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. 2026 Feb 28;18(5):794. doi: 10.3390/cancers18050794

Resveratrol and AG490 Overcome Glioblastoma Cells’ Resistance to Monotherapy by Inhibiting JAK2/STAT3 Signalling Pathway

Aziz Ullah 1,*, Chuanchun Han 1,*
Editor: William T Beck1
PMCID: PMC12984580  PMID: 41827729

Simple Summary

This study investigated RES and AG490 combination therapy to overcome treatment resistance in GBM, the most aggressive brain malignancy. Using LN428 and U251 cell lines, we found that while U251 cells were sensitive to monotherapy, LN428 cells exhibited resistance to RES alone. The combination therapy demonstrated superior efficacy, significantly reducing cell viability, migration, and proliferation while enhancing apoptosis through increased BAX/BCL-2 ratios. Importantly, combined treatment effectively suppressed JAK2/STAT3 signaling pathway activation in both cell lines, whereas monotherapy failed to inhibit STAT3/pSTAT3 expression in resistant LN428 cells. These findings demonstrate that concurrent administration of RES and AG490 overcomes GBM therapeutic resistance by targeting the JAK2/STAT3 pathway, representing a promising combinatorial strategy warranting further preclinical and clinical validation.

Keywords: glioblastoma, resveratrol, AG490, JAK2/STAT3

Abstract

Background: Glioblastoma (GBM) is the most aggressive malignancy of the central nervous system (CNS) and is characterized by poor prognosis and significant resistance to available treatments. Surgery, radiation therapy, and chemotherapy are the standard treatments; however, their efficacy is often limited by resistance. Resveratrol (RES), a naturally occurring polyphenol with antioxidant properties, has shown significant anticancer effects through inhibition of multiple cellular pathways. However, our earlier research revealed that the LN428 cell exhibited resistance, while the U251 cell showed sensitivity to RES monotherapy. Hence, RES and AG490, a JAK2 inhibitor, were used to overcome GBM cell resistance, which might enhance therapeutic efficacy. Methods: Human GBM cell lines LN428 and U251 were used. CCK-8, H&E staining, transwell, wound healing, calcein AM/PI, and flow cytometry assays were performed to evaluate cell proliferation, migration, and apoptosis. Molecular docking was performed to analyze the binding energy. Western blot, immunocytochemistry (ICC), and immunofluorescence (IF) were used to assess protein expression following treatment with RES, AG490, and their combination. Results: The results revealed that U251 cells were more sensitive to RES, AG490, and RES + AG490 than LN428 cells. Additionally, the combination of both compounds significantly reduced cell viability, proliferation, and migration, while increasing apoptosis in the LN428 and U251 cell lines. Moreover, the combination of RES and AG490 led to increased BAX protein expression while decreasing BCL-2 expression in LN428 and U251 cell lines. Notably, the monotherapy administration of RES did not significantly inhibit STAT3 or pSTAT3 protein expression in LN428 cells, while combination therapy significantly inhibited the expression of these proteins in LN428 and U251 cell lines. Conclusions: The concurrent administration of RES and AG490 effectively inhibited the JAK2/STAT3 signalling pathway and enhanced antitumor effects in GBM cells, indicating their potential as a therapeutic strategy.

1. Introduction

Glioblastoma (GBM) is the most frequently diagnosed primary malignant tumour of the CNS and remains a major clinical challenge. Patient survival averages 12.1–14.6 months, and only 3.5% of patients exceed three years of survival [1]. Despite advances in antitumor drug development, GBM patient survival has not improved substantially due to tumour resistance to standard therapies [1]. Temozolomide (TMZ) is a widely used chemotherapeutic agent for GBM. However, its effectiveness is limited by the development of resistance and notable side effects, including bone marrow suppression and infertility [2]. Moreover, continuous administration of TMZ results in secondary drug resistance, tumour recurrence, and increased patient mortality [3].

The Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signalling pathway is a tightly regulated cascade that converts extracellular signals into intracellular responses, including regulatory key processes such as cell proliferation [4]. Moreover, the JAK2/STAT3 pathway is essential for the regulation of cellular functions in GBM, including cell proliferation, survival, apoptosis, and immunological modulation [5]. The activation of STAT3 is mediated by the phosphorylation of its tyrosine 705 residue by JAK2. This process is triggered by several cytokines, including interleukin-6 and interleukin-11, along with various growth factors [6]. STAT3 is constitutively activated in GBM and serves as a critical oncogenic driver, promoting tumour development and resistance to chemotherapy [7]. Indeed, the dependence of several cancer cells on STAT3 for their survival emphasizes its importance as a major target for anticancer treatment [8]. Consequently, modulation of the JAK2/STAT3 pathway presents a compelling avenue for novel therapeutic interventions and for increasing the efficacy of current therapies [4]. Therefore, it is essential to elucidate the roles of the JAK2/STAT3 pathway in GBM progression and resistance, which could lead to more effective targeted therapies.

RES (3, 5, 4′-trihydroxystilbene) is a naturally occurring polyphenolic compound with antioxidant and anticancer properties that has shown promising therapeutic potential in GBM treatment [9,10]. RES influences various biological processes, including cell survival, migration, proliferation, and apoptosis. Numerous studies have shown that RES has antitumor effects by affecting various molecular pathways involved in tumor growth and progression, including the inhibition of PI3K/AKT, MAPK/ERK, and NF-κB signaling pathways, as well as modulation of cell cycle regulators and apoptotic proteins [10,11,12,13]. It can cross the blood–brain barrier, making it a suitable candidate for brain tumor therapy [14]. Additionally, RES can suppress GBM cell growth and increase apoptosis by reducing STAT3 activation and transcriptional activity [15]; however, it shows resistance in LN428 cells during monotherapy [16,17]. AG490, a well-known JAK2 inhibitor, suppresses cancer cell migration, invasion, and proliferation by directly targeting the JAK2/STAT3 pathway [18]. Furthermore, combination therapy administration in GBM has been found to improve efficacy in both clinical and experimental settings [19], For instance, the combination of TMZ with bevacizumab has demonstrated improved progression-free survival in clinical trials, although without significant overall survival benefit [20,21]. Additionally, various natural compounds such as curcumin and quercetin combined with conventional chemotherapeutic agents have demonstrated synergistic effects in enhancing therapeutic outcomes in GBM models [22,23]. Based on the resistance of LN428 cells to RES monotherapy, the critical role of JAK2/STAT3 signaling in GBM therapeutic resistance, and the established efficacy of AG490 in inhibiting JAK2-mediated STAT3 activation, we hypothesized that RES and AG490 combination therapy would synergistically overcome resistance and improve therapeutic outcomes in GBM. This study therefore investigated whether combined RES and AG490 treatment overcomes resistance by targeting the JAK2/STAT3 pathway and evaluated its effects on cell viability, migration, proliferation, and apoptosis in LN428 and U251 cell lines.

2. Materials and Methods

2.1. GBM Cell Lines and Cell Culture

The U251 GBM cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), while the LN428 cell line was provided by Professor Nicolas de Tribolet (Lausanne University, Lausanne, Switzerland). Both Cell lines (LN428 and U251) were cultured in high glucose DMEM (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (HyClone, Logan, UT, USA) at 37 °C under 5% CO2.

2.2. Cell Counting Kit-8 (CCK-8) Assay

CCK-8 assay was performed to analyze the LN428 and U251 cells. According to the manufacturer’s guidelines, the cells were seeded in 96-well plates at a density of 5 × 104 cells per well for 24, 48, and 72 h. Once the cells adhered, they were treated with various concentrations of RES (25, 50, 75, 100, and 200 µM) and AG490 (0.1, 0.25, 0.5, 0.75, and 1.00 µM). After 2 h of incubation at 37 °C in a CCK-8 solution, the absorbance was documented at 450 nm by a Multiskan GO (Thermo Fisher Scientific, Waltham, MA, USA).

2.3. Cell Treatments

RES (Sigma-Aldrich, St. Louis, MO, USA) and AG490 (Sigma, St. Louis, MO, USA) [24] were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich). According to CCK-8 (IC50) results, LN428 cells were treated with 100 µM RES and 56 µM AG490, while U251 cells were treated with 100 µM RES and 44 µM AG490, respectively. The treatments were administered for 48 or 72 h. The coverslips containing cells were treated with RES, AG490, and RES + AG490, then fixed with cold acetone or 4% paraformaldehyde (PFA, G1101, Servicebio, Wuhan, China) for haematoxylin and eosin, immunofluorescence, and immunocytochemistry staining. To ensure accuracy, each experiment was carried out three times.

2.4. Hematoxylin and Eosin (H&E) Staining

To observe the effects of RES, AG490, and RES + AG490 on LN428 and U251 cell morphology, H&E staining was performed on glass coverslips (Biosharp Life Sciences, Hefei, China) in 6-well plates. After incubation, the treatment groups were supplemented with DMEM containing RES, AG490, and the RES + AG490 combination. The cells were treated for 48 or 72 h, washed thrice with phosphate-buffered saline (PBS), and fixed with acetone, and incubated for 5 min at room temperature, and then again washed three times with PBS. The nuclei were stained with hematoxylin, rinsed with double-distilled water, and counterstained with eosin after differentiation. The coverslips were sequentially dehydrated with 75% and 95% alcohol, absolute ethanol, ethanol-xylene, and xylene solutions. The coverslips were mounted with natural gum, and their morphological features were observed and photographed using the DMI4000B (Leica, Wetzlar, Germany) microscope.

2.5. Cell Viability Assay

GBM cell viability after treatment with RES, AG490, and RES + AG490 was evaluated using a Calcein AM/PI assay kit (Cat No. C2015M, Beyotime Biotechnology, Shanghai, China). In 24-well plates, LN428 and U251 cells were seeded and treated with RES, AG490, and RES + AG490. The viability was calculated as follows: Cell viability (%) = (number of Calcein-AM + cells)/(number of Calcein-AM + cells + number of PI+ cells) × 100. The results for each experimental group were examined using a Leica (Leica, Wetzlar, Germany) microscope. The experiment was conducted three times to ensure consistent findings.

2.6. Wound Healing Assay

Wound-healing assays were performed to evaluate the inhibiting effects of RES, AG490, and RES + AG490 on GBM cell migration. GBM cells were cultured in six-well plates with DMEM containing 10% FBS until they reached 85 to 90% confluence. A uniform scratch was introduced into the monolayer using a sterile pipette tip, after which the cells were washed with PBS, cultured in DMEM containing 2% FBS, and exposed to RES, AG490, and a combination of RES + AG490. Photographs of the wound area were captured at 0, 24, and 48 h post-treatment, and changes in these areas were observed using light microscopy.

2.7. Transwell Assay

Cell migration after treatment with RES, AG490, and RES + AG490 was assessed using a Transwell assay (LABSELECT, Hefei, China). GBM cells (U251 and LN428) were introduced into the upper chambers at a concentration of 4 × 105 cells in 200 μL of serum-free medium and treated with RES, AG490, and RES + AG490. In the lower chamber, 600 μL of DMEM containing 10% FBS was administered. After treatment at 37 °C, the cells in the upper chambers were gently removed using cotton swabs. Migrated cells on the lower membrane were fixed with acetone and stained with 0.5% crystal violet. Furthermore, the cells were counted and recorded in five randomly chosen fields using a microscope.

2.8. Flow Cytometry

To assess apoptosis, an Annexin V-FITC assay kit (Cat No. C1062S, Beyotime, Shanghai, China) was used. LN428 and U251 GBM cells were seeded at a density of 4 × 105 cells per well in 6-well plates. After a 24-h incubation period, the cells were treated with RES, AG490, and RES + AG490. The cells were then trypsinized, collected into test tubes, and centrifuged in a pre-cooled PBS solution. Each tube was treated with 5 μL of Annexin V-FITC and incubated in the dark for 10 min at room temperature. Then, 10 μL of propidium iodide (PI) was added, and flow cytometry was performed. Data were analysed using FlowJo_v10.9.0 software (BD Biosciences, San Jose, CA, USA).

2.9. Molecular Docking

Molecular docking investigations were carried out with AutoDock Vina 1.5.6 (the Scripps Research Institute, La Jolla, CA, USA), a well-known open-source software for assessing the binding interactions between small compounds and target proteins. JAK2 (PDB ID: 3krr) structures were acquired from the Protein Data Bank (PDB) (https://www.rcsb.org, accessed on 18 July 2025), and the ligand structures of RES (CID: 445154) and AG490 (CID: 5328779) were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov, accessed on 18 July 2025). The input structures were prepared, and the scoring function was configured according to the software’s guidelines. A random seed was used to ensure the robustness of the search process, which involved analysing the binding sites and refining the results after the initial search was performed.

2.10. Immunocytochemistry (ICC) Staining

For ICC staining, glass coverslips from each experimental group were exposed to pSTAT3 antibody (Absin, Shanghai, China, 1:500). After three washes with PBS, the coverslips were permeabilized with 2% Triton X-100 (ST797, Beyotime, Shanghai, China) for 10 min. An endogenous peroxide inhibitor (reagent-1) was applied at 37 °C for 10 min. The coverslips were then incubated with diluted primary antibodies overnight at 4 °C. Colour development was facilitated by 3, 3′ diaminobenzidine tetrahydrochloride (DAB), and photographs were recorded using a microscope (DMI4000B; LEICA).

2.11. Immunofluorescence (IF) Staining

Cells were initially cultured on glass coverslips positioned in six-well plates and subjected to RES, AG490, or a combination of both. After treatment, the coverslips were rinsed and fixed with acetone for 15 min. Then, the cells were permeabilized with 0.1% Triton X-100 for 10 min. To block endogenous peroxidase activity, reagent-1 was applied for 10 min at 37 °C, and then incubated overnight with the primary antibody, pSTAT3 (diluted 1:500). After washing, the secondary antibody was applied at a 1:500 dilution for 1 h in the dark. Furthermore, the nuclei were stained with Hoechst for 5 min. LN428 and U251 cells were observed and photographed using an Axio Imager Z2 (ZEISS, Oberkochen, Germany) fluorescence microscope.

2.12. Protein Extraction and Western Blot Analysis

Proteins were extracted from LN428 and U251 cells treated with RES, AG490, and RES + AG490. The cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors for 30 min. A BCA kit (Beyotime Biotechnology, Shanghai, China) was used to measure protein concentration, and then separated by 10% SDS-PAGE (G2177-50T Servicebio), and transferred to PVDF membranes. Afterword, the membranes were blocked with 5% skim milk for 2 h, and washed thrice with Tris-buffered saline (TBS-T, 8 min each), and incubated overnight at 4 °C with primary antibodies, Rabbit polyclonal anti STAT3 (1:1000, Protein Tech, Rosemont, IL, USA 10253-2-AP), Rabbit polyclonal anti pSTAT3 (1:1000, abs118973), Rabbit polyclonal anti BAX (1:1000, Protein Tech, USA 50599-2-lg), Rabbit polyclonal anti BCL-2 (1:1000, Protein Tech, USA 26593-1-AP), and Rabbit polyclonal anti- GAPDH (1:5000, Proteintech, Wuhan, China 10494-1-AP). The membranes were then washed three times with TBS-T for 8 min and incubated with HRP-conjugated goat anti-rabbit (1:3000, SE134, Solarbio, Beijing, China) secondary antibody for 2 h at 4 °C. In densitometry analysis, GAPDH was used as the internal quantitative control. The ChemiDoc (Bio-Rad, Hercules, CA, USA) was used to visualize the protein bands, which were then quantified with ImageJ software (version: 1.54p).

2.13. Statistical Analysis

All quantitative data derived from the experiments are presented consistently as mean ± standard deviation (SD), as explicitly indicated in the respective figure legends. To determine whether observed differences between multiple experimental groups were statistically significant, a one-way analysis of variance (ANOVA) was performed, followed by Tukey’s or Dunnett’s post hoc test. Statistical significance was set at p < 0.05, and all tests were conducted with GraphPad Prism (version 10.1.2).

3. Results

3.1. Different Chemosensitivities of RES and AG490 to GBM Cells

The CCK-8 assay was employed to quantitatively determine the half-maximal inhibitory concentration (IC50) values of RES and AG490 in LN428 and U251 cell lines. Following treatment, cell viability in both LN428 and U251 cells decreased in a time and concentration-dependent manner. Notably, U251 and LN428 cells exhibited distinct chemotherapeutic response profiles. At 48 h post-treatment, the IC50 values for RES were 108.9 μM in U251 cells and 441.3 μM in LN428 cells, indicating a 4-fold difference in sensitivity. In contrast, AG490 demonstrated IC50 values of 44.32 μM in U251 cells and 56.05 μM in LN428 cells at 72 h (Figure 1a). These findings demonstrate that both cell lines exhibited sensitivity to AG490, with U251 cells displaying significantly enhanced susceptibility compared to LN428 cells. Furthermore, combinations IC50 Values and isobologram analyses can be found in Supplementary File S1.

Figure 1.

Figure 1

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Differential cytotoxic responses of GBM cells to RES and AG490 treatment. (a) concentration-response curves of LN428 and U251 cells treated with RES or AG490 at 24, 48, and 72 h assessed by CCK-8 assay. (b) Calcein AM/PI dual staining of LN428 and U251 cells treated with RES, AG490, and RES + AG490. Green fluorescence indicates viable cells; red fluorescence indicates nonviable cells. Bar graphs show quantitative analysis of cell viability ratio. (c) H&E staining of LN428 and U251 cells following treatment with RES, AG490, or RES + AG490. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. Ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar: 100 µm.

3.2. The Combination of RES and AG490 Inhibits GBM Cell Proliferation

To evaluate the impact of RES, AG490, and their combination on GBM cell viability and morphology, Calcein AM/PI (viable/nonviable cell labelling) and H&E staining assays were performed on LN428 and U251 cell lines. The Calcein AM/PI dual staining assay results revealed different cytotoxic responses between the cell lines (Figure 1b). U251 cells exhibited substantially reduced viability following treatment with RES and AG490, both as monotherapies and in combination, with a progressive decrease in viable cells and a corresponding increase in nonviable cells compared to untreated controls. In contrast, LN428 cells demonstrated decreased viability in response to AG490 treatment but displayed marked resistance to RES monotherapy. Notably, the combination treatment (RES + AG490) elicited enhanced cytotoxic effects in both cell lines compared to individual treatments. Quantitative analysis confirmed these observations, with statistically significant reductions in cell viability ratios for combination therapy compared to monotherapies in both cell lines.

Furthermore, H&E staining was performed to assess morphological alterations and growth suppression in U251 and LN428 cells following treatment with RES, AG490 and RES + AG490 (Figure 1c). Microscopic examination revealed that treated cells exhibited distinct morphological changes characteristic of cytotoxic stress. In U251 cells, RES, AG490, and RES + AG490 treatments induced profound morphological alterations, including marked cell detachment, increased intercellular spacing, nuclear and cellular fragmentation, and cytoplasmic deformation compared to untreated controls. LN428 cells treated with AG490 and RES + AG490 similarly displayed cell detachment, reduced density, and nuclear fragmentation. Importantly, consistent with the viability assays findings, LN428 cells exhibited no discernible morphological response to RES monotherapy, maintaining cellular morphology similar to untreated controls.

3.3. RES and AG490 Inhibit GBM Cell Migration

To evaluate the inhibitory effects of RES, AG490, and their combination (RES + AG490) on GBM cell migration in vitro, wound healing and transwell migration assays were performed. Wound healing assays assessed the migratory capacity of LN428 and U251 cells over 48 h. Representative phase-contrast images acquired at 0, 24, 48 h revealed progressive wound closure in controls, whereas treated cells exhibited impaired migration (Figure 2a). In U251 cells, all treatments significantly attenuated wound closure, with RES, AG490, and RES + AG490 demonstrating pronounced effects. In LN428 cells, AG490 and RES + AG490 effectively suppressed migration, while RES monotherapy showed no inhibitory effect. Quantitative analysis confirmed these observations (Figure 2c). Furthermore, the transwell assay revealed that RES reduced migration in U251 and LN428 cells to 40.42% and 93.53%, respectively, while AG490 decreased the migration rates to 44.16% and 45.12%, respectively. Notably, the combination treatment (RES + AG490) resulted in migration rates of 20.21% for U251 cells and 40.75% for LN428 cells (Figure 2b,d). These findings indicate a significant decrease in GBM cell migration following treatment with RES, AG490, or RES + AG490 compared to that in the control group. The inhibitory effects were particularly pronounced in U251 cells across all treatments, while AG490 and RES + AG490 significantly reduced LN428 cell migration. Both wound healing and transwell migration assays revealed distinct treatment-dependent effects. RES alone exhibited no inhibitory effect on LN428 cell migration, whereas the combination treatment significantly decreased migration in both cell lines.

Figure 2.

Figure 2

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RES and AG490 suppress GBM cell migration in a cell line-dependent manner. (a) Wound healing assay demonstrating the inhibitory effects of RES, AG490, and RES + AG490 on LN428 and U251 cells migration at 0, 24, and 48 h. The red line in subfigure (a) indicates the wound boundary at 0, 24, 48 h in the wound healing assay and serves as a reference point for measuring cell migration into the scratched area. (b) Transwell migration assay illustrating transmigrated cells following treatment with RES, AG490, and RES + AG490. (c) Quantitative analysis of wound closure percentage. (d) Quantitative analysis of migrated cell number normalized to control. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. Ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar: 100 µm.

3.4. The Combination of RES and AG490 Promotes Apoptosis in GBM Cells

Flow cytometry and Western blot were performed to investigate the apoptotic capabilities of RES, AG490, and their combination on LN428 and U251 cell lines. Annexin V-FITC/PI dual staining followed by flow cytometric analysis revealed differential apoptosis induction between the two cell lines (Figure 3a). In LN428 cells, treatment with RES, AG490, and RES + AG490 resulted in total apoptosis rates of 5.88%, 15.92%, and 24.2%, respectively. In contrast, U251 cells demonstrated higher sensitivity, with apoptosis rates of 22.15% following RES treatment, 22.61% following AG490 treatment, and 48.26% following combination treatment (Figure 3b). To further investigate the molecular mechanisms underlying treatment induced apoptosis, Western blot analysis was performed to assess the expression level of apoptosis-related proteins BAX and Bcl-2. Treatment with RES, AG490, and RES + AG490 resulted in upregulation of the pro-apoptotic protein BAX and downregulation of the anti-apoptotic protein Bcl-2 in both cell lines compared to untreated control (Figure 3c). Densitometric quantification revealed that the BAX/Bcl-2 ratio increased in a treatment-dependent manner, with the combination treatment (RES + AG490) producing the highest BAX/Bcl-2 ratios in both cell lines (Figure 3d). These findings corroborate the flow cytometry results, indicating that the treatments promoted an apoptotic environment by enhancing signals for programmed cell death.

Figure 3.

Figure 3

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RES and AG490 induce apoptosis in GBM cells. (a) Representative flow cytometry dot plots showing Annexin V-FITC/PI staining in LN428 and U251 cells treated with RES. AG490, and RES + AG490. (b) Quantification of total apoptotic cells (early + late apoptosis) in LN428 and U251 cells following treatment. (c) Representative Western blot showing BCL-2 and BAX protein expression in LN428 and U251 cells treated with RES, AG490, and RES + AG490. GAPDH serves as the loading control. (d) Densitometric quantification of BCL-2 and BAX protein expression levels normalized to GAPDH in LN428 and U251 cells. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test. ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar: 100 µm.

3.5. Potential Binding Affinity of RES and AG490 Towards JAK2

Molecular docking analysis was performed to evaluate the binding affinities and interaction of RES and AG490 with the JAK2 protein. The three-dimensional structure of JAK2 and the three-dimensional chemical structure of RES are shown in (Figure 4a,b). RES demonstrated a binding affinity of −7.9 kcal/mol to JAK2, with consistent affinity values observed across multiple binding modes. The RES-JAK2 interaction network revealed a conventional hydrogen bond with residue LEU A:932, along with additional interactions involving LEU A:983, ALA A:880, VAL A:863, and LEU A:855 (Figure 4c–e). The JAK2 protein and AG490 chemical structure are presented in (Figure 4f,g), respectively. AG490 exhibited a higher binding affinity of −8.2 kcal/mol to JAK2, also showing consistency across subsequent binding modes. The AG490-JAK2 interaction network displayed multiple conventional hydrogen bonds with residues LEU A:932 and GLU A:930 complemented by additional interactions with GLY A:858, LEU A:983, LEU A:855, and ALA A:880 (Figure 4h–j).

Figure 4.

Figure 4

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Schematic 2D illustration showing the interaction between RES and AG490 with the JAK2 protein. (a) three-dimensional structure of JAK2 protein, (b) Chemical structure of RES, (c,d) 3-D structure of protein ligand complex (RES with JAK2), (e) RES with JAK2, highlighting key amino acids and distances, while (f) three-dimensional structure of JAK2 protein, (g) chemical structure of AG490, (h,i) 3-D structure of protein ligand complex (AG490 with JAK2), (j) AG490 with JAK2, highlighting key amino acids and distances. These attachments provide insights into the potential binding modes and specific interactions that may influence the biological effects of these compounds on JAK2 activity.

3.6. RES and AG490 Enhance GBM Treatment by Targeting the JAK2/STAT3 Pathway

To investigate the effects of RES, AG490, and their combination on the JAK2/STAT3 signaling pathway, the expression and cellular localization of phosphorylated (pSTAT3) were examined using IF staining, ICC staining, and Western blot analysis in LN428 and U251 cell lines. IF staining revealed that pSTAT3 was predominantly localized in the nucleus and perinuclear regions of both LN428 and U251 cells under control conditions (Figure 5a). In U251 cells, treatment with RES, AG490, and RES + AG490 resulted in a marked reduction in pSTAT3 fluorescence intensity in both cell lines, with the combination treatment producing the most pronounced decrease. In LN428 cells, RES monotherapy showed minimal effect on pSTAT3 fluorescence intensity, while AG490 treatment resulted in moderate reduction and the RES + AG490 combination produced substantial decrease in pSTAT3 localization. These observations were corroborated by ICC staining, which similarly demonstrated nuclear and perinuclear localization of pSTAT3 in control cells, with progressive reduction in staining intensity in U251 cells across all treatments, while LN428 cells showed significant reduction only with AG490 and RES + AG490 treatments (Figure 5b).

Figure 5.

Figure 5

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RES and AG490 suppress STAT3 signaling pathway activation in GBM cells. (a) Representative immunofluorescence images showing pSTAT3 (green) and nuclear staining with Hoechst (blue) in LN428 and U251 cells treated with RES, AG490, or RES + AG490. Merged images display the overlay of pSTAT3 and nuclear signals. (b) Representative immunocytochemistry results demonstrating pSTAT3 protein expression. (c) Representative Western blot results showing total STAT3 and pSTAT3 protein expression in both cells treated with RES, AG490, and RES + AG490. GAPDH serves as the loading control. (d) Densitometric quantification of STAT3 and pSTAT3 protein expression levels normalized to GAPDH in LN428 and U251 cells. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test. * p < 0.05, ** p < 0.01, **** p < 0.0001. Scale bar: 100 µm.

To quantitatively validate these findings, Western blot analysis was performed to assess the modulatory effects of RES, AG490, and their combination on total STAT3 and pSTAT3 protein expression levels. Densitometric quantification revealed pronounced cell line specific treatment responses (Figure 5c,d). In U251 cells, all three treatment regimens significantly attenuated both STAT3 and pSTAT3 expression relative to untreated controls. RES monotherapy reduced STAT3 expression (* p < 0.05) and achieved more pronounced suppression of pSTAT3 levels (** p < 0.01). AG490 treatment similarly decreased STAT3 expression (* p < 0.05) while demonstrating robust pSTAT3 inhibition (*** p < 0.001). The combination therapy of RES + AG490 elicited the most substantial suppression, significantly reducing STAT3 (** p < 0.01) and achieving maximal pSTAT3 downregulation (**** p < 0.0001). In contrast, LN428 cells exhibited no statistically significant alterations in either STAT3 or pSTAT3 expression following RES monotherapy. AG490 monotherapy effectively suppressed both STAT3 (* p < 0.05) and pSTAT3 (** p < 0.01) expression. The RES + AG490 combination significantly reduced STAT3 expression (* p < 0.05) and elicited synergistic pSTAT3 inhibition (*** p < 0.001). These results demonstrate that the RES + AG490 combination treatment effectively suppresses JAK2/STAT3 signaling pathway activation in both GBM cell lines, with U251 cells exhibiting greater sensitivity to individual treatments compared to LN428 cells.

4. Discussion

GBM is the most common and aggressive primary CNS tumour in adults, with poor prognosis and limited survival despite advances in surgery, radiotherapy, and chemotherapy [19]. TMZ is a primary chemotherapeutic agent; however, it frequently fails to prevent resistance and life-threatening recurrence in patients, with approximately 50% of GBM patients exhibiting primary or acquired TMZ resistance [25,26]. This emphasizes the crucial need for novel therapeutic strategies that are both highly effective and exhibit nominal toxicity in the treatment of GBMs.

Combination therapy is a pivotal approach for addressing various cancers that are resistant to standard chemotherapy. Traditional approaches frequently lack specificity, affecting a wide variety of cells and causing significant side effects in healthy tissues [27]. Over the past decade, RES has become a prominent focus of research owing to its diverse therapeutic effects, including anticancer capabilities [28]. Moreover, extensive research has elucidated the inhibitory effects of RES on various malignancies, including GBM [29]. Furthermore, AG490 is known for its efficacy in inhibiting JAK-2 in both in vitro and in vivo, and its derivatives are frequently utilized to reduce STAT3 activity by directly targeting JAK-2 [27]. In this study, we combined AG490 and RES to overcome the limited efficacy of RES monotherapy in the LN428 cell line. Although RES exhibits strong anticancer activity, blood–brain barrier permeability, and low toxicity to normal cells, its therapeutic impact alone is insufficient due to cellular resistance. Moreover, by inhibiting JAK2, AG490 increases the GBM cell sensitivity to RES, and their combined administration will be more effective in suppressing tumour progression while overcoming the resistance properties of GBM.

To evaluate the effects of RES and AG490 on GBM cells, we initially assessed the viability of LN428 and U251 cells following treatment with these compounds using the CCK-8 assay. Our results indicated that treatment with RES and AG490 significantly reduced cell viability in a dose and time-dependent manner. Moreover, U251 cells exhibited greater sensitivity to both compounds as compared to LN428 cells. These findings align with previous studies demonstrating differential sensitivity between U251 and LN428 cell lines, where U251 cells showed growth arrest and extensive cell death after RES treatment at 100 µM for 48 h, while LN428 demonstrated resistance to the same treatment [30,31]. Moli Wu et al. (2023) similarly reported that LN428 cells were considerably less sensitive to RES compared to A172 cells, conforming the heterogeneous response of different GBM cell lines to RES [16]. Furthermore, AG490 significantly inhibited the growth and proliferation of LN428 cells, which have demonstrated resistance to RES in previous studies [16]. A Calcein AM/PI assay was performed to evaluate cell viability. The findings demonstrated that the combination of RES + AG490 significantly decreased cell viability compared to the administration of either compound separately. These observations indicate that the additive interaction between RES and AG490 enhances their anticancer efficacy, thereby improving their effectiveness in inhibiting GBM cell growth. Similar enhanced effects with combination therapy approaches have been reported in GBM cells, where combined treatment achieved superior growth inhibition compared to monotherapy, particularly in resistant cell line [16].

Early metastasis is a significant factor contributing to increased mortality rates in patients with GBM [32]. The initial stage of tumor metastasis is critically dependent on cell migration [33]. The wound healing and Transwell assays revealed that both RES and AG490, administered alone or in combination, significantly inhibited cell migration in the U251 cell line. Notably, while RES alone did not inhibit cell migration in LN428 cells, AG490 effectively inhibited migration in both U251 and LN428 cell lines. Furthermore, the combination of RES and AG490 effectively inhibited cell migration in both cell lines. These findings are consistent with previous studies demonstrating that RES significantly inhibit migration and invasion of GBM cells, including U251 [34,35]. Subsequent analyses revealed that RES and AG490 inhibited GBM cell proliferation, which is a critical factor in GBM progression.

Apoptosis is an essential process in cellular physiology that maintains homeostasis by balancing cell death and cellular proliferation [36]. This process effectively discards both superfluous and harmful cells, contributing significantly to cancer prevention [37]. Numerous compounds have been shown to possess anticancer effects by facilitating cellular apoptosis [33]. Thus, flow cytometry and Western blot were used to investigate the apoptotic effects of RES, AG490, and their combination on GBM cells. The results demonstrated significant increases in both early and late apoptosis in U251 cells treated with RES, AG490, or their combination, indicating that these treatments effectively induced programmed cell death, which is crucial for tumor control. These findings are consistent with previous studies demonstrating that RES treatment significantly increases the percentage of apoptotic U251 cells in a dose dependent manner, as measured by Annexin V/PI staining using flow cytometry [16]. In contrast, RES alone did not induce significant apoptosis in LN428 cells, suggesting that these cells possess distinct signalling pathways and resistance mechanisms. This differential response reflects the profound heterogeneity of GBM, which comprises diverse cellular states that drive therapeutic resistance [38]. Consequently, this shift favors apoptosis in GBM cells, which is critical for inhibiting tumor growth and progression.

Molecular docking plays a crucial role in network pharmacology by assessing the strength of the interactions between molecular ligands and protein receptors, thereby validating the outcomes of network pharmacology analyses [39]. Thus, our validation shows comprehensive binding forces between RES and AG490 with JAK2. RES –JAK2 interaction network, displaying conventional hydrogen bonds (green dotted line) with residues LEU A: 932, along with additional interactions involving LEU A: 983, ALA A: 880, VAL A: 863, and LEU A: 855. Moreover, the AG490-JAK2 interaction network displayed multiple conventional hydrogen bonds with residues LEU A: 932 and GLU A: 930, complemented by additional interactions with GLY A: 858, LEU A: 983, LEU A:855, and ALA A:880. The distribution of these interaction points across the binding site creates a network that effectively anchors RES and AG490 to JAK2. Hydrogen bonds with hinge region residues, particularly Leu932, are critical determinant of JAK2 inhibition and binding stability, as these conserved interactions are consistently observed across multiple JAK2 inhibitors [40]. These results emphasize the critical binding interactions between ligands and target proteins, offering essential insights into their potential as therapeutic agents.

STAT3, functioning as a downstream molecule of JAK2, a member of the signal transducer and activator of transcription (STAT) family and exhibits continuous activation in various cancers, including glioma [41]. Multiple studies report that pSTAT3 is highly overexpressed in the majority of GBM patients and is associated with mesenchymal differentiation and poor prognosis [42], making the STAT3 pathway an important therapeutic target in glioma [43,44]. The JAK2/STAT3 signaling pathway plays a significant role in multiple cellular activities, including migration, differentiation, proliferation, and apoptosis [41]. Given that JAK2/STAT3 pathway activation represents a focal point of tumorigenesis and immune escape in GBM, with constitutive STAT3 activation occurring in approximately 60% of primary malignant gliomas [42], targeting the JAK2/STAT signaling pathway has emerged as a rational therapeutic strategy. Consequently, targeting the JAK/STAT signaling pathway may benefit individuals with cancer [45]. However, GBM resistance and stemness are regulated by interconnected oncogenic networks rather than a single pathway. Substantial crosstalk exists between STAT3 and other key signaling cascades, including Notch, JAK/STAT3, NF-κB, and PI3K/AKT, which collectively contribute to tumor plasticity and therapeutic resistance [46]. Given the heightened activity of the JAK2/STAT3 signaling pathway observed in glioma cells [47], we speculate that RES, AG490, and their combination exert anti-glioblastoma effects by inhibiting the JAK2/STAT3 signaling pathway. Consistent with this hypothesis, our ICC and IF results demonstrated that combined treatment with RES and AG490 significantly downregulated the expression levels of both STAT3 and pSTAT3 in both cell lines, supporting the involvement of JAK2/STAT3 pathway suppression in the observed antitumor effects. These findings are supported by recent studies showing that RES effectively inhibits STAT3 phosphorylation in GBM cells, leading to reduced proliferation, migration, and enhanced apoptosis [48]. Similarly, AG490, has been demonstrated to suppress constitutive STAT3 activation in GBM models [16]. Furthermore, Western blot analysis confirmed that both RES and AG490, administered individually or in combination, significantly downregulated STAT3 and pSTAT3 expression in U251 cells. In contrast, in the LN428 cell line, RES alone exhibited limited effectiveness; however, when combined with AG490, it effectively reduced STAT3 and pSTAT3 protein expression levels. This differential response underscores a key advantage of combination therapy approaches in GBM: the ability to overcome single-agent resistance through synergistic targeting of multiple pathways, a strategy increasingly recognized as essential given GBM’s molecular heterogeneity and adaptive resistance mechanisms [49]. These findings indicate that the combined use of RES and AG490 effectively disrupts the JAK2/STAT3 signaling pathway in GBM, suggesting a potential therapeutic strategy in combination. Although this study was limited to in vitro experiments using two GBM cell lines without evaluation of TMZ combination with RES and AG490, or in vivo validation, future research should confirm these findings in diverse cell lines and animal models to establish clinical translatability.

5. Conclusions

In conclusion, our study investigated the effects of RES, AG490, and their combination on GBM cells, specifically on LN428 and U251 cell lines. The results revealed that the use of these compounds together significantly reduced cell viability, proliferation, and migration compared to their monotherapy administration, highlighting their synergistic anticancer effects. Furthermore, RES and AG490 induced apoptosis and suppressed the JAK2/STAT3 signaling pathway. Our findings suggest that the concurrent administration of RES and AG490 enhances antitumor effects in GBM cells and improves responsiveness in a relatively RES-insensitive cell line compared with either agent alone.

Acknowledgments

The author expresses sincere gratitude to the China Scholarship Council (CSC) for its financial support, which substantially contributed to the successful execution of this research.

Abbreviations

ANOVA One-way analysis of variance
CNS Central nervous system
CCK-8 Cell Counting Kit-8
DMSO Dimethyl sulfoxide
FBS Fetal bovine serum
GBM Glioblastoma
H&E Haematoxylin and Eosin
IF Immunofluorescence
ICC Immunocytochemistry
JAK2/STAT3 Janus kinase 2/signal transducer and activator of transcription 3
RES Resveratrol
TMZ Temozolomide
PBS Phosphate-buffered saline
SD Standard deviation
TBS-T Tris-buffered saline
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Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers18050794/s1.

cancers-18-00794-s001.zip (173.2KB, zip)

Author Contributions

Conceptualization, C.H.; methodology, C.H. and A.U.; validation, C.H. and A.U.; formal analysis, A.U.; investigation, A.U.; resources, C.H.; data curation, A.U.; writing—original draft, A.U.; writing—review & editing, C.H. and A.U.; supervision, C.H.; project administration, C.H.; funding acquisition, C.H. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was supported by the United Fund of Natural Sciences Foundation of Liaoning Province (Grant No. 2023-MSLH-030 to Chuanchun Han).

Footnotes

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Associated Data

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Supplementary Materials

cancers-18-00794-s001.zip (173.2KB, zip)

Data Availability Statement

Data will be made available upon request.

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