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Tangeretin inhibits extracellular-signal-regulated kinase (ERK) phosphorylation
FEBS 29358
FEBS Letters 579 (2005) 1665–1669
Tangeretin inhibits extracellular-signal-regulated kinase (ERK) phosphorylation Se´verine Van Slambroucka, Virinder S. Parmarb, Sunil K. Sharmab, Bart De Bondta, Fleur Fore´a, Peter Coopmanc, Barbara W. Vanhoecked, Tom Boterberga, Herman T. Depypered, Guy Leclercqe, Marc E. Brackea,* a
Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, B-9000 Ghent, Belgium b Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, India c CNRS UMR 5539, University of Montpellier II, 34095 Montpellier, France d Department of Gynaecological Oncology, Ghent University Hospital, B-9000 Ghent, Belgium e Laboratory J-C Heuson of Mammary Cancerology, Bordet Institute, B-1000 Brussels, Belgium Received 23 August 2004; revised 6 October 2004; accepted 14 October 2004 Available online 21 February 2005 Edited by Lukas Huber
Abstract Tangeretin is a methoxyflavone from citrus fruits, which inhibits growth of human mammary cancer cells and cytolysis by natural killer cells. Attempting to unravel the flavonoidÕs action mechanism, we found that it inhibited extracellularsignal-regulated kinases 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent way. In human T47D mammary cancer cells this inhibition was optimally observed after priming with estradiol. The spectrum of the intracellular signalling kinase inhibition was narrow and comparison of structural congeners showed that inhibition of ERK phosphorylation was not unique for tangeretin. Our data add tangeretin to the list of small kinase inhibitors with a restricted intracellular inhibition profile. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Tangeretin; Methoxyflavone; Citrus; Extracellularsignal-regulated kinases 1/2; Mammary cancer
1. Introduction Tangeretin (4 0 ,5,6,7,8-pentamethoxyflavone), a natural compound from citrus fruits, was found to inhibit a number of cell activities such as growth of mammary cancer cells in vitro [1] and cytolysis by natural killer (NK) cells in vivo [2]. For both activities a pivotal role of the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) pathway has been reported [3,4]. A variety of peptide growth factors and steroid hormones are able to activate ERK via upstream activation of cytoplasmic kinases, including Ras, Src, Raf, and MAPK kinase (MKK) [5]. The synthetic methoxyflavone PD98059 was identified as an inhibitor of MKK [6] and hence of MAPK-driven cell proliferation and NK-cell activity [7]. Inspired by its structure similarity with PD 98059 and its ability to suppress cell proliferation and NK-activity as well, we investigated the effect of tangeretin on the MAPK signalling cascade. The model we used consisted of human T47D breast cancer cells stimulated with estradiol [8] and treated with tan* Corresponding author. Fax: +32 9 240 4991. E-mail address: [email protected] (M.E. Bracke).
geretin. Human breast cancer cell lines exhibit elevated activation levels of ERK after estradiol treatment, and this activation, mediated by plasma membrane-bound estrogen receptors, involves transactivation of ErbB tyrosine kinase receptors [9]. The dual tyrosine/threonine phosphorylation status of ERK was selected as an activation marker for this signalling pathway.
2. Materials and methods 2.1. Cell culture T47D cells are derived from a pleural effusion of a human mammary ductal carcinoma. Cells were grown in DulbeccoÕs modified EagleÕs medium (DMEM) (Invitrogen, Merelbeke, Belgium), supplemented with 100 IU/ml penicillin, 100 lg/ml streptomycin, 2.5 lg/ml amphotericin B, and 10% heat-inactivated fetal bovine serum (FBS) (Greiner Bio-one, Wemmel, Belgium) at 37 C in an atmosphere of 10% CO2 in air. 2.2. Chemicals and antibodies Tangeretin was kindly provided by Dr. Bill Widmer (Department of Citrus, Winter Haven, FL), and dissolved in ethanol as a stock solution of 102 M. 17-b-Estradiol (E2) was from Sigma (St. Louis, MO, USA), PD 98059 from Calbiochem (La Jolla, CA, USA), and recombinant heregulin-b1 (rHRG-b1) from R&D Systems (Abingdon, UK). The primary antibody for Western blotting was a rabbit polyclonal anti-phospho-p42/44 MAPK (Thr202/Tyr204) from Cell Signalling Technology (Beverly, MA, USA). The secondary anti-rabbit antibody was from Amersham Pharmacia Biotech (Amsterdam, The Netherlands). The monoclonal mouse-anti-Syk antibody (clone 4D10) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), while the polyclonal rabbit-anti-Src CST.1 antibody was kindly provided by Dr. S. Roche (CNRS FRE2593, Montpellier, France). RU 58668 from Roussel Uclaf (Romainville, France), 4-hydroxytamoxifen (4OH-TAM) from Zeneca Pharmaceuticals (Macclesfield, UK) and [3H]estradiol (±95 Ci/mmol) from Amersham Pharmacia Biotech were used for estrogen receptor a-mediated luciferase induction and ligand binding assays. 2.3. Western blotting All lysates were made from cells at approximately 70% confluency. For phosphorylation experiments, cells were washed three times with phosphate-buffered saline (PBS), serum-starved overnight, washed again three times with PBS and treated in serum-free medium. Before making lysates, the cells were washed again three times with PBS. Cells were lysed with PBS containing 1% Triton X-100, 1% Nonidet P-40 and the following protease inhibitors: aprotinin (10 lg/ml), leupeptin (10 lg/ml), phenylmethylsulfonyl fluoride (1.72 mM), NaF (100 M),
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2004.10.114
1666 NaVO3 (500 lM), and Na4P2O7 (500 lg/ml). After incubation at 4 C for 5 min, the samples were sonicated for 15 s. After clearing the lysates by centrifugation at 14 000 rpm for 10 min, protein concentration was determined using the Rc Dc Protein Assay (Bio-Rad, Hercules, CA, USA) and samples were prepared in such a way that equal amounts of protein were loaded. Sample buffer [10] with 5% 2-mercaptoethanol and 0.012% bromophenol blue was added, followed by boiling for 5 min and separation of proteins by gel electrophoresis on a 10% polyacrylamide gel and transfer onto a nitrocellulose membrane (Amersham Pharmacia Biotech). Quenching and immunostaining of the blots was done in 4% bovine serum albumin in PBS containing 0.2% Tween-20. The membranes were quenched for 1 h, incubated with primary antibody for 1 h, washed four times for 10 min, incubated with horseradish peroxidase-conjugated secondary antibody for 45 min, and washed six times for 10 min. Detection was done using Enhanced Chemiluminescence reagent (Amersham Pharmacia Biotech) as a substrate. Tubulin and total p42/p44 immunostaining was used as a loading control, and variations were less than 5%. After scanning the autoradiogram, bands quantification (the sum of p42 and p44) was performed using the Quantity One software (Bio-Rad). Experimental results were confirmed by obtaining concordant data from experiments performed at least in duplo. 2.4. Phosphorylation status analysis of cytoplasmic signalling proteins Kinetworks technology (Kinexus, Vancouver BC, Canada) permits the quantitative detection of the phosphorylation status of cytoplasmic proteins. The Kinetworks KPSS 1.3 Phosphoprotein Screen was performed as described by Pelech et al. [11] (http://www.kinexus.ca). The screen required 350 lg of protein, and for some kinases results obtained with different antibodies are presented. For the measurement in vitro of Syk and Src tyrosine kinases, Simian Cos1 cells were transiently transfected with the pCAF1-Syk [12] or the pSG5-Src cDNA (kindly provided by Dr. S. Roche) using the SuperFect reagent (Qiagen Inc, Venlo, The Netherlands). Two days after transfection, cells were lysed and cleared lysates were used for immunoprecipitation with protein A-agarose beads linked to antibodies against Syk or Src for 3 h at 4 C under constant rotation. Then the beads were washed in lysis buffer and used for Western blot or autophosphorylation testing. For in vitro kinase assays, the beads were re-suspended in 50 lL reaction buffer (10 mM Tris–HCl, pH 8, 10 mM MgCl2, and 10 mM MnCl2). Serial dilutions from a 102 M tangeretin stock solution or piceatannol, a specific inhibitor of Syk kinase activity (Calbiochem), were made in reaction buffer and added to the beads together with 10 lCi [c-32P] ATP (4500 Ci/mmol, ICN Biomedicals) and agitated for 20 min at room temperature. After washing in reaction buffer, beads were boiled in Laemmli sample buffer, loaded on 10% SDS–PAGE gels, dried, exposed and autophosphorylated Syk or Src kinase levels were detected and quantified using phosphor-image analysis (Molecular Dynamics, Sunnyvale, CA). 2.5. Assay for growth of T47D cells in vitro T47D cells were seeded in microtiter plates at an initial density of 1.5 · 104 cells in 200-ll culture medium. After an incubation period of 4 days, the amount of cell protein in each well was estimated with the sulforhodamine B assay [13]. 2.6. Luciferase induction assay MVLN cells are human MCF-7 mammary carcinoma cells, stably transfected with the pVit-tk-LUC reporter plasmid. These cells allow quantification of the transcriptional activity from estrogen receptor a-mediated estrogen responsive elements (ERE), in accordance with the luciferase induction assay by Pons et al. [14]. They were cultured in 35 mm diameter plastic petri dishes (80 000 cells/dish) in DMEM containing 10% dextran coated charcoal-treated FBS for 3–4 days. 250 ll of 5-fold diluted lysis solution (Promega E153A, Promega, Madison, WI, USA) was then added to the dishes and maintained under mild agitation for 20 min to extract luciferase. Lysed cells were subsequently detached with a scraper and centrifuged for 5 s at 10 000 · g to clarify the extracts. Finally, 20 ll of extracts was mixed at room temperature with 100 ll of a luciferase reactant medium (Promega E151A/E152A) prepared in accordance with the manufacturerÕs protocol and induced light was measured with a luminometer.
S. Van Slambrouck et al. / FEBS Letters 579 (2005) 1665–1669 2.7. Statistics Statistical evaluation of the data was performed with the StudentÕs t test.
3. Results 3.1. Dose- and time-dependent effect of tangeretin on the phosphorylation status of ERK Western blots were performed to determine whether tangeretin could inhibit the E2-induced ERK phosphorylation. T47D cells were treated with 109 M E2 for 20 min, in the absence or presence of varying concentrations of tangeretin (107 to 104 M), and assayed for ERK phosphorylation by immunostaining with an anti-phospho-MAPK antibody that detects the dual phosphorylation state (Thr202/Tyr204) of ERK 1/2 (p42 and p44). No activation was observed in solvent (ethanol 0.1%) treated cells as compared to untreated cells, but addition of E2 induced the phosphorylation of ERK 1/2. Tangeretin was able to inhibit this E2-induced phosphorylation in a dose-dependent manner (Fig. 1A). To determine the kinetics, T47D cells were treated with E2 (109 M) and tangeretin (104 M), and cell lysates were prepared after various incubation periods (Fig. 1B). ERK phosphorylation inhibition by tangeretin was evident after 10–30 min. Since E2 can stimulate the MAPK pathway via ErbB receptors, direct stimulation by rHRG-b1, a ligand of ErbB3 and ErbB4 [15], was applied as an alternative method to phosphorylate ERK [16]. To confirm the responsiveness of T47D cells to rHRG-b1, different concentrations (0.1–10 ng/ml) were initially tested and 1 ng/ml for 10 min proved to be an optimal stimulus. Tangeretin effectively reduced this rHRG-b1 stimulated ERK phosphorylation to 24% at a concentration of 104 M (Fig. 1C). 3.2. Effect of tangeretin on growth T47D mammary cancer cells Tangeretin inhibited the growth of estradiol-stimulated human T47D cells in the sulforhodamine B assay. The effect was dose-dependent and could be demonstrated at concentrations from 104 down to 107 M (Fig. 2). 3.3. Phosphorylation screening of other signalling molecules To further investigate the specificity of the kinase inhibition by tangeretin, a screening of the phosphorylation status of different cytoplasmic proteins (KPSS 1.3, Kinexus) was performed. Three samples pretreated with 109 M E2 for 20 min were analysed: one was solvent-treated, a second one was treated with 104 M tangeretin and the third one with 2.5 · 105 M PD98059 (Table 1). The results showed that tangeretin is indeed able to inhibit phophorylation of ERK 1/2 and of MAPK kinases. In contrast to PD98059, tangeretin is also able to inhibit phosphorylation of other proteins like adducin a and c, protein kinase Cd, signal transducer and activator of transcription (STAT) 1 and 3, and stress-activated protein kinase (JNK). The phosphorylation of other proteins such as JUN, protein kinase Ba (Akt1), and retinoblastoma 1, however, were increased. Overall the spectrum of inhibition by tangeretin differed from the one by PD98059. Quercetin and epigallocatechin-3-gallate were recently found to down regulate the activity of the Syk kinase and
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Fig. 2. Effect of tangeretin on growth of estradiol-stimulated human T47D cells. The cells were treated with different tangeretin concentration for 4 days, and the amount of cellular protein was assessed with the sulforhodamine B assay and expressed as optical density units (OD). Bars indicate mean values + S.D., *, P < 0.01.
Table 1 Effect of 104 M tangeretin and 2.5 · 105 M PD98059 on the phosphorylation status of different cytoplasmic kinases and kinase substrates
Fig. 1. Effect of tangeretin on ERK1/2 phosphorylation. T47D cells were pretreated with estradiol (A and B) or with heregulin (C), and treated with tangeretin or with solvent. In these typical experiments shown, tangeretin inhibited ERK1/2 phosphorylation in a dose- (A and C) and time- (B) dependent way. Western blot analysis and immunostaining were done with anti-phospho ERK1/2, and results are presented as percentages of E2- or heregulin-treated cells. Mean values + S.D., *, P < 0.01.
the Lyn Src-family kinase. In order to test the effect of tangeretin on the Syk and Src kinase activities, their autophosphorylation capacity was tested in vitro by incorporation of [c-32P] ATP in presence of increasing tangeretin concentrations. On Syk and Src kinase autophosphorylation levels no effect of tangeretin could be observed, yet piceatannol at 104 M completely abolished Syk autophosphorylation (data not shown).
Phosphorylation of
Tangeretin % of solvent control
PD98059
Adducin a Adducin c cAMP response element binding protein Cyclin-dependent kinase 1 (cdc2) dsRNA dependent protein kinase Extracellular regulated kinase 1 Extracellular regulated kinase 2 Glycogen synthase kinase 3 a Glycogen synthase kinase 3 a Glycogen synthase kinase 3 b Glycogen synthase kinase 3 b MAP kinase kinase 1/2 MAP kinase kinase 6 N-Methyl-D -aspartate glutamate receptor unit 1 Oncogene JUN Oncogene Raf 1 Oncogene Raf 1 Oncogene SRC Oncogene SRC P38 a MAP kinase Protein kinase B a (Akt1) Protein kinase B a (Akt1) Protein kinase C a Protein kinase C a/b Protein kinase C d Protein kinase C e Retinoblastoma 1 Retinoblastoma 1 S6 kinase p70 STAT 1 STAT 3 Stress-activated protein kinase (JNK) Stress-activated protein kinase (JNK)
48.3 50.3 157.3 115.2 154.5 64.9 63.5 114.6 97.8 263.5 97.3 75.0 64.7 90.2
122.1 128.7 151.7 43.1 11.6 20.9 14.2 74.1 19.4 146.4 22.3 104.7 83.9 143.1
211.8 86.3 95.6 72.4 110.8 104.4 161.3 138.3 127.7 101.9 73.7 92.1 127.6 131.5 56.3 70.7 66.6 78.6 64.1
105.7 89.3 64.9 69.7 86.5 116.6 87.5 99.9 162.3 217.5 127.3 91.3 150.3 207.4 119.6 99.1 78.9 84.5 126.8
3.4. Effects of methoxyflavone congeners on ERK phosphorylation A number of methoxyflavones and methoxyisoflavones were tested for their ability to affect ERK phosphorylation in E2stimulated T47D cells (Fig. 3A). At 104 M 5,6,7,8,3 0 ,4 0 ,5 0 heptamethoxyflavone (349) and 2,7-dimethoxyflavone (345) were more potent than tangeretin (Fig. 3B).
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Fig. 3. Structures of methoxyflavone/methoxyisoflavone congeners of tangeretin (A), and their effect on ERK1/2 phosphorylation in T47D cells (B). In this typical experiment shown, E2-stimulated (20 min) T47D cells were treated in absence or presence of different methoxyflavones or methoxyisoflavones at a concentration of 104 M. Western Blot analysis and immunostaining were done with anti-phospho ERK1/2. Results are presented as percentages of phosphorylation in tangeretin-treated cells.
3.5. Absence of effect of tangeretin on the ERE-dependent transcription activity Because it has been reported that some flavonoids can bind to the estrogen receptor, the effect of tangeretin on ERE-dependent transcription activity was measured. Treatment of MVLN cells for 24 h was done in the presence of 1010 M E2, 107 M 4-OH-TAM, 107 M RU58668, and different concentrations of tangeretin. Increase of ERE-luciferase activity was observed with E2 (+110%), while partial (4-OH-TAM) and pure (RU58668) anti-estrogens decreased the basal transcriptional activity by 65% and 55%, respectively. With tangeretin (105 to 109 M) neither stimulatory nor inhibitory effects on luciferase induction could be observed (Fig. 4). No affinity neither for the intracellular estrogen receptor a nor for the membrane estrogen binding sites could be attributed to tangeretin in a ligand binding competition assay with [3H] estradiol (data not shown).
Fig. 4. Luciferase induced activity (%) of tangeretin (105 to 109 M) in MVLN cells.
4. Discussion This report adds tangeretin to the list of flavonoids that were previously described in the literature as kinase inhibitors.
S. Van Slambrouck et al. / FEBS Letters 579 (2005) 1665–1669
Notorious examples are the phosphorylation inhibition of pp60src by quercetin [17], and of receptor tyrosine kinases by high-dose genistein [18]. Moreover, several pharmacological inhibitors of kinases belong to the flavonoid family, and PD 98059, the gold standard inhibitor for MAPK phosphorylation in vitro, is a synthetic methoxyflavone structurally related to tangeretin. Tangeretin, however, differs from genistein [19] and PD 98059 [20] in that it is devoid of any estrogenic or anti-estrogenic activity. A number of tangeretinÕs effects on cell behaviour can be matched with inhibition of ERK signalling. This pathway has shown to be involved in cell proliferation [5], motility and invasion [21], and natural killer cell activity [4]. Inhibition of the pathway by tangeretin treatment could explain the effects observed in several functional assays in vitro [22]. Tangeretin can be considered as a small kinase inhibitor with a restricted activity spectrum, and some molecules of this class may become drugs with potential therapeutic applications in mammary cancer [23,24]. Major interest has resulted from successful therapeutic use of a tyrosine kinase inhibitor coined imatinib (Glivec, Gleevec), which has now received international approval as the first-line treatment for chronic myeloid leukemia and gastro-intestinal stromal cell tumors [25]. Imatinib shares one important feature with tangeretin in that oral treatment is the application route of choice to obtain an effect on (tumor) cell growth in vivo. Oral administration of tangeretin to laboratory mice has indeed been shown to exert an effect on cancer immune surveillance cells and significantly affects tumor growth [2]. Furthermore, a safety study with laboratory mice receiving tangeretin chronically in their drinking water has shown no effects on several blood parameters, except for a reduced number of circulating lymphocytes in the blood [2]. So, considering the effectiveness after oral administration, the safety in laboratory animals and the restricted kinase inhibition scope, tangeretin is a natural product with potential applications in medicine. Acknowledgements: This work was supported by the Department of Citrus of the State of Florida, USA. We thank Professor Marc Mareel for reviewing the manuscript.
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1669 [2] Bracke, M.E., Depypere, H.T., Boterberg, T., Van Marck, V.L., Vennekens, K.M., Vanluchene, E., Nuytinck, M., Serreyn, R. and Mareel, M.M. (1999) J. Natl. Cancer Inst. 91, 354–359. [3] Chang, F., Steelman, L.S., Shelton, J.G., Lee, J.T., Navolanic, P.M., Blalock, W.L., Franklin, R. and McCubrey, J.A. (2003) Int. J. Oncol. 22, 469–480. [4] Wei, S., Gilvary, D.L., Corliss, B.C., Sebti, S., Sun, J., Straus, D.B., Leibson, P.J., Trapani, J.A., Hamilton, A.D., Weber, M.J. and Djeu, J.Y. (2000) J. Immunol. 165, 3811–3819. [5] Stork, P.J.S. and Schmitt, J.M. (2002) Trends Cell Biol. 12, 258– 266. [6] Dudley, D.T., Pang, L., Decker, S.J., Bridges, A.J. and Saltiel, A.R. (1995) Proc. Natl. Acad. Sci. USA 92, 7686–7689. [7] Porcu, P., Baiocchi, R.A. and Magro, C. (2003) Curr. Opin. Oncol. 15, 353–362. [8] Levin, E.R. (2003) Endocrinology 17, 309–317. [9] Razandi, M., Pedram, A., Park, S.T. and Levin, E.R. (2003) J. Biol. Chem. 278, 2701–2712. [10] Laemmli, U.K. (1970) Nature 227, 680–685. [11] Pelech, S., Sutter, C. and Zhang, H. (2003) Meth. Mol. Biol. 218, 99–111. [12] Coopman, P.J.P., Do, M.T.H., Barth, M., Bowden, E.T., Hayes, A.J., Basyuk, E., Blancato, J.K., Vezza, P.R., McLeskey, S.W., Mangeat, P.H. and Mueller, S.C. (2000) Nature 406, 742–747. [13] Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J. and Vistica, D., et al. (1990) J. Natl. Cancer Inst. 1990, 1107– 1112. [14] Pons, M., Gagne, D., Nicolas, J.C. and Mehtali, M. (1990) Biotechniques 9, 450–459. [15] Olayioye, M.A., Neve, R.M., Lane, H.A. and Hynes, N.E. (2000) EMBO J. 19, 3159–3167. [16] deFazio, A., Chiew, Y.E., Sini, R.L., Janes, P.W. and Sutherland, R.L. (2000) Int. J. Cancer 87, 487–498. [17] Graziani, Y. (1986) in: Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological and Structure-Activity Relationships (Cody, V., Middleton, E. Jr.Jr. and Harborne, J.B., Eds.), pp. 301–313, Alan R. Liss Inc., New York. [18] Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M. and Fukami, Y. (1987) J. Biol. Chem. 262, 5592–5595. [19] Martin, P.M., Horwitz, K.B., Ryan, D.S. and McGuire, W.L. (1978) Endocrinology 103, 1860–1867. [20] Long, X., Gize, E.A., Nephew, K. and Bigsby, R.M. (2001) Endocrinology 142, 5390–5393. [21] Reddy, K.B., Nabha, S.M. and Atanaskova, N. (2003) Cancer Metastasis Rev. 22, 395–403. [22] Bracke, M.E., Vermeulen, S.J., Bruyneel, E.A., Vennekens, K. and Mareel, M.M. (1996) in: Hypernutritious Foods (Finley, D.J., Armstrong, D.J., Nagy, S. and Robinson, S.F., Eds.), pp. 205–217, Agscience, Inc., Florida. [23] Johnston, S.R., Head, J., Pancholi, S., Detre, S., Martin, L.A., Smith, I.E. and Dowsett, M. (2003) Clin. Cancer Res. 9, 524S– 532S. [24] Noble, M.E.M., Endicott, J.A. and Johnson, L.N. (2004) Science 303, 1800–1805. [25] Radford, I.R. (2002) Curr. Opin. Investig. Drugs 3, 492–499.
FEBS Letters 579 (2005) 1665–1669
Tangeretin inhibits extracellular-signal-regulated kinase (ERK) phosphorylation Se´verine Van Slambroucka, Virinder S. Parmarb, Sunil K. Sharmab, Bart De Bondta, Fleur Fore´a, Peter Coopmanc, Barbara W. Vanhoecked, Tom Boterberga, Herman T. Depypered, Guy Leclercqe, Marc E. Brackea,* a
Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, B-9000 Ghent, Belgium b Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, India c CNRS UMR 5539, University of Montpellier II, 34095 Montpellier, France d Department of Gynaecological Oncology, Ghent University Hospital, B-9000 Ghent, Belgium e Laboratory J-C Heuson of Mammary Cancerology, Bordet Institute, B-1000 Brussels, Belgium Received 23 August 2004; revised 6 October 2004; accepted 14 October 2004 Available online 21 February 2005 Edited by Lukas Huber
Abstract Tangeretin is a methoxyflavone from citrus fruits, which inhibits growth of human mammary cancer cells and cytolysis by natural killer cells. Attempting to unravel the flavonoidÕs action mechanism, we found that it inhibited extracellularsignal-regulated kinases 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent way. In human T47D mammary cancer cells this inhibition was optimally observed after priming with estradiol. The spectrum of the intracellular signalling kinase inhibition was narrow and comparison of structural congeners showed that inhibition of ERK phosphorylation was not unique for tangeretin. Our data add tangeretin to the list of small kinase inhibitors with a restricted intracellular inhibition profile. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Tangeretin; Methoxyflavone; Citrus; Extracellularsignal-regulated kinases 1/2; Mammary cancer
1. Introduction Tangeretin (4 0 ,5,6,7,8-pentamethoxyflavone), a natural compound from citrus fruits, was found to inhibit a number of cell activities such as growth of mammary cancer cells in vitro [1] and cytolysis by natural killer (NK) cells in vivo [2]. For both activities a pivotal role of the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) pathway has been reported [3,4]. A variety of peptide growth factors and steroid hormones are able to activate ERK via upstream activation of cytoplasmic kinases, including Ras, Src, Raf, and MAPK kinase (MKK) [5]. The synthetic methoxyflavone PD98059 was identified as an inhibitor of MKK [6] and hence of MAPK-driven cell proliferation and NK-cell activity [7]. Inspired by its structure similarity with PD 98059 and its ability to suppress cell proliferation and NK-activity as well, we investigated the effect of tangeretin on the MAPK signalling cascade. The model we used consisted of human T47D breast cancer cells stimulated with estradiol [8] and treated with tan* Corresponding author. Fax: +32 9 240 4991. E-mail address: [email protected] (M.E. Bracke).
geretin. Human breast cancer cell lines exhibit elevated activation levels of ERK after estradiol treatment, and this activation, mediated by plasma membrane-bound estrogen receptors, involves transactivation of ErbB tyrosine kinase receptors [9]. The dual tyrosine/threonine phosphorylation status of ERK was selected as an activation marker for this signalling pathway.
2. Materials and methods 2.1. Cell culture T47D cells are derived from a pleural effusion of a human mammary ductal carcinoma. Cells were grown in DulbeccoÕs modified EagleÕs medium (DMEM) (Invitrogen, Merelbeke, Belgium), supplemented with 100 IU/ml penicillin, 100 lg/ml streptomycin, 2.5 lg/ml amphotericin B, and 10% heat-inactivated fetal bovine serum (FBS) (Greiner Bio-one, Wemmel, Belgium) at 37 C in an atmosphere of 10% CO2 in air. 2.2. Chemicals and antibodies Tangeretin was kindly provided by Dr. Bill Widmer (Department of Citrus, Winter Haven, FL), and dissolved in ethanol as a stock solution of 102 M. 17-b-Estradiol (E2) was from Sigma (St. Louis, MO, USA), PD 98059 from Calbiochem (La Jolla, CA, USA), and recombinant heregulin-b1 (rHRG-b1) from R&D Systems (Abingdon, UK). The primary antibody for Western blotting was a rabbit polyclonal anti-phospho-p42/44 MAPK (Thr202/Tyr204) from Cell Signalling Technology (Beverly, MA, USA). The secondary anti-rabbit antibody was from Amersham Pharmacia Biotech (Amsterdam, The Netherlands). The monoclonal mouse-anti-Syk antibody (clone 4D10) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), while the polyclonal rabbit-anti-Src CST.1 antibody was kindly provided by Dr. S. Roche (CNRS FRE2593, Montpellier, France). RU 58668 from Roussel Uclaf (Romainville, France), 4-hydroxytamoxifen (4OH-TAM) from Zeneca Pharmaceuticals (Macclesfield, UK) and [3H]estradiol (±95 Ci/mmol) from Amersham Pharmacia Biotech were used for estrogen receptor a-mediated luciferase induction and ligand binding assays. 2.3. Western blotting All lysates were made from cells at approximately 70% confluency. For phosphorylation experiments, cells were washed three times with phosphate-buffered saline (PBS), serum-starved overnight, washed again three times with PBS and treated in serum-free medium. Before making lysates, the cells were washed again three times with PBS. Cells were lysed with PBS containing 1% Triton X-100, 1% Nonidet P-40 and the following protease inhibitors: aprotinin (10 lg/ml), leupeptin (10 lg/ml), phenylmethylsulfonyl fluoride (1.72 mM), NaF (100 M),
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2004.10.114
1666 NaVO3 (500 lM), and Na4P2O7 (500 lg/ml). After incubation at 4 C for 5 min, the samples were sonicated for 15 s. After clearing the lysates by centrifugation at 14 000 rpm for 10 min, protein concentration was determined using the Rc Dc Protein Assay (Bio-Rad, Hercules, CA, USA) and samples were prepared in such a way that equal amounts of protein were loaded. Sample buffer [10] with 5% 2-mercaptoethanol and 0.012% bromophenol blue was added, followed by boiling for 5 min and separation of proteins by gel electrophoresis on a 10% polyacrylamide gel and transfer onto a nitrocellulose membrane (Amersham Pharmacia Biotech). Quenching and immunostaining of the blots was done in 4% bovine serum albumin in PBS containing 0.2% Tween-20. The membranes were quenched for 1 h, incubated with primary antibody for 1 h, washed four times for 10 min, incubated with horseradish peroxidase-conjugated secondary antibody for 45 min, and washed six times for 10 min. Detection was done using Enhanced Chemiluminescence reagent (Amersham Pharmacia Biotech) as a substrate. Tubulin and total p42/p44 immunostaining was used as a loading control, and variations were less than 5%. After scanning the autoradiogram, bands quantification (the sum of p42 and p44) was performed using the Quantity One software (Bio-Rad). Experimental results were confirmed by obtaining concordant data from experiments performed at least in duplo. 2.4. Phosphorylation status analysis of cytoplasmic signalling proteins Kinetworks technology (Kinexus, Vancouver BC, Canada) permits the quantitative detection of the phosphorylation status of cytoplasmic proteins. The Kinetworks KPSS 1.3 Phosphoprotein Screen was performed as described by Pelech et al. [11] (http://www.kinexus.ca). The screen required 350 lg of protein, and for some kinases results obtained with different antibodies are presented. For the measurement in vitro of Syk and Src tyrosine kinases, Simian Cos1 cells were transiently transfected with the pCAF1-Syk [12] or the pSG5-Src cDNA (kindly provided by Dr. S. Roche) using the SuperFect reagent (Qiagen Inc, Venlo, The Netherlands). Two days after transfection, cells were lysed and cleared lysates were used for immunoprecipitation with protein A-agarose beads linked to antibodies against Syk or Src for 3 h at 4 C under constant rotation. Then the beads were washed in lysis buffer and used for Western blot or autophosphorylation testing. For in vitro kinase assays, the beads were re-suspended in 50 lL reaction buffer (10 mM Tris–HCl, pH 8, 10 mM MgCl2, and 10 mM MnCl2). Serial dilutions from a 102 M tangeretin stock solution or piceatannol, a specific inhibitor of Syk kinase activity (Calbiochem), were made in reaction buffer and added to the beads together with 10 lCi [c-32P] ATP (4500 Ci/mmol, ICN Biomedicals) and agitated for 20 min at room temperature. After washing in reaction buffer, beads were boiled in Laemmli sample buffer, loaded on 10% SDS–PAGE gels, dried, exposed and autophosphorylated Syk or Src kinase levels were detected and quantified using phosphor-image analysis (Molecular Dynamics, Sunnyvale, CA). 2.5. Assay for growth of T47D cells in vitro T47D cells were seeded in microtiter plates at an initial density of 1.5 · 104 cells in 200-ll culture medium. After an incubation period of 4 days, the amount of cell protein in each well was estimated with the sulforhodamine B assay [13]. 2.6. Luciferase induction assay MVLN cells are human MCF-7 mammary carcinoma cells, stably transfected with the pVit-tk-LUC reporter plasmid. These cells allow quantification of the transcriptional activity from estrogen receptor a-mediated estrogen responsive elements (ERE), in accordance with the luciferase induction assay by Pons et al. [14]. They were cultured in 35 mm diameter plastic petri dishes (80 000 cells/dish) in DMEM containing 10% dextran coated charcoal-treated FBS for 3–4 days. 250 ll of 5-fold diluted lysis solution (Promega E153A, Promega, Madison, WI, USA) was then added to the dishes and maintained under mild agitation for 20 min to extract luciferase. Lysed cells were subsequently detached with a scraper and centrifuged for 5 s at 10 000 · g to clarify the extracts. Finally, 20 ll of extracts was mixed at room temperature with 100 ll of a luciferase reactant medium (Promega E151A/E152A) prepared in accordance with the manufacturerÕs protocol and induced light was measured with a luminometer.
S. Van Slambrouck et al. / FEBS Letters 579 (2005) 1665–1669 2.7. Statistics Statistical evaluation of the data was performed with the StudentÕs t test.
3. Results 3.1. Dose- and time-dependent effect of tangeretin on the phosphorylation status of ERK Western blots were performed to determine whether tangeretin could inhibit the E2-induced ERK phosphorylation. T47D cells were treated with 109 M E2 for 20 min, in the absence or presence of varying concentrations of tangeretin (107 to 104 M), and assayed for ERK phosphorylation by immunostaining with an anti-phospho-MAPK antibody that detects the dual phosphorylation state (Thr202/Tyr204) of ERK 1/2 (p42 and p44). No activation was observed in solvent (ethanol 0.1%) treated cells as compared to untreated cells, but addition of E2 induced the phosphorylation of ERK 1/2. Tangeretin was able to inhibit this E2-induced phosphorylation in a dose-dependent manner (Fig. 1A). To determine the kinetics, T47D cells were treated with E2 (109 M) and tangeretin (104 M), and cell lysates were prepared after various incubation periods (Fig. 1B). ERK phosphorylation inhibition by tangeretin was evident after 10–30 min. Since E2 can stimulate the MAPK pathway via ErbB receptors, direct stimulation by rHRG-b1, a ligand of ErbB3 and ErbB4 [15], was applied as an alternative method to phosphorylate ERK [16]. To confirm the responsiveness of T47D cells to rHRG-b1, different concentrations (0.1–10 ng/ml) were initially tested and 1 ng/ml for 10 min proved to be an optimal stimulus. Tangeretin effectively reduced this rHRG-b1 stimulated ERK phosphorylation to 24% at a concentration of 104 M (Fig. 1C). 3.2. Effect of tangeretin on growth T47D mammary cancer cells Tangeretin inhibited the growth of estradiol-stimulated human T47D cells in the sulforhodamine B assay. The effect was dose-dependent and could be demonstrated at concentrations from 104 down to 107 M (Fig. 2). 3.3. Phosphorylation screening of other signalling molecules To further investigate the specificity of the kinase inhibition by tangeretin, a screening of the phosphorylation status of different cytoplasmic proteins (KPSS 1.3, Kinexus) was performed. Three samples pretreated with 109 M E2 for 20 min were analysed: one was solvent-treated, a second one was treated with 104 M tangeretin and the third one with 2.5 · 105 M PD98059 (Table 1). The results showed that tangeretin is indeed able to inhibit phophorylation of ERK 1/2 and of MAPK kinases. In contrast to PD98059, tangeretin is also able to inhibit phosphorylation of other proteins like adducin a and c, protein kinase Cd, signal transducer and activator of transcription (STAT) 1 and 3, and stress-activated protein kinase (JNK). The phosphorylation of other proteins such as JUN, protein kinase Ba (Akt1), and retinoblastoma 1, however, were increased. Overall the spectrum of inhibition by tangeretin differed from the one by PD98059. Quercetin and epigallocatechin-3-gallate were recently found to down regulate the activity of the Syk kinase and
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Fig. 2. Effect of tangeretin on growth of estradiol-stimulated human T47D cells. The cells were treated with different tangeretin concentration for 4 days, and the amount of cellular protein was assessed with the sulforhodamine B assay and expressed as optical density units (OD). Bars indicate mean values + S.D., *, P < 0.01.
Table 1 Effect of 104 M tangeretin and 2.5 · 105 M PD98059 on the phosphorylation status of different cytoplasmic kinases and kinase substrates
Fig. 1. Effect of tangeretin on ERK1/2 phosphorylation. T47D cells were pretreated with estradiol (A and B) or with heregulin (C), and treated with tangeretin or with solvent. In these typical experiments shown, tangeretin inhibited ERK1/2 phosphorylation in a dose- (A and C) and time- (B) dependent way. Western blot analysis and immunostaining were done with anti-phospho ERK1/2, and results are presented as percentages of E2- or heregulin-treated cells. Mean values + S.D., *, P < 0.01.
the Lyn Src-family kinase. In order to test the effect of tangeretin on the Syk and Src kinase activities, their autophosphorylation capacity was tested in vitro by incorporation of [c-32P] ATP in presence of increasing tangeretin concentrations. On Syk and Src kinase autophosphorylation levels no effect of tangeretin could be observed, yet piceatannol at 104 M completely abolished Syk autophosphorylation (data not shown).
Phosphorylation of
Tangeretin % of solvent control
PD98059
Adducin a Adducin c cAMP response element binding protein Cyclin-dependent kinase 1 (cdc2) dsRNA dependent protein kinase Extracellular regulated kinase 1 Extracellular regulated kinase 2 Glycogen synthase kinase 3 a Glycogen synthase kinase 3 a Glycogen synthase kinase 3 b Glycogen synthase kinase 3 b MAP kinase kinase 1/2 MAP kinase kinase 6 N-Methyl-D -aspartate glutamate receptor unit 1 Oncogene JUN Oncogene Raf 1 Oncogene Raf 1 Oncogene SRC Oncogene SRC P38 a MAP kinase Protein kinase B a (Akt1) Protein kinase B a (Akt1) Protein kinase C a Protein kinase C a/b Protein kinase C d Protein kinase C e Retinoblastoma 1 Retinoblastoma 1 S6 kinase p70 STAT 1 STAT 3 Stress-activated protein kinase (JNK) Stress-activated protein kinase (JNK)
48.3 50.3 157.3 115.2 154.5 64.9 63.5 114.6 97.8 263.5 97.3 75.0 64.7 90.2
122.1 128.7 151.7 43.1 11.6 20.9 14.2 74.1 19.4 146.4 22.3 104.7 83.9 143.1
211.8 86.3 95.6 72.4 110.8 104.4 161.3 138.3 127.7 101.9 73.7 92.1 127.6 131.5 56.3 70.7 66.6 78.6 64.1
105.7 89.3 64.9 69.7 86.5 116.6 87.5 99.9 162.3 217.5 127.3 91.3 150.3 207.4 119.6 99.1 78.9 84.5 126.8
3.4. Effects of methoxyflavone congeners on ERK phosphorylation A number of methoxyflavones and methoxyisoflavones were tested for their ability to affect ERK phosphorylation in E2stimulated T47D cells (Fig. 3A). At 104 M 5,6,7,8,3 0 ,4 0 ,5 0 heptamethoxyflavone (349) and 2,7-dimethoxyflavone (345) were more potent than tangeretin (Fig. 3B).
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Fig. 3. Structures of methoxyflavone/methoxyisoflavone congeners of tangeretin (A), and their effect on ERK1/2 phosphorylation in T47D cells (B). In this typical experiment shown, E2-stimulated (20 min) T47D cells were treated in absence or presence of different methoxyflavones or methoxyisoflavones at a concentration of 104 M. Western Blot analysis and immunostaining were done with anti-phospho ERK1/2. Results are presented as percentages of phosphorylation in tangeretin-treated cells.
3.5. Absence of effect of tangeretin on the ERE-dependent transcription activity Because it has been reported that some flavonoids can bind to the estrogen receptor, the effect of tangeretin on ERE-dependent transcription activity was measured. Treatment of MVLN cells for 24 h was done in the presence of 1010 M E2, 107 M 4-OH-TAM, 107 M RU58668, and different concentrations of tangeretin. Increase of ERE-luciferase activity was observed with E2 (+110%), while partial (4-OH-TAM) and pure (RU58668) anti-estrogens decreased the basal transcriptional activity by 65% and 55%, respectively. With tangeretin (105 to 109 M) neither stimulatory nor inhibitory effects on luciferase induction could be observed (Fig. 4). No affinity neither for the intracellular estrogen receptor a nor for the membrane estrogen binding sites could be attributed to tangeretin in a ligand binding competition assay with [3H] estradiol (data not shown).
Fig. 4. Luciferase induced activity (%) of tangeretin (105 to 109 M) in MVLN cells.
4. Discussion This report adds tangeretin to the list of flavonoids that were previously described in the literature as kinase inhibitors.
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Notorious examples are the phosphorylation inhibition of pp60src by quercetin [17], and of receptor tyrosine kinases by high-dose genistein [18]. Moreover, several pharmacological inhibitors of kinases belong to the flavonoid family, and PD 98059, the gold standard inhibitor for MAPK phosphorylation in vitro, is a synthetic methoxyflavone structurally related to tangeretin. Tangeretin, however, differs from genistein [19] and PD 98059 [20] in that it is devoid of any estrogenic or anti-estrogenic activity. A number of tangeretinÕs effects on cell behaviour can be matched with inhibition of ERK signalling. This pathway has shown to be involved in cell proliferation [5], motility and invasion [21], and natural killer cell activity [4]. Inhibition of the pathway by tangeretin treatment could explain the effects observed in several functional assays in vitro [22]. Tangeretin can be considered as a small kinase inhibitor with a restricted activity spectrum, and some molecules of this class may become drugs with potential therapeutic applications in mammary cancer [23,24]. Major interest has resulted from successful therapeutic use of a tyrosine kinase inhibitor coined imatinib (Glivec, Gleevec), which has now received international approval as the first-line treatment for chronic myeloid leukemia and gastro-intestinal stromal cell tumors [25]. Imatinib shares one important feature with tangeretin in that oral treatment is the application route of choice to obtain an effect on (tumor) cell growth in vivo. Oral administration of tangeretin to laboratory mice has indeed been shown to exert an effect on cancer immune surveillance cells and significantly affects tumor growth [2]. Furthermore, a safety study with laboratory mice receiving tangeretin chronically in their drinking water has shown no effects on several blood parameters, except for a reduced number of circulating lymphocytes in the blood [2]. So, considering the effectiveness after oral administration, the safety in laboratory animals and the restricted kinase inhibition scope, tangeretin is a natural product with potential applications in medicine. Acknowledgements: This work was supported by the Department of Citrus of the State of Florida, USA. We thank Professor Marc Mareel for reviewing the manuscript.
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