Abstract
Harmine (HM), a natural β-carboline alkaloid derived from the plant Peganum harmala, has a range of pharmacological effects, including anti-inflammatory, neuroprotective, antidiabetic, and antitumor effects. However, the potential targets involved in its therapeutic effects on gastric cancer (GC) remain unclear. In this study, the anti-gastric cancer effects of HM were investigated and HSP90AA1 was identified as its molecular target. In vitro experiments demonstrated that HM significantly inhibited the proliferation, migration, and invasion of GC cells and induced GC cell apoptosis. By integrating data from multiple databases and from pull-down assays and mass spectrometry analyses, 25 key GC-related targets were identified. A protein‒protein interaction (PPI) network was constructed, and ten core targets were prioritized using the maximal clique centrality (MCC) algorithm. Functional enrichment analysis revealed relevant biological processes and pathways, highlighting the multitarget anticancer mechanism of HM. Molecular docking analysis of the interactions between HM and the ten core targets resulted in the selection of heat shock protein 90 alpha family class A member 1 (HSP90AA1) as a candidate for further investigation. Quantitative real-time PCR (RT‒qPCR) and Western blot (WB) assays demonstrated that HM treatment significantly decreased the mRNA and protein expression levels of HSP90AA1 in GC cells. Immunofluorescence staining revealed high expression of the HSP90AA1 protein in tumor tissues from the HM-treated group in a mouse xenograft model. The binding affinity between HM and HSP90AA1 was validated as moderate using surface plasmon resonance (SPR) and microscale thermophoresis (MST) assays, further confirming that HSP90AA1 is a key binding target of HM in GC. To elucidate the functional role of HSP90AA1, lentivirus-mediated small interfering RNA (siRNA) was used to generate HSP90AA1-knockdown GC cells, and their functional responses to HM treatment were subsequently examined. The results showed that knockdown of HSP90AA1 inhibited the proliferation, migration, and invasion of GC cells. Combined treatment with HM and HSP90AA1 knockdown further suppressed cell migration, but no significant synergistic effects on proliferation or invasion were observed. In conclusion, the results of this study demonstrate that HM exerts significant anti-GC effects, and HSP90AA1 was identified as a critical binding target that mediates the anti-tumor activity of HM. These findings provide important insights into the potential therapeutic application of HM in GC and support further investigations into personalized treatment strategies.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
References
Sung, H. et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA. Cancer. J. Clin. 71, 209–249 (2021).
Smyth, E. C., Nilsson, M., Grabsch, H. I., van Grieken, N. C. & Lordick, F. Gastric cancer. Lancet 396, 635–648 (2020).
Matsuoka, T. & Yashiro, M. Biomarkers of gastric cancer: Current topics and future perspective. World J. Gastroenterol. 24, 2818–2832 (2018).
Winkler, J., Abisoye-Ogunniyan, A., Metcalf, K. J. & Werb, Z. Concepts of extracellular matrix remodelling in tumour progression and metastasis. Nat. Commun. 11, 5120 (2020).
Correa, P. Gastric cancer: Overview. Gastroenterol. Clin. North Am. 42, 211–217 (2013).
Research Overview of Camelthorn Foreign. Medicine (Herbal Medicine Volume)[in Chinese] 104–107. (1992).
Medicinal Plant Species and Distribution of the Genus Thalictrum in Northwest China. Chin. Med. Materials[in Chi-nese] 336–337. https://doi.org/10.13863/j.issn1001-4454.1996.07.005 (1996).
Patel, K., Gadewar, M., Tripathi, R., Prasad, S. & Patel, D. K. A review on medicinal importance, pharmacological activity and bioanalytical aspects of beta-carboline alkaloid “Harmine”. Asian Pac. J. Trop. Biomed. 2, 660–664 (2012).
Zhang, L., Li, D. & Yu, S. Pharmacological effects of harmine and its derivatives: A review. Arch. Pharm. Res. 43, 1259–1275 (2020).
Zhang, X.-F. et al. Synthesis and mechanisms of action of novel harmine derivatives as potential antitumor agents. Sci. Rep. 6, 33204 (2016).
Timbilla, A. A. et al. The anticancer properties of harmine and its derivatives. Phytochem. Rev. 24, 1535–1564 (2025).
Li, C. et al. Anticancer activities of harmine by inducing a pro-death autophagy and apoptosis in human gastric cancer cells. Phytomedicine 28, 10–18 (2017).
Hopkins, A. L. Network pharmacology: The next paradigm in drug discovery. Nat. Chem. Biol. 4, 682–690 (2008).
Boezio, B., Audouze, K., Ducrot, P. & Taboureau, O. Network-based approaches in pharmacology. Mol. Inform. https://doi.org/10.1002/minf.201700048 (2017).
Zhao, L. et al. Network pharmacology, a promising approach to reveal the pharmacology mechanism of Chinese medicine formula. J. Ethnopharmacol. 309, 116306 (2023).
Jiao, X. et al. A comprehensive application: Molecular docking and network pharmacology for the prediction of bioactive constituents and elucidation of mechanisms of action in component-based Chinese medicine. Comput. Biol. Chem. 90, 107402 (2021).
Englebienne, P., Hoonacker, A. V. & Verhas, M. Surface plasmon resonance: Principles, methods and applications in biomedical sciences. J. Spectrosc. 17, 372913 (2003).
Mariani, S. & Minunni, M. Surface plasmon resonance applications in clinical analysis. Anal. Bioanal. Chem. 406, 2303–2323 (2014).
Duhr, S. & Braun, D. Why molecules move along a temperature gradient. Proc. Natl. Acad. Sci. U.S.A. 103, 19678–19682 (2006).
Jerabek-Willemsen, M. et al. MicroScale Thermophoresis: Interaction analysis and beyond. J. Mol. Struct. 1077, 101–113 (2014).
Entzian, C. & Schubert, T. Studying small molecule–aptamer interactions using MicroScale Thermophoresis (MST). Methods 97, 27–34 (2016).
Wienken, C. J., Baaske, P., Rothbauer, U., Braun, D. & Duhr, S. Protein-binding assays in biological liquids using MicroScale Thermophoresis. Nat. Commun. 1, 100 (2010).
Jerabek-Willemsen, M., Wienken, C. J., Braun, D., Baaske, P. & Duhr, S. Molecular interaction studies using MicroScale Thermophoresis. Assay Drug Dev. Technol. 9, 342–353 (2011).
Zhang, L. et al. Harmine suppresses homologous recombination repair and inhibits proliferation of hepatoma cells. Cancer Biol. Ther. 16, 1585–1592 (2015).
Uhl, K. L., Schultz, C. R., Geerts, D. & Bachmann, A. S. Harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor induces caspase-mediated apoptosis in neuroblastoma. Cancer Cell Int. 18, 82 (2018).
Tarpley, M. et al. Identification of Harmine and β-carboline analogs from a high-throughput screen of an approved drug collection; profiling as differential inhibitors of DYRK1A and Monoamine oxidase A and for in vitro and in vivo anti-cancer studies. Eur. J. Pharm. Sci. 162, 105821 (2021).
Chen, Z.-Y. et al. Harmine reinforces the effects of regorafenib on suppressing cell proliferation and inducing apoptosis in liver cancer cells. Exp. Ther. Med. 23, 209 (2022).
Chen, L. et al. Gene ontology and KEGG pathway enrichment analysis of a drug target-based classification system. PLoS One 10, e0126492 (2015).
Kanehisa, M. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 28, 1947–1951 (2019).
Ziaka, K. & van der Spuy, J. The role of Hsp90 in retinal proteostasis and disease. Biomolecules 12, 978 (2022).
Deng, Z. H. et al. Mesenchymal stem cell-derived exosomes ameliorate hypoxic pulmonary hypertension by inhibiting the Hsp90aa1/ERK/pERK pathway. Biochem. Pharmacol. 226, 116382 (2024).
Yuan, Z., Wang, L. & Chen, C. Analysis of the prognostic, diagnostic and immunological role of HSP90α in malignant tumors. Front. Oncol. 12, 963719 (2022).
Zhang, M. et al. DAB2IP down-regulates HSP90AA1 to inhibit the malignant biological behaviors of colorectal cancer. BMC Cancer 22, 561 (2022).
Wang, Z. et al. HSP90AA1 is an unfavorable prognostic factor for hepatocellular carcinoma and contributes to tumorigenesis and chemotherapy resistance. Transl. Oncol. 50, 102148 (2024).
Xiang, S., Zhang, W., Wang, Z., Chen, H. & Yang, C. Evaluating HSP90AA1 as a predictive biomarker for prognosis in lung adenocarcinoma. Transl. Cancer Res. 14, 2580–2593 (2025).
Li, Y. et al. Mulberrin suppresses gastric cancer progression and enhances chemosensitivity to oxaliplatin through HSP90AA1/PI3K/AKT axis. Phytomedicine 139, 156441 (2025).
Pratt, W. B. The hsp90-based chaperone system: Involvement in signal transduction from a variety of hormone and growth factor receptors. Proc. Soc. Exp. Biol. Med. 217, 420–434 (1998).
Zuehlke, A. D., Beebe, K., Neckers, L. & Prince, T. Regulation and function of the human HSP90AA1 gene. Gene 570, 8–16 (2015).
Xiao, X. et al. HSP90AA1-mediated autophagy promotes drug resistance in osteosarcoma. J. Exp. Clin. Cancer. Res. 37, 201 (2018).
Tang, F. et al. HSP90AA1 promotes lymphatic metastasis of hypopharyngeal squamous cell carcinoma by regulating epithelial-mesenchymal transition. Oncol. Res. 31, 787–803 (2023).
Daina, A., Michielin, O. & Zoete, V. SwissTargetPrediction: Updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Res. 47, W357–W364 (2019).
Wang, X. et al. PharmMapper 2017 update: A web server for potential drug target identification with a comprehensive target pharmacophore database. Nucleic Acids Res. 45, W356–W360 (2017).
Safran, M. et al. GeneCards Version 3: the human gene integrator. Database (Oxford) baq020 (2010). (2010).
Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30 (2000).
Morris, G. M. et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).
Acknowledgements
The authors thanks Dr. Yang Yu for guidance with the pharmacology network analysis.
Funding
This work was supported by the Gansu Provincial Health and Health Industry Research Plan Management Project (Grant No. GSWSKY2020-60), the Gansu Provincial Science and Technology Department Key R&D Program - Social Development Project (Grant No. 23YFFA0067), the Gansu University of Chinese Medicine Open Subjects of Gansu Traditional Chinese Medicine Research Center (Grant No.zyzx-2023-15), and the Gansu Provincial Higher Education Innovation Fund Project (Grant No. 2022 A-071), the Lanzhou City Science and Technology Development Plan Project (Grant No. 2021-1-99), the Research Project on COVID-19 Prevention and Control Technologies (Grant No. 2020-XG-27), the Scientific Research and Innovation Fund Project of Gansu University of Chinese Medicine (Grant No. 2019KC2D-1), the Open Fund Project of the Provincial Key Laboratory for Molecular Medicine and TCM Prevention and Treatment of Major Diseases in Higher Education Institutions in Gansu Province (Grant No. FZYX15-8), the Research Project of Higher Education Institutions in Gansu Province (Grant No. 2016 A-045), the Gansu Provincial Natural Science Foundation (Grant No. 17JR5A169), the Technical Research and Development Special Plan of the Gansu Provincial Department of Science and Technology (Grant No. 1105TCYA019), the Gansu Province Clinical Medicine Research Centers Support Program (Grant Nos. 18JR2FA002 and 21JR7RA682), and the National-Level TCM Superior Specialty Construction Project for Gastroenterology and Ac.
Author information
Authors and Affiliations
Contributions
Y-HH and YL contributed equally to the work and should be regarded as co-first authors. Y-HH and HC are cocorresponding authors. The other authors have no conflicts of interest to declare.Y-HH: Conceptualization, Methodology, Writing – original draft, Writing – review & editing. YL: Conceptualization, Data curation, Writing – original draft, Writing – review & editing. H-XW: Writing – original draft. S-JM: Writing – original draft. J-HL Writing – review & editing. Q-LW: Writing – review & editing. H-YL: Writing – original draft & Software. Z-JH: Visualization, Writing – original draft, Writing – review & editing. HC: Writing – review & editing.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Ethics
The animal study was approved by the Animal Experimental Ethics Committee of Gansu University of Chinese Medicine (Approval Number 2021–821), and all procedures were conducted in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Hu, Y., Li, Y., Wang, H. et al. Network pharmacology analysis and experimental validation of the gastric cancer-related targets of harmine. Sci Rep (2026). https://doi.org/10.1038/s41598-026-45985-1
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-45985-1
