Exploring the underlying mechanism of action of a traditional Chinese medicine formula, Youdujing ointment, for cervical cancer treatment

Abstract

Background: A traditional Chinese medicine formula, Youdujing (YDJ) ointment, is widely used for treatment of human papilloma virus-related diseases, such as cervical cancer. However, the underlying mechanisms by which active compounds of YDJ alleviates cervical cancer are still unclear.

Methods: We applied a comprehensive network pharmacology approach to explore the key mechanisms of YDJ by integrating potential target identification, network analysis, and enrichment analysis into classical molecular docking procedures. First, we used network and enrichment analyses to identify potential therapeutic targets. Second, we performed molecular docking to investigate the potential active compounds of YDJ. Finally, we carried out a network-based analysis to unravel potentially effective drug combinations.

Results: Network analysis yielded four potential therapeutic targets: ESR1, NFKB1, TNF, and AKT1. Molecular docking highlighted that these proteins may interact with four potential active compounds of YDJ: E4, Y2, Y20, and Y21. Finally, we found that Y2 or Y21 can act alone or together with E4 to trigger apoptotic cascades via the mitochondrial apoptotic pathway and estrogen receptors.

Conclusion: Our study not only explained why YDJ is effective for cervical cancer treatment, but also lays a strong foundation for future clinical studies based on this traditional medicine.

Graphical abstract

Keywords

Youdujing / cervical cancer / traditional Chinese medicine / network pharmacological / molecular docking / synergy effect

References

[1] Cohen, P. A., Jhingran, A., Oaknin, A. and Denny, L. (2019) Cervical cancer. Lancet, 393, 169–182

[2] Wu, W., Song, L., Yang, Y., Wang, J., Liu, H. and Zhang, L. (2020) Exploring the dynamics and interplay of human papillomavirus and cervical tumorigenesis by integrating biological data into a mathematical model. BMC Bioinformatics, 21, 152

[3] Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A. and Jemal, A. (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, Ca-A Cancer J. Clin., 6, 394–424

[4] Cleveland, A. A., Gargano, J. W., Park, I. U., Griffin, M. R., Niccolai, L. M., Powell, M., Bennett, N. M., Saadeh, K., Pemmaraju, M., Higgins, K., (2020) Cervical adenocarcinoma in situ: Human papillomavirus types and incidence trends in five states, 2008-2015. Int. J. Cancer, 146, 810–818

[5] Roden, R. and Wu, T.-C. (2006) How will HPV vaccines affect cervical cancer? Nat. Rev. Cancer, 6, 753–763

[6] Vaccarella, S., Laversanne, M., Ferlay, J. and Bray, F. (2017) Cervical cancer in Africa, Latin America and the Caribbean and Asia: Regional inequalities and changing trends. Int. J. Cancer, 141, 1997–2001

[7] Toots, M., Ustav, M. Jr, Männik, A., Mumm, K., Tämm, K., Tamm, T., Ustav, E. and Ustav, M. (2017) Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay. PLoS Pathog., 13, e1006168

[8] Kolluru, S., Momoh, R., Lin, L., Mallareddy, J. R., and Krstenansky, J. L. (2019) Identification of potential binding pocket on viral oncoprotein HPV16 E6: a promising anti-cancer target for small molecule drug discovery. BMC Mol. Cell. Biol., 20, 30

[9] Celegato, M., Messa, L., Goracci, L., Mercorelli, B., Bertagnin, C., Spyrakis, F., Suarez, I., Cousido-Siah, A., Travé G., Banks, L., (2020) A novel small-molecule inhibitor of the human papillomavirus E6-p53 interaction that reactivates p53 function and blocks cancer cells growth. Cancer Lett., 470, 115–125

[10] Gyawali, B. and Iddawela, M. (2017) Bevacizumab in advanced cervical cancer: issues and challenges for low- and middle-income countries. J. Glob. Oncol., 3, 93–97

[11] Corr, B. R., Breed, C., Sheeder, J., Weisdack, S. and Behbakht, K. (2016) Bevacizumab induced hypertension in gynecologic cancer: Does it resolve after completion of therapy? Gynecol. Oncol. Rep., 17, 65–68

[12] Tewari, K. S., Sill, M. W., Penson, R. T., Huang, H., Ramondetta, L. M., Landrum, L. M., Oaknin, A., Reid, T. J., Leitao, M. M., Michael, H. E., (2017) Bevacizumab for advanced cervical cancer: final overall survival and adverse event analysis of a randomised, controlled, open-label, phase 3 trial (Gynecologic Oncology Group 240). Lancet, 390, 1654–1663

[13] Lin, J., Chen, L., Qiu, X., Zhang, N., Guo, Q., Wang, Y., Wang, M., Gober, H. J., Li, D. and Wang, L. (2017) Traditional Chinese medicine for human papillomavirus (HPV) infections: A systematic review. Biosci. Trends, 11, 267–273

[14] Chen, X., Hu, X., Liu, L., Liang, X. and Xiao, J. (2019) Extracts derived from a traditional Chinese herbal formula triggers necroptosis in ectocervical Ect1/E6E7 cells through activation of RIP1 kinase. J. Ethnopharmacol., 239, 111922

[15] Tang, F., Zhang, Q., Nie, Z., Yao, S. and Chen, B. (2009) Sample preparation for analyzing traditional Chinese medicines. Trends Analyt. Chem., 28, 1253–1262

[16] Gao, H., Wang, Z., Li, Y. and Qian, Z. (2011) Overview of the quality standard research of traditional Chinese medicine. Front. Med., 5, 195–202

[17] Xiao, J., Wu, J., and Yu, B. (2012) Therapeutic efficacy of Youdujing preparation in treating cervical high-risk human papilloma virus infection patients. Chinese journal of integrated traditional and Western medicine, 9, 1212–1215, in Chinese

[18] Cheng, F., Kovács, I. A. and Barabási, A.-L. (2019) Network-based prediction of drug combinations. Nat. Commun., 1, 1–11

[19] Zhang, L., Fu, C., Li, J., Zhao, Z., Hou, Y., Zhou, W. and Fu, A. (2019) Discovery of a ruthenium complex for the theranosis of glioma through targeting the mitochondrial DNA with bioinformatic methods. Int. J. Mol. Sci., 20, 4643

[20] Liu, G.-D., Li, Y.-C., Zhang, W. and Zhang, L. (2020) A brief review of artificial intelligence applications and algorithms for psychiatric disorders. Engineering (Beijing), 6, 462–467

[21] Kanehisa, M. and Goto, S. (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res., 28, 27–30

[22] Zhang, L., Qiao, M., Gao, H., Hu, B., Tan, H., Zhou, X. and Li, C. M. (2016) Investigation of mechanism of bone regeneration in a porous biodegradable calcium phosphate (CaP) scaffold by a combination of a multi-scale agent-based model and experimental optimization/validation. Nanoscale, 8, 14877–14887

[23] Zhang, L., Liu, Y., Wang, M., Wu, Z., Li, N., Zhang, J. and Yang, C. (2017) EZH2-, CHD4-, and IDH-linked epigenetic perturbation and its association with survival in glioma patients. J. Mol. Cell Biol., 9, 477–488

[24] Zhang, L. and Zhang, S. (2017) Using game theory to investigate the epigenetic control mechanisms of embryo development: Comment on: “Epigenetic game theory: How to compute the epigenetic control of maternal-to-zygotic transition” by Qian Wang et al. Phys. Life Rev., 20, 140–142

[25] Zhang, L., Zheng, C., Li, T., Xing, L., Zeng, H., Li, T., Yang, H., Cao, J., Chen, B. and Zhou, Z. (2017) Building up a robust risk mathematical platform to predict colorectal cancer. Complexity, 2017, 1–14

[26] Zhang, L., Xiao, M., Zhou, J. and Yu, J. (2018) Lineage-associated underrepresented permutations (LAUPs) of mammalian genomic sequences based on a Jellyfish-based LAUPs analysis application (JBLA). Bioinformatics, 34, 3624–3630

[27] Xiao, M., Yang, X., Yu, J., and Zhang, L. (2019) CGIDLA: Developing the Web Server for CpG Island related Density and LAUPs (Lineage-associated Underrepresented Permutations) Study, IEEE/ACM Trans. Comput. Biol. Bioinform. 17, 2148–2154

[28] Zhang, L., Bai, W., Yuan, N. and Du, Z. (2019) Comprehensively benchmarking applications for detecting copy number variation. PLOS Comput. Biol., 15, e1007069

[29] Zhang, L., Liu, G., Kong, M., Li, T., Wu, D., Zhou, X., Yang, C., Xia, L., Yang, Z. and Chen, L. (2019) Revealing dynamic regulations and the related key proteins of myeloma-initiating cells by integrating experimental data into a systems biological model. Bioinformatics, btz542

[30] Trott, O. and Olson, A. J. (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem, 31, 455–461

[31] Brake, T. and Lambert, P. F. (2005) Estrogen contributes to the onset, persistence, and malignant progression of cervical cancer in a human papillomavirus-transgenic mouse model. Proc. Natl. Acad. Sci. USA, 102, 2490–2495

[32] James, M. A., Lee, J. H. and Klingelhutz, A. J. (2006) Human papillomavirus type 16 E6 activates NF-kappaB, induces cIAP-2 expression, and protects against apoptosis in a PDZ binding motif-dependent manner. J. Virol., 80, 5301–5307

[33] Beevi, S. S., Rasheed, M. H. and Geetha, A. (2007) Evidence of oxidative and nitrosative stress in patients with cervical squamous cell carcinoma. Clin. Chim. Acta, 375, 119–123

[34] Cui, N., Yang, W. T. and Zheng, P. S. (2016) Slug inhibits the proliferation and tumor formation of human cervical cancer cells by up-regulating the p21/p27 proteins and down-regulating the activity of the Wnt/beta-catenin signaling pathway via the trans-suppression Akt1/p-Akt1 expression, Oncotarget. Article, 7, 26152–26167

[35] Rasul, A., Millimouno, F. M., Ali Eltayb, W., Ali, M., Li, J. and Li, X. (2013) Pinocembrin: a novel natural compound with versatile pharmacological and biological activities. BioMed Res. Int., 2013, 379850

[36] Rajavel, T., Mohankumar, R., Archunan, G., Ruckmani, K. and Devi, K. P. (2017) Beta sitosterol and Daucosterol (phytosterols identified in Grewia tiliaefolia) perturbs cell cycle and induces apoptotic cell death in A549 cells. Sci. Rep., 7, 3418

[37] Wang, G., Wang, Y.-Z., Yu, Y., Wang, J.-J., Yin, P.-H. and Xu, K. (2019) Triterpenoids extracted from Rhus chinensis Mill act against colorectal cancer by inhibiting enzymes in glycolysis and glutaminolysis: network analysis and experimental validation. Nutr. Cancer, 72, 293–319

[38] Kumar, M. S., Nair, M., Hema, P., Mohan, J., and Santhoshkumar, T. (2007) Pinocembrin triggers Bax-dependent mitochondrial apoptosis in colon cancer cells. Mol. Carcinog., 3, 231-241. Mol Carcinog., 46, 231–41

[39] Galluzzo, P., Martini, C., Bulzomi, P., Leone, S., Bolli, A., Pallottini, V. and Marino, M. (2009) Quercetin-induced apoptotic cascade in cancer cells: antioxidant versus estrogen receptor α-dependent mechanisms. Mol. Nutr. Food Res., 53, 699–708

[40] Miller, D. W. (2012) Improving potency and ADMET properties using matched molecular pair analysis. Abstracts of Papers of the American Chemical Society

[41] Safran, M., Dalah, I., Alexander, J., Rosen, N., Stein, T., Shmoish, M., Nativ, N., Bahir, I., Doniger, T., Krug, H., (2010) GeneCards Version 3: the human gene integrator. Database, 2010, baq020

[42] Huang, W., Sherman, B. T. and Lempicki, R. A. (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc., 4, 44–57

[43] Walboomers, J. M. M., Jacobs, M.V., Manos, M.M., Bosch, F.X., Kummer, J.A., Shah, K.V., Snijders, P.J., Peto, J., Meijer, C.J., and Muñoz, N., (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol., 189, 12–19

[44] Moktar, A., Singh, R., Vadhanam, M. V., Ravoori, S., Lillard, J. W., Gairola, C. G. and Gupta, R. C. (2011) Cigarette smoke condensate-induced oxidative DNA damage and its removal in human cervical cancer cells. Int J Oncol, 39, 941–947

[45] Wang, S. S., Zuna, R. E., Wentzensen, N., Dunn, S. T., Sherman, M. E., Gold, M. A., Schiffman, M., Wacholder, S., Allen, R. A., Block, I., (2009) Human papillomavirus cofactors by disease progression and human papillomavirus types in the study to understand cervical cancer early endpoints and determinants. Cancer Epidemiol. Biomarkers Prev., 18, 113–120

[46] Chung, S.-H., Franceschi, S. and Lambert, P. F. (2010) Estrogen and ERalpha: culprits in cervical cancer? Trends Endocrinol. Metab., 21, 504–511

[47] De Savi, C., Bradbury, R. H., Rabow, A. A., Norman, R. A., de Almeida, C., Andrews, D. M., Ballard, P., Buttar, D., Callis, R. J., Currie, G. S., (2015) Optimization of a novel binding motif to (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic Acid (AZD9496), a potent and orally bioavailable selective estrogen receptor downregulator and antagonist. J. Med. Chem., 58, 8128–8140

[48] He, M. M., Smith, A. S., Oslob, J. D., Flanagan, W. M., Braisted, A. C., Whitty, A., Cancilla, M. T., Wang, J., Lugovskoy, A. A., Yoburn, J. C., (2005) Small-molecule inhibition of TNF-α. Science, 310, 1022–1025

[49] Blake, J. F., Kallan, N. C., Xiao, D., Xu, R., Bencsik, J. R., Skelton, N. J., Spencer, K. L., Mitchell, I. S., Woessner, R. D., Gloor, S. L., (2010) Discovery of pyrrolopyrimidine inhibitors of Akt. Bioorg. Med. Chem. Lett., 20, 5607–5612

[50] Park, H., Shin, Y., Choe, H. and Hong, S. (2015) Computational design and discovery of nanomolar inhibitors of IκB kinase β. J. Am. Chem. Soc., 137, 337–348

[51] Chen, L., Du, J., Dai, Q., Zhang, H., Pang, W., and Hu, J., (2014) Prediction of anti-tumor chemical probes of a traditional Chinese medicine formula by HPLC fingerprinting combined with molecular docking. Eur. J. Med. Chem., 83, 294–306

[52] Rajavel, T., Banu Priya, G., Suryanarayanan, V., Singh, S. K. and Pandima Devi, K., (2019) Daucosterol disturbs redox homeostasis and elicits oxidative-stress mediated apoptosis in A549 cells via targeting thioredoxin reductase by a p53 dependent mechanism. Eur J Pharmacol, 855, 112–123

[53] Zhao, C., She, T., Wang, L., Su, Y., Qu, L., Gao, Y., Xu, S., Cai, S. and Shou, C. (2015) Daucosterol inhibits cancer cell proliferation by inducing autophagy through reactive oxygen species-dependent manner. Life Sci., 137, 37–43

[54] Wang, G., Wang, Y.-Z., Yu, Y. and Wang, J.-J. (2019) Inhibitory ASIC2-mediated calcineurin/NFAT against colorectal cancer by triterpenoids extracted from Rhus chinensis Mill. J Ethnopharmacol, 235, 255–267

[55] Zheng, Y., Wang, K., Wu, Y., Chen, Y., Chen, X., Hu, C. W. and Hu, F. (2018) Pinocembrin induces ER stress mediated apoptosis and suppresses autophagy in melanoma cells. Cancer Lett, 431, 31–42

[56] Cokol, M., Chua, H. N., Tasan, M., Mutlu, B., Weinstein, Z. B., Suzuki, Y., Nergiz, M. E., Costanzo, M., Baryshnikova, A., Giaever, G., (2011) Systematic exploration of synergistic drug pairs. Mol. Syst. Biol., 7, 544

[57] Zou, J., Ji, P., Zhao, Y. L., Li, L. L., Wei, Y. Q., Chen, Y. Z. and Yang, S. Y. (2012) Neighbor communities in drug combination networks characterize synergistic effect. Mol. Biosyst., 8, 3185–3196

[58] den Boon, J. A., Pyeon, D., Wang, S. S., Horswill, M., Schiffman, M., Sherman, M., Zuna, R. E., Wang, Z., Hewitt, S. M., Pearson, R., (2015) Molecular transitions from papillomavirus infection to cervical precancer and cancer: Role of stromal estrogen receptor signaling. Proc. Natl. Acad. Sci. USA, 112, E3255–E3264

[59] Chung, S.-H. and Lambert, P. F. (2009) Prevention and treatment of cervical cancer in mice using estrogen receptor antagonists. Proc. Natl. Acad. Sci. USA, 106, 19467–19472

[60] Naka, T., Sugamura, K., Hylander, B. L., Widmer, M. B., Rustum, Y. M. and Repasky, E. A. (2002) Effects of tumor necrosis factor-related apoptosis-inducing ligand alone and in combination with chemotherapeutic agents on patients’ colon tumors grown in SCID mice. Cancer Res, 62, 5800–5806

[61] Chang, K. J.Reid, T., Senzer, N., Swisher, S., Pinto, H., Hanna, N., Chak, A., and Soetikno, R., (2012) Phase I evaluation of TNFerade biologic plus chemoradiotherapy before esophagectomy for locally advanced resectable esophageal cancer. Gastrointes. Endos., 6, 1139–1146. e1132

[62] D’Archivio, M., Santangelo, C., Scazzocchio, B., Varì R., Filesi, C., Masella, R. and Giovannini, C. (2008) Modulatory effects of polyphenols on apoptosis induction: relevance for cancer prevention. Int. J. Mol. Sci., 9, 213–228

[63] Gopalakrishnan, A. and Tony Kong, A. N. (2008) Anticarcinogenesis by dietary phytochemicals: cytoprotection by Nrf2 in normal cells and cytotoxicity by modulation of transcription factors NF-κB and AP-1 in abnormal cancer cells. Food Chem. Toxicol., 46, 1257–1270

[64] Zhao, J., Dasmahapatra, A. K., Khan, S. I. and Khan, I. A. (2008) Anti-aromatase activity of the constituents from damiana (Turnera diffusa). J Ethnopharmacol, 120, 387–393

[65] Wang, L., Zhou, G. B., Liu, P., Song, J. H., Liang, Y., Yan, X. J., Xu, F., Wang, B. S., Mao, J. H., Shen, Z. X., (2008) Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia. Proc. Natl. Acad. Sci. USA, 105, 4826–4831

[66] Tian, P. (2011) Convergence: Where West meets East. Nature, 480, S84–S86

[67] Chen, Z., Rasul, A., Zhao, C., Millimouno, F. M., Tsuji, I., Yamamura, T., Iqbal, R., Malhi, M., Li, X. and Li, J., (2013) Antiproliferative and apoptotic effects of pinocembrin in human prostate cancer cells. Bangladesh J. Pharmacol., 3, 255–262

[68] Zhang, L., Dai, Z., Yu, J. and Xiao, M. (2020) CpG-island-based annotation and analysis of human housekeeping genes. Brief. Bioinform. bbz134

[69] Xu, H.-Y., Zhang, Y. Q., Liu, Z. M., Chen, T., Lv, C. Y., Tang, S. H., Zhang, X. B., Zhang, W., Li, Z. Y., Zhou, R. R., (2019) ETCM: an encyclopaedia of traditional Chinese medicine. Nucleic Acids Res., 47, D976–D982

[70] Xue, L., Godden, J. W., Stahura, F. L., Bajorath, J. (2003) Design and evaluation of a molecular fingerprint involving the transformation of property descriptor values into a binary classification scheme. J. Chem. Inf. Comput. Sci., 4, 1151–1157

[71] Cereto-Massagué A., Ojeda, M. J., Valls, C., Mulero, M., Garcia-Vallvé S. and Pujadas, G. J. M. (2015) Molecular fingerprint similarity search in virtual screening. Methods, 71, 58–63

[72] Wishart, D. S.,Feunang, Y.D., Guo, A.C., Lo, E.J., Marcu, A., Grant, J.R., Sajed, T., Johnson, D., Li, C., Sayeeda, Z., (2018) DrugBank 5.0: a major update to the DrugBank database for 2018. Nucleic Acids Res., 46(D1), D1074–D1082

[73] Szklarczyk, D., Gable, A. L., Lyon, D., Junge, A., Wyder, S., Huerta-Cepas, J., Simonovic, M., Doncheva, N. T., Morris, J. H., Bork, P., (2019) STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res., 47, D607–D613

[74] Shannon, P., Markiel, A., Ozier, O., Baliga, N. S., Wang, J. T., Ramage, D., Amin, N., Schwikowski, B. and Ideker, T. (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res., 13, 2498– 2504

[75] Barabási, A.-L. and Oltvai, Z. N. (2004) Network biology: understanding the cell’s functional organization. Nat. Rev. Genet., 5, 101–113

[76] Albert, R., Jeong, H. and Barabási, A.-L. (2000) Error and attack tolerance of complex networks. Nature. 6794, 378–382

[77] Lamb, J., Crawford, E.D., Peck, D., Modell, J.W., Blat, I.C., Wrobel, M.J., Lerner, J., Brunet, J.P., Subramanian, A., Ross, K.N., (2006) The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science, 5795, 1929–1935

[78] Yu, H., Kim, P. M., Sprecher, E., Trifonov, V. and Gerstein, M. (2007) The importance of bottlenecks in protein networks: correlation with gene essentiality and expression dynamics. PLOS Comput. Biol., 3, e59

[79] Goñi, J., Esteban, F. J., de Mendizábal, N. V., Sepulcre, J., Ardanza-Trevijano, S., Agirrezabal, I. and Villoslada, P. (2008) A computational analysis of protein-protein interaction networks in neurodegenerative diseases. BMC Syst. Biol., 2, 52

[80] Consortium, G. O. (2004) The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res., suppl_1, D258–D261

[81] Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S. and Olson, A. J. (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem., 30, 2785–2791

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