Antioxidant capacity, total phenolic content, and in vitro cytotoxic and oxidative effects of the methanol extract of Dunaliella sp. against breast cancer cells

Abstract

Microalgae are rich sources of essential biomolecules, including proteins, pigments, and a diverse array of secondary metabolites such as carotenoids, polyphenols, sterols, polyunsaturated fatty acids, and polysaccharides, which they secrete at various growth stages. Owing to this property, microalgae are gaining significant research interest as natural product producers with cell-protective potential and minimal or no side effects compared to synthetic drugs. This study explores the potential of the methanol extract of Dunaliella sp. (mD) as an in vitro therapeutic agent for breast cancer. The antioxidant activity and total phenolic content of mD were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocalteu methods. Additionally, its cytotoxic and oxidative effects were evaluated in vitro. mD exhibited a radical scavenging activity (RSA) of 40.27, 1.8% below the expected value, and a total phenolic content of 25.4 mg GAE/g dry extract. The MTT cell viability assay further indicated that this microalgal extract has cytotoxic potential against breast cancer cells. The IC50 values were 768 and 728 µg/mL for MCF-7 and MDA-MB-231 cells, respectively. While the use of H2O2 alone led to an increase in ROS production in the cells, treatment with mD reduced ROS production. According to the current study, mD could be an interesting candidate for the synthesis of drugs and nutraceuticals for the treatment of breast cancer.

Keywords

• Anticancer
• Cytotoxicity
• Dunaliella
• Lake Tuz
• Reactive oxygen species
• Secondary metabolites

References

1. Akar, B., Akar, Z., & Sahin, B. (2019). Identification of antioxidant activity by different methods of a freshwater alga Microspora sp. collected from a high mountain lake. Hittite Journal of Science and Engineering, 6(1), 25 29. https://doi.org/10.17350/HJSE19030000129
2. Alateyah, N., Ahmad, S.M.S., Gupta, I., Fouzat, A., Thaher, M.I., Das, P., Al Moustafa, A.E., & Ouhtit, A. (2022). Haematococcus pluvialis Microalgae Extract Inhibits Proliferation, Invasion, and Induces Apoptosis in Breast Cancer Cells. Frontiers in Nutrition, 9, 1-9. https://doi.org/10.3389/fnut.2022.882956
3. Altin-Celik, P., Eken, A., Derya-Andeden, M., Eciroglu, H., Uzen, R., & Donmez-Altuntas, H. (2024). Iturin A and Gramicidin A inhibit proliferation, trigger apoptosis, and regulate inflammation in breast cancer cells. Journal of Drug Delivery Science and Technology, 100, 106121. https://doi.org/10.1016/J.JDDST.2024.106121
4. Andriopoulos, V., Gkioni, M.D., Koutra, E., Mastropetros, S.G., Lamari, F.N., Hatziantoniou, S., & Kornaros, M. (2022). Total phenolic content, biomass composition, and antioxidant activity of selected marine microalgal species with potential as aquaculture feed. Antioxidants, 11(7), 1320. https://doi.org/10.3390/antiox11071320
5. Andeden, E.E., Ozturk, S., & Aslim, B. (2021). Evaluation of Thirty Microalgal Isolates as Biodiesel Feedstocks Based on Lipid Productivity and Triacylglycerol (TAG) Content. Current Microbiology, 78(2), 775-788. https://doi.org/10.1007/s00284-020-02340-5
6. Arun, N., & Singh, D.P. (2016). A review on pharmacological applications of halophilic alga Dunaliella. Indian Journal of Geo-Marine Science, 45(3).
7. Balboa, E.M., Conde, E., Moure, A., Falqué, E., & Domínguez, H. (2013). In vitro antioxidant properties of crude extracts and compounds from brown algae. Food Chemistry, 138, 1764–1785. https://doi.org/10.1016/j.foodchem.2012.11.026
8. Cakmak, Y.S., Kaya, M., & Asan-Ozusaglam, M. (2014). Biochemical composition and bioactivity screening of various extracts from Dunaliella salina, a green microalga. EXCLI Journal, 13, 679.

9. Cheng, Z., Moore, J., & Yu, L. (2006). High-throughput relative DPPH radical scavenging capacity assay. Journal of Agricultural and Food Chemistry, 54(20), 7429–7436. https://doi.org/10.1021/jf0611668
10. Christowitz, C., Davis, T., Isaacs, A., Van Niekerk, G., Hattingh, S., & Engelbrecht, A.M. (2019). Mechanisms of doxorubicin-induced drug resistance and drug resistant tumour growth in a murine breast tumour model. BMC Cancer, 19, 757 766. https://doi.org/10.1186/s12885-019-5939-z
11. Ciampi, F., Sordillo, L.M., Gandy, J.C., Caroprese, M., Sevi, A., Albenzio, M., & Santillo, A. (2020). Evaluation of natural plant extracts as antioxidants in a bovine in vitro model of oxidative stress. Journal of Dairy Science, 103(10), 8938 8947. https://doi.org/10.3168/jds.2020-18182
12. Demirel, Z. (2022). Monitoring of growth and biochemical composition of Dunaliella salina and Dunaliella polymorpha in different photobioreactors. Aquatic Research, 5(2), 136-145. https://doi.org/10.3153/ar22013
13. Derya Andeden, M., Altın Çelik, P., Çakır, M., Üzen, R., et al. (2024). Effects of Pervari Honey from Türkiye on Proliferation, Oxidative Stress, and Apoptosis of Human Breast Cancer Cells. Journal of Advanced Research in Natural and Applied Sciences, 10(3), 627-639. https://doi.org/10.28979/jarnas.1456528
14. Doğan, S.Ş., & Kocabaş, A. (2021). Metagenomic assessment of prokaryotic diversity within hypersaline Tuz Lake, Turkey. Microbiology (Russian Federation), 90(5), 647 655. https://doi.org/10.1134/S0026261721050118
15. Duan, J., Guo, H., Fang, Y., & Zhou, G. (2021). The mechanisms of wine phenolic compounds for preclinical anticancer therapeutics. Food and Nutrition Research, 65, 6507 6521. https://doi.org/10.29219/fnr.v65.6507
16. Ebrahimi Nigjeh, S., Yusoff, F.M., Mohamed Alitheen, N.B., Rasoli, M., Keong, Y.S., & Omar, A.R.B. (2013). Cytotoxic effect of ethanol extract of microalga, Chaetoceros calcitrans, and its mechanisms in inducing apoptosis in human breast cancer cell line. BioMed research international, 2013(1), 783690. https://doi.org/10.1155/2013/783690
17. El-fayoumy, E. A., Shanab, S.M.M., Gaballa, H.S., Tantawy, M.A., & Shalaby, E.A. (2021). Evaluation of antioxidant and anticancer activity of crude extract and different fractions of Chlorella vulgaris axenic culture grown under various concentrations of copper ions. BMC Complementary Medicine and Therapies, 21(1), 51-66. https://doi.org/10.1186/s12906-020-03194-x
18. Elshobary, M.E., El-Shenody, R.A., Ashour, M., Zabed, H.M., & Qi, X. (2020). Antimicrobial and antioxidant characterization of bioactive components from Chlorococcum minutum. Food Bioscience, 35, 100567. https://doi.org/10.1016/j.fbio.2020.100567
19. Fernández, A.B., Vera-Gargallo, B., Sánchez-Porro, C., Ghai, R., Papke, R.T., Rodriguez-Valera, F., & Ventosa, A. (2014). Comparison of prokaryotic community structure from Mediterranean and Atlantic saltern concentrator ponds by a metagenomic approach. Frontiers in Microbiology, 5(MAY). https://doi.org/10.3389/fmicb.2014.00196
20. Goiris, K., Muylaert, K., Fraeye, I., Foubert, I., De Brabanter, J., & De Cooman, L. (2012). Antioxidant potential of microalgae in relation to their phenolic and carotenoid content. Journal of Applied Phycology, 24, 1477-1486. https://doi.org/10.1007/s10811-012-9804-6
21. Haas, I.C. da S., Marmitt, D.J., Fedrigo, I.M.T., Goettert, M.I., & Bordignon-Luiz, M.T. (2020). Evaluation of antiproliferative and anti-inflammatory effects of non-pomace sediment of red grape juices (Vitis labrusca L.) in healthy and cancer cells after in vitro gastrointestinal simulation. PharmaNutrition, 13, 100204. https://doi.org/10.1016/j.phanu.2020.100204
22. Hajimahmoodi, M., Faramarzi, M.A., Mohammadi, N., Soltani, N., Oveisi, M.R., & Nafissi-Varcheh, N. (2010). Evaluation of antioxidant properties and total phenolic contents of some strains of microalgae. Journal of Applied Phycology, 22(1), 43 50. https://doi.org/10.1007/s10811-009-9424-y
23. Hamed, I., Ak, B., Isik, O., & Uslu, L. (2017). The Effects of Salinity and Temperature on the Growth of Dunaliella sp. Isolated from the Salt Lake (Tuz Gölü), Turkey. Turkish Journal of Fisheries and Aquatic Sciences, 17, 1367-1372. https://doi.org/10.4194/1303-2712-v17_6_29
24. Herrero, M., Mendiola, J.A., Plaza, M., & Ibañez, E. (2013). Screening for bioactive compounds from algae. In Advanced biofuels and bioproducts (pp. 833–872). Springer. https://doi.org/10.1007/978-1-4614-3348-4_35
25. Holliday, D.L., & Speirs, V. (2011). Choosing the right cell line for breast cancer research. Breast Cancer Research, 13(4), 215-221. https://doi.org/10.1186/bcr2889
26. Irigoien, X., Hulsman, J., & Harris, R.P. (2004). Global biodiversity patterns of marine phytoplankton and zooplankton. Nature, 429(6994). https://doi.org/10.1038/nature02593
27. Jayappriyan, K.R., Rajkumar, R., Venkatakrishnan, V., Nagaraj, S., & Rengasamy, R. (2013). In vitro anticancer activity of natural β-carotene from Dunaliella salina EU5891199 in PC-3 cells. Biomedicine and Preventive Nutrition, 3(2), 99 105. https://doi.org/10.1016/j.bionut.2012.08.003
28. Jerez-Martel, I., García-Poza, S., Rodríguez-Martel, G., Rico, M., Afonso-Olivares, C., & Gómez-Pinchetti, J.L. (2017). Phenolic profile and antioxidant activity of crude extracts from microalgae and cyanobacteria strains. Journal of Food Quality, 2017, 1 8. https://doi.org/10.1155/2017/2924508
29. Kabała-Dzik, A., Rzepecka-Stojko, A., Kubina, R., Iriti, M., Wojtyczka, R.D., Buszman, E., & Stojko, J. (2018). Flavonoids, bioactive components of propolis, exhibit cytotoxic activity and induce cell cycle arrest and apoptosis in human breast cancer cells MDA-MB-231 and MCF 7 A comparative study. Cellular and Molecular Biology, 64(8). https://doi.org/10.14715/cmb/2018.64.8.1
30. Kapoor, S., Singh, M., Srivastava, A., Chavali, M., Chandrasekhar, K., & Verma, P. (2022). Extraction and characterization of microalgae-derived phenolics for pharmaceutical applications: A systematic review. Journal of Basic Microbiology, 62(9), 1044-1063. https://doi.org/10.1002/jobm.202100458
31. Katzenellenbogen, B.S., Kendra, K.L., Norman, M.J., & Berthois, Y. (1987). Proliferation, Hormonal Responsiveness, and Estrogen Receptor Content of MCF-7 Human Breast Cancer Cells Grown in the Short-Term and Long-Term Absence of Estrogens. Cancer Research, 47(16), 4355-4360.
32. Kaurinovic, B., & Vastag, D. (2019). Flavonoids and Phenolic Acids as Potential Natural Antioxidants. Antioxidants. IntechOpen. https://doi.org/10.5772/intechopen.83731
33. Kiokias, S., & Oreopoulou, V. (2021). A review of the health protective effects of phenolic acids against a range of severe pathologic conditions (Including coronavirus-based infections). Molecules, 26(17), 5405-5422. https://doi.org/10.3390/molecules26175405
34. León-Vaz, A., León, R., Vigara, J., & Funk, C. (2023). Exploring Nordic microalgae as a potential novel source of antioxidant and bioactive compounds. New Biotechnology, 73, 1-8. https://doi.org/10.1016/j.nbt.2022.12.001
35. Liou, G.Y., & Storz, P. (2010). Reactive oxygen species in cancer. Free Radical Research, 44(5), 479-496. https://doi.org/10.3109/10715761003667554
36. Mailloux, R.J. (2015). Teaching the fundamentals of electron transfer reactions in mitochondria and the production and detection of reactive oxygen species. Redox Biology, 5, 381-398. https://doi.org/10.1016/j.redox.2015.02.001
37. Martínez, K.A., Saide, A., Crespo, G., Martín, J., Romano, G., Reyes, F., Lauritano, C., & Ianora, A. (2022). Promising Antiproliferative Compound from the Green Microalga Dunaliella tertiolecta Against Human Cancer Cells. Frontiers in Marine Science, 9, 1-10. https://doi.org/10.3389/fmars.2022.778108
38. Mofeed, J., Deyab, M., Sabry, A.E.N., & Ward, F. (2021). In vitro anticancer activity of five marine seaweeds extract from Egypt against human breast and colon cancer cell lines. https://doi.org/10.21203/rs.3.rs-462221/v1
39. Moghadassi, Z., Emtyazjoo, M., Rabanie, M., Labibie, F., Azarghashb, E., & Mosaffa, N. (2011). Study effect of anti cancer ethanol extract Dunaliella salina isolated from Hoz-Soltan against Squamous cell carcinoma in vitro. Iranian Journal of Medicinal and Aromatic Plants Research, 27(2), 306-315. https://doi.org/10.22092/ijmapr.2011.6418
40. Mozafari, A.A., Vafaee, Y., & Shahyad, M. (2018). Phytochemical composition and in vitro antioxidant potential of Cynodon dactylon leaf and rhizome extracts as affected by drying methods and temperatures. Journal of Food Science and Technology, 55(6), 2220-2229. https://doi.org/10.1007/s13197-018-3139-5
41. Mumtaz, M.Z., Kausar, F., Hassan, M., Javaid, S., & Malik, A. (2021). Anticancer activities of phenolic compounds from Moringa oleifera leaves: in vitro and in silico mechanistic study. Beni Suef University Journal of Basic and Applied Sciences, 10(1), 12 22. https://doi.org/10.1186/s43088-021-00101-2
42. Mutlu, M.B., Martínez-García, M., Santos, F., Peña, A., Guven, K., & Antón, J. (2008). Prokaryotic diversity in Tuz Lake, a hypersaline environment in Inland Turkey. FEMS Microbiology Ecology, 65(3), 474-483. https://doi.org/10.1111/j.1574-6941.2008.00510.x
43. Nagasawa, H., Fujii, Y., Kageyama, Y., Segawa, T., & Ben-Amotz, A. (1991). Suppression by beta-carotene-rich algae Dunaliella bardawil of the progression, but not the development, of spontaneous mammary tumours in SHN virgin mice. Anticancer research, 11(2), 713-717.
44. Newman, D.J., & Cragg, G.M. (2012). Natural products as sources of new drugs over the 30 years from 1981 to 2010. Journal of Natural Products, 75(3), 311 335. https://doi.org/10.1021/np200906s
45. Ozcan, M., Aydemir, D., Bacanlı, M., Anlar, H.G., Ulusu, N.N., & Aksoy, Y. (2021). Protective Effects of Antioxidant Chlorophyllin in Chemically Induced Breast Cancer Model In vivo. Biological Trace Element Research, 199(12), 4475-4488. https://doi.org/10.1007/s12011-021-02585-6
46. Pérez-gálvez, A., Viera, I., & Roca, M. (2020). Carotenoids and chlorophylls as antioxidants. Antioxidants, 9(6), 505-538. https://doi.org/10.3390/antiox9060505
47. Perillo, B., Di Donato, M., Pezone, A., Di Zazzo, E., Giovannelli, P., Galasso, G., Castoria, G., & Migliaccio, A. (2020). ROS in cancer therapy: the bright side of the moon. Experimental & Molecular Medicine, 52, 192–203. https://doi.org/10.1038/s12276-020-0384-2
48. Pezzuto, J.M., & Vang, O. (2020). Natural products for cancer chemoprevention: Single compounds and combinations. In Natural Products for Cancer Chemoprevention: Single Compounds and Combinations. https://doi.org/10.1007/978-3-030-39855-2
49. Pizzimenti, S., Toaldo, C., Pettazzoni, P., Dianzani, M.U., & Barrera, G. (2010). The “Two-Faced” effects of reactive oxygen species and the lipid peroxidation product 4-Hydroxynonenal in the hallmarks of cancer. Cancers, 2(2), 338 363. https://doi.org/10.3390/cancers2020338
50. Reuter, S., Gupta, S.C., Chaturvedi, M.M., & Aggarwal, B.B. (2010). Oxidative stress, inflammation, and cancer: How are they linked?. Free Radical Biology and Medicine. https://doi.org/10.1016/j.freeradbiomed.2010.09.006
51. Sabharwal, S.S., & Schumacker, P.T. (2014). Mitochondrial ROS in cancer: Initiators, amplifiers or an Achilles’ heel?. Nature Reviews Cancer, 14, 709 721. https://doi.org/10.1038/nrc3803
52. Salem, O., El Assi, R., & Saleh, M. (2020). Bioactive constituents of three algal species extracts and their anticancer activity against human cancer cell lines. Egyptian Journal of Phycology, 21(1), 1-18. https://doi.org/10.21608/egyjs.2020.132190
53. Sansone, C., & Brunet, C. (2019). Promises and challenges of microalgal antioxidant production. Antioxidants, 8(7), 199-207. https://doi.org/10.3390/antiox8070199
54. Savikin, K., Alimpic, A., Zdunic, G., Zivkovic, J., Jankovic, T., Menkovic, N., & Duletic, L. (2017). Antioxidant and antineurodegenerative properties of st. John’s-wort dry extract. Lekovite Sirovine, 37, 5-9. https://doi.org/10.5937/leksir1737005s
55. Sawasdee, N., Jantakee, K., Wathikthinnakon, M., Panwong, S., Pekkoh, J., Duangjan, K., Yenchitsomanus, P.-t., & Panya, A. (2023). Microalga Chlorella sp. extract induced apoptotic cell death of cholangiocarcinoma via AKT/mTOR signaling pathway. Biomedicine and Pharmacotherapy, 160, 1-14. https://doi.org/10.1016/j.biopha.2023.114306
56. Sheu, M.J., Huang, G.J., Wu, C.H., Chen, J.S., Chang, H.Y., Chang, S.J., & Chung, J.G. (2008). Ethanol extract of Dunaliella salina induces cell cycle arrest and apoptosis in A549 human non-small cell lung cancer cells. in vivo, 22(3), 369-378.
57. Siegel, R.L., Miller, K.D., Wagle, N.S., & Jemal, A. (2023). Cancer statistics, 2023. CA: A Cancer Journal for Clinicians, 73(1), 17-48. https://doi.org/10.3322/caac.21763
58. Silva, M.R.O.B. da, Moura, Y.A.S., Converti, A., Porto, A.L.F., Viana Marques, D. de A., & Bezerra, R.P. (2021). Assessment of the potential of Dunaliella microalgae for different biotechnological applications: A systematic review. Algal Research, 58, 1 14. https://doi.org/10.1016/j.algal.2021.102396
59. Singh, S., Kate, B.N., & Banecjee, U.C. (2005). Bioactive compounds from cyanobacteria and microalgae: An overview. Critical Reviews in Biotechnology, 25(3), 73 95. https://doi.org/10.1080/07388550500248498
60. Singleton, V.L., & Rossi, J.A. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16(3), 144-158.
61. Sosa, V., Moliné, T., Somoza, R., Paciucci, R., Kondoh, H., & LLeonart, M.E. (2013). Oxidative stress and cancer: An overview. Ageing Research Reviews, 12(1), 376-390. https://doi.org/10.1016/j.arr.2012.10.004
62. Tavares-Carreón, F., De la Torre-Zavala, S., Arocha-Garza, H.F., Souza, V., Galán-Wong, L.J., & Avilés-Arnaut, H. (2020). In vitro anticancer activity of methanolic extract of Granulocystopsis sp., a microalgae from an oligotrophic oasis in the Chihuahuan desert. PeerJ, 8, e8686. https://doi.org/10.7717/peerj.8686
63. Varshney, A., & Singh, V. (2013). Effects of Algal Compounds on Cancer Cell Line. Journal of Experimental Biology and Agricultural Sciences, 1(5), 337-352.
64. Wang, S., Meckling, K.A., Marcone, M.F., Kakuda, Y., & Tsao, R. (2011). Synergistic, additive, and antagonistic effects of food mixtures on total antioxidant capacities. Journal of Agricultural and Food Chemistry, 59(3), 960-968. https://doi.org/10.1021/jf1040977
65. Westley, B., & Rochefort, H. (1979). Estradiol induced proteins in the MCF7 human breast cancer cell line. Biochemical and Biophysical Research Communications, 90(2), 410-416. https://doi.org/10.1016/0006-291X(79)91250-6
66. White, T.J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. In PCR Protocols. https://doi.org/10.1016/b978-0-12-372180-8.50042-1
67. Widowati, I., Zainuri, M., Kusumaningrum, H. P., Susilowati, R., Hardivillier, Y., Leignel, V., ..., Mouget, J.L. (2017, February). Antioxidant activity of three microalgae Dunaliella salina, Tetraselmis chuii and Isochrysis galbana clone Tahiti. In IOP Conference Series: Earth and Environmental Science (Vol. 55, No. 1, p. 012067). IOP Publishing. https://doi.org/10.1088/1755-1315/55/1/012067
68. Xue, L.X. (1993). Experimental study on extract of Dunaliella salina in preventing NSAR-induced cancer of proventriculus in mice. Chinese Journal of Preventive Medicine, 27(6), 350-353.
69. Yersal, O., & Barutca, S. (2014). Biological subtypes of breast cancer: Prognostic and therapeutic implications. World Journal of Clinical Oncology, 5(3), 412 424. https://doi.org/10.5306/wjco.v5.i3.412