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Introduction

Quercetin is a member of the flavonoids family and one of the leading dietary antioxidants. It is present ubiquitously in vegetables and fruits and is considered to exert beneficial health effects (1,2). Quercetin is known to induce autophagy in several cell types (3–5) and has attracted much attention in recent years due to its anticancer effects in many types of cancer (6,7).

Apoptosis is a morphologically obvious form of programmed cell death that plays a critical role during homeostasis, development, and diseases including cancer, acquired immunodeficiency syndrome and neurodegenerative disorders (8). Apoptosis, also called programmed cell death type I, can be induced in cancer cells by anticancer agents (9,10). Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was first confirmed as one of the TNF super-family. It can induce apoptosis selectively in tumorigenic or transformed cells, but not in normal cells, highlighting its potential therapeutic application in cancer treatment (11).

Autophagy is a conserved trafficking pathway that is highly regulated by environmental conditions (12). The implication of autophagy as a cell death mechanism in cancer with inactivated apoptosis is not surprising. However, the role of autophagy in cancer and treatment responsiveness is complicated (13). Autophagy is a general term for the degradation of cytoplasmic ingredients within lysosomes (14). Macro-autophagy was first described in mammals as the isolation of complete portions of cytosol, including soluble proteins as well as complete organelles, in a double membrane vesicle called autophagosome (15,16). Once the 2 sides of the autophagosome fuse to each other, there is a second fusion event between the autophagosome and the lysosomes/vacuole that leads to the formation of the autolysosome (17). The anti-malarial drug, chloroquine (CQ) inhibits lysosomal acidification and prevents the degradation of autophagosomes, thereby suppressing the autophagy flux (18).

Microtubule-associated protein light chain 3 (LC3) is localized and aggregated on the autophagosome and is, therefore, considered as a marker of autophagy. LC3B undergoes lipidation and is recruited to the phagophore where it is essential for membrane elongation and closure (19). LC3B transforms from LC3B-I to LC3B-II during autophagosome formation (20). P62 is a multifunctional signaling molecule, associated with a variety of cellular pathways. It is one of the best-known autophagic substrates, and is, therefore, extensively employed as an indicator of autophagic degradation (21). P62 can deliver ubiquitinylated cargos to the proteasome, though they are mainly degraded by autophagy (21,22). P62 levels are generally inversely related to autophagic degradation, since the loss of Atg genes or factors required for the fusion of autophagosomes with lysosomes all result in a marked increase of P62-positive aggregates (23,24).

Apoptosis is blocked in various cancer cells, and autophagy may be a major contributing mechanism of cancer cell death; thus, the induction of autophagy may be employed as a promising therapeutic strategy in cancer treatment (25). A549, human lung cancer cells, are known to be TRAIL-resistant cells (26). We identified that treatment with TRAIL or quercetin alone did not exert an influence on A549 human cancer cells. We next investigated the effect of co-treatment of TRAIL and quercetin on human cancer cells and quercetin-mediated autophagy flux. We also examined the effect of quercetin-induced autophagy on TRAIL-induced apoptosis in human lung cancer cells.

Materials and methods

Cell culture

The human lung cancer cell line A549 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in RPMI-1640 (Invitrogen-Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen-Gibco), 100 U/ml penicillin, and 0.1 mg/ml gentamycin in a humidified incubator maintained at 37°C and 5% CO2. Cells were treated for 12 h with quercetin (Sigma-Aldrich, St. Louis, MO, USA) and then exposed for 3 h to 200 ng/ml TRAIL, with or without the autophagy inhibitor, chloroquine (10 µM) (Sigma-Aldrich).

Crystal violet assay

Cell morphology was assessed microscopically (inverted Microscope, Nikon Eclipse TS100; Nikon Corp., Tokyo, Japan) and cell viability was determined by crystal violet staining (C0775; Sigma-Aldrich), as previously described (27). Briefly, cells were stained for 10 min at RT with crystal violet solution (0.5% crystal violet in 30% ethanol and 3% formaldehyde), washed 5 times with water, and then dried. Subsequently, the cells were lysed with 1% SDS (sodium dodecyl sulphate) and the absorbance was measured at 550 nm. Cell viability was calculated from the relative dye intensity of the samples compared to the controls.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

TUNEL assay was carried out as previously described (28). TUNEL analysis was performed to measure the degree of cellular apoptosis using an in situ ApoBrdu DNA fragmentation assay kit (BioVision, Mountain View, CA, USA), following the manufacturer’s instructions. Cells were counterstained with propidium iodide (PI) to show cell nuclei.

Trypan blue exclusion assay

The number of viable cells was determined by trypan blue dye exclusion (Sigma-Aldrich) using a hemocytometer. The result was expressed as a percentage relative to vehicle-treated controls.

BacMam transduction

Wild-type or mutant GFP-tagged LC3B was expressed in cells by adding the appropriate concentrations of appropriate virus from the Premo Autophagy Sensor LC3B-GFP (BacMam 2.0) kit (P36235; Life Technologies) to the growth medium as indicated in the figure legends.

Western blot analysis

A549 cells were lysed in lysis buffer [25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 100 mM NaCl, 1 mM EDTA (ethylene diamine tetra acetic acid), 5 mM MgCl2, 0.1 mM DTT (dithiothreitol), and a protease inhibitor mixture]. Whole cell proteins were electrophoretically resolved on a 10–15% sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoreactivity was detected through sequential incubation with primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and enhanced chemiluminescence reagents i.e. West Save Gold detection kit (AbFrontier Co., Ltd., Seoul, Korea). The primary antibodies used for immunoblotting were anti-LC3B (#4108; Cell Signaling Technology, Danvers, MA, USA), anti-P62 (#MABC32; Millipore, Billerica, MA, USA), anti-phospho-AKT (#2118-1; Epitomics, Burlingame, CA, USA), anti-caspase-3 (#9665; Cell Signaling Technology), anti-cleaved caspase-3 (#9661; Cell Signaling Technology) and anti-β-actin (A5441; Sigma Aldrich). Images were examined using a Fusion FX7 imaging system (Vilber Lourmat, Marne La Vallee, France). Densitometry of the signal bands was analyzed using Bio-1D software (Vilber Lourmat).

Statistical analysis

The unpaired t-test or Welch’s correction was used for comparison between the 2 groups. The one-way ANOVA followed by the Tukey-Kramer test was used for multiple comparison. All statistical analyses were performed with GraphPad Prism software. Results were considered significant for values **P<0.01 or ***P< 0.001.

Results

Quercetin enhanced TRAIL-mediated cell death in human lung cancer cells

We conducted several types of cell viability assays to investigate the effect of co-treatment with quercetin and TRAIL on A549 human lung cancer cells. First, we examined the photographed image of cell amounts using a light microscope and the crystal violet assay. The cell viability of cells treated with quercetin only was comparable to that of untreated controls. Cell death resulted in 3–5% of TRAIL-treated A549 cells. Importantly, quercetin treatment enhanced cell death to ~70% on photographed images (Fig. 1A) and augmented cell death to ~50% in the crystal violet assay (Fig. 1B and C). We additionally performed a TUNEL assay (Fig. 1D) and trypan blue exclusion assay (Fig. 1E). As shown in Fig. 1D, apoptosis in quercetin and TRAIL treated cells emitted green fluorescence indicative of DNA strand breakage. In Fig. 1E, quercetin dose-dependently increased TRAIL-mediated cell death. These results indicated that quercetin was effective in promoting TRAIL-induced cell death in A549 human lung cancer cells.

Figure 1 Quercetin enhanced TRAIL-mediated cell death in human lung cancer cells. A549 human lung cancer cells were pretreated with quercetin (12 h) in a dose-dependent manner, and then exposed to 200 ng/ml TRAIL for 3 h. Cell viability was measured by photographed image with a light microscope (magnification, ×100) (A) and by the crystal violet assay (B). (C) Bar graph indicated that the averages of density of dyed crystal violet. (D) Representative fluorescence images of TUNEL in A549 cells after co-treatment with 80 µM of quercetin (12 h) and 200 mg/ml of TRAIL (3 h). The positive (green) cells were counterstained with PI (red) to show all cell nuclei. (E) The treated cells were also measured by the trypan blue dye exclusion assay. **P<0.01, ***P<0.001; significant differences between control and each treatment group. Quer, quercetin; TR, tumor necrosis factor (TNF)-related apoptosis-inducing ligand.

Quercetin treatment induces autophagy flux and apoptosis

We evaluated quercetin mediated autophagy flux by estimating LC3B transformation and P62 expression. Levels of the late autophagosome marker LC3-II increased in the quercetin-treated group in a dose-dependent manner as compared with the control group on western blot analysis (Fig. 2A). The activation of the autophagy through the formation of autophagosomes in A549 lung cancer cells was visualized by the Premo Autophagy Sensor (LC3B-FP) BacMam 2.0 system. LC3B-FP and LC3B (G120A)-FP viral vectors (MOI=30) were transduced in A549 cells, enabling the expression of fluorescent LC3B protein. We consequently monitored autophagosomes dynamics using inverted fluorescent microscopy. The mutant chimera LC3B (G120A)-FP was used as a negative control. According to the results shown in Fig. 2B, BacMam LC3B (G120A)-FP transduced cells showed a marked diffuse cytosolic expression pattern. A549 cells treated with quercetin, exhibited an extensive punctate fluorescent distribution pattern, suggesting LC3B-FP protein accumulation in autophagosome. P62 was also decreased dose-dependently by quercetin (Fig. 2A and C). These results suggested that quercetin dose-dependently mediated autophagy flux. Akt is a key signaling molecule that associates oncogenic receptors to many essential pro-survival cellular functions in human cancer (29). We determined that quercetin treatment interrupted Akt activation and enhanced caspase-3 cleavage (Fig. 2D). Caspase-3 plays a key role in regulating programmed cell death or apoptosis, a normal process required for regulatory maintenance of physiological functions (30). Thus, these results suggested that quercetin mediated autophagy flux and enhanced TRAIL-induced apoptosis in A549 human lung cancer cells.

Figure 2 Quercetin treatment induces autophagy flux and apoptosis. (A) A549 cells were treated with quercetin (12 h) in a dose-dependent manner. The treated cells were assessed for LC3B production and P62 expression by western blot analysis. (B) A549 cells were mixed with a titration (30 MOI) of BacMam GFP-LC3B virus for 18 h, and then treated with quercetin (12 h), negative control reagent and positive control reagent (CQ) at the same time. (C) Bar graph indicated the averages of P62/β-actin ratio. (D) Cells were pre-treated with quercetin (12 h) in a dose-dependent manner, and then exposed to 200 ng/ml TRAIL for 1 h. The treated cells were assessed for phosphorylation of Akt and caspase-3 cleavage by western blot analysis. Results were normalized with β-actin. ***P<0.001; significant differences between control and each treatment group. Quer, quercetin; TR, tumor necrosis factor (TNF)-related apoptosis-inducing ligand; p-Akt, phosphorylation of Akt; cas3, caspase-3; c-cas3, cleaved caspase-3; adj.volume, adjustment of volume (band volume minus background volume).

Quercetin regulates autophagy flux

We investigated whether quercetin induced autophagy flux or regulated chloroquine (CQ)-mediated autophagy flux. CQ is widely used to inhibit the maturation of autophagosome into degradative autolysosome (31,32). We showed that upregulation of LC3B-II by quercetin was potentiated by CQ, since CQ inhibited the fusion of autophagosome and autolysosome (Fig. 3A). A549 cells treated with quercetin, presented an extensive punctate fluorescent distribution pattern which was also augmented by CQ (Fig. 3B). P62 is a crucial mediator to target protein to the autophagy system in the removal of aggregated proteins. P62 is itself degraded during autophagy (33). Reduction of P62 level by quercetin treatment indicated autophagy flux activation, and also the activation of autophagy flux was inhibited by CQ treatment, given the increase of P62 levels (Fig. 3A and C). According to the results shown in Fig. 3D, A549 cells co-treated with CQ were augmented Akt activation and decreased caspase-3 cleavage, whereas caspase-3 activation were induced in cells treated with quercetin and TRAIL treatment, which indicated that CQ played a protective role against apoptotic cell death in A549 cells.

Figure 3 Quercetin regulates autophagy flux. (A) A549 cells were pretreated with autophagy inhibitor (chloroquine) (1 h) and then exposed to quercetin (12 h) in dose-dependent manner. The treated cells were assessed for LC3B production and P62 expression by western blot analysis. (B) A549 cells were mixed with a titration (30 MOI) of BacMam GFP-LC3B virus over 18 h, and then treated with chloroquine for 1 h and quercetin for 12 h. Negative control reagent and positive control reagent (CQ) was also treated at the same time. (C) Bar graph indicated the averages of P62/β-actin ratio. (D) Cells were pre-treated with autophagy inhibitor (chloroquine) (1 h) and then exposed to quercetin for 12 h and TRAIL for 1 h. The treated cells were assessed for phosphorylation of Akt and caspase-3 cleavage by western blot analysis. Results were normalized with β-actin. ***P<0.001; significant differences between control and each treatment group. Quer, quercetin; TR, tumor necrosis factor (TNF)-related apoptosis-inducing ligand; CQ, chloroquine; p-Akt, phosphorylation of Akt; cas3, caspase-3; c-cas3, cleaved caspase-3; adj.volume, adjustment of volume (band volume minus background volume).

Autophagy regulates quercetin-induced TRAIL sensitivity

We analyzed cell viability using CQ, to investigate the effect of quercetin-mediated autophagy on TRAIL-sensitivity. A549 cells treated with CQ recovered Akt activation and were protected against cleavage of caspase-3 (Fig. 3B). We accordingly investigated whether autophagy inhibition using CQ exerted an influence on cell viability. We examined the photographed image of cell amounts using light microscopy and performed the crystal violet assay. Quercetin treatment in TRAIL treated cells enhanced cell death to ~70% in the photographed image (Fig. 4A), and to 50% in the crystal violet assay (Fig. 4B and C). However, cell death recovered to ~60% cell survival in the photographed image (Fig. 4A) and 85% cell survival in the crystal violet assay (Fig. 4B and C). We performed a TUNEL assay (Fig. 1D) and trypan blue exclusion assay (Fig. 1E). As shown in Fig. 4D, the apoptotic process in quercetin and TRAIL treated cells emitted green fluorescence, indicative of DNA strand breakage, however, green fluorescence was weak in CQ-treated cells. In Fig. 1E, CQ treatment alleviated cell death by inhibiting quercetin-mediated TRAIL sensitivity. These results indicated that CQ was effective in inhibiting quercetin-induced TRAIL sensitivity by regulating autophagy flux in A549 human lung cancer cells. Collectively our data suggested that quercetin-induced autophagy flux had a harmful effect on TRAIL sensitivity and inhibition of autophagy flux played a protective role against quercetin-mediated TRAIL sensitivity in A549 human lung cancer cells.

Figure 4 Autophagy regulates quercetin-induced TRAIL sensitivity. A549 human lung cancer cells were pretreated with CQ (1 h) and then exposed to quercetin (12 h) and TRAIL (3 h). Cell viability was measured by photographed image with a light microscope (magnification, ×100) (A) and by the crystal violet assay (B). (C) Bar graph indicated the average density of dyed crystal violet. (D) Representative fluorescence images of TUNEL were implemented in A549 cells after the co-treatment 50 µM of CQ (1 h), 80 µM of quercetin (12 h) and 200 mg/ml of TRAIL (3 h). The positive (green) cells were counterstained with PI (red) to show all cell nuclei. (E) The treated cells were also measured by trypan blue dye exclusion assay. ***P<0.001; significant differences between control and each treatment group. Quer, quercetin; TR, tumor necrosis factor (TNF)-related apoptosis-inducing ligand; CQ, chloroquine.

Discussion

The purpose of the present study was to investigate the role of quercetin-induced autophagy flux and the regulation of TRAIL-mediated sensitivity by quercetin treatment in A549 human lung cancer cells. The results suggested that quercetin-induced autophagy and the resultant enhancement in quercetin-induced TRAIL sensitivity might be a key underlying mechanism of autophagy flux.

Quercetin belongs to an extensive class of polyphenolic flavonoid compounds and is practically ubiquitous in plants and plant food sources. It has been studied as a promising chemoprevention component in a variety of cancer models (34). Consistent with previous reports, the present study showed that quercetin promoted TRAIL-mediated apoptosis in lung cancer cells.

Autophagy is an evolutionary conserved, dynamic lysosome-mediated process that entails the sequestration and delivery of cytoplasmic material to the lysosome where it is degraded and recycled (35,36). Some studies have insisted that autophagy is a double-edged sword, with both useful and harmful potential in cancer (37). Wang et al (38) suggested that quercetin induces protective autophagy in gastric cancer cells. Their study indicated that quercetin-induced apoptosis and autophagy played a protective role during apoptosis. The present study, on the other hand, showed that quercetin-induced autophagy induced TRAIL-sensitivity, and then mediated apoptosis. We demonstrated that quercetin-induced autophagy did not play a protective function by using an autophagy inhibitor in A549 lung cancer cells. However, this experimental evidence was insufficient to elucidate function of autophagy flux.

Some reports confirmed that TRAIL induces autophagy in several types of cancer cells (39,40). However, our results indicated that TRAIL treatment did not mediate transformation from LC3B-I to LC3B-II (data not shown). On the contrary, TRAIL-treated LC3-II transformation decreased slightly than control. We concluded that TRAIL was possibly not associated with autophagy in A549 lung cancer cells.

According to the results reported in Figs. 2D and 3D, Akt signaling was activated by TRAIL treatment and decreased activation of Akt was observed on treatment with quercetin. Akt signal is activated by TRAIL-resistance in breast cancer cells (41) and TRAIL phosphorylated PI3k and Akt in leukemic T Jurkat cells (42). Akt or protein kinase B (PKB) is one of the most critical kinases in the regulation of cell survival. Enhanced activity of the PI3K/Akt pathway is found in many malignancies and is associated with the stimulation of cell growth and cell survival (43).

Cross-talk between autophagy and apoptosis is complicated and sometimes contradictory; however, it is a critical determinant of the overall fate of the cell. This study determined that quercetin-induced autophagy flux might play a crucial role in TRAIL sensitivity in A549 human lung cancer cells. Quercetin may thus be a useful regulator for TRAIL-mediated cancer therapy in lung cancer.

Acknowledgments

The present study was supported by a grant from the National Research Foundation of Korea (NRF), funded by the Korean government (MISP) (2013R1A4A1069486).

References