Human liver cancer cells (Huh7 and Hep3B) were obtained from the American Type Culture Collection (Global Bioresource Center, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). For experimentation, the medium of the cells was changed to DMEM containing 1% FBS. Cells were cultured at 37°C with 5% CO₂ in humidified incubator.

Luteolin (solvent, DMSO) was acquired from Cayman Chemical Co. (Ann Arbor, MI 48108, USA), TRAIL [solvent, phosphate-buffered saline (PBS)] was acquired from AbFrontier Co., Ltd. (Seoul, Korea). Chloroquine (CQ) diphosphate salt was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and CQ was dissolved in water to produce a 10 mM stock solution. SP600125 (10 µM) was acquired from the Beyotime Institute of Biotechnology (Shanghai, China). SP600125 and CQ added to the cells 1 h prior to treatment luteolin or TRAIL.

The liver cancer cells were plated in 12-well plates and treated with luteolin (0, 5, 10 and 20 µM) for 18 h and exposed to TRAIL (200 ng/ml) for a further 2 h. Cell morphology was assessed under a microscope (Nikon, Tokyo, Japan, magnification, ×100), and cell viability was evaluated by the crystal violet staining solution kit from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) according to the manufacturer’s instructions.

Cell viability was evaluated by trypan blue exclusion assay (Sigma-Aldrich, St. Louis, MO, USA) using a hemocytometer (#02-671-10; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Following each treatment, cells were stained with 0.4% trypan blue for 5 min at room temperature. Unstained cells were regarded as viable, and stained cells were regarded as dead. The total cell number and the number of trypan blue-positive cells were counted using a light microscope in a blinded manner. The percentage of surviving cells was calculated using the formula: Number of stained cells/number of total cells ×100. Each experiments was performed in triplicate.

Following fixation of the cells in 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and 2% paraformaldehyde (Electron Microscopy Sciences) in 0.05 M sodium cacodylate (pH 7.2; Electron Microscopy Sciences) for 2 h at 4°C specimens were fixed in 1% osmium tetroxide (Electron Microscopy Sciences) for 1 h at 4°C, dehydrated with increasing ethanol (25, 50, 70, 90 and 100%) for 5 min each and embedded in epoxy resin (Embed 812; Electron Microscopy Sciences) for 48 h at 60°C according to the manufacturers’ instructions. Ultrathin sections (60 nm) were prepared using an LKB-III ultratome (Leica Microsystems GmbH, Wetzlar, Germany) and were stained with 0.5% uranyl acetate (Electron Microscopy Sciences) for 20 min and 0.1% lead citrate (Electron Microscopy Sciences) for 7 min at room temperature. Images were recorded on a Hitachi H7650 electron microscope (Hitachi, Ltd., Tokyo, Japan; magnification, ×10,000) installed at the Center for University-Wide Research Facilities (CURF) at Chonbuk National University.

The Huh7 cells were cultured on poly-L-lysine-coated coverslips. Following differentiation and specific treatment, the cells were adjusted with 4% paraformaldehyde and permeablized with 0.1% Triton X-100. The cells were then incubated in a blocking solution followed by overnight incubation at 4°C with anti-p62 (cat. no. 5114; 1:1,000) and p-c-Jun N-terminal kinase (JNK, cat. no. 9255s) antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, USA). After washing with PBS, the cells were incubated with secondary antibodies (Alexa Fluor^® 488-conjugate; donkey polyclonal anti-rabbit, 1:500; Thermo Fisher Scientific, Inc.; cat. no. A-21206) and Texas-Red-X-conjugate (goat poly-clonal anti-mouse, 1:500; Thermo Fisher Scientific, Inc.; cat. no. T-6390) for 2 h in the dark. In addition, 4′,6′-diamidino-2-phenyl-indole (DAPI; D9564; Sigma-Aldrich, St. Louis, MO, USA) was used to non-specifically stain the nuclei. Finally, immunostaining was visualized under a fluorescence microscope (#451203, Nikon ECLIPSE 80i; Nikon Corporation, Tokyo, Japan; magnification, ×400).

Western blot analysis was carried out as previously described (32). Briefly, RIPA lysis buffer was used to extract total proteins. The supernatant was collected by centrifuging at 13,282 × g at 4°C for 10 min. Protein concentration was tested using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). The samples (30 µg) were separated on SDS-PAGE (10%) and then blotted onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Bedford, MA, USA). The membrane was blocked with 5% non-fat dry milk at 25°C for 1 h, and then incubated with primary antibodies overnight for 1 h at 4°C. The β-actin antibody was from Sigma-Aldrich (cat. no. A2228; 1:2,000; Merck KGaA, Darmstadt, Germany), The antibodies against DR4 (cat. no. ab8414; 1:1,000) and DR5 (cat. no. ab181846; 1:1,000) were from Abcam (Cambridge, MA, USA); against LC3A/B (cat. no. 3868; 1:1,000), cleaved caspase-3 (cat. no. 9661; 1:500), p-JNK (cat. no. 9255s, 1:1,000), p62 (cat. no. 5114; 1:1,000), ATG5 (cat. no. 2630; 1:1,000) were from Cell Signaling Technology (Danvers, MA, USA) and the antibody against cleaved caspase-8 (cat. no. 551242; 1:1,000) was from BD Pharmingen/BD Biosciences (San Jose, CA, USA). The membrane was incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (cat. no. 4410; Cell Signaling Technology; 1:2,000) at 25°C for 1 h. The immunoreactive protein bands were visualized using an enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Chalfont, UK).

The Huh7 cells were seeded into 6-well plates at a density of 1×10⁴ cells/well. Following 48 h of incubation the cells reached 80% confluence and were transiently transfected with ATG5 siRNA using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The sequences for ATG5 siRNA (siRNA; oligo ID HSS114103; Invitrogen/Thermo Fisher Scientific) were as follows: Sense, 5′-GGCCUUUCAUUCAGAAGCUTT-3′ and antisense, 5′-AGCUUCUGAAUGAAAGGCCTT-3′; the sequences for negative control (NC) were sense, 5′-UCUCCGAACGUGUCACGUTT-3′ and anti-sense, 5′-ACGUGACACGUUCGGAGAATT-3′. The oligodeoxynucleotides for the NC were obtained following scrambling of the siRNA oligodeoxynucleotide for ATG5, and were determined to not be associated with any mRNA sequence by BLAST. Cell transfection was performed, according to the protocol of the transfection kit manufacturer. Briefly, each sequence of MIF siRNA and 10 µl Lipofectamine 2000 was diluted in serum-free medium (250 µl) at room temperature for 5 min, mixed together, and incubated for 30 min at room temperature. The mixture was subsequently administered to the Tca8113, SCC25 and HN5 cells, and after 5 h of incubation, the medium was replaced with complete medium.

Statistical analyses were carried out using GraphPad Prism software (version 5.03; GraphPad Software, Inc., La Jolla, CA, USA). All experiments were performed 3 times, and the data are expressed as the means ± standard error. Significant differences between the control and treated samples were analyzed using one-way factorial analysis of variance (ANOVA), followed by Duncan’s post-hoc test. A value of P<0.05 was considered to indicate a statistically significant difference.

Treatment with TRAIL or luteolin alone induced minimal or no cell death (Fig. 1). In addition, the treated cells exhibited no morphological differences from those of the control cells, revealing that the HCC cells were immensely resistant to TRAIL-mediated apoptosis. Co-treatment with TRAIL and increasing concentrations of luteolin markedly attenuated cell viability, increasing apoptotic bodies and decreasing attached cells compared to treatment with luteolin or TRAIL alone (Fig. 1A and E). Luteolin treatment sensitized both cells to TRAIL, but the sensitivity was higher in Huh7 cells than in Hep3B cells, and we focused on the Huh7 cells in this study. These findings suggested that luteolin effectively enhanced the sensitivity of human HCC cells to TRAIL-induced apoptosis.

As shown in Fig. 2A and B, luteolin treatment significantly upregulated LC3-II and downregulated p62 expression, which is consistent with DR5 upregulation. The results of TEM analysis revealed that numerous autophagic and vacant vacuoles were secreted in the luteolin-treated cells (Fig. 2C). The cells co-treated with luteolin and TRAIL also exhibited a higher expression of cleaved caspase-3 and cleaved caspase-8 (Fig. 2D). These outcomes suggested that luteolin can initiate autophagy in Huh7 cells.

Co-treatment with luteolin, chloroquine and TRAIL suppressed cell death. Cell morphological analysis revealed that the apoptotic bodies were markedly reduced and the attaching cells were increased in the luteolin-, chloroquine- and TRAIL-treated cells. These results also confirmed that treatment with chloroquine attenuated the cell death induced by combined treatment with luteolin and TRAIL (Fig. 3A). Co-treatment with luteolin, TRAIL and chloroquine markedly increased the viability of the human HCC Huh7 cells (Fig. 3B-D). These outcomes suggested that chloroquine blocked TRAIL-induced and luteolin-mediated liver cancer cell death.

The origination of autophagy was further confirmed based on the suppression of the autophagic flux following chloroquine treatment, which resulted in an increase in the accumulation of membrane-bound LC3-II, an increase in p62 levels, and the partial suppression of DR5 upregulation (Fig. 4A). The results of immunofluorescence staining also confirmed the increased p62 levels following treatment with chloroquine (Fig. 4B). Chloroquine also partially inhibited the upregulation DR5 which had been induced by luteolin and TRAIL (Fig. 4C). In addition, co-treatment with luteolin, TRAIL and chloroquine inhibited the increase in cleaved caspase-8 and cleaved caspase-3 induced by luteolin and TRAIL (Fig. 4D). These outcomes suggested that the luteolin-induced augmentation of TRAIL-initiated apoptosis and the promotion of the autophagic flux were suppressed by chloroquine.

Co-treatment with luteolin, ATG5 siRNA and TRAIL prevented cell death. Cell morphological analysis revealed that the apoptotic bodies were markedly reduced and the attaching cells were increased in the luteolin-, ATG5 siRNA- and TRAIL-treated cells. The results confirmed that autophagy inhibition suppressed the cell death induced by treatment with luteolin and TRAIL, and also compared to transfection with the negative control (NC) (Fig. 5A). Co-treatment with luteolin and TRAIL, and transfection with ATG5 siRNA markedly increased the viability of the Huh7 HCC cells and significantly inhibited cell death (Fig. 5B-D). These outcomes suggested that autophagy inhibition blocked TRAIL- and luteolin-induced liver cancer cell death.

ATG5 knockdown decreased the luteolin-induced expression of LC3-II and DR5 and markedly increased the p62 protein levels (Fig. 6A). The p62 protein levels determined based on immunofluorescence staining were consistent with the results of western blot analysis (Fig. 6B). However, co-treatment with luteolin, ATG5 siRNA and TRAIL attenuated the increase in Ac-cas3 and Ac-cas8 expression (Fig. 6C). These findings suggested that the luteolin-induced augmentation of TRAIL-initiated apoptosis mediated by the activation of the autophagic flux was suppressed by the genetic attenuation of autophagy.

Cell morphological analysis revealed that the apoptotic bodies were markedly reduced and the attaching cells were increased in JNK inhibitor (SP600125)-, luteolin- and TRAIL-treated cells (Fig. 7A). Co-treatment with luteolin, SP600125 and TRAIL reduced the cell apoptosis induced by luteolin and TRAIL (Fig. 7A and B). The results revelaed that luteolin increased JNK and decreased Akt phosphorylation (Fig. 7C). Pre-treatment with the JNK inhibitor (SP600125) significantly attenuated JNK phosphorylation and inhibited DR5 upregulation (Fig. 7D). The results of immunofluorescence staining also confirmed that SP600125 treatment significantly reduced JNK phosphorylation (Fig. 7E).