Effects of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and metastasis-associated properties in A431 cells overexpressing epidermal growth factor receptor

Abstract

1. Flavonoids display a wide range of pharmacological properties including anti-inflammatory. Anti-mutagenic, anti-carcinogenic and anti-cancer effects. Here, we evaluated the effects of eight flavonoids on the tumour cell proliferation, cellular protein phosphorylation, and matrix metalloproteinase (MMPs) secretion. 2. Of the flavonoids examined, luteolin (Lu) and quercetin (Qu) were the two most potent agents, and significantly inhibited A431 cell proliferation with IC50 values of 19 and 21 micronM, respectively. 3. The epidermal growth factor (EGF) (10 nM) promoted growth of A431 cells (+25+/-4.6%) and mediated epidermal growth factor receptor (EGFR) tyrosine kinase activity and autophosphorylation of EGFR were inhibited by Lu and Qu. At concentration of 20 micronM, both Lu and Qu markedly decreased the levels of phosphorylation of A431 cellular proteins, including EGFR. 4. A431 cells treated with Lu or Qu exhibited protuberant cytoplasmic blebs and progressive shrinkage morphology. Lu and Qu also time-dependently induced the appearance of a ladder pattern of DNA fragmentation, and this effect was abolished by EGF treatment. 5. The addition of EGF only marginally diminished the inhibitory effect of luteolin and quercetin on the growth rate of A431 cells, treatment of cellular proteins with EGF and luteolin or quercetin greatly reduced protein phosphorylation, indicating Lu and Qu may act effectively to inhibit a wide range of protein kinases, including EGFR tyrosine kinase. 6. EGF increased the levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), while Lu and Qu appeared to suppress the secretion of these two MMPs in A431 cells. 7. Examination of the relationship between the chemical structure and inhibitory effects of eight flavonoids reveal that the double bond between C2 and C3 in ring C and the OH groups on C3' and C4' in ring B are critical for the biological activities. 8. This study demonstrates that the inhibitory effects of Lu and Qu, and the stimulatory effects of EGF, on tumour cell proliferation, cellular protein phosphorylation, and MMP secretion may be mediated at least partly through EGFR. This study supports the idea that Lu and Qu may have potential as anti-cancer and anti-metastasis agents.

Figure 1

The chemical structures of flavonoids. Flavone: luteolin and wogonin; Flavonol: quercetin and kaempferol; Flavanones: taxifolin and naringenin; Catechin: d-catechin; Isoflavone: genistein.

Figure 2

Effect of flavonoids and EGF on the kinase activity of A431 cellular proteins. (A) Cell lysates of 50 μg proteins were incubated with various flavonoids at 20 μM or with 100 nM EGF in the presence of [γ-³²P]-ATP. Lanes: 1, control; 2, EGF; 3, luteolin; 4, quercetin; 5, genistein; 6, Taxifolin; 7, d-catechin; 8, naringenin; 9, kaempferol; 10, wogonin. Note that luteolin and quercetin both dramatically inhibited the phosphorylation of most protein bands, including the EGF receptor band (170-kDa), whereas genistein produced only a marginal (12%) decrease of the phosphorylation level of the 170-kDa band. (B) Cell lysates with equivalent amounts of protein (50 μg) were immunoprecipitated with anti-EGFR antibodies. After centrifugation, the supernatants were collected and then subjected to total kinase activity assay as described above. Lanes: 1, cell lysate; 2, EGF+cell lysate; 3. EGFR-removed cell lysate.

Figure 3

Effects of flavonoids on the kinase activity of liver tumour proteins. Liver tumour extracts of 50 μg proteins were incubated with various flavonoids (20 μM) in the presence of [γ-³²P]-ATP. Lanes: 1, control; 2, luteolin; 3, quercetin; 4, d-catechin; 5, genistein; 6, taxifolin.

Figure 4

Effect of luteolin (Lu) and EGF on the tyrosine kinase activity of A431 cellular proteins. Cell lysates of 50 μg proteins, in the presence of ATP, were incubated with or without various concentrations of luteolin and EGF. The reaction mixtures were subjected to immunoblotting analysis. Lanes: 1, Control; 2, 100 nM EGF; 3–6, luteolin of 10, 20, 40 and 80 μM in the presence of 100 nM EGF, respectively. Anti-phosphotyrosine protein antibody was used to detect phosphotyrosyl protein (A). The same blot was then stripped off primary and secondary antibodies, and reprobed with anti-EGFR antibody (B). Note that similar amounts of the 170-kDa EGFR band were observed in all six lanes (B), while the phosphorylation levels were varied (A).

Figure 5

Effects of flavonoids on the growth (A) unstimulated and (B) EGF-stimulated A431 cells. Cells were treated with flavonoids (20 μM) and/or EGF (10 nM) for 24 h. Cell numbers of the culture were then determined. Each bar represents the mean (±s.e.mean) of six replicates from a representative experiment. Three separate experiments were performed and all showed similar results. The mean cell number of control groups was 6.08×10⁵. Con, control; Lu, luteolin; Qu, quercetin; Gen, genistein; Tax, taxifolin; d-Cat. d-catechin; Nar, naringenin; Kae, kaempferol; Wog. wogonin.

Figure 6

Effects of (A) luteolin and (B) quercetin on the growth of A431 cells. Cells were treated with various concentrations of flavonoids for 12–72 h. At the indicated time, cell numbers of the cultures were determined. Each point represents the mean (±s.e.mean) of triplicate wells from one of three separate representative experiments, all of which gave similar results, Lu, luteolin; Qu, quercetin.

Figure 7

Effects of EGF, luteolin (Lu) and quercetin (Qu) on the growth of HepG2, MCF-7 and MiaPaCa-2 tumour cell lines. Ten nM EGF, 20 μM Lu or Qu were added to the cultures at time zero. After 48 h, the cells were harvested and the numbers were determined. The cell numbers of control groups in HepG2, MCF-7, and MiaPaCa 2 were 6.23×10⁵, 6.18×10⁵, and 6.90×10⁵, respectively. Each bar represent the mean (±s.e.mean) of triplicate wells from one of three separate experiments, all of which gave similar results.

Figure 8

Effects of EGF, luteolin and quercetin on the morphology of A431 cells. Cells treated with 10 nM EGF, 20 μM luteolin, or 20 μM quercetin for 48 h. Cells were photographed under phase contrast microscopy. (A) control; (B) EGF; (C) luteolin and (D) quercetin. Magnification 200×.

Figure 9

Effects of luteolin, quercetin and genistein on DNA integrity of A431 cells. Cells were treated with 0, 20, 50 and 100 μM of luteolin (lanes 1–4), quercetin (lanes 5–8) or genistein (lanes 9–12) for 24 h. Lane 13, A431 cells treated with 50 μM luteolin for 24 h and then treated with 10 nM EGF for 24 h. Lane 14, A431 cells treated with 50 μM for 48 h. At the end of the culture, approximately 2×10⁶ cells were harvested. DNA was isolated from each sample and subjected to electrophoresis in a 1.8% agarose gel. EGF was shown to abrogate DNA degradation caused by luteolin (lane 13) and quercetin (data not shown). This ladder pattern of DNA fragments is a characteristic of apoptosis.

Figure 10

Effects of flavonoids and EGF on the secretion of gelatinases in A431 cells. Cells at 40% confluence were treated with vehicle (lane 1), 20 μM genistein, luteolin, or quercetin (lanes: 2–4, respectively), 10 nM EGF (lane 5) in serum-free medium for 48 h. The conditioned or without flavonoids and EGF (lane 1) in serum-free medium for 48 h. The conditioned media were collected and subjected to gelatin zymography analysis. Representative zymograms of gels in the absence (A) or the presence (B) of a metalloproteinase inhibitor 5 mM 1,10-phenanthroline (or EDTA) during substrate buffer incubation. Trypsin (lane 6), a serine protease, was not inhibited by metalloproteinase inhibitor. The conditioned media were collected and normalized by cell numbers prior to gelatin zymography analysis. (C), Immunoblotting analysis using anti-MMP-9 and MMP-2 indicated that the identity of 92- and 72-kDa gelatinases were MMP-9 and MMP-2, respectively.

Figure 11

Effect of luteolin on gelatinase activity of A431 cell lysates. Cells were treated with EGF or luteolin for 24 h. Cell lysates containing 50 μg proteins were analysed by gelatin zymography. Lanes: 1, control; 2, EGF; lanes 3–5; 10, 20 and 50 μM luteolin, respectively. Similar results were observed in A431 cells using the same concentration of quercetin in the same experiments.