Temozolomide (TMZ) and eriodictyol (purity ≥ 98%) were purchased from Dalian Meilun Biotechnology, Co., Ltd. (Dalian, China) and dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in culture medium was ≤ 1 ‰. LY294002 and 740 Y-P were bought from MedChemExpress (MCE, United States). Hoechst 33342, the TUNEL apoptosis assay kit, and Lipo8000 were obtained from Beyotime Biotechnology, Co., Ltd. (Shanghai, China). The Annexin V–FITC/PI Apoptosis Detection Kit was purchased from Vazyme Biotech, Co., Ltd. (Nanjing, China). Antibodies against PI3K, phospho-PI3K (Y607), and Caspase-9 and goat anti-rabbit IgG H&L (IRDye 800CW) pre-adsorbed secondary antibodies were obtained from Abcam (Cambridge, United Kingdom). Antibodies against Akt, phospho-Akt (S473), Caspase-3, cleaved Caspase-3, cleaved Caspase-8, Bax, Bcl-2, Bcl-xL, and NF-κB were bought from Cell Signaling Technology (Danvers, MA, United States). Antibodies against Ki67, phospho-NF-κB (S536) and phospho-IκBα (S32/S36) were obtained from Affinity Biosciences (Zhenjiang, China). Antibodies against IκBα and PARP were purchased from ProteinTech Group, Inc. (Chicago, IL, United States). Antibodies against β-actin and the Cell Counting Kit-8 (CCK-8) were purchased from Bimake (Houston, TX, United States).

Human glioma cell lines U87MG, A172, T98-G, the non-small cell lung cancer cell line NCI-H1975, the colorectal cancer cell line HCT116, the hepatoma cell line HepG2, and the pancreatic cancer cell line PANC1 were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The human breast cancer cell line CAL148, the glioma cell line CHG-5, and primary mouse astrocytes were a gift from Associate Professor Li Xiaoli (College of Pharmacy, Chongqing Medical University, Chongqing, China). All cells were cultured in DMEM medium with 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit-Haemek, Israel) and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO₂ at 37°C.

The effects of eriodictyol on viability were assessed using the CCK-8 assay. Cells were plated at 3 ×10³ cells per well in 96-well plates. On the following day, 0–400 μM eriodictyol was added to the medium. After 24–72 h, cells were incubated with 10 μl CCK-8 for 2 h at 37°C with 5% CO₂. Absorbance was measured at 450 nm using a SynergyH1 microplate spectrophotometer (BioTek).

U87MG and CHG-5 cells were plated in six-well plates (500 cells/well). After the cells were allowed to attach for 12 h, the cells were treated with different concentrations (0, 10, 20, 40 or μM) of eriodictyol, and the medium was changed every 3 days. After 12 days, the medium was discharged. The cells were washed with cold PBS, fixed in 4% paraformaldehyde for 30 min, and stained with 0.5% crystal violet solution for 15 min. After washing away excessive crystal violet and drying, the colonies were observed, and the results were analyzed using Image J software.

U87MG and CHG-5 cells were plated in six-well plates with DMEM containing 10% FBS. After the cells had 100% confluent, a 200 μl pipette tip was used to scrape the wells to mimic wounds. The cells were gently washed twice with PBS to remove the detached cells and cultured in DMEM containing 2% FBS. The cells were treated with various concentrations (0, 25, 50, 100 μM) of eriodictyol. After 0, 12, and 24 h, the cells were photographed using a light microscope (Nikon, Japan) and the area of the wound was measured.

Transwell chambers (8-μm pore size; 24-well) were used for migration assays (Costar; Corning Incorporated, Corning, NY, United States). U87MG and CHG-5 cells were plated into the upper chamber at 1 × 10⁴ cells/well in 200 μl of serum-free DMEM, while the lower chamber was filled with DMEM containing 10% FBS. Various concentrations (0, 25, 50, 100 μM) of eriodictyol were added to the upper chambers. After 24 h incubation at 37°C, non-migrated cells were removed from the upper side of each chamber with a cotton swab. Cells on the lower side of the chamber were fixed with 4% paraformaldehyde for 30 min at room temperature and then stained with 0.5% crystal violet for 15 min. Migrated cells were counted at 100× magnification using a microscope (Nikon).

The Transwell chamber was pretreated with Matrigel (Corning, NY, United States) and dried at 37°C for 1 h. Other procedures were the same as for the Transwell migration assay. The results of the Transwell invasion assay were also calculated according to the number of transferred cells.

U87MG and CHG-5 cells were plated in six-well plates at a density of 2 × 10⁵ cells per well. After 12 h, various concentrations (0, 25, 50, 100 μM) of eriodictyol were added to each well, and cells were incubated for an additional 48 h. Then both floating and adherent cells were collected and washed with PBS twice. Subsequently, the cells were fixed in 70% ethanol solution overnight and finally suspended in 50 μg/ml of PI solution containing 0.5% Triton X-100 and 2% RNase A. Finally, the cell cycle was determined using a CytoFLEX flow cytometer (Beckman Coulter, United States).

Similar to the cell cycle analysis, U87MG and CHG-5 cells were plated in six-well plates (2 × 10⁵ cells/well), and treated with different concentrations (0, 25, 50, or 100 μM) of eriodictyol. After 48 h treatment, the cells were collected and washed with cold PBS and suspended in 100 μl of 1× binding buffer, followed by the addition of 5 μl of FITC Annexin V and 5 μl of PI and incubation for 10 min at room temperature in the dark. Finally, after the addition of 400 μl of 1× binding buffer, samples were analyzed with a CytoFLEX flow cytometer (Beckman Coulter).

U87MG and CHG-5 cells were plated in 24-well plates at a density of 1 × 10⁴ cells per well. Different concentrations (0, 25, 50, or 100 μM) of eriodictyol were added to each well, and cells were incubated for 48 h. Subsequently, the cells were stained with Hoechst 33342 (10 μg/ml) for 10 min at 37°C in the dark. Subsequently, the cells were washed twice with PBS and observed by fluorescence microscopy at 200× magnification (Nikon).

U87MG and CHG-5 cells were seeded into 24-well plates (1 × 10⁴ cells/well) and incubated overnight. The cells were treated with different concentrations of eriodictyol for 48 h. Apoptosis was determined by TUNEL assay. The TUNEL assay was performed according to the instruction manual (Vazyme Biotech, China). Cells were photographed by fluorescence microscopy at 200× magnification (Nikon).

Cells and tumor tissues were lysed in RIPA buffer containing 1 mM protease inhibitor and 1 mM phosphatase inhibitor (Bimake, Houston, TX, USA). The protein concentration was measured by bicinchoninic acid (BCA) assay (BCA kit, Beyotime Biotechnology, China). Subsequently, proteins were separated by (6%, 8%, and 12%) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and incubated with primary antibodies overnight at 4°C. Subsequently, the PVDF membranes were washed with TBST for three times and then incubated with secondary antibodies for 2 h at room temperature. Finally, protein bands were visualized by an Odyssey^® CLx Imaging System (LI‐COR Biosciences, United States).

Briefly, total RNA was extracted following the instruction manual of the RNA extraction kit (TIANGEN Biotech, China), and 1 μg of total RNA was reverse transcribed to cDNA by using the All-in-One cDNA Synthesis SuperMix kit (Bimake). Then, qPCR was performed using the PCR kit according to the instructions. Expression values were normalized to the expression of β-actin. Primers used in this study were the following: Bax: 5′-TGCGTCCACCAAGAAGC-3′ (forward), 5′-TCCAGTTCGTCCCCGAT-3′ (reverse); Bcl-2: 5′-GCGGATTGACATTTCTGTG-3′ (forward), 5′-CATAAGGCAACGATCCCA-3′ (reverse); Bcl-xL: 5′-CCTGGGTTCCCTTTCCTT-3′ (forward), 5′-TCCTGGTCCTTGCATCTTT-3′ (reverse); β-actin: 5′-AGGGTGTTGTGGAGATGGG-3′ (forward), 5′-TGGCCTTGAGTTTCCTGCT-3′ (reverse).

Tissue sections were dewaxed, soaked in ethanol, and then blocked with 3% H₂O₂ for 10 min. Next, the tissue sections were washed carefully by distilled water and blocked with 5% goat serum. Then the sections were incubated with primary antibodies overnight at 4°C. Subsequently, sections were washed with PBS and further incubated with secondary antibodies. Finally, tissue sections were stained with DAB and observed with a light microscope at 200× magnification (Nikon).

Nude mice (50% male and 50% female, 4–5 weeks old) were obtained from the Animal Ethics Committee of Chongqing Medical University and housed in a specific pathogen-free laboratory environment. The nude mice were injected subcutaneously with U87MG cells (5 × 10⁶) to establish the xenograft model. When tumors had grown to about 50 mm³, mice were divided randomly into five groups and injected intraperitoneally with normal saline, 50, 100, or 200 mg/kg eriodictyol, or 50 mg/kg temozolomide once per day for 21 days. Tumor size and mouse body weight were measured every three days. After three weeks, all mice were sacrificed, and the tumors were excised, weighed, and fixed in 4% paraformaldehyde for further analysis.

Statistical analysis was carried out using GraphPad 6.0 software. The results are presented as means ± SD or SEM. Data were analyzed using Student's t-test or ANOVA, and P < 0.05 was considered to indicate statistical significance.

To evaluate the potential anti-cancer effect of eriodictyol on cancer cells, we treated several cancer cell lines (NCI-H1975 lung cancer, HCT116 colon cancer, CAL148 breast cancer, PANC1 pancreatic cancer, U87MG glioma, and HepG2 liver cancer cell lines) with different concentrations of eriodictyol (0, 25, 50, 100, 200, or 400 μM). After 48 h, the proliferation of cancer cell lines was examined through the CCK-8 assay. Our data demonstrate that eriodictyol could suppress cancer cell proliferation, especially in U87MG glioma cells ( Figure 1B ). Then, in order to further explore the anti-proliferation effect of eriodictyol on glioma cells, the CCK-8 assay was repeated with four glioma cell lines (U87MG, CHG-5, A172, and T98-G). The results are shown in Figure 1C . The growth of U87MG and CHG-5 glioma cells was significantly inhibited by eriodictyol treatment in a dose- and time-dependent manner ( Figures 1D, E ). IC₅₀ values of eriodictyol for U87MG and CHG-5 cells were presented in Table 1 . Moreover, the anti-proliferation effect of eriodictyol was strong on glioma cells but very weak on normal mouse astrocytes ( Figures 1F, G ).

U87MG and CHG-5 cells were seeded into six-well plates and cultured with different concentrations of eriodictyol (0, 10, 20, or 40 μM) for 12 days. As shown in Figures 2A, B , the colony formation ability of U87MG and CHG-5 cells was dramatically inhibited by eriodictyol.

The anti-migration and anti-invasion effects of eriodictyol on U87MG and CHG-5 cells were evaluated by wound healing and Transwell assays. Eriodictyol significantly inhibited the wound healing ability of U87MG and CHG-5 cells in a dose- and time-dependent manner ( Figures 3A, B ). Moreover, the Transwell assay showed that (i) eriodictyol markedly inhibited the migration ability of U87MG and CHG-5 cells, consistent with the wound healing assay, and (ii) the number of cells which passed through the membrane was obviously reduced with increasing eriodictyol concentrations (0, 25, 50, and 100 μM) ( Figures 3C, D ).

To investigate the effects of eriodictyol on the cell cycle, we treated U87MG and CHG-5 cells with eriodictyol for 48 h, and their cell cycle status was determined by flow cytometry. The data indicate that eriodictyol arrests the cell cycle at the S phase ( Figures 4A, B ).

The effects of eriodictyol on glioma cell apoptosis were investigated by flow cytometry, Hoechst 33342 assay, and TUNEL assay. Firstly, the apoptotic effects on glioma cells (U87MG and CHG-5) were detected by Hoechst 33342 assay. The cell brightness, the degree of chromatin condensation, and nuclear fragmentation increased upon treatment with increasing eriodictyol concentrations (0, 25, 50, and 100 μM) ( Figure 5A ). Then, we used flow cytometry to further evaluate the anti-cancer effects of eriodictyol. As shown in Figures 5B, C , the number of apoptotic cells increased with increasing eriodictyol concentrations. Finally, we examined the apoptotic effects of eriodictyol by TUNEL assay. The fluorescence intensity of U87MG and CHG-5 increased upon eriodictyol treatment ( Figure 5D ).

To further explore the apoptotic effects of eriodictyol on U87MG and CHG-5 cells, expression levels of a series of apoptosis-related proteins were measured by Western blot. As shown in Figure 6A , the expression levels of cleaved Caspase-3, 8, and 9, cleaved PARP, and Bax in U87MG and CHG-5 cells were increased upon eriodictyol treatment, while the expression levels of anti-apoptotic proteins (Bcl-2 and Bcl-xL) were decreased. Next, we detected the mRNA levels of Bax, Bcl-2, and Bcl-xL in U87MG and CHG-5 cells after treatment with eriodictyol. The changes in mRNA levels were consistent with our Western blot results ( Figure 6B ).

To investigate the mechanisms by which eriodictyol induces apoptosis in glioma cells, we measured the levels of proteins in the PI3K/Akt/NF-κB signaling pathway by Western blot. The expression levels of p-PI3K, p-Akt, p-IκBα, and p-NF-κB were downregulated upon treatment with eriodictyol ( Figure 7A ).

To further investigate the mechanisms underlying eriodictyol-induced apoptosis in glioma cells, we measured cell viability by CCK-8 assay after treatment with eriodictyol, 740 Y-P (PI3K agonist), LY294002 (PI3K inhibitor), eriodictyol combined with 740 Y-P, or eriodictyol combined with LY294002. As shown in Figure 7B , eriodictyol significantly inhibited the cell viability of U87MG and CHG-5 cells. Moreover, 740 Y-P (25 μg/ml) reversed the eriodictyol-induced decline in cell viability, and LY294002 (30 μM) enhanced the effects of eriodictyol on cell viability. Next, we measured the rate of apoptotic cells by flow cytometry, and the results were consistent with our CCK-8 assay results ( Figures 7C, D ). The expression levels of apoptosis-related and PI3K/Akt/NF-κB signaling pathway proteins were measured by Western blot. The expression levels of Bax and cleaved PARP were increased by eriodictyol, while the opposite was observed for 740 Y-P ( Figure 7E ). Moreover, 740 Y-P significantly reversed the eriodictyol-inhibited phosphorylation of PI3K, Akt, IκBα, and NF-κB. Collectively, these results suggest that the eriodictyol-induced apoptosis of U87MG and CHG-5 cells might be correlated with the PI3K/Akt/NF-κB signaling pathway.

We also investigated the anti-cancer activities of eriodictyol in vivo. A xenograft mouse model was established using U87MG cells and nude mice ( Figure 8A ). We found that eriodictyol dose-dependently reduced the tumor volume and weight ( Figures 8B, C ). Furthermore, the weight loss of mice in the eriodictyol group was significantly smaller than that of the temozolomide group compared with the control group ( Figure 8D ). However, the anti-cancer effects of eriodictyol were weaker than those of temozolomide, even at high doses. After TUNEL staining, the number of TUNEL-positive cells in tumor tissue sections was increased after eriodictyol treatment. The number of Ki67-positive cells, however, decreased after eriodictyol treatment in a dose-dependent manner ( Figures 9A, B ). The expression levels in tumor tissue of Caspase-3 and Bax were measured by Western blot. As shown in Figure 9C , the expression levels of cleaved Caspase-3 and Bax were increased upon eriodictyol treatment. These results illustrate that eriodictyol could inhibit glioma growth in vivo.