Hispidulin (Shanghai Aladdin Bio-Chem Technology Co., Ltd.) was dissolved in 0.1% DMSO and 0.1% DMSO alone was used as the vehicle control. Tauroursodeoxycholic acid (TUDCA; Merck KGaA) was dissolved in saline. ROS probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was obtained from Beyotime Institute of Biotechnology and glutathione (GSH) was purchased from Sigma-Aldrich; Merck KGaA.

Human NSCLC cell lines NCI-H460 and A549 (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) were cultured in RPMI-1640 media (Thermo Fisher Scientific, Inc.) with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were grown at 37°C and 5% CO₂ (v/v) in a humidified cell incubator.

Cell viability was determined by methyl thiazolyl tetrazolium (MTT). DMSO was used to dissolve the formazan product. The optical density of viable NCI-H460 and A549 cells was measured using a spectrophotometer at 450 nm (Tecan Group, Ltd.) following treatment with different concentrations of hispidulin (0, 4, 8, 15, 30 and 60 µM) for 24 or 48 h. Cell viability assays were performed in triplicate.

NCI-H460 and A549 cells (1,000 cells/well) were cultured in a six-well plate. The cultures were maintained for 12 h upon 5 and 10 µM hispidulin treatment, and cells were grown for 8–11 days with fresh medium (RPMI-1640 with 10% FBS) at 37°C in a humidified 5% CO₂ atmosphere. The cells were subsequently fixed with 4% paraformaldehyde for 15 min at room temperature, stained with crystal violet for 30 min, and photographed using a digital camera (Nikon DXM-1200; Nikon Corporation).

FITC Annexin V/PI apoptosis kit (BD Pharmingen; BD Biosciences) was used to determine cell apoptosis. Cells were pretreated with 2.5 mM TUDCA prior to treatment with 30 µM hispidulin for 24 h. The NCI-H460 and A549 cells were collected, washed, and resuspended in 195 µl Annexin V-FITC-binding buffer. Following this, cells were incubated with Annexin V-FITC and PI (5 µl each) for 15 min at 4°C. The cells were analyzed by Accuri C6 plus flow cytometry (BD Biosciences). The flow cytometry data were quantified using Flow Jo 7.6.1 software (Tree Star, Ashland, Inc.).

Cell nuclear staining was performed using a Hoechst 33342 Staining kit (Beyotime Institute of Biotechnology). The NCI-H460 and A549 cells were incubated in Hoechst 33342 (2 µg/ml) for 30 min at 37°C 24 h post hispidulin treatment. Then, the stained cells were examined by using a fluorescence microscope.

NCI-H460 cells were seeded in six-well plates and incubated for 24 h. After pretreatment with 5 mM GSH for 1 h and treatment with the 15 and 30 µM hispidulin for 3 h, the cells were incubated with 10 µM DCFH-DA for 20 min at 37°C in the dark. The cells were subsequently washed twice with PBS and the levels of intracellular ROS were analyzed immediately using a fluorescence microscope (Nikon Corporation). The fluorescence intensity measurements were performed using ImageJ (National Institutes of Health).

NCI-H460 cells were cultured with hispidulin (15 or 30 µM) for 4 or 24 h and collected. Cells were pretreated with 2.5 mM TUDCA prior to treatment with 30 µM hispidulin for 24 h. Proteins were isolated from the hispidulin-treated or control DMSO-treated cells and the protein concentrations were determined using the bicinchoninic acid protein assay kit (cat. no. P0010; Beyotime Institute of Biotechnology). Total proteins (~50 µg/lane) were subjected to 10% SDS-PAGE and transferred to a PVDF membrane which was blocked with 5% (w/v) low-fat milk for 1 h at room temperature. The membrane was subsequently incubated with the following primary antibodies, cleaved-poly [ADP-ribose] polymerase (PARP; cat. no. sc-56196; 1:200), caspase3 (cat. no. sc-56053; 1:1,000) and GAPDH (cat. no. sc-32233; 1:1,000) purchased from Santa Cruz Biotechnology, Inc. and ATF4 (cat. no. 11815; 1:1,000), phospho (p)-eukaryotic translation initiation factor 2 subunit α (EIF2α; cat. no. 3398; Ser51; 1:1,000), EIF2α (cat. no. 5324; 1:1,000), C/EBP-homologous protein (CHOP; cat. no. 2895; 1:1,000), cleaved-caspase3 (cat. no. 9664; 1:1,000) purchased from Cell Signaling Technology, Inc. overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. nos. sc-2317 and sc-2318; 1:2,000) purchased from Santa Cruz Biotechnology Inc. were incubated with membrane for 1.5 h at room temperature. The blot signal was detected using a chemiluminescent substrate (KPL, Inc.). BandScan software (Glyko Biomedical, Ltd.) was used for densitometry. All assays were performed in triplicate.

A total of 18 male athymic BALB/c mice (8 weeks old; 18–19 g) were housed at a constant room temperature with a 12/12-h light/dark cycle and fed a standard rodent diet under standard pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Kunming Medical University. NCI-H460 cells (1×10⁶) were subcutaneously injected into the left dorsal flanks of the BALB/c mice. A total of 15 days after cell injection, mice were divided randomly into three groups (6 mice/group): Vehicle group (0.9% sodium chloride with 1% DMSO), low dose group (20 mg/kg hispidulin) and high dose group (40 mg/kg hispidulin) once every 2 days for 20 days. Mice body weight and tumor volumes were measured every 3 days for 21 days. All mice were euthanized with CO₂ and serum samples were aliquoted into sterile tubes, centrifuged at 1,100 × g for 10 min at room temperature for subsequent analysis. The activities of aspartate transaminase (AST) and alanine transaminase (ALT) were analyzed by using a VetScan analyzer (Abaxis, Inc.). Assay kits of AST and ALT were purchased from Abcam (cat. nos. ab105134 and ab105135). The harvested heart, liver and kidney tissues of mice were used for hematoxylin and eosin (H&E) staining. Immunohistochemical (IHC) staining were performed on NCI-H460 cell-derived xenograft tumors as previously described (20). The following primary antibodies were used for IHC: Proliferation marker protein Ki-67 (1:50; cat. no sc-7846; Santa Cruz Biotechnology, Inc.), cleaved-caspase 3 (1:100; cat. no. 9664; Cell Signaling Technology, Inc.) and p-EIF2α (1:1,000; cat. no. 3398; Cell Signaling Technology, Inc). HRP-conjugated secondary antibodies (cat. nos. sc-2317 and sc-2031) were purchased from Santa Cruz Biotechnology, Inc. Caspase-3 activity in tumor lysates was determined using a caspase-3 activity kit (Beyotime Institute of Biotechnology) according to the kit instructions.

Data are presented as the mean ± SEM from three separate experiments. Statistical comparisons between cells were performed by using one-way ANOVA and Dunnett's t test when comparing more than two groups of data with control group, two-way ANOVA, non-parametric Kruskal-Wallis test, followed by Dunn's post hoc test when comparing multiple independent groups and one-way repeated measures ANOVA followed by Dunnett's post hoc test when comparing tumor volume and animal body weight. Experimental data were analyzed by using GraphPad Prism software (version 8.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

To explore the effect of hispidulin treatment on cell viability, human NSCLC cells (NCI-H460 and A549) were treated with various concentrations of hispidulin (0, 4, 8, 15, 30 and 60 µM/l) for 24 and 48 h. The MTT assay results showed that hispidulin markedly decreased the viability of A549 and NCI-H460 cell lines in a time- and concentration-dependent manner (Fig. 1B and C). Additionally, 5 and 10 µM hispidulin was used to treat NCI-H460 and A549 cells for 12 h followed by a culture in a new medium for 8–11 days. Colony formation results showed that the colony formation ability was inhibited following treatment with 5 and 10 µM hispidulin compared with the control group (Fig. 1D and E). These results revealed that hispidulin inhibited the viability of NCI-H460 and A549 cells in a time and dose-dependent manner.

To explore whether hispidulin induced apoptosis in NCI-H460 and A549 cells, flow cytometric analysis and Hoechst 33342 staining was performed. Hoechst staining results showed that 15 and 30 µM hispidulin treatment for 24 h lead to cell apoptosis in a dose-dependent manner in both cell lines (Fig. 2A). Flow cytometry results indicated that treatment with hispidulin (15 and 30 µM) for 24 h lead to a significant increase in the percentage of apoptotic cells (Annexin V-FITC⁺/PI⁻ and Annexin V-FITC⁺/PI⁺ rates). The proapoptotic effect induced by hispidulin occurred in a concentration-dependent manner (Figs. 2B and S1). Additionally, NCI-H460 cells were treated with 15 and 30 µM hispidulin for 24 h and western blotting results showed that the expression levels of caspase 3 were markedly downregulated after exposure to the increasing concentrations of hispidulin compared with the DMSO group. However, the expression levels of proteolytic cleaved forms of caspase-3 as well as cleaved PARP were found to be upregulated (Fig. 2C and D), which revealed that hispidulin treatment could induce caspase-3 activation and apoptosis in NCI-H460 cells.

To investigate the role of ROS in hispidulin-induced apoptosis, the ROS level was measured in NCI-H460 cells treated with various concentrations of hispidulin for 3 h. The results showed that the ROS level was significantly increased by hispidulin in a concentration-dependent manner (Fig. 3A and B). However, pre-treatment of NCI-H460 cells with 5 mM ROS scavenger GSH showed a significantly decreased level of ROS compared with the hispidulin-treated cells (Fig. 3C and D). Further western blotting results indicated that GSH pre-treatment attenuated hispidulin-induced expression of apoptosis-related proteins in NCI-H460 cells (Fig. 3E and F). The same results were observed using the Annexin V/PI staining assay (Fig. 3G and H). These results demonstrated that ROS generation was significantly involved in hispidulin-induced apoptosis.

To explore the relationship between ER stress and cancer cell apoptosis, the expression levels of ER stress-related proteins including p-eIF2α and cyclic AMP-dependent transcription factor ATF4 were detected in NCI-H460 cells treated with hispidulin. Western blot analysis results revealed that the expression of p-eIF2α and ATF4 were increased upon 4-h hispidulin treatment in a dose-dependent manner (Figs. 4A and B and S2). CHOP is an important factor mediating ER stress-induced apoptosis (21). The current study found that 10-h hispidulin treatment lead to a significant increase in the protein expression level of CHOP (Fig. 4A and B). Then the role of ROS in hispidulin-induced ER stress progress was evaluated. The expression of p-eif2α, ATF4 and CHOP was significantly decreased upon 5 mM GSH pre-treatment for 1 h compared with hispidulin treatment alone in NCI-H460 cells (Figs. 4C and D and S2). TUDCA, a chemical chaperone frequently used for block the ER stress process (22), was used to investigate whether ER stress was involved in the anticancer effect of hispidulin. Pre-treatment with TUDCA (2.5 mM) for 1 h in NCI-H460 and A549 cells effectively reversed the increase in CHOP protein expression levels induced by treatment with hispidulin for 10 h (Figs. 4E and F and S3A and B). Besides, Pre-treatment of NCI-H460 or A549 cells with TUDCA significantly decreased apoptosis rate induced by Hispidulin treatment for 24 h (Figs. 4G and S3C and D). All these findings demonstrate that hispidulin-induced cell apoptosis may be mediated through activation of the ER stress pathway.

The in vivo effect of hispidulin was assessed using an NSCLC xenograft mouse model. NCI-H460 cells were implanted in BALB/c mice and then the mice were injected with different doses of hispidulin (20 and 40 mg/kg) or vehicle control. Hispidulin attenuated the xenograft tumor growth compared with vehicle group at both doses 21 days after the first treatment (Fig. 5A). However, there were no significant differences in the body weights of the mice in different treatment groups (Fig. 5B). Moreover, the side effects of hispidulin were evaluated using normal hepatocytes of tumor-bearing mice. The effects on serum makers of liver function including ALT and AST were tested immediately at the end of the treatment. There was no significant difference in these indices between the vehicle group and hispidulin-treated groups (Fig. 5C and D). Furthermore, H&E staining showed that hispidulin treatment did not induce marked histological changes compared with the vehicle group (Fig. 5E). IHC staining was used to assess the protein expression of representative tumor progression (Ki-67), ER stress (p-eIF2α) or apoptosis marker (cleaved caspase-3) markers in the xenograft tumors which were harvested from the three groups. The results revealed that Ki-67 expression was decreased in the His-treated groups compared with the vehicle group, while the cleaved caspase-3 and p-eIF2α expression levels were increased (Fig. 5F). Furthermore, hispidulin effectively induced cell apoptosis in vivo, as verified by caspase-3 activity assay (Fig. 5G). These results suggested that hispidulin induced NSCLC xenograft tumor growth inhibition as well as apoptosis in vivo.