Enhanced anti-tumor activity by the combination of the natural compounds (-)-epigallocatechin-3-gallate and luteolin: potential role of p53

Abstract

Natural dietary agents have drawn a great deal of attention toward cancer prevention because of their wide safety margin. However, single agent intervention has failed to bring the expected outcome in clinical trials; therefore, combinations of chemopreventive agents are gaining increasingly popularity. In the present study, we investigated a combinatorial approach using two natural dietary polyphenols, luteolin and EGCG, and found that their combination at low doses (at which single agents induce minimal apoptosis) synergistically increased apoptosis (3-5-fold more than the additive level of apoptosis) in both head and neck and lung cancer cell lines. This combination also significantly inhibited growth of xenografted tumors in nude mice. The in vivo findings also were supported by significant inhibition of Ki-67 expression and increase in TUNEL-positive cells in xenografted tissues. Mechanistic studies revealed that the combination induced mitochondria-dependent apoptosis in some cell lines and mitochondria-independent apoptosis in others. Moreover, we found more efficient stabilization and ATM-dependent Ser(15) phosphorylation of p53 due to DNA damage by the combination, and ablation of p53 using shRNA strongly inhibited apoptosis as evidenced by decreased poly(ADP-ribose) polymerase and caspase-3 cleavage. In addition, we observed mitochondrial translocation of p53 after treatment with luteolin or the combination of EGCG and luteolin. Taken together, our results for the first time suggest that the combination of luteolin and EGCG has synergistic/additive growth inhibitory effects and provides an important rationale for future chemoprevention trials of head and neck and lung cancers.

FIGURE 1.

Synergistic apoptosis induced by the combination of EGCG and luteolin. A, Tu212 cells were treated with 10 μm luteolin (L), 30 μm EGCG (E), or a combination of 10 μm luteolin and 30 μm EGCG (C) for 24, 48, and 72 h. Apoptosis was measured by annexin V-phycoerythrin staining. B, SCCHN cell lines were treated with 10 μm luteolin, 30 μm EGCG, or a combination of 10 μm luteolin and 30 μm EGCG for 72 h, and apoptosis was measured as above. C, lung cancer cell lines expressing wild-type p53 were treated with luteolin, 30 μm EGCG, or a combination of luteolin and EGCG, and apoptosis was measured. The number after L indicates the dose of luteolin in μm. D, lung cancer cell lines expressing mutant or no p53 were treated with luteolin, 30 μm EGCG, or a combination of luteolin and EGCG, and apoptosis was measured. E, noncancerous BEAS-2B and BJ cells were treated with the compounds for 72 h, and apoptosis was measured. F, H460 cells were seeded at a concentration of 250 cells/well in six-well plates and treated with the indicated concentration of luteolin, EGCG, or a combination of 5 μm luteolin plus varying concentrations of EGCG until colonies were visible. Finally, colonies were counted after methylene blue staining. G, Tu212, H292, and BEAS-2B cells were seeded at a density of 2.5 × 10⁵ cells/well in six-well plates, treated twice (72 h each) with 10 μm luteolin, 30 μm EGCG, or their combination, and cultured in drug-free media for an additional 2 weeks and stained with methylene blue. Apoptosis occurring in untreated cells was subtracted from apoptosis occurring after compound treatment, and net apoptosis occurring after treatment with the compounds was presented. All experiments were repeated at least three times. Error bars represent S.D. from at least three independent experiments. NT, no treatment.

FIGURE 2.

Cleavage of PARP after treatment with the combination of EGCG and luteolin. Tu212, H358, and Tu686 cells were treated with luteolin (L), EGCG (E), or their combination (C) for different time periods. Total cell lysates were immunoblotted with antibody that detects both full-length and cleaved PARP (Cell Signaling Technologies, Danvers, MA). Representative data from three independent experiments are shown. NT, no treatment.

FIGURE 3.

Combination of EGCG and luteolin induced both mitochondria-dependent and -independent apoptosis. A, Tu212 cells were treated with 10 μm luteolin (L), 30 μm EGCG (E), or their combination (C) for various time periods, and total cell lysates were immunoblotted with anti-caspase-8 (detects both full-length and cleaved form, Cell Signaling Technologies, Danvers, MA) and anti-caspase-3 (detects only cleaved forms at 19 and 17 kDa, Cell Signaling Technologies). B, cells were treated with luteolin, EGCG, or their combination, and total cell lysates were immunoblotted with anti-DR5 (ProSci, Poway, CA). C–D, Tu212 (C), H460 (D, upper panel), and A549 (D, lower panel) cells were treated with luteolin, EGCG, or their combination. Cytoplasmic and mitochondrial fractions were separated and immunoblotted with cytochrome c (Cyto C) antibody. COX4 (a mitochondrial protein) was used to show efficiency of cell fractionation. For A–D, all experiments were repeated at least three times, and representative data are presented. NT, no treatment.

FIGURE 4.

Phosphorylation and stabilization of p53 by the combination of luteolin and EGCG. H460 (A) and A549 (B) cells were treated with luteolin (L), EGCG (E), or their combination (C), and total cell lysates were immunoblotted with anti-p53 (Santa Cruz Biotechnology) and anti-phospho p53 (Ser¹⁵, Cell Signaling Technologies). Representative data from three independent experiments are shown. Numbers below each lane represent fold change. C, H460 cells were pretreated with 10 μm Ku55933 for 1 h, followed by treatment with 20 μm luteolin, 30 μm EGCG, their combination, or 2 μg/ml camptothecin (Cp) for 48 h. Cells were treated with capmtothecin for 24 h. Total cell lysates were immunoblotted with antiphospho p53 Ser¹⁵. Representative data from three independent experiments are shown. D, H460 cells were treated with luteolin, EGCG, their combination, or camptothecin for 24 h. Total cell lysates were immunoblotted with phospho-H2AX (γ-H2AX) and p53. Experiments were repeated three times. NT, no treatment.

FIGURE 5.

Mitochondrial translocation of p53. H460 (A) and A549 (B) cells were treated with luteolin (L), EGCG (E), or their combination (C) for 48 h. Cytoplasmic (Cyto) and mitochondrial (Mito) fractions were immunoblotted with p53, Bax, and Erk2 antibodies (Santa Cruz Biotechnology). Erk2 (a cytoplasmic protein) was used to show efficiency of separation. C. H460 cells were treated with luteolin, EGCG, or their combination for 24 h, and immunofluorescence staining was done as described under “Experimental Procedures.” Arrows in the combination lane indicate nuclear condensation due to apoptosis. For all panels, representative data from at least three independent experiments are shown. NT, no treatment.

FIGURE 6.

Role of p53 in apoptosis induced by the combination of luteolin and EGCG. p53 was knocked down in H460 (A) and A549 (B) cells as described under “Experimental Procedures” and treated with luteolin (L), EGCG (E), or their combination (C). Total cell lysates were immunoblotted with p53, p21, Bax, PARP, and caspase-3 antibodies. Each experiment was repeated at least three times. NT, no treatment; CF, cleaved form.

FIGURE 7.

Inhibition of tumor growth by the combination of luteolin and EGCG in nude mice. Four groups of animals were orally gavaged with vehicle control, luteolin, EGCG, or their combination as described under “Experimental Procedures.” Growth curves were obtained for Tu212 (A) and A549 (B) xenografted tumors. Tu212 tumor growth was significantly inhibited in the combination group as compared with the control (p = 0.031), EGCG (E; p = 0.025), or luteolin (L; p = 0.05) groups. For A549 tumors, the combination of luteolin and EGCG significantly inhibited tumor growth as compared with control group (p = 0.026). Other comparisons were insignificant (p > 0.05). C, quantification of Ki-67 staining. *, statistically significant p values (*, control versus luteolin, p = .0071; **, control versus EGCG, p = .0047; ***, control versus combination (C), p = .0000074; ****, EGCG versus combination, p = .0032; *****, luteolin versus combination, p = .0011). D, quantification of TUNEL staining. An asterisk indicates statistically significant p values (*, control versus luteolin, p = .001; **, control versus EGCG, p = .002; ***, control versus combination, p = .00000019; ****, luteolin versus combination, p = .000069). EGCG versus combination was not statistically significant (p = .69) because one of the mice treated with EGCG had a high number of TUNEL-positive cells. NT, no treatment.