Solasodine inhibited the proliferation of gastric cancer cells through suppression of Hedgehog/Gli1 signaling.

Introduction
Gastric cancer remains a global health issue, ranking fifth in incidence (4.9%) and fourth in cancer-related mortality (6.8%) among all cancer types (Bray et al., 2024). Due to its asymptomatic nature in the early stages, gastric cancer is often diagnosed at an advanced stage, resulting in a poor prognosis despite advancements in diagnosis and treatment (Machlowska et al., 2020). The overall five-year survival rate remains at 36%, dropping to just 7.0% for cases with distant metastases (Li et al., 2022b). Despite significant advancements in gastric cancer treatment, further research is needed to enhance early detection through specific molecular biomarkers, overcome drug resistance, and reduce adverse side effects (Guan et al., 2023). These unresolved challenges underscore the need for novel therapeutic agents to prevent gastric cancer.
Hedgehog (Hh) signaling plays a crucial role in embryonic development, tissue homeostasis, and cellular differentiation (Jiang, 2022). It is activated upon the presence of ligands such as Desert hedgehog (Dhh), Indian hedgehog (Ihh), and Sonic hedgehog (Shh). Upon ligand binding to the receptor Patched (Ptch), Smoothened (Smo) is released, triggering the activation of glioma-associated oncogene (Gli) transcription factors, Gli1 and Gli2, which then translocate to the nucleus to regulate downstream target genes (Zhou et al., 2022a). However, constitutive Hh signaling activation has been mechanistically linked to gastric cancer. Gli1, the key mediator of Hh signaling, is closely associated with gastric cancer malignancy, particularly in cases with lymph node metastasis (Dong et al., 2019). The overexpression of Gli1 and Shh is essential for CD44⁺ gastric cancer stem cells to sustain their stem-like phenotype and malignant transformation potential (Xu et al., 2015). Hh signaling also plays a key role in gastric cancer resistance, as studies have shown that its inhibition can overcome resistance to chemotherapy drugs, such as cisplatin (Yao et al., 2019). Hence, targeting the Hh signaling represents a promising strategy for suppressing gastric cancer progression.
Fruit and vegetable consumption has been inversely associated with gastric cancer risk, largely due to their rich content of phytochemicals with antioxidant and anticancer properties (Ferro et al., 2020; Mi et al., 2025; Park et al., 2025). In fact, phytochemicals that inhibit Hh signaling have been shown to suppress cancer progression (Bao et al., 2018). For instance, sulforaphene from cruciferous vegetables has been reported to inhibit breast cancer migration and invasion through Hh signaling (Bao et al., 2016), while garcinone C from mangosteen has been demonstrated to suppress the growth of colon cancer stem cells (Zhou et al., 2022b). Notably, solasodine, a naturally occurring steroidal alkaloid abundant in Solanaceae plants such as eggplant, tomato, and potato, has shown potent antitumor effects in colorectal cancer, breast cancer, and ovarian cancers through diverse mechanisms (Chen et al., 2022; Xu et al., 2017; Zhuang et al., 2017). However, the mechanisms underlying the effects of solasodine in gastric cancer remain unclear. In this study, we investigated the effects of solasodine on gastric cancer cells and elucidated its regulatory impact on Hh signaling pathway.

Materials and methods

Reagents and cell culture
Solasodine (purity > 98%) was purchased from Chengdu Alpha Company (Chengdu, China). Gant61 and vismodegib were ordered from Millipore (Darmstadt, Germany) and LC Laboratories (Woburn, MA, USA), respectively. Solasodine, Gant61, and vismodegib were dissolved in DMSO and diluted in culture medium before use. The final DMSO concentration was kept below 0.1% in all experiments. AGS and MKN74 gastric cancer cell lines were acquired from the Korean Cell Line Bank. Cells were maintained in RPMI-1640 medium (Gibco, MD, USA) supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaillé, France) and 1% penicillin–streptomycin (P/S, Gibco, MD, USA) and incubated at 37 °C in a humidified incubator with 5% CO₂. The Ptch⁻/⁻ mouse embryonic fibroblast (MEF) cells were cultured in high-glucose DMEM (Gibco) supplemented with 10% FBS (Biowest) and 1% P/S (Gibco) and incubated at 37°C in a humidified incubator with 5% CO₂ (Bao et al., 2014).

Cell viability assay
Cells were seeded into 96-well plates at a density of 3,000 cells per well in 200 μL of culture medium. After 24 h of incubation at 37 °C, cells were treated with various concentrations of compounds for 72 h. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and viability was calculated relative to the control group.

Colony formation assay
AGS and MKN74 cells were seeded at a density of 1,000 cells per well in 12-well plates and allowed to adhere for 24 h. Cells were then treated with the respective compounds for 1–2 weeks, with media changes every three days. After treatment, cells were washed twice with phosphate-buffered saline (PBS), fixed with 10% formalin for 10 min, and stained with 0.5% crystal violet for 5 min at room temperature. Visible colonies were counted for analysis.

Western blot analysis
Protein samples were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon®-P, 0.45 µm, Merck Millipore, Darmstadt, Germany; Cat# IPVH00010) using a wet transfer system, according to the manufacturer’s protocol. Then, the membranes were blocked with 5% skim milk in TBST (1 × TBST containing 0.1% Tween-20) for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C with gentle agitation, followed by three washes with TBST. Membranes were then incubated with the appropriate secondary antibodies for 2 h at room temperature, washed three more times, and developed using HRP substrate (Thermo fisher, MA, USA). Primary antibodies for Cyclin D1, p27, Smo and secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for CDK2, Gli1, Gli2, Bax, Bcl-2, and β-Actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Confocal microscopy analysis
AGS and MKN74 cells were seeded in 3.5 cm glass-bottom dishes at a density of 4 × 106 cells per dish and incubated for 24 h. After overnight starvation, cells were treated with garcinone C, Gant61, or vismodegib for 24 h. Cells were then washed once with PBS and fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. Following two washes with PBS, cells were blocked with 5% bovine serum albumin for 1 h and incubated overnight with the primary antibody. After three PBS washes, cells were incubated with fluorescently labeled secondary antibodies for 1 h in the dark. After an additional PBS wash, cells were counterstained with DAPI mounting solution (Vector Laboratories, USA). Images were captured using a confocal microscope (Zeiss, Germany) at 400 × magnification.

Pharmacology networking
Solasodine target prediction was predicted using the SuperPred 3 online platform (http://www.swisstargetprediction.ch/) and PharmMapper (https://www.lilab-ecust.cn/pharmmapper/). Gastric cancer-related targets were retrieved from GeneCards (https://www.genecards.org/) and OMIM (https://www.omim.org/). The identified target proteins were standardized using the UniProtKB database (https://www.uniprot.org/id-mapping). To identify key target protein of solasodine in gastric cancer, the intersection of predicted solasodine targets and gastric cancer-related targets was determined using the jvenn website (https://jvenn.toulouse.inrae.fr/app/example.html). These overlapping targets were then analyzed via the STRING database (https://string-db.org/) to obtain the protein–protein interaction (PPI) network, Homo sapiens was selected as the species. The compound-target network was visualized using Cytoscape 3.10.3 (Boston, MA, USA) with the “Network Analyzer” plugin.

Molecular docking
The 3D X-ray structure of Gli1 (PDB ID: 2GLI1) was obtained from the Protein Data Bank (https://www.rcsb.org), and the structure of solasodine (SDF format) was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov). Molecular docking was carried out using CB-Dock2, an online docking platform based on AutoDock Vina (Liu et al., 2022; Yuan et al., 2025). Protein preparation, including the addition of hydrogen atoms and the removal of water molecules, was performed within the CB-Dock2 platform. The binding affinity (kcal/mol) of the solasodine-Gli1 complexes was calculated, and the optimal docking model was selected based on the lowest binding energy. The docking grid was constructed with the center defined by the coordinates of the original ligand bound to the target pocket.

Statistical analysis
All data are presented as mean ± standard deviation (SD). Statistical comparisons between groups were analyzed using one-way analysis of variance (ANOVA) post hoc Dunnett’s test. p < 0.05* and p < 0.01** were considered statistically significant.

Results and discussion

Solasodine significantly inhibited the proliferation of gastric cancer cells
Steroidal alkaloids are an important class of alkaloids and secondary metabolites predominantly found in plant families such as Solanaceae, Apocynaceae, Liliaceae, and Buxaceae (Xiang et al., 2022). They are characterized by a basic steroidal skeleton that incorporates a nitrogen atom within the rings or side chains (Jiang et al., 2016). Notably, solasodine with different carbohydrate moieties can form various glycoalkaloids, such as solasonine and solamargine, which have demonstrated potential as therapeutic agents for gastric cancer (Fu et al., 2019; Jiang et al., 2016; Li et al., 2022a). Here, we investigated the effects of solasodine on AGS and MKN74 gastric cancer cells. AGS cells, derived from poorly differentiated gastric adenocarcinoma, exhibit high proliferative activity and moderate migratory capacity, making them a common model for drug response studies (Slefarska-Wolak et al., 2022). MKN74 cells, from moderately differentiated intestinal-type gastric carcinoma, show greater cellular differentiation and invasive potential (Koike et al., 1997). Both cell lines display elevated Hh signaling activity (Zhou et al., 2025), which make them valuable tools for assessing the therapeutic efficacy of solasodine targeting Hh-driven gastric carcinogenesis. The results showed that solasodine inhibited the growth of both cell lines in a dose-dependent manner (Fig. 1B). In the colony formation assay, solasodine significantly reduced the number of colonies per well in both AGS and MKN74 cell lines (Fig. 1C). Specifically, in AGS cells, the number of colonies decreased from 85.5 ± 3.5 colonies per well in the control group to 27.0 ± 2.8 colonies per well in the 10 μM solasodine-treated group. Similarly, in MKN74 cells, colony numbers were reduced from 184.0 ± 2.8 to 59.0 ± 2.8 colonies per well under the same treatment conditions. These results demonstrate the potent antiproliferative effects of solasodine on gastric cancer cells.

Solasodine regulated cell cycle and apoptosis-related proteins in gastric cancer cells
Cell proliferation is primarily regulated by the cell cycle and apoptosis (Evan and Vousden, 2001). To determine how solasodine inhibits the growth of AGS and MKN74 cells, we performed western blot analysis to assess key markers involved in cell cycle regulation and apoptosis. The results demonstrated that solasodine significantly modulated the expression of cell cycle regulators Cyclin D1 and p27 in both cell lines. At 10 μM, Cyclin D1 expression decreased to 0.7 ± 0.1-fold in AGS cells and 0.5 ± 0.02-fold in MKN74 cells, while p27 expression increased approximately 2.7 ± 0.1-fold and 1.55 ± 0.1-fold, respectively, compared to the control group (Fig. 2). However, CDK2 was not affected by solasodine treatment. In addition, solasodine influenced apoptosis-related proteins by upregulating Bax and significantly reducing Bcl-2 levels in a dose-dependent manner in both cell lines. Previous studies have shown that gramicidin inhibits gastric cancer proliferation by inducing cell cycle arrest and apoptosis, leading to the downregulation of CyclinD1 and Bcl-2 (Chen et al., 2019). Gant61, the Gli1/2 inhibitor, exerted similar effects on Cyclin D1, p27, Bax, and Bcl-2, whereas vismodegib, which targets Smo, was ineffective. Following the previous studies demonstrating cyclin D1, p21, Bax, and Bcl-2 as direct targets of Gli1 (Bigelow et al., 2004; Tu et al., 2022; Zheng et al., 2013), the current results suggest that solasodine suppresses gastric cancer cell proliferation by modulating key proteins involved in cell cycle regulation and apoptosis through a Gli1-dependent mechanism.

Solasodine inhibited gastric cancer cell proliferation by regulating Hh/Gli1 signaling
Hh signaling plays a crucial role in cancer proliferation, invasion, and chemo-resistance (Sari et al., 2018). Hh signaling can be classified into canonical and non-canonical pathways based on their activation mechanisms (Sigafoos et al., 2021). The canonical pathway involves the Hh ligand binding to Ptch1, relieving its inhibition of Smo, which then activates downstream Gli transcription factors. Non-canonical activation bypasses the Ptch1–Smo axis and can activate Gli proteins via alternative mechanisms. The accumulating evidences underscore the importance of the non-canonical Hh pathway in gastric cancer with Smo-independent activation of Gli1 (Pietrobono et al., 2019). Previous results showed that Smo-independent, non-canonical Hh signaling promotes proliferation in AGS and MKN74 cells (Chong et al., 2016). Here we found that solasodine exhibited similar effects to Gant61 (the Gli1/2 inhibitor) rather than vismodegib (the Smo inhibitor) in suppressing cell proliferation (Fig. 1B, C). In addition, solasodine showed a similar effect to Gant61 in reducing the protein levels of Hh signaling components, whereas vismodegib only affected Smo expression. Specifically, solasodine and Gant61 significantly downregulated the protein levels of Gli1 and Gli2, while Smo expression remained largely unchanged. At the highest concentration, Gli1 levels decreased by approximately 2.2 ± 0.02-fold in AGS cells and 2.7 ± 0.05-fold in MKN74 cells, while Gli2 levels were reduced by approximately 1.6 ± 0.05-fold and 1.7 ± 0.02-fold, respectively (Fig. 3A). The activation of the Hh signaling relies on the nuclear translocation of Gli1 (Jiang, 2022). Confocal microscopic results showed that treatment with 10 μM solasodine or Gant61 effectively blocked the nuclear localization of Gli1, leading to its accumulation in the cytoplasm in both cell lines (Fig. 3B), while vismodegib had no such effect. Gant61 is a small molecule inhibitor that directly binds to the Gli transcription factors, specifically targeting the Gli1 protein between zinc fingers 2 and 3 at residues E119 and E167, as demonstrated by computational docking and validated by surface plasmon resonance (Agyeman et al., 2014). This binding occurs independently of the Gli-DNA interaction domain and is conserved in Gli1 and Gli2, which explains why Gant61 reduces the protein levels and transcriptional activity of multiple downstream Hh components such as Gli1 and Gli2. In contrast, vismodegib is a well-characterized antagonist of Smo leading to inactivation of Smo and blockage of canonical Hh signaling stimulated by Hh ligands (Cirrone and Harris, 2012). Previous studies showed that vismodegib failed to improve progression-free survival in gastric cancer patients (Cohen et al., 2013) and that solasodine inhibited tumorsphere growth by suppressing Gli1 expression and preventing its nuclear translocation in breast cancer stem cells (Chen et al., 2022). These results demonstrate that solasodine inhibits Gli1-dependent Hh signaling by modulating the expression and translocation of Gli1 in AGS and MKN74 gastric cancer cells.

Gli1 is a potential target of solasodine in gastric cancer
To explore the potential targets of solasodine in gastric cancer, we conducted network pharmacology analysis. Target prediction through SuperPred 3 and PharmMapper found 366 solasodine target genes. Additionally, 2,695 gastric cancer-associated targets were obtained from GeneCards and OMIM. Cross these datasets revealed 178 key targets specific to gastric cancer (Fig. 4A, B). Compound-target-gastric cancer network analysis further refined these to 53 core targets, among which Gli1 was identified as a critical node (Fig. 4C). These results indicate that solasodine may exert its antiproliferative effects on gastric cancer by targeting Gli1. Notably, non-canonical activation of Hh signaling can bypass the Ptch–Smo axis, with Gli transcription factors being regulated by alternative pathways such as PI3K/AKT/mTOR, AMPK, or ERK2 (Dong et al., 2019; Jiang, 2022). Consistently, compound-target-gastric cancer network also predicted that solasodine may interact with several key molecules within the PI3K/AKT/mTOR axis (Fig. 4C), raising the possibility that it modulates Gli1 through the alternative signaling pathways. However, further experiments are required to confirm whether solasodine directly targets these pathways to regulate Gli1.

Solasodine inhibited gastric cancer cell proliferation by targeting Gli1
Molecular docking analysis revealed that solasodine binds stably within the C3 pocket of Gli1 with a binding affinity of −7.4 kcal/mol, as predicted by CB-Dock2 (Fig. 5A). Visualization of the docking model showed that solasodine fits well into the pocket and forms multiple interactions with key amino acid residues, including TRP108, SER127, GLU128, HIS129, ILE130, HIS131, GLY132, GLU133, ARG134, LYS135, and GLU136. Notably, solasodine formed hydrogen bonds with residues such as HIS131 and GLU133, while additional hydrophobic and electrostatic interactions with surrounding residues further stabilized the ligand–protein complex. Consistent with these findings, previous studies have reported that solasodine can suppress Gli1 expressions by specifically binding to the zinc finger domain of Gli1 (Chen et al., 2022). To further validate these findings, we examined the effect of solasodine in Ptch (−/−) MEF cells, which exhibit higher expression levels of both Smo and Gli1 compared to AGS and MKN74 cells (Fig. 5B). As shown in Fig. 5C, solasodine significantly reduced Gli1 protein levels by approximately 1.9 ± 0.3-fold, while having no effect on Smo expression. These results further support the role of solasodine in modulating Gli1 activity by directly targeting its functional domain.
In conclusion, our study demonstrated that solasodine inhibited the proliferation of gastric cancer cells by targeting Gli1-dependent Hh signaling. Solasodine suppressed AGS and MKN74 cell growth, regulated cell cycle and apoptosis markers, and disrupted Gli1 expression and nuclear translocation, similar to the effects of the Gli1 inhibitor Gant61. In contrast, the Smo inhibitor vismodegib showed limited effects, suggesting a Smo-independent mechanism. Network pharmacology further demonstrated Gli1 as a key target of solasodine. These findings highlight solasodine as a potential agent for the prevention and/or suppression of gastric cancer by targeting Hh/Gli1 signaling. However, further studies, especially in vivo animal experiments, are necessary to fully validate its therapeutic potential and elucidate the underlying molecular mechanism.