Cytotoxic and apoptotic effects of Prunus spinosa fruit extract on HT-29 colon cancer line

Fulya Dilek Gökalp; Egzona Qipa

doi:10.62313/ijpbp.2024.161

Authors

Fulya Dilek Gökalp Trakya University, Science Faculty, Biology Department, 22030, Edirne, Türkiye https://orcid.org/0000-0001-8219-6657
Egzona Qipa Trakya University, Science Faculty, B https://orcid.org/0000-0002-1828-4123

DOI:

https://doi.org/10.62313/ijpbp.2024.161

Keywords:

Prunus spinosa, Ethidium bromide/Acridine Orange, MTT, Apoptosis, HT-29

Abstract

Colon cancer holds the position of the third most common type of cancer and stands as the third leading cause of cancer-related deaths for both men and women. Modern strategies in cancer prevention center around the use of natural compounds, which demonstrate a range of effects, including preventive, inhibitory, and latency-inducing impacts on the progression of cancer. In the present study, aqueous extracts derived from the fruits of Prunus spinosa L. (blackthorn, Rosaceae) are employed to assess their cytotoxic potential against the HT-29 colon cancer cell line. The fruit extract is administered to the HT29 cell line in different concentrations over 24 and 48-hours to evaluate the induction of apoptosis. The MTT cell viability test is employed to quantify the cytotoxic effect, indicating the extent of the impact. Additionally, the EB/AO (ethidium bromide/acridine orange) dual staining method is utilized to gather supplementary information regarding the cytotoxic effects. Observations after 24 hours of exposure showed no significant cytotoxic effect; however, 48-hour exposure revealed IC₂₀, IC₅₀, and IC₈₀ values of 1.27, 173.7, and > 1000 µg/ml, respectively, as determined by MTT analysis. Correspondingly, values of 5.06, 123.8, and > 1000 µg/ml were recorded by the EB/AO dual staining method. Our results show that P. spinosa fruit water extract has an inhibitory effect on the HT-29 cell viability by exerting cytotoxic and apoptotic effects in a concentration-dependent and time-dependent manner. Toxicity studies have shown that MTT and EB/AO support each other and achieve similar results. Further extensive research into the metabolic and functional effects of P. spinosa could illuminate its potential and increase its economic importance in the field of anticancer treatments as a natural drug.

References

Aviram, M., Kaplan, M., Rosenblat, M., & Fuhrman, B. (2005). Dietary antioxidants and paraoxonases against LDL oxidation and atherosclerosis development. In A. von Eckardstein (Ed.), Atherosclerosis: Diet and Drugs. Handbook of Experimental Pharmacology, vol 170 (pp. 263-300): Springer, Berlin, Heidelberg. DOI: https://doi.org/10.1007/3-540-27661-0_9

Badal, S., Miller, G., & Sattley, W. (2024). Plant metabolites for treating diseases. In S. McCreath & Y. Clement (Eds.), Pharmacognosy (Second Edition) (pp. 377-395): Elsevier. DOI: https://doi.org/10.1016/B978-0-443-18657-8.00010-4

Calabriso, N., Scoditti, E., Massaro, M., Pellegrino, M., Storelli, C., Ingrosso, I., Giovinazzo, G., & Carluccio, M. A. (2016). Multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts: differential role of hydroxycinnamic acids, flavonols and stilbenes on endothelial inflammatory gene expression. European Journal of Nutrition, 55, 477-489. https://doi.org/10.1007/s00394-015-0865-6 DOI: https://doi.org/10.1007/s00394-015-0865-6

Carocho, M., & Ferreira, I. C. (2013). The role of phenolic compounds in the fight against cancer–a review. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents), 13(8), 1236-1258. https://doi.org/10.2174/18715206113139990301 DOI: https://doi.org/10.2174/18715206113139990301

Chunarkar-Patil, P., Kaleem, M., Mishra, R., Ray, S., Ahmad, A., Verma, D., Bhayye, S., Dubey, R., Singh, H. N., & Kumar, S. (2024). Anticancer Drug Discovery Based on Natural Products: From Computational Approaches to Clinical Studies. Biomedicines, 12(1), 201. https://doi.org/10.3390/biomedicines12010201 DOI: https://doi.org/10.3390/biomedicines12010201

Condello, M., & Meschini, S. (2021). Role of natural antioxidant products in colorectal cancer disease: A focus on a natural compound derived from Prunus spinosa, Trigno ecotype. Cells, 10(12), 3326. https://doi.org/10.3390/cells10123326 DOI: https://doi.org/10.3390/cells10123326

Dedić, A., Džudžević-Čančar, H., Stanojković, T., Roje, M., Damjanović, A., Alispahić, A., & Jerković-Mujkić, A. (2023). HPLC Analysis of Phytosterols in Prunus spinosa L. Extracts and Their Antiproliferative Activity on Prostate Cancer Cell Lines. Kemija u Industriji, 72(5-6), 323-330. https://doi.org/10.15255/KUI.2022.077 DOI: https://doi.org/10.15255/KUI.2022.077

del Rio, D., Rodriguez-Mateos, A., Spencer, J. P., Tognolini, M., Borges, G., & Crozier, A. (2013). Dietary (poly) phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases. Antioxidants & Redox Signaling, 18(14), 1818-1892. https://doi.org/10.1089/ars.2012.4581 DOI: https://doi.org/10.1089/ars.2012.4581

Dewi, S., Ramadhan, M. F., Abdullah, M., & Sadikin, M. (2022). The Effect of Avidin on Viability and Proliferation of Colorectal Cancer Cells HT-29. Asian Pacific Journal of Cancer Prevention, 23(6), 1967-1973. DOI: https://doi.org/10.31557/APJCP.2022.23.6.1967

Fraternale, D., Giamperi, L., Bucchini, A., Sestili, P., Paolillo, M., & Ricci, D. (2009). Prunus spinosa fresh fruit juice: antioxidant activity in cell-free and cellular systems. Natural Product Communications, 4(12). https://doi.org/10.1177/1934578X0900401211 DOI: https://doi.org/10.1177/1934578X0900401211

Gasiorowski, K., Brokos, B., Kulma, A., Ogorzałek, A., & Skórkowska, K. (2001). Impact of four antimutagens on apoptosis in genotoxically damaged lymphocytes in vitro. Cellular & Molecular Biology Letters, 6(3), 649-675.

Gerardi, C., Frassinetti, S., Caltavuturo, L., Leone, A., Lecci, R., Calabriso, N., Carluccio, M. A., Blando, F., & Mita, G. (2016). Anti-proliferative, anti-inflammatory and anti-mutagenic activities of a Prunus mahaleb L. anthocyanin-rich fruit extract. Journal of Functional Foods, 27, 537-548. https://doi.org/10.1016/j.jff.2016.09.024 DOI: https://doi.org/10.1016/j.jff.2016.09.024

Ghasemi, M., Liang, S., Luu, Q. M., & Kempson, I. (2023). The MTT assay: a method for error minimization and interpretation in measuring cytotoxicity and estimating cell viability. In D. Gilbert & O. Friedrich (Eds.), Cell Viability Assays: Methods and Protocols (pp. 15-33): Springer. DOI: https://doi.org/10.1007/978-1-0716-3052-5_2

Guimarães, R., Barros, L., Calhelha, R. C., Carvalho, A. M., Queiroz, M. J. R., & Ferreira, I. C. (2014). Bioactivity of different enriched phenolic extracts of wild fruits from Northeastern Portugal: A comparative study. Plant Foods for Human Nutrition, 69, 37-42. https://doi.org/10.1007/s11130-013-0394-5 DOI: https://doi.org/10.1007/s11130-013-0394-5

Guimarães, R., Barros, L., Dueñas, M., Carvalho, A. M., Queiroz, M. J. R., Santos-Buelga, C., & Ferreira, I. C. (2013). Characterisation of phenolic compounds in wild fruits from Northeastern Portugal. Food Chemistry, 141(4), 3721-3730. https://doi.org/10.1016/j.foodchem.2013.06.071 DOI: https://doi.org/10.1016/j.foodchem.2013.06.071

Gunnar, S., & Bohlin, L. (1999). Drugs of Natural Origin. In G. Kumar & K. Jayaveera (Eds.), A Textbook of Pharmacognosy and Phytochemistry (pp. 99-121): S. Chand & Company Pvt. Ltd.

Halliwell, B. (1996). Antioxidants in human health and disease. Annual Review of Nutrition, 16(1), 33-50. https://doi.org/10.1146/annurev.nu.16.070196.000341 DOI: https://doi.org/10.1146/annurev.nutr.16.1.33

Herrmann, K., & Nagel, C. W. (1989). Occurrence and content of hydroxycinnamic and hydroxybenzoic acid compounds in foods. Critical Reviews in Food Science & Nutrition, 28(4), 315-347. https://doi.org/10.1080/10408398909527504 DOI: https://doi.org/10.1080/10408398909527504

Huang, H. M., Johanning, G. L., & O'Dell, B. L. (1986). Phenolic acid content of food plants and possible nutritional implications. Journal of Agricultural and Food Chemistry, 34(1), 48-51. https://doi.org/10.1021/jf00067a013 DOI: https://doi.org/10.1021/jf00067a013

Janicke, B., Hegardt, C., Krogh, M., Önning, G., Åkesson, B., Cirenajwis, H. M., & Oredsson, S. M. (2011). The antiproliferative effect of dietary fiber phenolic compounds ferulic acid and p-coumaric acid on the cell cycle of Caco-2 cells. Nutrition and Cancer, 63(4), 611-622. https://doi.org/10.1080/01635581.2011.538486 DOI: https://doi.org/10.1080/01635581.2011.538486

Karakas, N., Okur, M. E., Ozturk, I., Ayla, S., Karadag, A. E., & Polat, D. Ç. (2019). Antioxidant activity of blackthorn (Prunus spinosa L.) fruit extract and cytotoxic effects on various cancer cell lines. Medeniyet Medical Journal, 34(3), 297-304. https://doi.org/10.5222/MMJ.2019.87864 DOI: https://doi.org/10.5222/MMJ.2019.87864

Kasibhatla, S., Amarante-Mendes, G. P., Finucane, D., Brunner, T., Bossy-Wetzel, E., & Green, D. R. (2006). Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosis. Cold Spring Harbor Protocols, 2006(3), pdb. prot4493. DOI: https://doi.org/10.1101/pdb.prot4493

Kolodziej, H., Haberland, C., Woerdenbag, H., & Konings, A. (1995). Moderate cytotoxicity of proanthocyanidins to human tumour cell lines. Phytotherapy Research, 9(6), 410-415. https://doi.org/10.1002/ptr.2650090605 DOI: https://doi.org/10.1002/ptr.2650090605

Kurata, R., Adachi, M., Yamakawa, O., & Yoshimoto, M. (2007). Growth suppression of human cancer cells by polyphenolics from sweetpotato (Ipomoea batatas L.) leaves. Journal of Agricultural and Food Chemistry, 55(1), 185-190. https://doi.org/10.1021/jf0620259 DOI: https://doi.org/10.1021/jf0620259

Liu, R. H. (2013). Health-promoting components of fruits and vegetables in the diet. Advances in Nutrition, 4(3), 384S-392S. https://doi.org/10.3945/an.112.003517 DOI: https://doi.org/10.3945/an.112.003517

Manach, C., Scalbert, A., Morand, C., Rémésy, C., & Jiménez, L. (2004). Polyphenols: food sources and bioavailability. The American Journal of Clinical Nutrition, 79(5), 727-747. https://doi.org/10.1093/ajcn/79.5.727 DOI: https://doi.org/10.1093/ajcn/79.5.727

Mazza, G. (1993). Anthocyanins in Fruits, Vegetables, and Grains (1st Edition). In: Florida: CRC Press. https://doi.org/10.1201/9781351069700 DOI: https://doi.org/10.1201/9781351069700

Meschini, S., Pellegrini, E., Condello, M., Occhionero, G., Delfine, S., Condello, G., & Mastrodonato, F. (2017). Cytotoxic and apoptotic activities of Prunus spinosa Trigno ecotype extract on human cancer cells. Molecules, 22(9), 1578. https://doi.org/10.3390/molecules22091578 DOI: https://doi.org/10.3390/molecules22091578

Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65(1-2), 55-63. https://doi.org/10.1016/0022-1759(83)90303-4 DOI: https://doi.org/10.1016/0022-1759(83)90303-4

Murati, T., Miletić, M., Kolarić, J., Lovrić, V., Bursać Kovačević, D., Putnik, P., Landeka Jurčević, I., Đikić, D., Dragović-Uzelac, V., & Kmetič, I. (2019). Toxic activity of Prunus spinosa L. flower extract in hepatocarcinoma cells. Archives of Industrial Hygiene and Toxicology, 70(4), 303-308. https://doi.org/10.2478/aiht-2019-70-3322 DOI: https://doi.org/10.2478/aiht-2019-70-3322

Murati, T., Miletić, M., Štefanko, A., Kolarić, J., Dragović-Uzelac, V., & Kmetič, I. (2016). In vitro prooxidant and antiproliferative activity of Prunus spinosa L. plant extract in liver cancer cell lines. Paper presented at the 16th Ruzicka days “Today Science–Tomorrow Industry”, Vukovar, Croatia.

Nabende, P. (2015). Anti-proliferative Activity of Prunus africana, Warburgia stuhlmannii and Maytenus senegalensis Extracts in Breast and Colon Cancer Cell Lines. Jomo Kenyatta University of Agriculture and Technology. DOI: https://doi.org/10.9734/EJMP/2015/14081

Natić, M., Pavlović, A., Bosco, F. L., Stanisavljević, N., Zagorac, D. D., Akšić, M. F., & Papetti, A. (2019). Nutraceutical properties and phytochemical characterization of wild Serbian fruits. European Food Research and Technology, 245, 469-478. https://doi.org/10.1007/s00217-018-3178-1 DOI: https://doi.org/10.1007/s00217-018-3178-1

Nile, S. H., & Park, S. W. (2014). Edible berries: Bioactive components and their effect on human health. Nutrition, 30(2), 134-144. https://doi.org/10.1016/j.nut.2013.04.007 DOI: https://doi.org/10.1016/j.nut.2013.04.007

Nirmala, M. J., Samundeeswari, A., & Sankar, P. D. (2011). Natural plant resources in anti-cancer therapy-a review. Research in Plant Biology, 1(1), 1-14.

Orhan, D. D., Hartevioğlu, A., Küpeli, E., & Yesilada, E. (2007). In vivo anti-inflammatory and antinociceptive activity of the crude extract and fractions from Rosa canina L. fruits. Journal of Ethnopharmacology, 112(2), 394-400. https://doi.org/10.1016/j.jep.2007.03.029 DOI: https://doi.org/10.1016/j.jep.2007.03.029

Parks, D. R., Bryan, V. M., Oi, V. T., & Herzenberg, L. A. (1979). Antigen-specific identification and cloning of hybridomas with a fluorescence-activated cell sorter. Proceedings of the National Academy of Sciences, 76(4), 1962-1966. https://doi.org/10.1073/pnas.76.4.1962 DOI: https://doi.org/10.1073/pnas.76.4.1962

Petit, J. M., Denis‐Gay, M., & Ratinaud, M. H. (1993). Assessment of fluorochromes for cellular structure and function studies by flow cytometry. Biology of the Cell, 78(1‐2), 1-13. https://doi.org/10.1016/0248-4900(93)90109-R DOI: https://doi.org/10.1016/0248-4900(93)90109-R

Pinacho, R., Cavero, R. Y., Astiasarán, I., Ansorena, D., & Calvo, M. I. (2015). Phenolic compounds of blackthorn (Prunus spinosa L.) and influence of in vitro digestion on their antioxidant capacity. Journal of Functional Foods, 19, 49-62. https://doi.org/10.1016/j.jff.2015.09.015 DOI: https://doi.org/10.1016/j.jff.2015.09.015

Popović, B., Blagojević, B., Pavlović, R. Ž., Mićić, N., Bijelić, S., Bogdanović, B., Mišan, A., Duarte, C. M., & Serra, A. T. (2020). Comparison between polyphenol profile and bioactive response in blackthorn (Prunus spinosa L.) genotypes from north Serbia-from raw data to PCA analysis. Food Chemistry, 302, 125373. https://doi.org/10.1016/j.foodchem.2019.125373 DOI: https://doi.org/10.1016/j.foodchem.2019.125373

Puangpraphant, S., Berhow, M. A., Vermillion, K., Potts, G., & Gonzalez de Mejia, E. (2011). Dicaffeoylquinic acids in Yerba mate (Ilex paraguariensis St. Hilaire) inhibit NF‐κB nucleus translocation in macrophages and induce apoptosis by activating caspases‐8 and ‐3 in human colon cancer cells. Molecular Nutrition & Food Research, 55(10), 1509-1522. https://doi.org/10.1002/mnfr.201100128 DOI: https://doi.org/10.1002/mnfr.201100128

Scalbert, A., Johnson, I. T., & Saltmarsh, M. (2005). Polyphenols: antioxidants and beyond. The American Journal of Clinical Nutrition, 81(1), 215S-217S. https://doi.org/10.1093/ajcn/81.1.215S DOI: https://doi.org/10.1093/ajcn/81.1.215S

Slatnar, A., Jakopic, J., Stampar, F., Veberic, R., & Jamnik, P. (2012). The effect of bioactive compounds on in vitro and in vivo antioxidant activity of different berry juices. PLoS ONE, 7(10), e47880. https://doi.org/10.1371/journal.pone.0047880 DOI: https://doi.org/10.1371/journal.pone.0047880

Spencer, J. P., Abd El Mohsen, M. M., Minihane, A. M., & Mathers, J. C. (2008). Biomarkers of the intake of dietary polyphenols: strengths, limitations and application in nutrition research. British Journal of Nutrition, 99(1), 12-22. https://doi.org/10.1017/S0007114507798938 DOI: https://doi.org/10.1017/S0007114507798938

Stanković, M. I., SaviĆ, V. L., ŽIvković, J. V., Tadić, V. M., & Arsić, I. A. (2019). Tyrosinase inhibitory and antioxidant activity of wild Prunus spinosa L. fruit extracts as natural source of bioactive compounds. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 47(3), 651-657. https://doi.org/10.15835/nbha47311425 DOI: https://doi.org/10.15835/nbha47311425

Ürkek, B., Şengül, M., Akgül, H. İ., & Kotan, T. E. (2019). Antioxidant activity, physiochemical and sensory characteristics of ice cream incorporated with sloe berry (Prunus spinosa L.). International Journal of Food Engineering, 15(11-12), 20180029. https://doi.org/10.1515/ijfe-2018-0029 DOI: https://doi.org/10.1515/ijfe-2018-0029

Zhang, Y., Seeram, N. P., Lee, R., Feng, L., & Heber, D. (2008). Isolation and identification of strawberry phenolics with antioxidant and human cancer cell antiproliferative properties. Journal of Agricultural and Food Chemistry, 56(3), 670-675. https://doi.org/10.1021/jf071989c DOI: https://doi.org/10.1021/jf071989c