30:1379–83. doi: 10.1248/ bpb.30.1379. 28. Kijjoa A, Wattanadilok R, Campos N, Nascimento MS, Pinto M, Herz W. Anticancer activity evaluation of kuanoniamines A and C isolated from the marine sponge Oceanapia sagittaria, collected from the Gulf of Thailand. Mar Drugs 2007; 5:6–22. doi: 10.3390/md502006. 29. Smith V, Raynaud F, Workman P, Kelland LR. Characterization of a human colorectal carcinoma cell line with acquired resistance to flavopiridol. Mol Pharmacol 2001; 60:885–93. doi: 10.1124/mol.60.5.885. 30. Choubey S, Varughese LR, Kumar V, Beniwal V. Medical importance of gallic acid and its ester derivatives: A patent review. Pharm Pat Anal 2015; 4:305–15. doi: 10.4155/ppa.15.14. 31. Singh BN, Singh BR, Singh RL, Prakash D, Dhakarey R, Upadhyay G, et al. Oxidative DNA damage protective activity, antioxidant and anti-quorum sensing potentials of Moringa oleifera. Food Chem Toxicol 2009; 47:1109–16. doi: 10.1016/j. fct.2009.01.034. 32. Wang K, Zhu X, Zhang K, Zhu L, Zhou F. Investigation of gallic acid induced anticancer effect in human breast carcinoma MCF-7 cells. J Biochem Mol Toxicol 2014; 28:387–93. doi: 10.1002/jbt.21575. 33. Imbalzano KM, Tatarkova I, Imbalzano AN, Nickerson JA. Increasingly transformed MCF-10A cells have a progressively tumor-like phenotype in three-dimensional basement membrane culture. Cancer Cell Int 2009; 9:7. doi: 10.1186/1475- 2867-9-7. 34. Byju K, Anuradha V, Vasundhara G, Nair SM, Kumar NC. In vitro and in silico studies on the anticancer and apoptosis- inducing activities of the sterols identified from the soft coral, Subergorgia reticulata. Pharmacogn Mag 2014; 10:S65–71. doi: 10.4103/0973-1296.127345. 35. Andrianasolo EH, Haramaty L, White E, Lutz R, Falkowski P. Mode of action of diterpene and characterization of related metabolites from the soft coral, Xenia elongata. Mar Drugs 2014; 12:1102–15. doi: 10.3390/md12021102. http://dx.doi.org/10.3390/molecules18043641 http://dx.doi.org/10.1248/cpb.50.1297 http://dx.doi.org/10.1080/14786410310001622059 http://dx.doi.org/10.1016/j.mycmed.2008.11.003 http://dx.doi.org/10.1080/08927010290013026 http://dx.doi.org/10.1002/9781118336144 http://dx.doi.org/10.1186/1475-2867-13-55 http://dx.doi.org/10.1155/2010/214186 http://dx.doi.org/10.1007/978-3-319-07145-9_18 http://dx.doi.org/10.1248/bpb.30.1379 http://dx.doi.org/10.1248/bpb.30.1379 http://dx.doi.org/10.3390/md502006 http://dx.doi.org/10.1124/mol.60.5.885 http://dx.doi.org/10.4155/ppa.15.14 http://dx.doi.org/10.1016/j.fct.2009.01.034 http://dx.doi.org/10.1016/j.fct.2009.01.034 http://dx.doi.org/10.1002/jbt.21575%20 http://dx.doi.org/10.1186/1475-2867-9-7 http://dx.doi.org/10.1186/1475-2867-9-7 http://dx.doi.org/10.4103/0973-1296.127345 http://dx.doi.org/10.3390/md12021102