In vitro and In silico Studies on Leonotis nepetifolia (L.) R. Br. Root
Extract against Cancer Cells

Anna      Merecz-Sadowska; Przemysław      Sitarek; Tomasz      Śliwiński; Karolina      Zajdel; Katarzyna      Malinowska; Hanna      Zielińska-Bliźniewska; Ewa      Kucharska; Radosław      Zajdel

doi:10.2174/1389201023666220304095225

Abstract

Background: Leonotis nepetifolia (L.) R. Br. (Lamiaceae) is a shrub traditionally used to alleviate inflammatory conditions.

Objectives: The present study aimed at investigating the biological activity of methanolic nontransformed and transformed Rhizobium rhizogenes root extracts from L. nepetifolia against human melanoma cells.

Methods: Cytotoxicity and genotoxicity properties, the impact on topoisomerase I activity, and proapoptotic activity were evaluated by the MTT test, comet assay, topoisomerase I assay, and fluorescence-activated cell sorting analysis. Moreover, the expressions of p53 were examined by qPCR and Western blot analysis. Docking studies were conducted to assess the potential interactions of the identified phytochemicals with the p53 binding protein Mdm-2, and computational analyses exhibited their antioxidant potential.

Results: Both extracts showed cytotoxic potential against human melanoma cells, but generally the activity was more potent for transformed roots than untransformed (IC₅₀ 760 μg/mL and 980 μg/mL, respectively). A similar effect was revealed during the evaluation of genotoxic and proapoptotic properties. Moreover, the expression of p53 was also found to be increased after extract treatment. The most dominant identified compounds in both extracts were as follows: (+)- catechin, p-coumaric acid, m-coumaric acid, and (+)-rosmarinic acid. Docking studies and computational analysis showed that (+)-rosmarinic acid possesses the highest binding affinity to the p53 binding protein, Mdm-2, and exhibits the best antioxidant property from the most commonly identified phytochemicals.

Conclusion: Our findings revealed the potential of L. nepetifolia transformed root extract as a source of bioactive compounds with cytotoxic, genotoxic, and proapoptotic activity against human melanoma cells as well as antioxidant properties.

Keywords: L. nepetifolia transformed root extract, phenolic compounds, A375, human melanoma cell line, antioxidant activity, proapoptotic properties.

« Previous Next »

Graphical Abstract

[1] 
de Oliveira, D.P.; de Almeida, L.; Marques, M.J.; de Carvalho, R.R.; Dias, A.L.T.; da Silva, G.A.; de Pádua, R.M.; Braga, F.C.; da Silva, M.A. Exploring the bioactivity potential of Leonotis nepetifolia: Phytochemical composition, antimicrobial and antileishmanial activities of extracts from different anatomical parts. Nat. Prod. Res.,  2019, 1-6.
[PMID:  31691582] 
[2] 
Sobolewska, D.; Pasko, P.; Galanty, A.; Makowska-Was, J.; Padło, K.; Wasilak, W. Preliminary phytochemical and biological screening of methanolic and acetone extracts from Leonotis nepetifolia (L.) R. Br. J. Med. Plants Res.,  2012, 6, 4582-4585.
[http://dx.doi.org/10.5897/JMPR12.578] 
[3] 
Kowalczyk, T.; Merecz-Sadowska, A.; Rijo, P.; Isca, V.M.S.; Picot, L.; Wielanek, M.; Śliwiński, T.; Sitarek, P. Preliminary phytochemical analysis and evaluation of the biological activity of leonotis nepetifolia (L.) R. Br rhizogenes -mediated transformation. Cells,  2021, 10, 1242.
[http://dx.doi.org/10.3390/cells10051242] [PMID:  34070057] 
[4] 
Lawrence, T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb. Perspect. Biol.,  2009, 1(6)a001651
[http://dx.doi.org/10.1101/cshperspect.a001651] [PMID:  20457564] 
[5] 
Ueda, F.; Iizuka, K.; Tago, K.; Narukawa, Y.; Kiuchi, F.; Kasahara, T.; Tamura, H.; Funakoshi-Tago, M. Nepetaefuran and leonotinin isolated from Leonotis nepetaefolia R. Br. potently inhibit the LPS signaling pathway by suppressing the transactivation of NF-κB. Int. Immunopharmacol.,  2015, 28(2), 967-976.
[http://dx.doi.org/10.1016/j.intimp.2015.08.015] [PMID:  26319953] 
[6] 
Manimegalai, T.; Raguvaran, K.; Kalpana, M.; Maheswaran, R. Green synthesis of silver nanoparticle using Leonotis nepetifolia and their toxicity against vector mosquitoes of Aedes aegypti and Culex quinquefasciatus and agricultural pests of Spodoptera litura and Helicoverpa armigera. Environ. Sci. Pollut. Res. Int.,  2020, 27(34), 43103-43116.
[http://dx.doi.org/10.1007/s11356-020-10127-1] [PMID:  32725570] 
[7] 
Sitarek, P.; Skała, E.; Toma, M.; Wielanek, M.; Szemraj, J.; Skorski, T.; Białas, A.J.; Sakowicz, T.; Kowalczyk, T.; Radek, M.; Wysokińska, H.; Śliwiński, T. Transformed root extract of Leonurus sibiricus induces apoptosis through intrinsic and extrinsic pathways in various grades of human glioma cells. Pathol. Oncol. Res.,  2017, 23(3), 679-687.
[http://dx.doi.org/10.1007/s12253-016-0170-6] [PMID:  28032310] 
[8] 
Sieuwerts, A.M.; Klijn, J.G.M.; Peters, H.A.; Foekens, J.A. The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival. Eur. J. Clin. Chem. Clin. Biochem.,  1995, 33(11), 813-823.
[http://dx.doi.org/10.1515/cclm.1995.33.11.813] [PMID:  8620058] 
[9] 
Singh, N.P.; McCoy, M.T.; Tice, R.R.; Schneider, E.L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res.,  1988, 175(1), 184-191.
[http://dx.doi.org/10.1016/0014-4827(88)90265-0] [PMID:  3345800] 
[10] 
Sitarek, P.; Skała, E.; Wysokińska, H.; Wielanek, M.; Szemraj, J.; Toma, M.; Śliwiński, T. The effect of Leonurus sibiricus plant extracts on stimulating repair and protective activity against oxidative DNA damage in CHO cells and content of phenolic compounds. Oxid. Med. Cell. Longev.,  2016, 20165738193
[http://dx.doi.org/10.1155/2016/5738193] [PMID:  26788249] 
[11] 
Dikilitas, M.; Kocyigit, A. Mononuclear leukocyte DNA damage on higher cells caused by eco-friendly pesticides and their analysis using CAPS® programme. J. Agric. Fac.,  2010, 14, 47-56.
[12] 
Nitiss, J.L.; Soans, E.; Rogojina, A.; Seth, A.; Mishina, M. Topoisomerase assays. Curr. Protoc. Pharmacol., 2012, Chapter 3: Unit 3.3; , 2012. 
[13] 
Chaves, N.; Santiago, A.; Alías, J.C. Quantification of the antioxidant activity of plant extracts: Analysis of sensitivity and hierarchization based on the method used. Antioxidants,  2020, 9(1), 76.
[http://dx.doi.org/10.3390/antiox9010076] [PMID:  31952329] 
[14] 
Crowley, L.C.; Marfell, B.J.; Scott, A.P.; Waterhouse, N.J. Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harb. Protoc.,  2016, 2016(11)
[http://dx.doi.org/10.1101/pdb.prot087288] 
[15] 
Schmittgen, T.D.; Livak, K.J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc.,  2008, 3(6), 1101-1108.
[http://dx.doi.org/10.1038/nprot.2008.73] [PMID:  18546601] 
[16] 
Skała, E.; Sitarek, P.; Toma, M.; Szemraj, J.; Radek, M.; Nieborowska-Skorska, M.; Skorski, T.; Wysokińska, H.; Śliwiński, T. Inhibition of human glioma cell proliferation by altered Bax/Bcl-2-p53 expression and apoptosis induction by Rhaponticum carthamoides extracts from transformed and normal roots. J. Pharm. Pharmacol.,  2016, 68(11), 1454-1464.
[http://dx.doi.org/10.1111/jphp.12619] [PMID:  27696406] 
[17] 
Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem.,  2009, 30(16), 2785-2791.
[http://dx.doi.org/10.1002/jcc.21256] [PMID:  19399780] 
[18] 
Discovery Studio Visualizer, V.  19. Available from:
                    https://www.3dsbiovia.com/products/collaborative-science/biovia-discovery-studio/visualization-download.php(Accessed  on 18 May 2021).
[19] 
Hunter, A.D. ACD/ChemSketch 1.0 (freeware); ACD/ChemSketch 2.0 and its Tautomers, Dictionary, and 3D Plug-ins; ACD/HNMR 2.0; ACD/CNMR 2.0. J. Chem. Educ.,  1997, 74, 905.
[http://dx.doi.org/10.1021/ed074p905] 
[20] 
Morris, G.M.; Goodsell, D.S.; Halliday, R.S.; Huey, R.; Hart, W.E.; Belew, R.K.; Olson, A.J. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem.,  1998, 19, 1639-1662.
[http://dx.doi.org/10.1002/(SICI)1096-987X(19981115)19:14<1639:AID-JCC10>3.0.CO;2-B] 
[21] 
Schmidt, M.W.; Baldridge, K.K.; Boatz, J.A.; Elbert, S.T.; Gordon, M.S.; Jensen, J.H.; Koseki, S.; Matsunaga, N.; Nguyen, K.A.; Su, S. General atomic and molecular electronic structure system. J. Comput. Chem.,  1993, 14, 1347-1363.
[http://dx.doi.org/10.1002/jcc.540141112] 
[22] 
Pantsar, T.; Poso, A. Binding affinity via docking: Fact and fiction. Molecules,  2018, 23(8), 1899.
[http://dx.doi.org/10.3390/molecules23081899] [PMID:  30061498] 
[23] 
Stewart, J.J.P. Stewart Computational Chemistry; Colorado Springs, CO, USA, 2016. 
[24] 
Meng, Q.; Yang, Z.; Jie, G.; Gao, Y.; Zhang, X.; Li, W.; Li, B.; Tu, Y. Evaluation of Antioxidant activity of tea polyphenols by a quantum chemistry calculation method - PM6. J. Food Nutr. Res.,  2014, 2, 965-972.
[http://dx.doi.org/10.12691/jfnr-2-12-17] 
[25] 
Veerabadran, U.; Venkatraman, A.; Souprayane, A.; Narayanasamy, M.; Perumal, D.; Elumalai, S.; Sivalingam, S.; Devaraj, V.; Perumal, A. Evaluation of antioxidant potential of leaves of Leonotis nepetifolia and its inhibitory effect on MCF7 and Hep2 cancer cell lines. Asian Pac. J. Trop. Dis.,  2013, 3, 103-110.
[http://dx.doi.org/10.1016/S2222-1808(13)60053-5] 
[26] 
Gurunagarajan, S.; Pemaiah, B. Available online through comparative studies on cytotoxic effect of Hyptis suaveolens Poit. and Leonotis nepeatefolia R. Br. against EAC cell lines. J. Pharm. Res.,  2011, 4, 1222-1224.
[27] 
Damasceno, L.M.O.; Silva, A.L.N.; dos Santos, R.F.; Feitosa, T.A.; Viana, L.G.F.; de Oliveira, R.G.; Silva, M.G.; Rolim, L.A.; Araújo, C.S.; Araújo, E.C.C. Cytotoxic activity of chemical constituents and essential oil from the leaves of Leonotis nepetifolia (Lamiaceae). Rev. Virtual Quim.,  2019, 11, 517-528.
[http://dx.doi.org/10.21577/1984-6835.20190039] 
[28] 
Tonisi, S.; Okaiyeto, K.; Hoppe, H.; Mabinya, L. V.; Nwodo, U.U.; Okoh, A.I. Chemical constituents, antioxidant and cytotoxicity properties of Leonotis leonurus used in the folklore management of neurological disorders in the Eastern Cape, South Africa. 3 Biotech, 2020. 10, 141.
[29] 
Saeed, M.E.M.; Meyer, M.; Hussein, A.; Efferth, T. Cytotoxicity of South-African medicinal plants towards sensitive and multidrug-resistant cancer cells. J. Ethnopharmacol.,  2016, 186, 209-223.
[http://dx.doi.org/10.1016/j.jep.2016.04.005] [PMID:  27058630] 
[30] 
El-Ansari, M.A.; Aboutabl, E.A.; Farrag, A.R.H.; Sharaf, M.; Hawas, U.W.; Soliman, G.M.; El-Seed, G.S. Phytochemical and pharmacological studies on Leonotis leonurus. Pharm. Biol.,  2009, 47, 9.
[http://dx.doi.org/10.1080/13880200902942428] 
[31] 
Sitarek, P.; Kowalczyk, T.; Santangelo, S.; Białas, A.J.; Toma, M.; Wieczfinska, J.; Śliwiński, T.; Skała, E. The extract of Leonurus sibiricus transgenic roots with atpap1 transcriptional factor induces apoptosis via dna damage and down regulation of selected epigenetic factors in human cancer cells. Neurochem. Res.,  2018, 43(7), 1363-1370.
[http://dx.doi.org/10.1007/s11064-018-2551-6] [PMID:  29786770] 
[32] 
Chen, G.; Guo, M. Screening for natural inhibitors of topoisomerases i from Rhamnus davurica by affinity ultrafiltration and high-performance liquid chromatography–mass spectrometry. Front. Plant Sci.,  2017, 8, 1521.
[http://dx.doi.org/10.3389/fpls.2017.01521] [PMID:  28919906] 
[33] 
Rice-Evans, C.A.; Miller, N.J.; Paganga, G. Antioxidant properties of phenolic compounds. Trends Plant Sci.,  1997, 2, 152-159.
[http://dx.doi.org/10.1016/S1360-1385(97)01018-2] 
[34] 
Vasuki, K.; Senthamarai, R.; Sudhakar, P. Investigation of antioxidant potential of an ethanolic extract of whole plant of Leonotis nepetifolia (L). R. Br. Eur. J. Pharm. Med. Res.,  2016, 3, 219-222.
[35] 
Oyedemi, S.O.; Afolayan, A.J. In vitro and in vivo antioxidant activity of aqueous leaves extract of leonotis leonurus (L.) R. Br. Int. J. Pharmacol.,  2011, 7, 248-256.
[http://dx.doi.org/10.3923/ijp.2011.248.256] 
[36] 
Angeloni, S.; Spinozzi, E.; Maggi, F.; Sagratini, G.; Caprioli, G.; Borsetta, G.; Ak, G.; Sinan, K.I.; Zengin, G.; Arpini, S.; Mombelli, G.; Ricciutelli, M. Phytochemical profile and biological activities of crude and purified leonurus cardiaca extracts. Plants,  2021, 10(2), 195.
[http://dx.doi.org/10.3390/plants10020195] [PMID:  33494336] 
[37] 
Chinwala, M.G.; Gao, M.; Dai, J.; Shao, J. In vitro anticancer activities of Leonurus heterophyllus sweet (Chinese motherwort herb). J. Altern. Complement. Med.,  2003, 9(4), 511-518.
[http://dx.doi.org/10.1089/107555303322284802] [PMID:  14499027] 
[38] 
Zhou, X.; Huang, N.; Chen, W.; Xiaoling, T.; Mahdavi, B.; Raoofi, A.; Mahdian, D.; Atabati, H. HPLC phenolic profile and induction of apoptosis by Linum usitatissimum extract in LNCaP cells by caspase3 and Bax pathways. AMB Express,  2020, 10(1), 203.
[http://dx.doi.org/10.1186/s13568-020-01138-9] [PMID:  33169228] 
[39] 
Sun, L.R.; Zhou, W.; Zhang, H.M.; Guo, Q.S.; Yang, W.; Li, B.J.; Sun, Z.H.; Gao, S.H.; Cui, R.J. Modulation of multiple signaling pathways of the plant-derived natural products in cancer. Front. Oncol.,  2019, 9, 1153.
[http://dx.doi.org/10.3389/fonc.2019.01153] [PMID:  31781485] 
[40] 
Singh, D.; Kumar, A.; Bhatia, A.; Singh, H.; Kukreja, S.; Singh, B.; Arora, S.; Arora, R. Role of phytochemicals in modulating signaling cascades in cancer cells. Pharmacotherapeutic Botanicals for Cancer Chemoprevention; Springer: Singapore, 2020. 
[http://dx.doi.org/10.1007/978-981-15-5999-0_2] 
[41] 
Krishnan, V.; Subramaniam, S.; Chia–Chuan, C.; Venkatachalam, B.; Thomas Cheeran, A.; Chi-Ying, F.H. Anticancer activity of leonurus sibiricus l.: Possible involvement of intrinsic apoptotic pathway. Nutr. Cancer,  2020, 12, 1-12.
[42] 
Moll, U.M.; Petrenko, O. The MDM2-p53 interaction. Mol. Cancer Res.,  2003, 1(14), 1001-1008.
[PMID:  14707283] 
[43] 
Nag, S.; Qin, J.; Srivenugopal, K.S.; Wang, M.; Zhang, R. The MDM2-p53 pathway revisited. J. Biomed. Res.,  2013, 27(4), 254-271.
[PMID:  23885265] 
[44] 
Riaz, M.; Ashfaq, U.A.; Qasim, M.; Yasmeen, E.; Ul Qamar, M.T.; Anwar, F. Screening of medicinal plant phytochemicals as natural antagonists of p53-MDM2 interaction to reactivate p53 functioning. Anticancer Drugs,  2017, 28(9), 1032-1038.
[http://dx.doi.org/10.1097/CAD.0000000000000548] [PMID:  28723868] 
[45] 
Chen, L.; Yin, H.; Farooqi, B.; Sebti, S.; Hamilton, A.D.; Chen, J. p53 α-Helix mimetics antagonize p53/MDM2 interaction and activate p53. Mol. Cancer Ther.,  2005, 4(6), 1019-1025.
[http://dx.doi.org/10.1158/1535-7163.MCT-04-0342] [PMID:  15956260] 
[46] 
Pereira, D.M.; Valentão, P.; Pereira, J.A.; Andrade, P.B. Phenolics: From chemistry to biology. Molecules,  2009, 14, 2202-2211.
[http://dx.doi.org/10.3390/molecules14062202] 
[47] 
Velioglu, Y.S.; Mazza, G.; Gao, L.; Oomah, B.D. Antioxidant
activity and total phenolics in selected fruits, vegetables, and grain
products. J. Agric. Food Chem., 1998, 46, 4113-4117. 
[http://dx.doi.org/10.1021/jf9801973] 

Rights & Permissions Print Cite

Article Metrics

19

1

Journal Information

For Authors

For Editors

For Reviewers

Explore Articles

Open Access

Open Access Articles

For Visitors

DOI https://dx.doi.org/10.2174/1389201023666220304095225	Print ISSN 1389-2010
Publisher Name Bentham Science Publisher	Online ISSN 1873-4316

Current Pharmaceutical Biotechnology

In vitro and In silico Studies on Leonotis nepetifolia (L.) R. Br. Root Extract against Cancer Cells

Abstract Play Pause

Graphical Abstract

Related Journals

Related Books

Abstract