The HepG2 human hepatic cancer cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin; Beyotime Institute of Biotechnology, Haimen, China) and incubated at 37°C under a humidified atmosphere of 5% CO₂.

HepG2 cells were seeded in 96-well culture plates at a density of 1×10⁴ cells/well. Subsequent to overnight growth, the cells were incubated with 0, 5, 10, 25 50 and 100 µM kaempferol (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. A separate group of HepG2 cells was treated with 100 µM kaempferol for time periods of 3, 6, 12 and 24 h. Kaempferol was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich), and the final concentration of DMSO in the culture medium was maintained at <0.1% (v/v). The vehicle (VE) and blank control (BC) groups were established. For the viability assay, at the end of each treatment, 200 µl culture medium containing 0.5 mg/ml 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Amresco LLC, Solon, OH, USA) was added to each well and the mixture was incubated at 37°C for 4 h. The supernatant was removed, formazan crystals were dissolved in 150 µl DMSO and the absorbance was measured at 570 nm using a microplate reader (Multiskan MK3; Bio-Rad Laboratories, Inc., Hercules, CA, USA). To determine how kaempferol triggers apoptosis via molecular signaling pathways, the following three additional treatment groups were established: i) 4-Phenyl butyric acid (4-PBA; Sigma-Aldrich) pretreatment group, where cells were pretreated with 4-PBA at 1 mM for 30 min; ii) transfection group with CHOP small interfering (si) RNA; and iii) plasmid group with a CHOP overexpressing plasmid (Shanghai GenePharma Co., Ltd., Shanghai, China). HepG2 cells were then exposed to 100 µM kaempferol for 24 h, and an MTT assay was performed as abovementioned. Cell viability was calculated as follows: [(A_{kaempferol treatment group}−A_BC/A_VE−A_BC)] ×100, where A represents absorbance.

A colorimetric lactate dehydrogenase (LDH) activity assay kit (Applygen Technologies, Inc., Beijing, China) was used to quantify the level of LDH released into the supernatant from the damaged cells. This assay was performed subsequent to treatment application. The supernatants from each treatment group were collected and the assay was performed according to the manufacturer's protocol.

An Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining assay (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China) was used to quantify apoptosis. HepG2 cells were cultivated in 60 mm culture plates for 24 h at 37°C. Following exposure to the indicated treatments, cells were harvested by trypsinization (Sigma-Aldrich), washed with phosphate-buffered saline (PBS), pelleted by centrifugation at 800 × g and 4°C for 5 min, and resuspended in 0.5 ml binding buffer (Nanjing KeyGen Biotech, Co., Ltd.). The cells were incubated with 5 ml annexin V-FITC and 5 ml PI working solution for 15 min at room temperature in the dark. The samples were analyzed on a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using FlowJo software (version 7.6; FlowJo LLC, Ashland, OR, USA). Double staining of cells with annexin V-FITC and PI enabled the identification of different cell populations based on their staining patterns, as follows: Lower left quadrant, live cells (FITC⁻PI⁻); lower right quadrant, early apoptotic cells (FITC⁺PI⁻); upper right quadrant, late apoptotic cells (FITC⁺PI⁺); upper left quadrant, necrotic cells (FITC⁻PI⁺).

At 24 h prior to transfection, HepG2 cells were prepared in 12-well culture plates. Cells were transfected with 20 µM human CHOP siRNA and negative control siRNA using Lipofectamine 2000 reagent for 6 h according to the manufacturer's protocol (Shanghai GenePharma Co., Ltd.). CHOP siRNA sequences were designed as follows: Forward (F) 5′-GAGCUCUGAUUGACCGAAUTT-3′ and reverse (R) 5′-AUUCGGUCAAUCAGAGCUCTT-3′; control siRNA, F: 5′-UGA GAG UGU CAG CAA UTT CCU-3′ and R: 5′-AUC GCU UCA AAG UUA GCG CTT-3′. The transfected cells were then treated with 100 µM kaempferol for 24 h.

HepG2 cells were cultured in 12-well culture plates of 1 ml volumes. CHOP overexpression plasmid (Shanghai GenePharma Co., Ltd.) and the empty vector control plasmid were transfected into HepG2 cells using Lipofectamine 2000 reagent, according to the manufacturer's protocol (Shanghai GenePharma Co., Ltd.). Following a 24 h transfection, HepG2 cells were treated with ER stress inhibitor 4-PBA at 1 mM for 30 min at 37°C, and cultured with 100 µM kaempferol for an additional 24 h.

The mRNA levels of target genes were evaluated using RT-qPCR. HepG2 cells were cultured at a concentration of 1×10⁵ cells/well in 12-well culture plates. Cells were cultured for 24 h prior to treatments. Total RNA was extracted from HepG2 cells using TRIzol reagent and then reverse-transcribed into cDNA by PrimeScript First Strand cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan), following the manufacturer's protocol. The hypoxanthine phosphoribosyl transferase (HPRT) gene was selected as an endogenous control. PCR was performed in a reaction mixture (20 µl) containing 4 µl cDNA, 0.4 µl each primer (10 µM), 5.2 µl diethylpyrocarbonate water and 10 µl SYBR Green (Takara Bio, Inc.) using a quantitative PCR instrument (ABI Prism 7500; Applied Biosystems Inc., Waltham, MA, USA). The reaction was processed with an initial 2 min denaturation step at 50°C, followed by 95°C for 5 min, 95°C for 15 sec, and 60°C for 30 sec, for 40 cycles, and then 55°C for 4 sec for 41 cycles. The mRNA levels were calculated using the 2^−ΔΔCq method (21). The specific primer sequences for these genes were as follows: HPRT, F 5′-TCAACGGGGGACATAAAAGT-3′ and R 5′-TGCATTGTTTTACCAGTGTCAA-3′; CHOP, F 5′-CCTAGCTTGGCTGACAGAGG-3′ and R 5′-CTGCTCCTTCTCCTTCATGC-3′; GPR78, F 5′-AGTGGTGCCTACCAAGAAGTCTCA-3′ and R 5′-TGTCAGGGGTCTTTCACCTTCATA-3′; GPR94, F 5′-ACGTGGTCTGTTTGACGAATATGG-3′ and R 5′-TACACGGCGCACATAGAGCTTAAT-3′.

Subsequent to the indicated treatments, cells were scraped off and washed with ice-cold PBS, and then lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing a mixture of protease inhibitors. A total of 30 µg of protein from each sample was separated by 12% sodium dodecyl sulfate-polyacrylamide gel (Sigma-Aldrich) electrophoresis at 80 v for 30 min and 120 v for 1 h, and then electrotransferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.) using the Bio-Rad Laboratories, Inc. transfer blotting system. The membranes were subsequently incubated with 5% skimmed milk in Tris-buffered saline with Tween-20 (Beyotime Institute of Biotechnology) for 1 h to block nonspecific binding and then overnight with the following antibodies: Monoclonal rabbit anti-human GRP78 (1:1000; cat. no. 3177; Cell Signaling Technology, Inc., Danvers, MA, USA), monoclonal rabbit anti-human GRP94 (1:1,000; cat. no. 20292; Cell Signaling Technology, Inc.), monoclonal rabbit anti-human PERK (1:1,000; cat. no. 5683; Cell Signaling Technology, Inc.), monoclonal mouse anti-human partial ATF-6 (1:1,000; cat. no. IMG-273; Imgenex Corporation, San Diego, CA, USA), monoclonal rabbit anti-human IRE1α (1:1,000; cat. no. 3294; Cell Signaling Technology, Inc.), monoclonal rabbit anti-human caspase-4 (1:1,000; cat. no. 4450; Cell Signaling Technology, Inc.), monoclonal mouse anti-human CHOP (1:1,000; cat. no. 2895; Cell Signaling Technology, Inc.), monoclonal rabbit anti-human cleaved caspase-3 (1:1,000; cat. no. 9664; Cell Signaling Technology, Inc.) and monoclonal rabbit anti-human β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) at 4°C, followed by incubation with monoclonal goat anti-rabbit IgG (1:2,000; cat. no. 14708; Cell Signaling Technology, Inc.) secondary antibody for 1 h at room temperature. Proteins were visualized using an enhanced chemiluminescence commercial kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA). The absorbance of each well containing the protein and reagent was determined at a wavelength of 630 nm using a microplate reader (Multiskan MK3; Thermo Fisher Scientific, Inc.) and a protein concentration standard curve was established to calculate the concentration of each protein sample.

Statistical analyses were performed using SPSS statistical software (version 16.0: SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation from a minimum of three separate experiments. Statistical comparisons between groups were performed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of different concentrations of kaempferol on apoptosis, HepG2 cells were treated with 0–100 µM kaempferol for 24 h. Cell viability was significantly reduced at 100 µM kaempferol concentration compared with the control group (P<0.0001; Fig. 1B). Additionally, LDH activity (P=0.0006) and the rate of apoptosis significantly increased at 100 µM kaempferol (P<0.0001; Fig. 1C–E). Western blotting also indicated that kaempferol triggers expression of cleaved caspase-3 (Fig. 1F). The results suggest that kaempferol induces apoptosis in HepG2 cells in a dose-dependent manner.

To examine whether kaempferol triggers HepG2 cells apoptosis in a time-dependent manner, HepG2 cells were treated with 100 µM kaempferol for 0, 3, 6, 12 and 24 h. Kaempferol gradually inhibited the proliferation of HepG2 cells and promoted cell death between 12 and 24 h. Following 24 h of 100 µM kaempferol treatment, cell viability was significantly reduced (P<0.0001; Fig. 2A). There was greater LDH activity (P<0.05; Fig. 2B) and an increased rate of apoptosis (P<0.0001; Fig. 2C and D) in cells exposed to kaempferol for ≥3 h compared with the control group. The expression of cleaved caspase-3 was also elevated (Fig. 2E). These data indicate that kaempferol induces apoptosis in HepG2 cells in a time-dependent manner.

In order to explore the molecular mechanisms of kaempferol-induced apoptosis, the protein and mRNA expression levels of ER stress markers, including GRP78, GRP94, PERK, partial ATF-6, IRE1α, caspase-4 and CHOP were determined, using western blotting and RT-qPCR, respectively. Kaempferol treatment led to an increase of these protein and mRNA levels in a dose-and time-dependent manner (Fig. 3A and B). The protein and mRNA levels of GRP78, GRP94 and CHOP were also significantly increased with in a dose-dependant manner (Fig. 3C and D). Therefore, the results indicate that kaempferol-induced apoptosis in HepG2 cells is associated with the induction of the ER stress response.

To confirm the role of the ER stress response as the underlying molecular mechanism of kaempferol-induced apoptosis in HepG2 cells, the cells were pretreated with the ER stress inhibitor 4-PBA to suppress ER stress. Compared with the kaempferol only treatment group, cell viability was significantly higher in the 4-PBA pretreatment group (P=0.0066; Fig. 4A). Additionally, LDH activity and apoptotic rates were lower in the 4-PBA pretreatment group compared with the kaempferol group (P=0.037 and P=0.002; Fig. 4A–C). Furthermore, the protein expression levels of ER stress markers were evaluated. The protein expression levels of CHOP, caspase-4 and cleaved caspase-3 were observed to be high in the kaempferol treatment group, whereas they were reduced subsequent to the inhibition of ER stress by 4-PBA (Fig. 4D). The results suggest that kaempferol-induced apoptosis in HepG2 cells is mediated by the ER stress response.

CHOP is an extensively-characterized factor in the transition of ER stress to apoptosis (16); therefore, its function in kaempferol-induced apoptosis in HepG2 cells was investigated by limiting its expression. Knockdown of CHOP with siRNA significantly attenuated the reduction of cell viability compared with the kaempferol only treatment group (P=0.0024; Fig. 5A). LDH activity (P=0.0006), apoptotic rate (P<0.0001); and protein expression levels of CHOP and cleaved caspase-3 were markedly reduced in the CHOP siRNA treatment group compared with the kaempferol group (Fig. 5A–D). Following transfection with the CHOP-overexpressing plasmid, the protein expression levels of CHOP were markedly increased, resulting in a reversal of the protective effect of 4-PBA on kaempferol-induced cell apoptosis (Fig. 6A–D). These results indicate that kaempferol promotes apoptosis of HepG2 cells via the ER stress-CHOP pathway.