Luteolin was obtained from Sigma-Aldrich (St. Louis, MO, USA). A549 and H460 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Both control shRNA and Axl shRNA which were annealed to gold nanoparticles were synthesized by the domestic company, Bioneer Corp. (Daejeon, Korea). Lipofectamine 2000 and G418 were obtained from Roche Diagnostics Corp. (Indianapolis, IN, USA) and Gibco-BRL (Gaithersburg, MD, USA), respectively. The plasmid, pGL3-basic vector, and the Dual-Glo luciferase assay kit were purchased from Promega Corp. (Madison, WI, USA). For western blot analysis, specific antibodies against Axl, phosphor-Axl, MerTK, Tyro3 and GAPDH, as well as secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

The A549 and H460 cells were grown in RPMI-1640 medium (Gibco-BRL) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 U/ml penicillin and 10 g/ml streptomycin at 37°C in 5% CO₂ in a water-saturated atmosphere. Cisplatin-resistant cells, A549/CisR and H460/CisR, were established by stepwise exposure of parental cells to escalating concentrations of cisplatin (ranging from 0.5 to 2 µM).

Cells (3×10⁵) were seeded in a 60 mm culture dish and grown overnight. They were then treated with the indicated concentrations of luteolin for 24 h. Total RNA was extracted using TRI reagent and subjected to cDNA synthesis and PCR. The specific primers were as follows: Axl sense, 5′-AACCTTCAACTCCTGCCTTCTCG-3′ and antisense, 5′-CAGCTTCTCCTTCAGCTCTTCAC-3′; GAPDH sense, 5′-GGAGCCAAAAGGGTCATCAT-3′ and antisense, 5′-GTGATGGCATGGACTGTGGT-3′.

The promoter reporter plasmid, pGL3-Axl, which contains the Axl promoter region ranging from −887 to +7 bp of the transcriptional start site was amplified by PCR and subcloned into the pGL3-basic vector, the luciferase reporter plasmid. The constructed promoter-reporter plasmid was co-transfected into cells (3×10⁵ cells in a 60-mm dish) with Renilla luciferase vectors, pRL-SV40, as an internal control. Luciferase activity was asessed using a Dual-Glo luciferase assay system.

Total cell lysates were prepared from the parental or chemoresistant cells treated with the indicated concentrations (0, 20, 40 and 80 µM) of luteolin using lysis buffer [1% Triton X-100, 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM Na₃VO₄ and protease inhibitor cocktail]. Untreated cells were used as controls. Protein concentrations were determined using Bio-Rad protein assays. Proteins from the cell lysates (20–40 µg) were separated by 12% SDS-PAGE, and electrotransferred onto nitrocellulose membranes. The membranes were blocked for 30 min at room temperature in Tris-buffered saline with 0.05% Tween-20 (TTBS) containing 5% non-fat dry milk, and then incubated with TTBS containing a primary antibody for 4 h at room temperature. After 3×10 min washes in TTBS, the membranes were incubated with a peroxidase-conjugated secondary antibody for 1 h. Following three additional 10-min washes with TTBS, the protein bands of interest were visualized using an enhanced chemiluminescence detection system (Amersham™ ECL™ Prime Western Blotting Detection Reagent; GE Healthcare, Piscataway, NJ, USA). The density of each protein level was measured by LAS-3000 FujiFilm Image Reader and Multi-Gauge 3.0 software and the Axl protein level was normalized with that of GAPDH.

To assess cell viability, the number of viable cells was counted after trypan blue staining. Briefly, 3×10³ cells were seeded into a 60-mm culture dish, grown overnight and then treated with the indicated concentrations (0, 20, 40 and 80 µM) of luteolin for 24 h. After luteolin treatment, cells were harvested and stained with 0.4% trypan blue solution. Dye-excluding viable cells were counted under the microscope. Cell viability was also expressed as a percentage of the viable cells with respect to the untreated control cells. Additionally, the viability of cells was assessed using Cell Counting Kit-8 (CCK-8) assay kit (Dojindo Laboratories, Kumamoto, Japan). Cells (1×10³ cells/well) were seeded in 96-well plates and grown overnight, and then treated with the indicated concentrations of luteolin with or without 4 µM cisplatin for the 24 h. At the end of the treatment, 10 µl of CCK-8 solution was added and further incubated for 4 h. The absorbance at 450 nm was measured using a microplate reader (Model 680 microplate reader; Bio-Rad Laboratories, Hercules, CA, USA). Values are expressed as the mean ± SD for triplicate wells and normalized to that of the control group to determine the % of viability.

Cells were seeded into 24-well plates (1×10² cells/well) and treated with the indicated concentrations (0, 20, 40 and 80 µM) of luteolin for 24 h. Luteolin-treated cells were then cultured for the next 7–10 days to form colonies. Colonies of >50 cells were stained with crystal violet (in 60% methanol; Junsei Chemical Co., Ltd., Tokyo, Japan) and images were acquired using the RAS-3000 Image Analysis System (FujiFilm, Tokyo, Japan).

To ectopically express Axl, the recombinant plasmid, pcDNA3-Axl, was constructed by cloning the Axl cDNA into the EcoRI and BamHI sites of the pcDNA3 vector and 2 µg of purified plasmids were transfected into the A549 or A549/Cis cells (3×10⁵ cells in a 100-mm dish) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). To establish stable cell lines, which constitutively express Axl, the transfected cells were cultured in the presence of 400 µg/ml of G418. The RPMI-1640 medium containing G418 was refreshed every three days. After three to four weeks, the Axl-expressing cells were enriched and the Axl expression in these cells was analyzed by western blot analysis.

Chemically functionalized gold nanoparticles (AuNPs) were annealed with the shRNA targeting Axl gene (5′-TAATACGACTCACTATAGGGAAGAUUUGGAGAACACACUGA-3′) and used as a gene delivery system (GDS) to decrease Axl expression. Briefly, cells (3×10⁵) were seeded in 60-mm culture dishes, grown overnight and then incubated with 10 nM AuNPs-Axl or control AuNPs. The cells were harvested for 24 and 48 h after transfection and used to evaluate protein expression and cell proliferation, respectively.

Data are expressed as the means ± SD of triplicate samples or at least three independent experiments. To determine statistical significance, the Student's t-test was used with a P-value threshold of <0.05.

The antiproliferative effect of luteolin was first examined in parental non-small cell lung cancer (NSCLC) A549 and H460 cells, as well as cisplatin-resistant A549/CisR and H460/CisR cells. As shown in Fig. 1A, the viability of these cells was found to be decreased in a dose-dependent manner. Next, a clonogenic assay was further performed to confirm the cytotoxicity of luteolin in A549, H460, A549/CisR and H460/CisR cells. These cells were treated with the indicated concentrations of luteolin, and then allowed to grow for the next 10 days. Luteolin treatment resulted in the dose-dependent decrease in the colony formation (Fig. 1B). Notably, H460 and H460/CisR cells that were incubated with 80 µM luteolin failed to form visible colonies, suggesting that luteolin seems to be more cytotoxic to H460 cells than to A549 cells. We also examined the effect of co-treatment of luteolin and cisplatin on cell proliferation using H460/CisR cells. While 4 µM cisplatin alone decreased the viability of the H460/CisR cells to 61% and luteolin decreased that to 67, 41 and 27% in proportion to the concentration of luteolin, co-treatment of cells with luteolin and cisplatin reduced that to 36, 27 and 18%, respectively (Fig. 1C). The results demonstrated that in the presence of cisplatin, the cytotoxicity of luteolin was additively increased in the cisplatin-resistant cells.

Since TAM RTKs have been reported to be associated with oncogenesis, proliferation, survival and anti-apoptosis (9,10,42,43), we next assessed the effect of luteolin on the expression of TAM RTKs. Cells were incubated with the indicated concentrations of luteolin for 24 h and the protein levels of each TAM RTK was measured by western blot analysis. We found that in the A549 and A549/CisR cells, luteolin treatment dose-dependently decreased the expression levels of all three TAM RTKs, Tyro3, Axl and MerTK. Similarly in the H460 and H460/CisR cells, the expression levels of both Axl and Tyro3 were decreased by luteolin (Fig. 2A). Notably, in the H460 cells, MerTK was undetectable by western blot analysis.

Consistent with the western blot results, the inhibitory effect of luteolin on the Axl gene expression was further demonstrated by RT-PCR and assessment of Axl promoter activity. As shown in Fig. 2B, the mRNA level of Axl was decreased by luteolin treatment in a dose-dependent manner. Additionally, H460 cells transfected with pGL3-Axl, the Axl promoter-luciferase reporter plasmid, were treated with 0, 20, 40 and 80 µM of luteolin for the indicated time periods and luciferase activity was found to be dose-dependently decreased by luteolin treatment (Fig. 2C), indicating that luteolin suppresses Axl expression at the transcriptional level.

Since growth arrest-specific gene 6 (Gas6) binds to all three TAM RTKs and is highly specific to Axl (5), we next observed the effect of luteolin on Axl activation upon ligand binding. Serum-starved H460 cells were pre-treated with luteolin and then stimulated with Gas6. As illustrated in Fig. 2D, Gas6-induced Axl phosphorylation was significantly inhibited by luteolin, indicating that luteolin suppresses tyrosine kinase activity of Axl which is mediated by autophosphorylation of tyrosine residues in the intracellular kinase domain in response to ligand binding (6).

To validate the association of Axl in the antiproliferative effects of luteolin, we examined its cytotoxicity toward the H460 cells in which the Axl protein was overexpressed or knocked down, respectively. As shown in Fig. 3A, cells which were transfected with pcDNA3-Axl containing Axl cDNA for ectopic expression of Axl were found to be a less sensitive to luteolin than cells transfected with the pcDNA3 vector (Fig. 3A). Colony formation assay results also showed that clonogenicity of Axl-overexpressing cells was less affected by luteolin treatment compared with the cells transfected with pcDNA3, since luteolin treatment led H460/pc-DNA3 cells to form less colonies and the size of each colony was smaller than that of the H460/pc-DNA3-Axl cells (Fig. 3B). Consistent with the cell viability and colony formation assays, the Axl protein level in the H460/pcDNA3-Axl cells was found to be higher than that in the control cells even after luteolin treatment (Fig. 3C). These results point to the fact that overexpression of Axl attenuates the antiproliferative effect of luteolin.

Next, we examined whether knockdown of Axl increases the antiproliferative effect of luteolin. To decrease Axl expression, we used gold nanoparticle (AuNP)-assisted gene delivery systems (GDS) (44,45). H460 cells were incubated with AuNP GDS conjugates which were annealed with Axl-specific shRNA, or control shRNA. As shown in Fig. 4A, AuNP-GDS-Axl conjugates resulted in the additional decrease in Axl expression following luteolin treatment. The cell viability assay also showed that the AuNP-GDS-Axl conjugates increased cytotoxicity of luteolin (Fig. 4B). These results indicate that the amount of Axl protein was tightly correlated with cell viability and imply that luteolin exerts an antiproliferative effect via the downregulation of Axl expression. Collectively, these results indicate that luteolin inhibits Axl expression, concomitantly induces the protein level of p21, and subsequently abrogates cell proliferation.

Since IL-8, a multifunctional inflammatory cytokine, has been reported to be associated with cell survival, growth, angiogenesis and metastasis in various types of cancers such as melanoma, lung cancer, nasopharyngeal, hepatocellular, ovarian, colorectal and prostate cancer (33,46–49), we observed the effect of luteolin on IL-8 expression. ELISA results showed that the IL-8 production in cells treated with 80 µM luteolin was decreased, whereas 20 and 40 µM luteolin rather increased IL-8 level (Fig. 5A). Notably, IL-8 expression/cell was found to be fairly increased by luteolin treatment (Fig. 5B), indicating that the antiproliferative effect of luteolin was not associated with dysregulation of IL-8 expression.