Evaluation of antioxidant, antiangiogenic and antitumor properties of Anogeissus leiocarpus against colon cancer

Loiy Elsir Ahmed Hassan ^{a, b*}, Foud S. Al-Suade ^a,  Samah M.Fadul ^c, Amin Malik Shah Abdul Majid ^a

Anogeissus leiocarpus is a traditional medicinal plant, used to treat diarrhea, fever, coughs, rheumatism, leprosy, wounds and skin diseases and psoriasis. This study was aimed to investigate the antiangiogenic and cytotoxic effects of six extracts from Anogeissus leiocarpus (leaves and bark).

Abstract

Anogeissus leiocarpus is a traditional medicinal plant, used to treat diarrhea, fever, coughs, rheumatism, leprosy, wounds and skin diseases and psoriasis. This study was aimed to investigate the antiangiogenic and cytotoxic effects of six extracts from Anogeissus leiocarpus (leaves and bark). Further, the tested biological activities were correlated with antioxidant capabilities, total phenolic and total flavonoid contents. In order to confirm the effect of the extract on motility of human endothelial cells, cell migration assay was conducted. In addition, VEGF suppressive effect of the extracts was assessed in endothelial cells. Finally, the antitumor effect of the leaves ethanolic extract was evaluated using in vivo human tumor xenograft model. A. leiocarpus extracts significantly inhibited the outgrowth of new vessels from rat aortic tissue explants by more than 50%.  Hexane extract of the stem bark and ethanolic leaves extract profoundly inhibited the sprouting of microvessels (by 89.56% and 87.12% respectively) in rat aortic ring assay. The ethanol extract of the leaves inhibited HUVEC migration by 87.57 and 65.23% after 18 hours at 80µg/ml and 40µg/ml concentrations respectively; it also demonstrated significant inhibition of VEGF levels (45.32% and 30.52 % respectively) in treated endothelial cells. The ethanol extract of the leaves showed remarkable DPPH scavenging effect with IC₅₀ = 29.87µg/ml. Furthermore, it displayed higher levels of total phenolics and flavonoids compared to other extracts. Finally the ethanol extract of the leaves exhibited potent anti-tumorigenic effect in anthymic mice with ?T/?C =2.97 and 7.54% at doses 400 and 200mg/kg, respectively. These results may provide novel guidelines towards improved strategies using Anogeissus leiocarpus extracts based on the suppression of angiogenesis to curb tumors growth. The plant can be used as promising candidate for anti-neoplasic drug development.

Key words: antiangiogic, antitumor, colon cancer, Anogeissus leiocarpus, antioxidant, total phenolic and flavonoid contents, ring assay, VEGF, cell migration

References

Ahmed Hassan, L. E., Khadeer Ahamed, M. B., Abdul Majid, A. S., Iqbal, M. A., Al Suede, F. S. R., Haque,  Majid, A. M. S. A. (2014). Crystal Structure Elucidation and Anticancer Studies of (-)-Pseudosemiglabrin: A Flavanone Isolated from the Aerial Parts of Tephrosia apollinea. PLoS ONE, 9(3), e90806. doi: 10.1371/journal.pone.0090806

Albrecht, I., Kopfstein, L., Strittmatter, K., Schomber, T., Falkevall, A., Hagberg, C. E., . . . Pietras, K. (2010). Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis. PloS one, 5(11), e14109. Retrieved from https://doi.org/10.1371/journal.pone.0014109 doi:10.1371/journal.pone.0014109

Baghel, S., Shrivastava, N., RS Baghel, P., & Rajput, S. (2012). A review of quercetin: antioxidant and anticancer properties. World J Pharm Pharmaceutical Sci, 1(1), 146 - 160. Balasundram, N., Sundram, K., & Samman, S. (2006). Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chem, 99(1), 191 - 203.

Blacher, S., Devy, L., Noel, A., & Foidart, J.-M. (2011). quantification of angiogenesis on the rat aortic ring assay. [angiogenesis; complexity; fibroblast-like cells; microvessels; quantification]. 2011, 22(1), 6. https://doi.org/10.5566/ias.v22.p43-48

Carmeliet, P. (2005). Angiogenesis in life, disease and medicine. Nature, 438(7070), 932-936. https://doi.org/10.1038/nature04478

Carmeliet, P., & Jain, R. K. (2011). Molecular mechanisms and clinical applications of angiogenesis. Nature, 473(7347), 298-307. https://doi.org/10.1038/nature10144

Chen, D., Wan, S. B., Yang, H., Yuan, J., Chan, T. H., & Dou, Q. P. (2011). egcg, green tea polyphenols and their synthetic analogs and prodrugs for human cancer prevention and treatment. Advances in Clinical Chemistry, 53, 155-177. 

Chirinos, R., Pedreschi, R., Rogez, H., Larondelle, Y., & Campos, D. (2013). Phenolic compound contents and antioxidant activity in plants with nutritional and/or medicinal properties from the Peruvian Andean region. Industrial Crops and Products, 47(Supplement C), 145-152. https://doi.org/10.1016/j.indcrop.2013.02.025

Corbett, T., Polin, L., LoRusso, P., Valeriote, F., Panchapor, C., Pugh, S., . . . Jones, J. (2004). In vivo methods for screening and preclinical testing Anticancer drug development guide (pp. 99-123): Springer.

De Flora, S., & Ferguson, L. R. (2005). Overview of mechanisms of cancer chemopreventive agents. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 591(1–2), 8-15. https://doi.org/10.1016/j.mrfmmm.2005.02.029

De Palma, M., Biziato, D., & Petrova, T. V. (2017). Microenvironmental regulation of tumour angiogenesis. Nat Rev Cancer, 17(8), 457-474. https://doi.org/10.1038/nrc.2017.51

Eccles, S. A., & Welch, D. R. (2007). Metastasis: recent discoveries and novel treatment strategies. Lancet, 369(9574), 1742-1757. https://doi.org/10.1016/S0140-6736(07)60781-8

Folkman, J., & Klagsbrun, M. (1987). Angiogenic factors. Science, 235(4787), 442-447. 

Galati, G., & O'Brien, P. (2004). Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties. Free Radic Biol Med, 37(3), 287 - 303. 

Gupta, M. K., & Qin, R.-Y. (2003). Mechanism and its regulation of tumor-induced angiogenesis. World Journal of Gastroenterology : WJG, 9(6), 1144-1155. https://doi.org/10.3748/wjg.v9.i6.1144

Hassan, L. E., Ahamed, M. B., Majid, A. S., Baharetha, H. M., Muslim, N. S., Nassar, Z. D., & Majid, A. M. (2014). Correlation of antiangiogenic, antioxidant and cytotoxic activities of some Sudanese medicinal plants with phenolic and flavonoid contents. BMC Complement Altern Med, 14, 406. https://doi.org/10.1186/1472-6882-14-406

Hassan, L. E. A., Dahham, S. S., Saghir, S. A. M., Mohammed, A. M. A., Eltayeb, N. M., Majid, A. M. S. A., & Majid, A. S. A. (2016). Chemotherapeutic potentials of the stem bark of Balanite aegyptiaca (L.)

Delile: an antiangiogenic, antitumor and antioxidant agent. BMC Complementary and Alternative Medicine, 16, 396. https://doi.org/10.1186/s12906-016-1369-5

Hassan, L. E. A., Iqbal, M. A., Dahham, S. S., Tabana, Y. M., Ahamed, M. B. K., & Majid, A. (2017). Colorectal, Prostate and Pancreas Human Cancers Targeted Bioassay-guided Isolations and Characterization of Chemical Constituents from Tephrosia apollinea. Anticancer Agents Med Chem, 17(4), 590-598. https://doi.org/10.2174/1871520616666160926113711

Hennenberg, K. J., Goetze, D., Minden, V., Traoré, D., & Porembski, S. (2005). Size-class distribution of Anogeissus leiocarpus (Combretaceae) along forest–savanna ecotones in northern Ivory Coast. Journal of Tropical Ecology, 21(3), 273-281. https://doi.org/10.1017/S0266467404002160

Jiang, C., Agarwal, R., & Lü, J. (2000). Anti-Angiogenic Potential of a Cancer Chemopreventive Flavonoid Antioxidant, Silymarin: Inhibition of Key Attributes of Vascular Endothelial Cells and Angiogenic Cytokine Secretion by Cancer Epithelial Cells. Biochemical and Biophysical Research Communications, 276(1), 371-378. https://doi.org/10.1006/bbrc.2000.3474

Kim, Y.-W., West, X. Z., & Byzova, T. V. (2013). Inflammation and oxidative stress in angiogenesis and vascular disease. [journal article]. Journal of Molecular Medicine, 91(3), 323-328. https://doi.org/10.1007/s00109-013-1007-3

Konaté, K., Kiendrébéogo, M., Ouattara, M. B., Souza, A., Lamien-Meda, A., Nongasida, Y., . . . Nacoulma, O. G. (2011). Antibacterial Potential of Aqueous Acetone Extracts from Five Medicinal Plants used Traditionally to Treat Infectious Diseases in Burkina Faso. Current Research Journal of Biological Sciences, 3(5), 435-442. 

Kumamoto, M., Nakashima, Y., & Sueishi, K. (1995). Intimal neovascularization in human coronary atherosclerosis: Its origin and pathophysiological significance. Human Pathology, 26(4), 450-456. https://doi.org/10.1016/0046-8177(95)90148-5

Liang, C.-C., Park, A. Y., & Guan, J.-L. (2007). In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nature protocols, 2(2), 329-333. 

Liou, G.-Y., & Storz, P. (2010). Reactive oxygen species in cancer. Free radical research, 44(5), 10.3109/10715761003667554. https://doi.org/10.3109/10715761003667554

Luna, L. G., & Armed Forces Institute of, P. (1968). Manual of histologic staining methods of the Armed Forces Institute of Pathology. New York: Blakiston Division, McGraw-Hill.

Mammen, D., & Daniel, M. (2012). A critical evaluation on the reliability of two aluminum chloride chelation methods for quantification of flavonoids. Food Chemistry, 135(3), 1365-1368. https://doi.org/10.1016/j.foodchem.2012.05.109

Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 65(1-2), 55-63. 

Murakami, M., & Simons, M. (2008). Fibroblast growth factor regulation of neovascularization. Current opinion in hematology, 15(3), 215-220. https://doi.org/10.1097/MOH.0b013e3282f97d98

Oak, M. H., El Bedoui, J., & Schini-Kerth, V. B. (2005). Antiangiogenic properties of natural polyphenols from red wine and green tea. J Nutr Biochem, 16(1), 1-8. https://doi.org/10.1016/j.jnutbio.2004.09.004

Okpekon, T., Yolou, S., Gleye, C., Roblot, F., Loiseau, P., Bories, C., . . . Hocquemiller, R. (2004). Antiparasitic activities of medicinal plants used in Ivory Coast. Journal of Ethnopharmacology, 90(1), 91-97. https://doi.org/10.1016/j.jep.2003.09.029

Ottu, O. J., Atawodi, S. E., & Onyike, E. (2013). Antioxidant, hepatoprotective and hypolipidemic effects of methanolic root extract of Cassia singueana in rats following acute and chronic carbon tetrachloride intoxication. Asian Pacific Journal of Tropical Medicine, 6(8), 609-615. https://doi.org/10.1016/S1995-7645(13)60105-4

Papiewska-Pajak, I., Balcerczyk, A., Stec-Martyna, E., Koziolkiewicz, W., & Boncela, J. (2017). Vascular endothelial growth factor-D modulates oxidant–antioxidant balance of human vascular endothelial cells. Journal of Cellular and Molecular Medicine, 21(6), 1139-1149. https://doi.org/10.1111/jcmm.13045

Phaniendra, A., Jestadi, D. B., & Periyasamy, L. (2015). Free Radicals: Properties, Sources, Targets, and Their Implication in Various Diseases. Indian Journal of Clinical Biochemistry, 30(1), 11-26. https://doi.org/10.1007/s12291-014-0446-0

Quail, D. F., & Joyce, J. A. (2013). Microenvironmental regulation of tumor progression and metastasis. Nature medicine, 19(11), 1423-1437. https://doi.org/10.1038/nm.3394

Rini, B. I., & Small, E. J. (2005). Biology and Clinical Development of Vascular Endothelial Growth Factor–Targeted Therapy in Renal Cell Carcinoma. Journal of Clinical Oncology, 23(5), 1028-1043. https://doi.org/10.1200/jco.2005.01.186

Russell, W., & Duthie, G. (2011). Plant secondary metabolites and gut health: the case for phenolic acids. Proceedings of the Nutrition Society, 70(3), 389-396. https://doi.org/10.1017/S0029665111000152

Sagar, S. M., Yance, D., & Wong, R. K. (2006). Natural health products that inhibit angiogenesis: a potential source for investigational new agents to treat cancer—Part 1. Current Oncology, 13(1), 14-26. 

Saggar, J. K., Yu, M., Tan, Q., & Tannock, I. F. (2013). The Tumor Microenvironment and Strategies to Improve Drug Distribution. Frontiers in Oncology, 3, 154. https://doi.org/10.3389/fonc.2013.00154

Samoylenko, A., Hossain, J. A., Mennerich, D., Kellokumpu, S., Hiltunen, J. K., & Kietzmann, T. (2013). Nutritional Countermeasures Targeting Reactive Oxygen Species in Cancer: From Mechanisms to Biomarkers and Clinical Evidence. Antioxidants & Redox Signaling, 19(17), 2157-2196. https://doi.org/10.1089/ars.2012.4662

Seong, H., Ryu, J., Jeong, J. Y., Chung, I. Y., Han, Y. S., Hwang, S. H., . . . Seo, S. W. (2015). Resveratrol suppresses vascular endothelial growth factor secretion via inhibition of CXC-chemokine receptor 4 expression in ARPE-19 cells. Mol Med Rep, 12(1), 1479-1484. https://doi.org/10.3892/mmr.2015.3518

Shahidi, F., & Naczk, M. (1995). Food phenolics : sources, chemistry, effects, applications. Lancaster, Pa.: Technomic Pub. Co.
Shao, Z. M., Shen, Z. Z., Liu, C. H., Sartippour, M. R., Go, V. L., Heber, D., & Nguyen, M. (2002). Curcumin exerts multiple suppressive effects on human breast carcinoma cells. Int J Cancer, 98(2), 234-240. 

Sharma, O. P., & Bhat, T. K. (2009). DPPH antioxidant assay revisited. Food Chemistry, 113(4), 1202-1205. https://doi.org/10.1016/j.foodchem.2008.08.008

Singh, D., Baghel, U. S., Gautam, A., Baghel, D. S., Yadav, D., Malik, J., & Yadav, R. (2016). The genus Anogeissus: A review on ethnopharmacology, phytochemistry and pharmacology. Journal of Ethnopharmacology, 194(Supplement C), 30-56. https://doi.org/ https://doi.org/10.1016/j.jep.2016.08.025

Staton, C. A., Stribbling, S. M., Tazzyman, S., Hughes, R., Brown, N. J., & Lewis, C. E. (2004). Current methods for assaying angiogenesis in vitro and in vivo. International Journal of Experimental Pathology, 85(5), 233-248. https://doi.org/ 10.1111/j.0959-9673.2004.00396.x

Subbaraju, G., & Trimurtulu, G. (2004). History of Natural Supplements in Cancer Therapy and Prevention Phytopharmaceuticals in Cancer Chemoprevention: CRC Press.

Theresa Ibibia, E., Oghenerobor Benjamin, A., & Stephen Olugbemiga, O. (2016). Determination of antibacterial activity, total phenolic, flavonoid and saponin contents in leaves of Anogeissus leiocarpus (DC.) Guill and Perr. Journal of Coastal Life Medicine, 4(4), 310-314. https://doi.org/10.12980/jclm.4.2016J5-218

Therond, P. (2006). [Oxidative stress and damages to biomolecules (lipids, proteins, DNA)]. Ann Pharm Fr, 64(6), 383-389. 
Tomayko, M. M., & Reynolds, C. P. (1989). Determination of subcutaneous tumor size in athymic (nude) mice. Cancer chemotherapy and pharmacology, 24(3), 148-154. 

Ucuzian, A. A., Gassman, A. A., East, A. T., & Greisler, H. P. (2010). Molecular Mediators of Angiogenesis. Journal of burn care & research : official publication of the American Burn Association, 31(1), 158. https://doi.org/10.1097/BCR.0b013e3181c7ed82

Upadhyay, S., & Dixit, M. (2015). Role of Polyphenols and Other Phytochemicals on Molecular Signaling. Oxidative Medicine and Cellular Longevity, 2015, 15. https://doi.org/ 10.1155/2015/504253

Van Zijl, F., Krupitza, G., & Mikulits, W. (2011). Initial steps of metastasis: Cell invasion and endothelial transmigration. Mutation Research, 728(1-2), 23-34. https://doi.org/ 10.1016/j.mrrev.2011.05.002

Vauzour, D., Rodriguez-Mateos, A., Corona, G., Oruna-Concha, M. J., & Spencer, J. P. E. (2010). Polyphenols and Human Health: Prevention of Disease and Mechanisms of Action. Nutrients, 2(11), 1106-1131. https://doi.org/10.3390/nu2111106

Veltmann, M., Hollborn, M., Reichenbach, A., Wiedemann, P., Kohen, L., & Bringmann, A. (2016). Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells. PLoS ONE, 11(1), e0147312. https://doi.org/10.1371/journal.pone.0147312

Walter, A., Etienne-Selloum, N., Brasse, D., Schleiffer, R., Bekaert, V., Vanhoutte, P., . . . Schini-Kerth, V. (2009). Red wine polyphenols prevent acceleration of neovascularization by angiotensin II in the ischemic rat hindlimb. J Pharmacol Exp Ther, 329(no. 2), 329 - 699. 

Yance, D. R., & Sagar, S. M. (2006). Targeting Angiogenesis With Integrative Cancer Therapies. Integrative Cancer Therapies, 5(1), 9-29. https://doi.org/10.1177/1534735405285562