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.