Wogonin (Chengdu Institute of Biology, Chinese Academy of Science, Chengdu, China) was dissolved in dimethyl sulfoxide (DMSO; 100 mg/ml) and stored at −20°C. The solution was diluted as required using RPMI-1640 medium. 5-Fluorouracil (5-Fu) and MTT were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). SGC-7901 and A549 cell lines were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). RPMI-1640 medium, Fetal Bovine Serum (cat. no. 16000-044) and trypsin-EDTA 0.25% (cat. no. 25200-072) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Bicinchoninic acid (BCA) Protein Assay kit (cat. no. P0010), RIPA Lysis Buffer (cat. no. P0013B) and Trypan blue Staining Cell Viability Assay kit (cat. no. C0011) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Hexokinase (HK) assay kit (cat. no. A007-1), pyruvate kinase (PK) assay kit (cat. no. A076-1), lactate dehydrogenase (LDH) assay kit (cat. no. A020-1) and succinate dehydrogenase (SDH) activity assay kit (cat. no. A022). ATP assay kit (cat. no. A059-1) and Hematoxylin-Eosin stain kit (cat. no. D006) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Rabbit HIF-1α (cat. no. PB0245) and MCT-4 (cat. no. PB0269) antibodies were obtained from Boster Biological Technology (Pleasanton, CA, USA). Mouse β-actin (cat. no. 60008-1), horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no. SA00001-2) and goat anti-mouse IgG (cat. no. SA00001-1) antibodies were obtained from ProteinTech Group, Inc. (Chicago, IL, USA). The diaminobenzidine (DAB) kit (cat. no. ZLI-9018) was obtained from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (OriGene Technologies, Inc., Beijing. China). The enhanced chemiluminescence (ECL) kit (cat. no. 32109) was obtained from Thermo Fisher Scientific, Inc.

SGC-7901 and A549 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified incubator in an atmosphere of 5% CO₂ at 37°C as previously described (22,23). Cells were seeded in 96-well plates at a density of 5×10⁴ cells/well and incubated with 5% CO₂ at 37°C for 24 h. Subsequently, the cells were treated with wogonin at varying concentrations (5, 10, 15, 20, 25 and 30 µg/ml) at 37°C for 48 h. An untreated control experiment was also performed. Cell viability was assessed by adding MTT in PBS to a final concentration of 5 mg/ml. Plates were incubated at 37°C for a further 4 h. DMSO (150 µl) was added to each well and incubated for 10 min at room temperature. The amount of MTT-formazan was directly proportional to the number of living cells and was determined by measuring the optical density at 490 nm. Cell survival rate was calculated using the following equation: Cell survival rate (%) = (Average OD_wogonin - Average OD_control)/(Average OD_control - Average OD_blank) × 100%. IC₅₀ were calculated using SPSS 20.0 (IBM Corp., Armonk, NY, USA). Each experiment was repeated 4 times.

SGC-7901 and A549 cells were seeded into 24-well plates at a density of 5×10⁴ cells/well and incubated with 5% CO₂ at 37°C for 24 h. Following pretreatment with wogonin (15 µg/ml) or 5-Fu (10 µg/ml) at 37°C for 24 h, a cell suspension was prepared at a suitable dilution (1.0×10⁵ cells/ml) in PBS. A total of 50 µl of cell suspension was taken and mixed with an equal volume of 0.4% Trypan blue. The number of viable cells was counted under a light microscope at a magnification of ×20 once daily for 7 days, and the growth curve was constructed. An untreated control group was analyzed additionally. Each experiment was repeated 4 times and the results were expressed as the mean ± standard deviation for each treatment group.

SGC-7901 and A549 cells were seeded at a density of 5×10⁴ cells/well on coverslips, which were placed in 6-well plates and incubated with 5% CO₂ at 37°C for 24 h. Subsequently, the cells were treated with wogonin (15 µg/ml) at 37°C for 48 h. An untreated control experiment was performed in parallel. Cells were washed with ice-cold PBS and fixed with 95% ethanol for 15 min at room temperature, followed by staining with hematoxylin (5 mg/ml) for 5 min and eosin (5 mg/ml) for 3 min at room temperature. Then the coverslips were then dehydrated with graded ethanol for 1–2 sec at room temperature and soaked in xylene for 5 min, mounted at room temperature and dried at 37°C for 24 h. Morphological changes of the cells were observed under a light microscope at a magnification of ×40. Furthermore, the number of cells in each group were counted at a magnification of ×20 and evaluated. Each experiment was repeated 4 times.

After SGC-7901 and A549 cells were seeded into 6-well plates at a density of 5×10⁵ cells/well, cells were pretreated with wogonin (15 µg/ml) for 48 h at 37°C. An untreated control experiment was also performed. Following pretreatment, cells were trypsinized with Trypsin-EDTA and suspended in 0.9% physiological saline, then centrifuged at 2,000 × g for 5 min at 4°C. HK, PK, LDH and SDH activities were assessed with activity assay kits by measuring the absorbance at 340, 440 and 600 nm, respectively. Enzyme activities were normalized against the protein concentration, determined using the BCA Protein Assay kit at 562 nm. Each experiment was repeated 4 times.

HIF-1α and MCT-4 expression were examined by western blot analysis. After SGC-7901 and A549 cells reached a confluence of 70%, they were incubated with wogonin (15 µg/ml) for 48 h with 5% CO₂ at 37°C in cell culture flasks. Cells were then washed with ice-cold PBS 3 times and the total protein was extracted with RIPA Lysis Buffer. Samples were then centrifuged at 13,500 × g for 15 min at 4°C to remove cell debris. An untreated control experiment was also performed. The protein concentration was measured using the BCA Protein Assay kit. Protein (~30 µg) was loaded and separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Following 1 h of blocking with 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) at room temperature, the membranes were incubated with primary antibodies with gentle shaking overnight at 4°C; HIF-1α (1:100), MCT-4 (1:100) and β-actin (1:1,000) prepared in 5% non-fat dried milk in TBS-T. Membranes were further incubated with secondary antibodies for 1 h at room temperature, then developed using an ECL kit and scanned densitometry. The band densities were quantified via densitometry using Quantity One Software 4.4 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The intensities of the protein bands were normalized against β-actin. Each experiment was repeated 4 times.

Data are expressed as mean ± standard deviation. Statistical analysis was performed with SPSS (version 20.0; IBM Corp., Armonk, NY, USA). Differences between the conditions tested were identified using one-way analysis of variance and differences among groups were analyzed for significance using a Tukey's test. Comparisons between two groups were performed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the effect of wogonin on cell proliferation, SGC-7901 and A549 cells were treated with varying concentrations of wogonin and viability was evaluated by MTT assays. As presented in Fig. 1A, the results suggested that the inhibition rates of wogonin in A549 cells were stable at high concentrations (10–30 µg/ml). Measured rates were all similar to the IC₅₀ (18.17 µg/ml) for A549 cells. To achieve high efficiency and low toxicity, 15 µg/ml wogonin were used in subsequent experiments with A549 cells. In SGC-7901 cells, the inhibitory effect of wogonin on cell proliferation was dose-dependent between 5 and 20 µg/ml. Although the inhibitory rate was higher at 20 µg/ml compared with the 15 µg/ml treatment, to achieve lower toxicity and to be consistent with the A549 cells, 15 µg/ml wogonin in SGC-7901 cells was selected for further research. The inhibitory rates of SGC-7901 and A549 cells following wogonin treatment (15 µg/ml) were 35.00±0.12 and 54.17±0.24%, respectively, indicating a stronger inhibitory effect of wogonin on A549 cell proliferation compared with SGC-7901 cells.

To investigate whether the effect of wogonin on cell proliferation was time-dependent, SGC-7901 and A549 cells were treated with 15 µg/ml wogonin for 7 days and the cell numbers were counted daily. Growth curves were prepared to describe the effect of wogonin on cell proliferation over time. As presented in Fig. 1B, wogonin decreased the cell proliferation in the cell lines in a time-dependent manner compared with the untreated control. Treatment with wogonin markedly decreased cell number in a time-dependent manner in each cell line, which was similar in strength to the effect exhibited by 10 µg/ml 5-Fluorouracil (5-Fu). 5-Fu is a potent anti-tumor agent that affects pyrimidine synthesis by inhibiting thymidylate synthetase, thus depleting intracellular dTTP pools (24). The results suggested that wogonin significantly inhibited the proliferation of SGC-7901 and A549 cells over time compared with an untreated control.

Morphological changes induced by Wogonin (15 µg/ml) were observed by H&E staining. As presented in Fig. 2, the majority of treated cells exhibited a round in shape, loss of cell volume, hyperchromatic nuclei and cytoplasm agglutination compared with the untreated controls (Fig. 2A-D). Compared with controls, the cell numbers were significantly decreased following treatment with wogonin in SGC-7901 and A549 cells (Fig. 2E and F). These results indicated that wogonin may induce cell morphological changes in the studied cell lines.

To investigate the effects of wogonin on energy metabolism, activities of enzymes participating in glucose anaerobic glycolysis, including HK, PK and LDH, and the activity of SDH, a contributor to the tricarboxylic acid cycle (TAC), were measured. As presented in Table I, SDH activity and ATP levels were significantly decreased by wogonin in SGC-7901 cells when compared with the untreated control (P<0.05). The inhibitory effect of wogonin significantly impacted on LDH activity when compared to the untreated control (P<0.01). Compared with the controls, A549 cells treated with wogonin significantly inhibited LDH activity (P<0.01) and ATP content was decreased (P<0.05). HK, SDH and PK activities were not significantly affected. The results indicated that wogonin may affect lactic acid and cell energy generation in SGC-7901 cells. Furthermore, wogonin treatment only regulated lactic acid and ATP generation in A549 cells.

To further investigate the effect of wogonin on energy metabolism-associated enzymes, HIF-1α and MCT-4 expression were determined by western blot. As presented in Fig. 3, following treatment with wogonin for 48 h, HIF-1α and MCT-4 expression significantly decreased in SGC-7901 cells compared with the control (P<0.01; Fig. 3A), but no significant changes were observed in A549 cells (Fig. 3B).