Multiple Targets Directed Multiple Ligands: An In Silico and In Vitro Approach to Evaluating the Effect of Triphala on Angiogenesis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Methods
2.2.1. Docking Studies
Target Selection and Preparation
Ligand Preparation
Molecular Docking
Prediction Model
Screening Targets
2.2.2. Angiogenesis Assay
2.2.3. Cell Migration Assay
2.2.4. Enzyme-Linked Immunosorbent Assay (ELISA)
2.2.5. Western Blot
2.2.6. Statistical Analysis
3. Results
3.1. In Silico Identification of Drug Targets against VEGF-Mediated Angiogenesis
3.2. In Silico Identification of Drug Targets against Inflammation
3.3. Effect of Ethanolic Extract of Triphala Churna on Markers of Angiogenesis
3.4. Effect of Ethanolic Extract of Triphala Churna on the Production of Angiogenic Growth Factors by HUVECs in Culture
3.5. Effect of Punicalagin on Markers of Angiogenesis
3.6. Effect of Punicalagin on the Production of Angiogenic Growth Factors by HUVECs in Culture
3.7. Effect of Triphala Extract and Punicalagin on Endothelial Cell Migration
3.8. Binding of Punicalagin with Crucial Targets
3.9. Effect of Punicalagin on the Levels and Activation of Akt in HUVECs
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
- Oklu, R.; Walker, T.G.; Wicky, S.; Hesketh, R. Angiogenesis and current antiangiogenic strategies for the treatment of cancer. J. Vasc. Interv. Radiol. 2010, 21, 1791–1805. [Google Scholar] [CrossRef] [PubMed]
- Moserle, L.; Casanovas, O. Anti-angiogenesis and metastasis: A tumor and stromal cell alliance. J. Intern. Med. 2013, 273, 128–137. [Google Scholar] [CrossRef] [PubMed]
- Dor, Y.; Porat, R.; Keshet, E. Vascular endothelial growth factor and vascular adjustments to perturbations in oxygen homeostasis. Am. J. Physiol. Cell Physiol. 2001, 280, 367–374. [Google Scholar] [CrossRef]
- Lohela, M.; Bry, M.; Tammela, T.; Alitalo, K. VEGFs and receptors involved in angiogenesis versus lymphangiogenesis. Curr. Opin. Cell Biol. 2009, 21, 154–165. [Google Scholar] [CrossRef] [PubMed]
- Takahashi, Y.; Kitadai, Y.; Bucana, C.D.; Cleary, K.R.; Ellis, L.M. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res. 1995, 55, 3964–3968. [Google Scholar] [PubMed]
- Moghaddam, S.M.; Amini, A.; Morris, D.L.; Pourgholami, M.H. Significance of vascular endothelial growth factor in growth and peritoneal dissemination of ovarian cancer. Cancer Metastasis Rev. 2012, 31, 143–162. [Google Scholar] [CrossRef] [Green Version]
- Ferrara, N. VEGF and the quest for tumor angiogenesis factors. Nat. Rev. Cancer. 2002, 2, 795–803. [Google Scholar] [CrossRef]
- Sagar, S.M.; Yance, D.; Wong, R.K. Natural health products that inhibit angiogenesis: A potential source for investigational new agents to treat cancer-Part 1. Curr. Oncol. 2006, 13, 14–26. [Google Scholar]
- Abhinand, C.S.; Raju, R.; Soumya, S.J.; Arya, P.S.; Sudhakaran, P.R. VEGF-A/VEGFR2 signaling network in endothelial cells relevant to angiogenesis. J. Cell Commun. Signal. 2016, 10, 347–354. [Google Scholar] [CrossRef] [Green Version]
- Lu, K.; Basu, S. The natural compound chebulagic acid inhibits vascular endothelial growth factor A mediated regulation of endothelial cell functions. Sci. Rep. 2015, 10, 9642. [Google Scholar] [CrossRef] [Green Version]
- Singh, D.P.; Govindarajan, R.; Rawat, A.K. High-performance liquid chromatography as a tool for the chemical standardisation of Triphala—an Ayurvedic formulation. Phytochem. Anal. 2008, 19, 164–168. [Google Scholar] [CrossRef] [PubMed]
- Pawar, V.; Lahorkar, P.; Anantha Narayana, D.B. Development of a RP-HPLC Method for Analysis of Triphala Curna and its Applicability to Test Variations in Triphala Curna Preparations. Indian J. Pharm. Sci. 2009, 71, 382–386. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Baliga, M.S. Triphala, Ayurvedic formulation for treating and preventing cancer: A review. J. Altern. Complement. Med. 2010, 16, 1301–1308. [Google Scholar] [CrossRef] [PubMed]
- Lu, K.; Chakroborty, D.; Sarkar, C.; Lu, T.; Xie, Z.; Liu, Z.; Basu, S. Triphala and its active constituent chebulinic acid are natural inhibitors of vascular endothelial growth factor-a mediated angiogenesis. PLoS ONE 2012, 7, 43934. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Athira, A.P.; Helen, A.; Saja, K.; Reddanna, P.; Sudhakaran, P.R. Inhibition of Angiogenesis In Vitro by Chebulagic Acid: A COX-LOX Dual Inhibitor. Int. J. Vasc. Med. 2013, 843897. [Google Scholar] [CrossRef] [Green Version]
- Reddy, D.B.; Reddy, T.C.; Jyotsna, G.; Sharan, S.; Priya, N.; Lakshmipathi, V.; Reddanna, P. Chebulagic acid, a COX-LOX dual inhibitor isolated from the fruits of Terminalia chebula Retz., induces apoptosis in COLO-205 cell line. J. Ethnopharmacol. 2009, 124, 506–512. [Google Scholar] [CrossRef]
- Athira, A.P.; Abhinand, C.S.; Saja, K.; Helen, A.; Reddanna, P.; Sudhakaran, P.R. Anti-angiogenic effect of chebulagic acid involves inhibition of the VEGFR2- and GSK-3β-dependent signaling pathways. Biochem. Cell Biol. 2017, 95, 563–570. [Google Scholar] [CrossRef]
- Sandhya, T.; Lathika, K.M.; Pandey, B.N.; Mishra, K.P. Potential of traditional ayurvedic formulation, Triphala, as a novel anticancer drug. Cancer Lett. 2006, 231, 206–214. [Google Scholar] [CrossRef]
- Gu, J.; Chen, L.; Yuan, G.; Xu, X. A Drug-Target Network-Based Approach to Evaluate the Efficacy of Medicinal Plants for Type II Diabetes Mellitus. Evid. Based Complement. Alternat. Med. 2013, 203614. [Google Scholar] [CrossRef] [Green Version]
- Kiran, M.S.; Sameer Kumar, V.B.; Viji, R.I.; Sudhakaran, P.R. Temporal relationship between MMP production and angiogenic process in HUVECs. Cell Biol. Int. 2006, 30, 704–713. [Google Scholar] [CrossRef]
- Liang, C.C.; Park, A.Y.; Guan, J.L. In vitro scratch assay: A convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2007, 2, 329–333. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Engvall, E.; Perlmann, P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971, 8, 871–874. [Google Scholar] [CrossRef]
- Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275. [Google Scholar] [PubMed]
- Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 1979, 76, 4350–4354. [Google Scholar] [CrossRef] [Green Version]
- Karar, J.; Maity, A. PI3K/AKT/mTOR Pathway in Angiogenesis. Front. Mol. Neurosci. 2011, 4, 51. [Google Scholar] [CrossRef] [Green Version]
- Koch, S.; Claesson-Welsh, L. Signal transduction by vascular endothelial growth factor receptors. Cold Spring Harb. Perspect. Med. 2012, 2, a006502. [Google Scholar] [CrossRef]
- Zhao, X.; Guan, J.L. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis. Adv. Drug Deliv. Rev. 2011, 63, 610–615. [Google Scholar] [CrossRef] [Green Version]
- Yu, J.; Bian, D.; Mahanivong, C.; Cheng, R.K.; Zhou, W.; Huang, S. p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression. J. Biol. Chem. 2004, 279, 50446–50454. [Google Scholar] [CrossRef] [Green Version]
- Mor, F.; Quintana, F.J.; Cohen, I.R. Angiogenesis-inflammation cross-talk: vascular endothelial growth factor is secreted by activated T cells and induces Th1 polarization. J. Immunol. 2004, 172, 4618–4623. [Google Scholar] [CrossRef] [Green Version]
- Coussens, L.M.; Werb, Z. Inflammation and cancer. Nature 2002, 420, 860–867. [Google Scholar] [CrossRef]
- Todoric, J.; Antonucci, L.; Karin, M. Targeting Inflammation in Cancer Prevention and Therapy. Cancer Prev. Res. (Phila) 2016, 9, 895–905. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rasool, M.; Sabina, E.P. Antiinflammatory effect of the Indian Ayurvedic herbal formulation Triphala on adjuvant-induced arthritis in mice. Phytother. Res. 2007, 21, 889–894. [Google Scholar] [CrossRef] [PubMed]
- Kalaiselvan, S.; Rasool, M. Triphala exhibits anti-arthritic effect by ameliorating bone and cartilage degradation in adjuvant-induced arthritic rats. Immunol. Invest. 2015, 44, 411–426. [Google Scholar] [CrossRef] [PubMed]
- Yang, M.H.; Ali, Z.; Khan, I.A.; Khan, S.I. Anti-inflammatory activity of constituents isolated from Terminalia chebula. Nat. Prod. Commun. 2014, 9, 965–968. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- BenSaad, L.A.; Kim, K.H.; Quah, C.C.; Kim, W.R.; Shahimi, M. Anti-inflammatory potential of ellagic acid, gallic acid and punicalagin A&B isolated from Punica granatum. BMC Complement. Altern. Med. 2017, 17, 47. [Google Scholar] [CrossRef] [Green Version]
- Loizou, S.; Lekakis, I.; Chrousos, G.P.; Moutsatsou, P. Beta-sitosterol exhibits anti-inflammatory activity in human aortic endothelial cells. Mol. Nutr. Food Res. 2010, 54, 551–558. [Google Scholar] [CrossRef] [PubMed]
- Rabelo, T.K.; Guimarães, A.G.; Oliveira, M.A.; Gasparotto, J.; Serafini, M.R.; de Souza Araújo, A.A.; Quintans-Júnior, L.J.; Moreira, J.C.F.; Gelain, D.P. Shikimic acid inhibits LPS-induced cellular pro-inflammatory cytokines and attenuates mechanical hyperalgesia in mice. Int. Immunopharmacol. 2016, 39, 97–105. [Google Scholar] [CrossRef]
- Huang, L.; Guan, T.; Qian, Y.; Huang, M.; Tang, X.; Li, Y.; Sun, H. Anti-inflammatory effects of maslinic acid, a natural triterpene, in cultured cortical astrocytes via suppression of nuclear factor-kappa B. Eur. J. Pharmacol. 2011, 672, 169–174. [Google Scholar] [CrossRef]
- Jin, F.; Cheng, D.; Tao, J.Y.; Zhang, S.L.; Pang, R.; Guo, Y.J.; Ye, P.; Dong, J.H.; Zhao, L. Anti-inflammatory and anti-oxidative effects of corilagin in a rat model of acute cholestasis. BMC Gastroenterol. 2013, 13, 79. [Google Scholar] [CrossRef] [Green Version]
- Jang, S.A.; Park, D.W.; Kwon, J.E.; Song, H.S.; Park, B.; Jeon, H.; Sohn, E.H.; Koo, H.J.; Kang, S.C. Quinic acid inhibits vascular inflammation in TNF-α-stimulated vascular smooth muscle cells. Biomed. Pharmacother. 2017, 96, 563–571. [Google Scholar] [CrossRef]
- DeLisser, H.M.; Christofidou-Solomidou, M.; Strieter, R.M.; Burdick, M.D.; Robinson, C.S.; Wexler, R.S.; Kerr, J.S.; Garlanda, C.; Merwin, J.R.; Madri, J.A.; et al. Involvement of endothelial PECAM-1/CD31 in angiogenesis. Am. J. Pathol. 1997, 151, 671–677. [Google Scholar] [PubMed]
- Nishiwaki, Y.; Yoshida, M.; Iwaguro, H.; Masuda, H.; Nitta, N.; Asahara, T.; Isobe, M. Endothelial E-selectin potentiates neovascularization via endothelial progenitor cell-dependent and -independent mechanisms. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 512–518. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lamalice, L.; Le Boeuf, F.; Huot, J. Endothelial cell migration during angiogenesis. Circ. Res. 2007, 100, 782–794. [Google Scholar] [CrossRef] [PubMed]
- Ucuzian, A.A.; Gassman, A.A.; East, A.T.; Greisler, H.P. Molecular mediators of angiogenesis. J. Burn Care Res. 2010, 31, 158–175. [Google Scholar] [CrossRef] [PubMed]
- Ribatti, D. Tumor refractoriness to anti-VEGF therapy. Oncotarget 2016, 7, 46668–46677. [Google Scholar] [CrossRef] [PubMed] [Green Version]
L1 | L2 | L3 | L4 | L5 | L6 | L7 | L8 | L9 | L10 | L11 | L12 | L13 | L14 | L15 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
T1 | −14.3 | −12.1 | −6 | −16.1 | −14.7 | −7.1 | −7.4 | −6.7 | −5.9 | −5.6 | −6 | −5 | −8.6 | −7.6 | −7.7 |
T2 | −13.6 | −11.6 | −5.7 | −14.4 | −13.1 | −6.5 | −6.9 | −6.9 | −5.5 | −5.5 | −5.6 | −4.2 | −7.8 | −7.4 | −8.1 |
T3 | −15.4 | −15.1 | −5 | −15.7 | −15.3 | −8 | −7.8 | −6 | −4.8 | −5 | −5 | −5.3 | −7.9 | −8.2 | −7.8 |
T4 | −17.9 | −15.2 | −6.2 | −17.4 | −16.6 | −7.5 | −8.4 | −6.7 | −5.7 | −5.7 | −5.6 | −5 | −8.7 | −7.7 | −7.4 |
T5 | −14.6 | −12.9 | −5.6 | −15 | −14.1 | −7.3 | −7.2 | −6.9 | −5.2 | −5.2 | −5.7 | −4.2 | −9 | −8.5 | −7.4 |
T6 | −13 | −12.6 | −7.1 | −15.3 | −14.5 | −6 | −10.6 | −6.8 | −7.1 | −7.2 | −7.6 | −6.2 | −8.1 | −8 | −7.5 |
T7 | −13 | −12 | −5.4 | −13.7 | −13.8 | −6.1 | −6.7 | −6.1 | −4.8 | −5 | −5.1 | −4 | −7.9 | −6.4 | −6.2 |
T8 | −20.4 | −14.6 | −6.2 | −15.1 | −14.1 | −8.6 | −8.7 | −8.2 | −6.7 | −6.7 | −6.4 | −6.8 | −7.6 | −8.9 | −8 |
T9 | −14.5 | −14.7 | −6.2 | −16.2 | −16 | −7.1 | −8.7 | −7.7 | −5.8 | −5.8 | −5.8 | −4.8 | −9.9 | −8.3 | −7.8 |
T10 | −14 | −12.7 | −6.5 | −14.4 | −13.1 | −7.7 | −7.9 | −6.9 | −6.5 | −6.5 | −6.9 | −5.5 | −8.5 | −7.9 | −7.5 |
T11 | −16.6 | −14.4 | −5.9 | −17.9 | −16.8 | −7.7 | −7.3 | −6.4 | −5.3 | −5.8 | −5.7 | −4.7 | −8.7 | −9.4 | −8.8 |
T12 | −13.7 | −12.2 | −5.7 | −15.8 | −14.9 | −7.2 | −9.2 | −6.7 | −5.2 | −5.9 | −5.3 | −5.3 | −8.7 | −8.3 | −7.9 |
T13 | −14.3 | −12.7 | −5.8 | −15 | −14.2 | −6.9 | −7.7 | −6.6 | −5.5 | −5.9 | −5.8 | −6.9 | −8.2 | −8.1 | −7.7 |
T14 | −15.3 | −13.2 | −5.6 | −15 | −14.2 | −8 | −8.4 | −7.4 | −5.6 | −5.6 | −5.9 | −6.6 | −8.1 | −9.1 | −8.2 |
T15 | −16.5 | −12.4 | −5.9 | −20 | −17.8 | −7.5 | −9.2 | −6.6 | −5.6 | −6.1 | −5.4 | −4.6 | −7.3 | −8.5 | −8.5 |
T16 | −14.9 | −12.7 | −5.5 | −15.2 | −13.6 | −6.5 | −8.1 | −7.4 | −5.4 | −5.1 | −5.7 | −4.3 | −8.8 | −7.9 | −7.9 |
T17 | −18.4 | −15.2 | −6.3 | −17.2 | −16.1 | −8.7 | −8.8 | −8.5 | 6.4 | −6.3 | −6.6 | −5.7 | −10 | −9.2 | −9.4 |
T18 | −12.7 | −10.8 | −5 | −13.3 | −12.5 | −6.7 | −7 | −6 | −4.8 | −4.8 | −5.4 | −3.4 | −7.1 | −7.2 | −7.3 |
T19 | −17.8 | −15.3 | −6.2 | −17.9 | −17.3 | −8 | −8.3 | −7.3 | −6 | −6.1 | −6.5 | −5.1 | −9.9 | −9.4 | −9.2 |
T20 | −18 | −16.9 | −7.1 | −19.5 | −17.3 | −9.4 | −8.8 | −7.6 | −6.6 | −7 | −7 | −6.3 | −9.7 | −9.1 | −9 |
T21 | −19.1 | −13.9 | −6.4 | −16.8 | −15.7 | −8.8 | −10.3 | −7.8 | −6.2 | −6.7 | −6.1 | −6.2 | −8.7 | −9.1 | −8.8 |
T22 | −12.1 | −11.1 | −5.1 | −12.6 | −12.3 | −6.2 | −7 | −5.5 | 4.6 | −5.2 | −4.5 | −5.2 | −6.9 | −7 | −6.9 |
T23 | −13.4 | −12.3 | −4.9 | −14.2 | −13.7 | −6.4 | −7 | −5.9 | −5 | −4.7 | −4.9 | −3.8 | −7.9 | −7.8 | −7.4 |
T24 | −17.1 | −14.9 | −6.9 | −18.4 | −18.4 | −8.8 | −8.2 | −7 | −7 | −7.1 | −7.4 | −5.9 | −9.4 | −9.5 | −8.7 |
T25 | −14.4 | −12 | −5.6 | −12.2 | −11.1 | −5.1 | −6.1 | −5.6 | −4.9 | −5 | −4.8 | −3.9 | −6.6 | −6.1 | −5.9 |
T26 | −16.5 | −13.7 | −7.2 | −17.1 | −16.5 | −7.9 | −9 | −7.7 | −6.9 | −6.9 | −7.1 | −5.5 | −8.7 | −8.5 | −8.3 |
T27 | −13.9 | −13.1 | −5.4 | −15.7 | −15.3 | −7.1 | −7 | −6.4 | −5 | −5.2 | −5 | −4.6 | −8.6 | −8.2 | −8.2 |
SI. No. | Phytocompound | PubChem ID | Prediction Efficacy (kcal/mol) |
---|---|---|---|
1. | Punicalagin | 44584733 | −424.8 |
2. | Chebulagic acid | 442674 | −414.3 |
3. | Isoterchebulin | 16143735 | −400.9 |
4. | Chebulinic acid | 72284 | −360.1 |
5. | Corilagin | 73568 | −226.3 |
6. | Ellagic acid | 5281855 | −217.1 |
7. | Maslinic acid | 73659 | −207.3 |
8. | Arjunolic acid | 73641 | −206.4 |
9. | Beta sitosterol | 222284 | −192.7 |
10. | Chebulic acid | 12302892 | −167.7 |
11. | Gallic acid | 370 | −72.3 |
12. | Dehydroshikimic acid | 5460360 | −66.6 |
13. | Quinic acid | 6508 | −61.6 |
14. | Shikimic acid | 8742 | −59.4 |
15. | Triacontanoic acid | 10471 | −39 |
Anti-Angiogenic Prediction Efficacy of Triphala | −436.7 |
L1 | L2 | L3 | L4 | L5 | L6 | L7 | L8 | L9 | L10 | L11 | L12 | L13 | L14 | L15 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
T1 | 16.1 | 13.7 | 6.1 | 17.1 | 15.8 | 6.7 | 23.8 | 6.9 | 6 | 6.1 | 6 | 4.2 | 9.5 | 8.9 | 7.9 |
T2 | 14.6 | 14.3 | 6.4 | 16.7 | 16.2 | 7.9 | 9 | 7 | 6.5 | 6.3 | 6.1 | 6.7 | 9.2 | 8.4 | 8.1 |
SI. No. | Phytocompound | PubChem ID | Prediction Efficacy (kcal/mol) |
---|---|---|---|
1. | Punicalagin | 44584733 | −33.8 |
2. | Isoterchebulin | 16143735 | −32 |
3. | Chebulagic acid | 442674 | −30.7 |
4. | Chebulinic acid | 72284 | −28 |
5. | Corilagin | 73568 | −18.7 |
6. | Maslinic acid | 73659 | −17.3 |
7. | Ellagic acid | 5281855 | −17 |
8. | Arjunolic acid | 73641 | −16 |
9. | Beta sitosterol | 222284 | −14.6 |
10. | Chebulic acid | 12302892 | −13.9 |
11. | Gallic acid | 370 | −12.5 |
12. | Shikimic acid | 8742 | −12.5 |
13. | Dehydroshikimic acid | 5460360 | −12.4 |
14. | Quinic acid | 6508 | −12.1 |
15. | Triacontanoic acid | 10471 | −6.7 |
Anti-Inflammation Prediction Efficacy of Triphala | −33.8 |
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Share and Cite
Abhinand, C.S.; Athira, P.A.; Soumya, S.J.; Sudhakaran, P.R. Multiple Targets Directed Multiple Ligands: An In Silico and In Vitro Approach to Evaluating the Effect of Triphala on Angiogenesis. Biomolecules 2020, 10, 177. https://doi.org/10.3390/biom10020177
Abhinand CS, Athira PA, Soumya SJ, Sudhakaran PR. Multiple Targets Directed Multiple Ligands: An In Silico and In Vitro Approach to Evaluating the Effect of Triphala on Angiogenesis. Biomolecules. 2020; 10(2):177. https://doi.org/10.3390/biom10020177
Chicago/Turabian StyleAbhinand, Chandran S., Prabhakaran A. Athira, Sasikumar J. Soumya, and Perumana R. Sudhakaran. 2020. "Multiple Targets Directed Multiple Ligands: An In Silico and In Vitro Approach to Evaluating the Effect of Triphala on Angiogenesis" Biomolecules 10, no. 2: 177. https://doi.org/10.3390/biom10020177