A549 (human lung adenocarcinoma cells), NCI-H460 (human lung adenocarcinoma cells, MCF-7 (human breast carcinoma cells), and PC3M (human prostate cancer cells) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Delphinidin was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA).

Cells were plated in 96-well culture plates at a density of 1×10⁴ cells/well in RPMI-1640 culture medium and allowed to attach for 24 h. The media were then discarded and replaced with 100 µl of new medium containing various concentrations of delphinidin and cultured for 24 h. A WST-1 assay kit (Cayman Chemical Co.) was added to each well. The amount of formazan deposits was quantified according to the supplier's protocol after 4 h of incubation with WST-1 test solution at 37°C in a 5% CO₂ incubator.

A549 cells were pre-incubated for 24 h, then stimulated with CoCl₂ (200 µM) and EGF (20 ng/ml) in the presence of delphinidin. After incubation, the cells were collected and washed twice with cold PBS. The cells were lysed in a lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO₃, 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml aprotinin, and 25 µg/ml leupeptin] and maintained on ice for 30 min. The cell lysates were obtained via centrifugation, and the protein concentrations were determined using a protein assay kit. Aliquots of the lysates were separated on 12% SDS-polyacrylamide gel and transferred onto a nitrocellulose transfer membrane (Whatman, Dassel, Germany) with a glycine transfer buffer [192 mM glycine, 25 mM Tris-HCl (pH 8.8), 20% methanol (v/v)]. After blocking the nonspecific site with 1% bovine serum albumin (BSA), the membrane was incubated overnight with a specific primary antibody at 4°C. The membrane was then incubated for an additional 1 h with a peroxidase-conjugated secondary antibody (1:1,000; Vector Laboratories, Inc., Burlingame, CA, USA) at room temperature. The immunoactive proteins were detected using an enhanced chemiluminescence (ECL) western blot analysis detection kit (Advansta, Inc., Menlo Park, CA, USA).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was carried out using a commercial kit (SuperScript II RNase H-reverse transcriptase; Invitrogen) and total RNA (1 µg) from A549 cells, according to the manufacturer's instructions. The sequences of the primers were as follows: for HIF-1α, 5′-CTCAAAGTCGGACAGCCTCA-3′ (sense) and 5′-AATGAGCCACCAGTGTCCAA-3′ (antisense); for VEGF, 5′-CTACCTCCACCATGCCAAGT- 3′ (sense) and 5′-TCTCTCCTATGTGCTGGCCT-3′ (antisense); for β-actin, 5′-GCCATCGTCACCAACTGGGAC-3′ (sense) and 5′-CGATTTCCCGCTCGGCCGTGG-3′ (antisense). PCR products were visualized using 1% agarose gel electrophoresis with ethidium bromide staining.

Cells were plated in 6-well culture plates at 2×10⁵ cells/well in RPMI-1640 culture medium and treated with various concentrations of delphinidin for 12 h in the presence of EGF. The VEGF levels in the culture supernatant were determined by ELISA using the VEGF ELISA development kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's instructions.

The ability of delphinidin to inhibit HIF-1α transcription was determined by the reporter gene assay dependent on the HRE. In brief, at 50–80% confluency, A549 cells were co-transfected with pGL3-HRE-luciferase, which contained six copies of HRE derived from the human VEGF gene, and pRL-CMV (Promega Corp., Madison, WI, USA), which encoded Renilla luciferase (Rluc) under the control of a constitutive promoter, using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions.

Total RNA was extracted from the cells with TRIzol reagent (Invitrogen), and reverse-transcriptase reactions were performed with a commercial kit (SuperScript II RNase H-reverse transcriptase; Invitrogen) using 50 µg/ml RNA. Quantitative PCRs were conducted on a real-time PCR system (LightCycler; Roche Diagnostics, Basel, Switzerland) with a commercial kit (SYBR-Green with low ROX; Enzynomics, Daejeon, Korea) in a reaction mixture containing diluted cDNA (1/5) as a template and the real-time PCR primers. The sequences of the primers were as follows: for HIF-1α, 5′-CTCAAAGTCGGACAGCCTCA-3′ (sense) and 5′-AATGAGCCACCAGTGTCCAA-3′ (antisense); for VEGF, 5′-CTACCTCCACCATGCCAAGT-3′ (sense) and 5′-TCTCTCCTATGTGCTGGCCT-3′ (antisense); for β-actin, 5′-ACAGGAAGTCCCTTGCCATC-3′ (sense) and 5′-AGGGAGACCAAAAGCCTTCA-3′ (antisense). The relative mRNA expression levels were normalized to the value of β-actin for each reaction.

C57BL/6N mice (male, 6 weeks) were purchased from Samtako (Osan, Korea) and maintained in pathogen-free conditions. A549 cells at subconfluence were harvested, washed with PBS, and re-suspended in serum-free medium. Aliquots of cells (3×10⁶) were mixed with 0.5 ml of Matrigel in the presence or absence of EGF (200 ng/ml) and delphinidin. Immediately, the mixture was subcutaneously injected into mice. The mice were sacrificed when tumors were visible, and the Matrigel plugs were carefully separated from adjacent tissue and photographed.

The separated Matrigel plugs were excised, and then were placed in cold PBS at 4°C overnight to liquefy the Matrigel. Specimens were subjected to centrifugation at 14,000 rpm and the supernatants were collected. The hemoglobin content was determined using Drabkin's reagent kit (Sigma Chemical Co., St. Louis, MO, USA), as previously described (21). All surgical and experimental procedures used in this study were approved by the Institutional Review Board Committee at Daegu Catholic University Medical Center which conforms to the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

All results are representative of at least three independent experiments performed in triplicate. The statistical significance between experimental and control values was calculated using a one-way ANOVA test.

HIF-1 is a master regulator of numerous genes involved in cell survival, adaptation to hypoxia, metabolism, and angiogenesis (22). Before investigating the inhibitory potential of delphinidin (Fig. 1A), the cytotoxic effects of delphinidin were examined by the WST-1 assay. Delphinidin did not significantly affect the viability of A549 or NCI-H460 lung cancer cells at the indicated concentrations (Fig. 1B). Delphinidin did not show any cytotoxic effects at concentrations of up to 40 µM; therefore, 10, 20 and 40 µM delphinidin was used in the subsequent experiments. To determine if delphinidin inhibits HIF-1α expression, we investigated the HIF-1α and HIF-1β protein levels under various conditions using western blot analysis. CoCl₂ (200 µM) and EGF (20 ng/ml) treatment greatly induced HIF-1α protein expression levels in the various cancer cells. Delphinidin dose-dependently decreased CoCl₂-stimulated HIF-1α protein expression in the A549 (lung carcinoma), NCI-H460 (lung carcinoma), MCF-7 (breast carcinoma), and PC3M (prostate cancer) cells. In particular, at a concentration of 40 µM, delphinidin completely abrogated HIF-1α protein expression. However, CoCl₂ and delphinidin did not affect HIF-1β protein expression (Fig. 1C). Under the EGF-induced conditions, delphinidin decreased HIF-1α protein expression without affecting HIF-1β protein expression (Fig. 1D). As the inhibitory effects of delphinidin on CoCl₂- and EGF-stimulated HIF-1α protein expression in A549 cells was higher than that in the other cell lines, A549 cells were used in the subsequent experiments.

VEGF, the main target gene of HIF-1α, directly participates in angiogenesis (23). To determine if the decrease in HIF-1α expression by delphinidin affects VEGF expression, the VEGF mRNA levels were evaluated using RT-PCR. Delphinidin dose-dependently decreased the VEGF mRNA levels under the CoCl₂- and EGF-stimulated conditions (Fig. 2A). Moreover, treatment with 40 µM delphinidin dramatically reduced the VEGF mRNA levels in both conditions.

Next, we examined the effects of delphinidin on the secretion of the VEGF protein in CoCl₂- and EGF-stimulated conditions via ELISA assay. Secretion of VEGF protein was significantly increased by up to 2-fold in cells treated with CoCl₂ or EGF compared with untreated cells (Fig. 2B). Delphinidin treatment dose-dependently decreased the secretion of VEGF protein in the CoCl₂- and EGF-stimulated A549 cells. Because delphinidin inhibited HIF-1α protein expression in various cancer cells, we sought to confirm that delphinidin regulated VEGF secretion in the other cancer cell lines. In MCF-7 and PC3M cells, CoCl₂- and EGF-stimulated VEGF protein secretion was also decreased by delphinidin in a dose-dependent manner (Fig. 2C). Although non-treated H460 cells secreted high levels of VEGF protein, treatment with 40 µM delphinidin decreased CoCl₂- and EGF-induced VEGF protein secretion levels when compared with the non-treated H460 cells. These results suggest that the inhibitory effect of delphinidin on VEGF protein production is regulated by decreasing the VEGF mRNA level in CoCl₂- and EGF-stimulated conditions in various cell lines.

The binding of HIF-1 to HRE in the VEGF promoter is a predominant enhancer of VEGF production (13). Thus, to investigate if delphinidin suppresses HRE promoter activity by decreasing HIF-1α protein expression, we performed an HRE promoter reporter gene assay. A549 cells were co-transfected with the pGL3-HRE-luciferase and β-galactosidase-luciferase plasmids for 24 h and then treated with the indicated concentration of delphinidin for 12 h. HRE promoter activity was increased up to 4.5-fold in the CoCl₂-stimulated cells compared with the untreated cells (Fig. 2D). Delphinidin dose-dependently decreased CoCl₂-stimulated HRE promoter activity. In addition, EGF-induced HRE promoter activity was increased up to ~5-fold compared with the untreated cells and was rapidly decreased after treatment with 10 µM delphinidin. These results showed that the inhibitory effects of delphinidin on VEGF transcriptional activity are related to the regulation of HRE promoter activity by inhibiting HIF-1α expression.

To determine if delphinidin decreased HIF-1α protein expression by regulating the HIF-1α mRNA levels, we examined the effects of delphinidin on CoCl₂- and EGF-induced HIF-1α transcriptional activity using real-time PCR. CoCl₂ and EGF treatment did not induce HIF-1α mRNA levels (Fig. 3A). Furthermore, delphinidin treatment did not change the HIF-1α mRNA level in CoCl₂- or EGF-stimulated A549 cells. In addition, the results of the RT-PCR assay showed that delphinidin did not affect the HIF-1α mRNA levels (Fig. 3B). These results showed that HIF-1α mRNA levels were not responsible for the inhibition of HIF-1α protein expression by delphinidin in the CoCl₂- and EGF-induced conditions. Next, to determine the effect of delphinidin on HIF-1α protein proteasomal degradation, proteasome inhibitor MG132 was used. MG132 significantly increased HIF-1α protein accumulation (Fig. 3C). Delphinidin also inhibited MG132-induced HIF-1α, which suggests that delphinidin did not appear to affect the proteasomal degradation of the HIF-1α protein. Therefore, we anticipated that the inhibitory effects of delphinidin on CoCl₂- and EGF-induced HIF-1α protein expression are regulated by a synthesis pathway.

The MAPK and Akt/mTOR pathways are associated with the regulation of HIF-1α protein synthesis at the translational levels (24,25). Thus, to confirm that MAPK, Akt, and mTOR pathway activity is related to CoCl₂- and EGF-induced HIF-1α protein expression, A549 cells were exposed to various kinase inhibitors. PD98059 (a MEK inhibitor), wortmannin (a PI3K inhibitor), and rapamycin (an mTOR inhibitor) blocked CoCl₂-induced HIF-1α expression, similarly to delphinidin (Fig. 4A). By contrast, SB203589 (a p38 inhibitor) did not affect HIF-1α protein expression in A549 cells. In EGF-stimulated cells PD98059, wortmannin, and rapamycin also blocked HIF-1α expression (Fig. 4B). These results indicate that CoCl₂- and EGF-induced HIF-1α protein expression is regulated by the ERK, PI3K and mTOR pathways, whereas the p38-mediated pathways are not involved.

To determine the mechanisms underlying HIF-1α inhibition by delphinidin, the phosphorylated forms of ERK, PI3K, Akt and mTOR were detected via western blot analysis. The phosphorylation of ERK, PI3K, Akt, mTOR and p70S6K were increased at 10 min after CoCl₂- and EGF-treatment, while delphinidin reduced phosphorylation of ERK, PI3K, Akt, mTOR, and p70S6K in CoCl₂-stimulated A549 cells (Fig. 4C and D). Similarly, delphinidin dose-dependently inhibited EGF-induced phosphorylation of ERK, PI3K, Akt, mTOR, and p70S6K. Phosphorylation of tyrosine residue 1068 (Tyr¹⁰⁶⁸) of EGFR is part of the initial activation process and these phosphotyrosine residues subsequently serve as docking sites for intracellular signaling molecules (26). Thus, to determine if the inhibition of HIF-1α protein synthesis was mediated by the downregulation of the EGFR tyrosine residue, the effect of delphinidin on the phosphorylation of EGFR (Tyr¹⁰⁶⁸) was determined. The results showed that delphinidin inhibited EGF-induced EGFR phosphorylation. These results suggest that delphinidin suppresses HIF-1α protein synthesis by the inhibiting EGFR-dependent and -independent activation of the ERK and PI3K/Akt/mTOR/p70S6K signaling pathways.

To further determine if delphinidin inhibits angiogenesis by blocking the expression of HIF-1α and VEGF, we performed a Matrigel plug assay in vivo. Matrigel plugs mixed with only cancer cells did not induce blood vessel formation. New blood vessel formation was induced by EGF-treated cancer cells (Fig. 5A). Delphinidin considerably suppressed EGF-induced angiogenesis, and it was almost completely abrogated upon treatment with 80 µM delphinidin. Moreover, delphinidin dose-dependently reduced the EGF-increased hemoglobin content (Fig. 5B). These results indicate that delphinidin is an angiogenesis inhibitor and an antitumor therapeutic agent in human lung adenocarcinoma A549 cells.