Evaluation of cytotoxic, apoptotic effects and phenolic compounds of sea cucumber Holothuria tubulosa Gmelin, 1791 extracts

This study aimed to investigate the potential cytotoxic effects and phenolic contents of aqueous HTS and methanolic HTM extracts of Holothuria tubulosa gathered from Muğla Turkey against different cancer cells for the first time. An MTT assay was carried out to determine cellular viability. Apoptotic cell death was determined using flow cytometry. DNA fragmentation assay and caspase-3 activity analysis were carried out. Both extracts were analyzed by high performance liquid chromatography HPLC for quantification of phenolic compounds. Both HTS and HTM extracts generally caused a decrease in the cell viability in a concentration- and timedependent manner. The lowest IC$_{50}$ value was found in the HeLa cell line with 21.01 μg/mL for the HTS extract at 72 h. Both extracts are capable of significantly enhancing the apoptotic process in HeLa and A549 cells, causing DNA fragmentation, and also increasing activity of caspase-3 in both these cell lines, especially after treatment with at 500 μg/mL. Based on HPLC analysis, the most abundant component was epicatechin 790.091 μg/g extract and 2.5 dihydroxybenzoic acid 153.890 μg/g extract in the HTS and HTM extracts, respectively. This is the first study showing potential cytotoxic activity of H. tubulosa extracts from Muğla, Turkey against human cancer cells. Our results suggest that H. tubulosa may be a good source for development of anticancer agents.

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  • 1. Rayan A, Raiyn J, Falah M. Nature is the best source of anticancer drugs: Indexing natural products for their anticancer bioactivity. Plos One 2017; 12 (11): e0187925. doi: 10.1371/journal.pone.0187925
  • 2. Kratz F, Müller IA, Ryppa C, Warnecke A. Prodrug strategies in anticancer chemotherapy. Chemistry Enabling Drug Discovery 2008; 3: 20-53. doi: 10.1002/cmdc.200700159
  • 3. Housman G, Byler S, Heerboth S, Lapinska K, Longacre M et al. Drug Resistance in cancer: an overview. Cancers 2014; 6 (3): 1769-1792. doi: 10.3390/cancers6031769
  • 4. Demain AL, Vaishnav P. Natural products for cancer chemotherapy. Microbial Biotechnology 2011; 4 (6): 687-699. doi: 10.1111/j.1751-7915.2010.00221.x
  • 5. Li Y, Himaya SWA, Kim SK. Triterpenoids of marine origin as anti-cancer agents. Molecules 2013; 18 (7): 7886-7909. doi: 10.3390/molecules18077886
  • 6. Basli A, Belkacem N, Amrani I. Health benefits of phenolic compounds against cancers. Available from http://dx.doi. org/10.5772/67232
  • 7. Sarfaraj HM, Sheeba F, Saba A, Mohd SK. Marine natural products: a lead for anticancer. Indian Journal of Geo-Marine Sciences 2012; 41 (1): 27-39.
  • 8. Altmann KH. Drugs from the Oceans: Marine natural products as leads for drug discovery. Chimia 2017; 71 (10): 646-652. doi: 10.2533/chimia.2017.646
  • 9. Suarez-Jimenez GM, Burgos-Hernandez A, Ezquerra-Brauer JM. Bioactive peptides and depsipeptides with anticancer potential: sources from marine animals. Marine Drugs 2012; 10 (5): 963-986. doi: 10.3390/md10050963
  • 10. Blunt JW, Carroll AR, Copp BR, Davis RA, Keyzers RA et al. Marine natural products. Natural Product Reports 2018; 35: 8-53. doi: 10.1039/c7np00052a
  • 11. Aminin DL, Menchinskaya ES, Pisliagin EA, Silchenko SA, Avilov SA et al. Anticancer activity of sea cucumber triterpene glycosides. Marine Drugs 2015; 13 (3): 1202-1223. doi: 10.3390/md13031202
  • 12. Bordbar S, Anwar F, Saari N. High-value components and bioactives from sea cucumbers for functional foods—a review. Marine Drugs 2011; 9 (10): 1761-1805. doi: 10.3390/ md9101761
  • 13. Khotimchenko Y. Pharmacological potential of sea cucumbers. International Journal of Molecular Sciences 2018; 19 (5): 1342. doi: 10.3390/ijms19051342
  • 14. Ridhowati S, Zakaria F, Syah D, Chasanah E. Sea cucumber as anticancer agents and its development for functional food products. Squalen Bulletin of Marine & Fisheries Postharvest & Biotechnology 2014; 9 (2): 85-96.
  • 15. Aydın M. Biometry, density and the biomass of the commercial sea cucumber population of the aegean sea. Turkish Journal Fisheries and Aquatic Science 2018; 19 (6): 463-474. doi: 10.4194/1303-2712-v19_6_02
  • 16. Lahlou M. The success of natural products in drug discovery. Pharmacology & Pharmacy 2013; 4: 17-31. doi: 10.4236/ pp.2013.43A003
  • 17. Kazanidis G, Antoniadou C, Lolas AP, Neofitou N, Vafidis D et al. Population dynamics and reproduction of Holothuria tubulosa (Holothuroidea: Echinodermata) in the Aegean Sea. Journal of the Marine Biological Association of the United Kingdom 2010; 90 (5): 895-901. doi: 10.1017/ S0025315410000251
  • 18. Luparello C, Ragona D, Asaro DML, Lazzara V, Affranchi F, Celi M, Arizza V, Vazzana M. Cytotoxic potential of the coelomic fluid extracted from the sea cucumber Holothuria tubulosa against triple-negative MDA-MB231 breast cancer cells. Biology 2019; 8 (4): E76. doi: 10.3390/biology8040076
  • 19. Herencia F, Ubeda A, Ferrandiz ML, Terencio MC, Alcaraz MJ et al. Anti-inflammatory activity in mice of extracts from Mediterranean marine invertebrates. Life Sciences 1998; 62 (9): 115-120. doi: 10.1016/s0024-3205(97)01188-0
  • 20. Künili İE, Çolakoğlu FA. Antioxidant and antimicrobial activity of sea cucumber (Holothuria tubulosa, Gmelin 1791) extracts. COMU Journal of Marine Science And Fisheries 2018; 1 (2): 66-71.
  • 21. Schillaci D, Cusimano MG, Cunsolo V, Saletti R, Russo D et al. Immune mediators of sea-cucumber Holothuria tubulosa (Echinodermata) as source of novel antimicrobial and antistaphylococcal biofilm agents. AMB Express 2013; 3 (1): 35. doi: 10.1186/2191-0855-3-35
  • 22. Aydın M, Sevgili H, Tufan B, Emre Y, Köse S. Proximate composition and fatty acid profile of three different fresh and dried commercial sea cucumbers from Turkey. International Journal of Food Science & Technology 2011; 46 (3): 500-508. doi: 10.1111/j.1365-2621.2010.02512.x
  • 23. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 1983; 65 (1-2): 55-63. doi: 10.1016/0022-1759(83)90303-4
  • 24. Nguyen MN, Ho-Huynh TD. Selective cytotoxicity of a Vietnamese traditional formula, Nam Dia long, against MCF-7 cells by synergistic effects. BMC Complementary and Alternative Medicine 2016; 16: 220. doi: 10.1186/s12906-016- 1212-z
  • 25. Gong J, Traganos F, Darzynkiewicz Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Analytical Biochemistry 1994; 218 (2): 314-319. doi: 10.1006/abio.1994.1184
  • 26. Bradford MM. A Rapid and Sensitive Method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 1976; 72: 248-254. doi: 10.1006/abio.1976.9999
  • 27. Caponio F, Alloggio V, Gomes T. Phenolic compounds of virgin olive oil; influence of paste preparation techniques. Food Chemistry 1999; 64 (2): 203-209. doi: 10.1016/S0308- 8146(98)00146-0
  • 28. Ioannou YA, Chen FW. Quantitation of DNA fragmentation in apoptosis. Nucleic Acid Research 1996; 24 (5): 992-993. doi: 10.1093/nar/24.5.992
  • 29. Janakiram NB, Mohammed A, Rao CV. Sea cucumbers metabolites as potent anti-cancer agents. Marine Drugs 2015; 13 (5): 2909-2923. doi: 10.3390/md13052909
  • 30. Dakrory AI, Fahmy SR, Soliman MA, Mohamed AS, Amer SAM. Protective and curative effects of the sea cucumber Holothuria atra extract against DMBA-induced hepatorenal diseases in rats. BioMed Research International 2015. doi: 10.1155/2015/563652
  • 31. Fahmy SR, Amer MA, Al-killidar MH. Ameliorative effect of the sea cucumber Holothuria arenicola extract against gastric ulcer in rats. The Journal of Basic & Applied Zoology 2015; 72: 16-25. doi: 10.1016/j.jobaz.2015.03.001
  • 32. Khademvatan S, Eskandari A, Saki J, Foroutan-Rad M. Cytotoxic activity of Holothuria leucospilota extract against Leishmania infantum in vitro. Advances in Pharmacological Sciences 2016. doi: 10.1155/2016/8195381
  • 33. Zou ZR, Yi YH, Wu HM, Wu JH, Liaw CC et al. Intercedensides A–C, three new cytotoxic triterpene glycosides from the sea cucumber Mensamaria intercedens Lampert. Journal of Natural Products 2003; 66 (8): 1055-1060. doi: 10.1021/np030064yf
  • 34. Ogushi M, Yoshie-Stark M, Suzuki T. Cytostatic activity of hot water extracts from the sea cucumber in Caco-2. Food Science and Technology Research 2005; 11 (2): 202-206. doi: 10.3136/ fstr.11.202
  • 35. Althunibat OY, Hashim RB, Taher M, Daud JM, Ikeda MA et al. In vitro antioxidant and antiproliferative activities of three Malaysian sea cucumber species. European Journal of Scientific Research 2009; 37 (3): 376-387.
  • 36. Mashjoor S, Yousefzadi M. Cytotoxic effects of three Persian Gulf species of Holothurians. Iranian Journal of Veterinary Research 2019; 20 (1): 19-26.
  • 37. Han H, Li L, Yi YH, Wang XH, Pan, MX. Triterpene glycosides from sea cucumber Holothuria scabra with cytotoxic activity. Chinese Herbal Medicines 2012; 4 (3): 183-188. doi: 10.3969/j. issn.1674-6384.2012.03.002
  • 38. Dhinakaran DI, Lipton AP. Bioactive compounds from Holothuria atra of Indian ocean. Springerplus 2014; 3: 673. doi: 10.1186/2193-1801-3-673
  • 39. Zhang JJ, Zhu KQ. A novel antitumor compound nobiliside D isolated from sea cucumber (Holothuria nobilis Selenka). Experimental and Therapeutic Medicine 2017; 14: 1653-1658. doi: 10.3892/etm.2017.4656
  • 40. Kareh M, El Nahas R, Al Aaraj L, Al Ghadban S, Naser Al Deen N et al. Anti-proliferative and anti-inflammatory activities of the sea cucumber Holothuria polii aqueous extract. SAGE Open Medicine 2018; 6: 2050312118809541. doi: 10.1177/2050312118809541
  • 41. Gerl R, Vaux DL. Apoptosis in the development and treatment of cancer. Carcinogenesis 2005; 26 (2): 263-270. doi: 10.1093/ carcin/bgh283
  • 42. Wong RS. Apoptosis in cancer: from pathogenesis to treatment. Journal of Experimental & Clinical Cancer Research 2011; 30: 87. doi: 10.1186/1756-9966-30-87
  • 43. Pistritto G, Trisciuoglio D, Ceci C, Garufi A, D’Orazi G. Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging 2016; 8 (4): 603-619. doi: 10.18632/ aging.100934
  • 44. Sachet M, Liang YY, Oehler R. The immune response to secondary necrotic cells. Apoptosis 2017; 22 (10): 1189-1204. doi: 10.1007/s10495-017-1413-z
  • 45. Vanden Berghe T, Vanlangenakker N, Parthoens E, Deckers W, Devos M et al. Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features. Cell Death and Differentiation 2010; 17 (6): 922-930. doi: 10.1038/ cdd.2009.184
  • 46. Baharara J, Amini E, Afzali M, Nikdel N, Mostafapour A et al. Apoptosis inducing capacity of Holothuria arenicola in CT26 colon carcinoma cells in vitro and in vivo. Iranian Journal of Basic Medical Sciences 2016; 19 (4): 358-365.
  • 47. Sangpairoj K, Chaithirayanon K, Vivithanaporn P, Siangcham T, Jattujan P et al. Extract of the sea cucumber, Holothuria scabra, induces apoptosis in human glioblastoma cell lines. Functional Foods in Health & Disease 2016; 6 (7): 452-468. doi: 10.31989/ffhd.v6i7.264
  • 48. Nursid M, Marraskuranto E, Chasanah E. Cytotoxicity and apoptosis induction of sea cucumber Holothuria atra extracts. Pharmacognosy Research 2019; 11 (1): 41-46. doi: 10.4103/ pr.pr_3_18
  • 49. Saraste A, Pulkki K. Morphologic and biochemical hallmarks of apoptosis. Cardiovascular Research 2000; 45 (3): 528-537. doi: 10.1016/s0008-6363(99)00384-3
  • 50. Elmore S. Apoptosis: a review of programmed cell death. Toxicologic Pathology 2007; 35 (4): 495-516. doi: 10.1080/01926230701320337
  • 51. Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death and Differentiation 1999; 6 (2): 99-104. doi: 10.1038/sj.cdd.4400476
  • 52. Baharara J, Nikdel N, Nezhad Shahrokhabadi K, Amini E. The synergistic influence of Holothuria arenicola extract and imidazole carboxamide on cellosaurus cell line B16-F10. Iranian Journal of Fisheries Sciences 2019; 18 (1): 173-187. doi: 10.22092/ijfs.2018.118806
  • 53. Carocho M, Ferreira ICFR. The role of phenolic compounds in the fight against cancer – A Review. Anti-Cancer Agents in Medicinal Chemistry 2013; 13: 1236-1258.
  • 54. Widyawati PS, Budianta TDW, Kusuma FA, Wijaya EL. Difference of solvent polarity to phytochemical content and antioxidant activity of Pluchea indica less leaves extracts. International Journal of Pharmacognosy and Phytochemical Research 2014; 6 (4): 850-855.
  • 55. Sepahpour S, Selamat J, Abdul Manap MY, Khatib A, Abdull Razis AF. Comparative analysis of chemical composition, antioxidant activity and quantitative characterization of some phenolic compounds in selected herbs and spices in different solvent extraction systems. Molecules 2018; 23 (2): 402. doi: 10.3390/molecules23020402
  • 56. Kahkeshani N, Farzaei F, Fotouhi M, Alavi SS, Bahramsoltani R et al. Pharmacological effects of gallic acid in health and diseases: a mechanistic review. Iranian Journal of Basic Medical Sciences 2018; 22 (3): 225-237. doi: 10.22038/ ijbms.2019.32806.7897
  • 57. Altinoz MA, Elmaci I, Ozpinar A. Gentisic acid, a quinonoid aspirin metabolite in cancer prevention and treatment. new horizons in management of brain tumors and systemic cancers. Journal of Cancer Research and Oncobiology 2018; 1 (2): 109. doi: 10.31021/jcro.20181109
  • 58. Khadem S, Marles RJ. Monocyclic phenolic acids; hydroxy-and polyhydroxybenzoic acids: occurrence and recent bioactivity studies. Molecules 2010; 15: 7985-8005. doi: 10.3390/ molecules15117985
  • 59. Zhang HM, Zhao L, Li H, Xu H, Chen WW et al. Research progress on the anticarcinogenic actions and mechanisms of ellagic acid. Cancer Biology & Medicine 2014; 11 (2): 92-100. doi: 10.7497/j.issn.2095-3941.2014.02.004
  • 60. Abdulkhaleq LA, Assi MA, Noor MHM, Abdullah R, Saad MZ et al. Therapeutic uses of epicatechin in diabetes and cancer. Veterinary World 2017; 10 (8): 869-872. doi: 10.14202/ vetworld.2017.869-872