In vitro REGULATION OF THE EXPRESSION OF THE SARS-CoV-2 RECEPTOR ANGIOTENSIN-CONVERTING ENZYME (ACE2) IN LUNG CANCER CELLS BY NATURAL PRODUCTS

Abstract

The COVID-19 pandemic continues infecting people causing deaths globally. Although various medicines have been tried to combat with COVID-19, there is no medicine or treatment that has been validated yet. People have been using natural products for centuries against bacterial and viral illnesses. This study aimed to test the effects of the biomolecule oleuropein, whey collected from industrial waste and soaproot extracts obtained from Gypsophila arrostii Guss. var. nebulosa Boiss. & Heldr. and Saponaria officinalis L. on the expression of the human ACE2 gene as SARS-CoV-2 receptor on the A549 adenocarcinoma cell-line by Real-Time Quantitative Polymerase Chain Reaction (qPCR). According to the cytotoxicity tests, G. arrostii var. nebulosa and S. officinalis extract treatments showed a dose dependent cytotoxic effect on the cells. The EC50 values of G. arrostii var. nebulosa and S. officinalis were found to be 54.3 μg/ml and 17.3 μg/ml, respectively. Oleuropein showed moderate cytotoxic effects with the EC50 value over 250 μg/ml. Whey (fermented and non-fermented) did not show any cytotoxic effect at the applied doses. The qPCR results showed that the ACE2 mRNA level decreased by 89.8% and 35.2% due to the fermented and non-fermented whey extracts, respectively. Similarly, G. arrostii var. nebulosa and S. officinalis downregulated ACE2 by 79.8% and 90.1%, respectively. In contrast, oleropein upregulated ACE2 (102.8%). Our results showed that the natural supporting products produced from soaproot extracts and fermented whey can be used against COVID-19 by both cancer patients and people in potential risk groups. 

Keywords

• Oleuropein
• soaproot
• whey
• COVID-19
• A549 adenocarcinoma cell-line

Öz

COVID-19 pandemisi tüm dünyada küresel çapta insanları enfekte etmeye ve ölümlere neden olmaya devam etmektedir. COVID-19 ile mücadelede birçok ilaç denenmiş olmasına karşın henüz herhangi bir ilaç veya tedavi yöntemi onaylanmamıştır. İnsanlar yüzyıllardan bu yana hastalıklara karşı doğal ürünleri kullanmışlardır. Bu çalışmadaki amacımız bir biyomolekül olan oleuropein, endüstriyel atık olarak bertaraf edilen peynir altı suyu ve Gypsophila arrostii Guss. var. nebulosa Boiss. & Heldr. ve Saponaria officinalis L. bitkilerinden elde edilen ekstraktların A549 kanserli hücre hatlarında ACE2 reseptörünü kodlayan ACE2 geninin anlatım seviyesi üzerine etkilerini Gerçek Zamanlı Kantitatif Polimeraz Zincir Reaksiyonu (qPCR) ile belirlemektedir. Yaptığımız sitotoksisite testlerine göre G. arrostii var. nebulosa ve S. officinalis ekstraktları sırası ile 54,3 μg/ml ve 17,3 μg/ml EC50 değerleri ile doza bağımlı sitotoksik etki gösterirken, oleuropein 250 μg/ml’nin üzerinde bir değerle orta dereceli sitotoksik etki göstermiştir. Öte yandan peynir altı suyu (ferment eve fermente edilmeyen), çalışmada kullanılan dozlarda sitotoksik etki göstermemiştir. qPCR sonuçlarına göre fermente edilmiş ve edilmemiş peynir altı suyunun ACE2 genine ait mRNA seviyesini sırası ile %89,8 ve %35,2 oranlarında düşürdüğü belirlenmiştir. Benzer şekilde G. arrostii var. nebulosa ve S. officinalis ekstraktlarının ACE2 geni mRNA seviyesini sırası ile %79,8 ve %90,1 oranında düşürdüğü belirlenmiştir. Bu sonuçların aksine oleuropein biyomolekülünün ACE2 mRNA seviyesini %102,8 oranında arttırdığı belirlenmiştir. Çalışma sonuçlarına göre kullanılan bitki ekstraktlarının ve fermente edilmiş peynir altı suyunun COVID-19 ile mücadelede kanser hastalarında ve risk gruplarında kullanılabilecek doğal destek ürünlerinin üretilmesinde kullanılabileceğini göstermektedir.

References

1. 1. Almehdar, H.A., El-Fakharany, E.M., Uversky, V.N. & Redwan, E.M. 2015. Disorder in milk proteins: structure, functional disorder, and biocidal potentials of lactoperoxidase. Current Protein & Peptide Science, 16(4): 352-365.
2. 2. Arslan, I., Celik, A. & Melzig, M.F. 2013. Nebulosides A-B, novel triterpene saponins from under-ground parts of Gypsophila arrostii Guss. var. nebulosa. Bioorganic & Medicinal Chemistry, 21(5): 1279-1283.
3. 3. Arslan, I. & Cenzano, A.M. 2020. N-triterpene saponins in cancer therapy: A review of mode of action. Revista Brasileira de Farmacognosia, 30(1): 1-6. https://doi.org/10.1007/s43450-020-00033-5
4. 4. Carrera-González, M.P., Ramírez-Expósito, M.J., Mayas, M.D. & Martínez-Martos, J.M. 2013. Protective role of oleuropein and its metabolite hydroxytyrosol on cancer. Trends in Food Science & Technology, 31(2): 92-99.
5. 5. Carzaniga, R., Sinclair, L., Fordham-Skelton, A.P., Harris, N. & Croy, R.R.D. 1994. Cellular and subcellular distribution of saporins, type-1 ribosome-inactivating proteins, in soapwort (Saponaria officinalis L.). Planta, 194(4): 461-470.
6. 6. Chen, N., Zhou, M., Dong, X., Qu, J., Gong, F., Han, Y., Qiu, Y., Wang, J., Liu, Y., Wei, Y., Xia, J., Yu, T., Zhang, X. & Zhang, L. 2020. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet (London, England), 395(10223): 507-513.
7. 7. Cheng, G., Gao, F., Sun, X., Bi, H. & Zhu, Y. 2016. Paris saponin VII suppresses osteosarcoma cell migration and invasion by inhibiting MMP‑2/9 production via the p38 MAPK signaling pathway. Molecular Medicine Reports, 14(4): 3199-3205.
8. 8. Davis, P.H. 1970. Flora of Turkey and the east aegean islands. Vol. 3. Edinburgh: University Press, 629 p.

9. 9. Ebbesen, P., Dalsgaard, K. & Madsen, M. 1976. Prolonged survival of AKR mice treated with the saponin adjuvant Quil A. Acta Pathologica et Microbiologica Scandinavica. Section A, Pathology, 84(4): 358-360.
10. 10. Feng, Y., Ni, L., Wan, H., Fan, L., Fei, X., Ma, Q., Gao, B., Xiang, Y., Che, J. & Li, Q. 2011. Overexpression of ACE2 produces antitumor effects via inhibition of angiogenesis and tumor cell invasion in vivo and in vitro. Oncology Reports, 26(5): 1157-1164.
11. 11. Goulter, A. B., Goddard, M. J., Allen, J. C., & Clark, K. L. 2004. ACE2 gene expression is up-regulated in the human failing heart. BMC Medicine, 2(1), 1-7. https://doi.org/10.1186/1741-7015-2-19
12. 12. Guo, J.W., Li, L.M., Qiu, G.Q., Deng, Z.J., Fu, Y.H., Yang, M., Pan, J.Q. & Liu, R.X. 2010. Effects of Panax notoginseng saponins on ACE2 and TNF-alpha in rats with post-myocardial infarction-ventricular remodelling. Journal of Chinese Medicinal Materials, 33(1): 89-92.
13. 13. Haris Omar, S. 2010. Oleuropein in olive and its pharmacological effects. Scientia Pharmaceutica, 78(2): 133-154. http://www.mdpi.com/2218-0532/78/2/133
14. 14. Hofmann, H., Geier, M., Marzi, A., Krumbiegel, M., Peipp, M., Fey, G.H., Gramberg, T. & Pöhlmann, S. 2004. Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor. Biochemical and Biophysical Research Communications, 319(4): 1216-1221.
15. 15. Jia, H.P., Look, D.C., Hickey, M., Shi, L., Pewe, L., Netland, J., Farzan, M., Wohlford-Lenane, C., Perlman, S. & McCray, P.B.J. 2006. Infection of human airway epithelia by SARS coronavirus is associated with ACE2 expression and localization. Advances in Experimental Medicine and Biology, 581: 479-484.
16. 16. Jia, X., Yin, C., Lu, S., Chen, Y., Liu, Q., Bai, J. & Lu, Y. 2020. Two things about COVID-19 might need attention. https://www.preprints.org/manuscript/202002.0315/v1 (Date accessed 14 July 2020).
17. 17. Kanwar, J.R., Kanwar, R.K., Sun, X., Punj, V., Matta, H., Morley, S.M., Parratt, A., Puri, M. & Sehgal, R. 2009. Molecular and biotechnological advances in milk proteins in relation to human health. Current Protein & Peptide Science, 10(4): 308-338.
18. 18. Kell, D.B., Heyden, E.L. & Pretorius, E. 2020. The biology of lactoferrin, an iron-binding protein that can help defend against viruses and bacteria. Frontiers in Immunology, 11: 1221.
19. 19. Koike, K., Jia, Z. & Nikaido, T. 1999. New triterpenoid saponins and sapogenins from Saponaria officinalis. Journal of Natural Products, 62(12): 1655-1659. https://doi.org/10.1021/np990311r
20. 20. Koyuncu, M., Kiliç, C.S. & Güvenç, A. 2008. Soaproot yielding plants of East Anatolia and their potential in nature | Doǧu Anadolu’da Çöven Elde Edilen Bitkiler ve Bunlarin Doǧadaki Potansiyeli. Turkish Journal of Botany, 32(6): 489-494.
21. 21. Li, W., Sui, J., Huang, I.C., Kuhn, J.H., Radoshitzky, S.R., Marasco, W.A., Choe, H. & Farzan, M. 2007. The S proteins of human coronavirus NL63 and severe acute respiratory syndrome coronavirus bind overlapping regions of ACE2. Virology, 367(2): 367-374.
22. 22. Liang, W., Guan, W., Chen, R., Wang, W., Li, J., Xu, K., Li, C., Ai, Q., Lu, W., Liang, H., Li, S. & He, J. 2020. Cancer patients in SARS-CoV-2 infection: a nationwide analysis in China. The Lancet Oncology, 21(3): 335-337.
23. 23. Livak, K.J. & Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods, 25. http://dx.doi.org/10.1006/meth.2001.1262
24. 24. Mostad, H.B. & Doehl, J. 1987. Separation and characterization of oleanene-type pentacyclic triterpenes from Gypsophila arrostii by liquid chromatography-mass spectrometry. Journal of Chromatography A, 396: 157-168.
25. 25. Msomi, N.Z. & Simelane, M.B.C. 2017. Olea europaea subsp. africana (Oleaceae). H.A. El-Shemy, ed., active ingredients from aromatic and medicinal plants. Rijeka: IntechOpen. https://doi.org/10.5772/65725
26. 26. Ng, T.B., Cheung, R.C.F., Wong, J.H., Wang, Y., Ip, D.T.M., Wan, D.C.C. & Xia, J. 2015. Antiviral activities of whey proteins. Applied Microbiology and Biotechnology, 99(17): 6997-7008.
27. 27. Parekh, S.L., Balakrishnan, S., Hati, S. & Aparnathi, K.D. 2017. Biofunctional properties of cultured buttermilk prepared by incorporation of fermented paneer whey. International Journal of Current Microbiology and Applied Sciences, 6(2): 933-945.
28. 28. Press, J.B., Reynolds, R.C., May, R.D. & Marciani, D.J. 2000. Structure/Function relationships of immunostimulating saponins. Studies in Natural Products Chemistry, Vol: 24, 131-174.
29. 29. Ruijter, J.M., Ramakers, C., Hoogaars, W.M.H., Karlen, Y., Bakker, O., van den hoff, M.J.B. & Moorman, A.F.M. 2009. Amplification efficiency: Linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Research, 37(6): e45.
30. 30. Serkedjieva, J., Manolova, N., Zgórniak-Nowosielska, I., Zawilińska, B. & Grzybek, J. 1990. Antiviral activity of the infusion (SHS-174) from flowers of Sambucus nigra L., aerial parts of Hypericum perforatum L., and roots of Saponaria officinalis L. against influenza and herpes simplex viruses. Phytotherapy Research, 4(3): 97-100. https://doi.org/10.1002/ptr.2650040305
31. 31. Teo, A., Goh, K.K.T., Wen, J., Oey, I., Ko, S., Kwak, H.-S. & Lee, S.J. 2016. Physicochemical properties of whey protein, lactoferrin and Tween 20 stabilised nanoemulsions: Effect of temperature, pH and salt. Food Chemistry, 197(Pt A): 297-306.
32. 32. Tian, S., Hu, W., Niu, L., Liu, H., Xu, H. & Xiao, S.-Y. 2020. Pulmonary pathology of early-phase 2019 novel coronavirus (COVID-19) pneumonia in to patients with lung cancer. Journal of Thoracic Oncology, 15(5): 700-704. https://doi.org/10.1016/j.jtho.2020.02.010
33. 33. Wang, H. & Zhang, L. 2020. Risk of COVID-19 for patients with cancer. The Lancet Oncology, 21(4): e181. http://dx.doi.org/10.1016/S1470-2045(20)30149-2
34. 34. Wang, Y., Zhou, S., Yang, F., Qi, X., Wang, X., Guan, X., Shen, C., Duma, N., Vera Aguilera, J., Chintakuntlawar, A., Price, K.A., Molina, J.R., Pagliaro, L.C., Halfdanarson, T.R., Grothey, A., Markovic, S.N., Nowakowski, G.S., Ansell, S.M. & Wang, M.L. 2019. Treatment-Related adverse events of PD-1 and PD-L1 inhibitors in clinical trials: A systematic review and meta-analysis. JAMA Oncology, 5(7): 1008-1019.
35. 35. Xia, Y., Jin, R., Zhao, J., Li, W. & Shen, H. 2020. Risk of COVID-19 for patients with cancer. The Lancet Oncology, 21(4): e180.
36. 36. Xu, X., Chen, P., Wang, J., Feng, J., Zhou, H., Li, X., Zhong, W. & Hao, P. 2020. Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission. Science China Life Sciences, 63(3): 457-460. https://doi.org/10.1007/s11427-020-1637-5
37. 37. Zhang, H., Penninger, J.M., Li, Y., Zhong, N. & Slutsky, A.S. 2020. Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intensive Care Medicine, 46(4): 586-590. https://doi.org/10.1007/s00134-020-05985-9
38. 38. Zhou, P., Yang, X.-L., Wang, X.-G., Hu, B., Zhang, L., Zhang, W., Si, H.-R., Zhu, Y., Li, B., Huang, C.-L., Chen, H.-D., Chen, J., Luo, Y., Guo, H., Jiang, R.-D., Liu, M.-Q., Chen, Y., Shen, X.-R., Wang, X., Zheng, X.-S., Zhao, K., Chen, Q.-J., Deng, F., Liu, L.-L., Yan, B., Zhan, F.-X., Wang, Y.-Y., Xiao, G.-F. & Shi, Z.-L. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 579(7798): 270-273. https://doi.org/10.1038/s41586-020-2012-7
39. 39. Zimecki, M. & Kruzel, M.L. 2007. Milk-derived proteins and peptides of potential therapeutic and nutritive value. Journal of Experimental Therapeutics & Oncology, 6(2): 89-106.