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Apigenin Induces Apoptosis through Proteasomal Degradation of HER2/neu in HER2/neu-overexpressing Breast Cancer Cells via the Phosphatidylinositol 3-Kinase/Akt-dependent Pathway

2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês

10.1074/jbc.m305529200

ISSN

1083-351X

Autores

Tzong‐Der Way, Ming‐Ching Kao, Jen‐Kun Lin,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

Apigenin is a low toxicity and non-mutagenic phytopolyphenol and protein kinase inhibitor. It exhibits anti-proliferating effects on human breast cancer cells. Here we examined several human breast cancer cell lines having different levels of HER2/neu expression and found that apigenin exhibited potent growth-inhibitory activity in HER2/neu-overexpressing breast cancer cells but was much less effective for those cells expressing basal levels of HER2/neu. Induction of apoptosis was also observed in HER2/neu-overexpressing breast cancer cells in a dose- and time-dependent manner. However, the one or more molecular mechanisms of apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells remained to be elucidated. A cell survival pathway involving phosphatidylinositol 3-kinase (PI3K), and Akt is known to play an important role in inhibiting apoptosis in response to HER2/neu-overexpressing breast cancer cells, which prompted us to investigate whether this pathway plays a role in apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells. Our results showed that apigenin inhibits Akt function in tumor cells in a complex manner. First, apigenin directly inhibited the PI3K activity while indirectly inhibiting the Akt kinase activity. Second, inhibition of HER2/neu autophosphorylation and transphosphorylation resulting from depleting HER2/neu protein in vivo was also observed. In addition, apigenin inhibited Akt kinase activity by preventing the docking of PI3K to HER2/HER3 heterodimers. Therefore, we proposed that apigenin-induced cellular effects result from loss of HER2/neu and HER3 expression with subsequent inactivation of PI3K and AKT in cells that are dependent on this pathway for cell proliferation and inhibition of apoptosis. This implies that the inhibition of the HER2/HER3 heterodimer function provided an especially effective strategy for blocking the HER2/neu-mediated transformation of breast cancer cells. Our results also demonstrated that apigenin dissociated the complex of HER2/neu and GRP94 that preceded the depletion of HER2/neu. Apigenin-induced degradation of mature HER2/neu involves polyubiquitination of HER2/neu and subsequent hydrolysis by the proteasome. Apigenin is a low toxicity and non-mutagenic phytopolyphenol and protein kinase inhibitor. It exhibits anti-proliferating effects on human breast cancer cells. Here we examined several human breast cancer cell lines having different levels of HER2/neu expression and found that apigenin exhibited potent growth-inhibitory activity in HER2/neu-overexpressing breast cancer cells but was much less effective for those cells expressing basal levels of HER2/neu. Induction of apoptosis was also observed in HER2/neu-overexpressing breast cancer cells in a dose- and time-dependent manner. However, the one or more molecular mechanisms of apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells remained to be elucidated. A cell survival pathway involving phosphatidylinositol 3-kinase (PI3K), and Akt is known to play an important role in inhibiting apoptosis in response to HER2/neu-overexpressing breast cancer cells, which prompted us to investigate whether this pathway plays a role in apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells. Our results showed that apigenin inhibits Akt function in tumor cells in a complex manner. First, apigenin directly inhibited the PI3K activity while indirectly inhibiting the Akt kinase activity. Second, inhibition of HER2/neu autophosphorylation and transphosphorylation resulting from depleting HER2/neu protein in vivo was also observed. In addition, apigenin inhibited Akt kinase activity by preventing the docking of PI3K to HER2/HER3 heterodimers. Therefore, we proposed that apigenin-induced cellular effects result from loss of HER2/neu and HER3 expression with subsequent inactivation of PI3K and AKT in cells that are dependent on this pathway for cell proliferation and inhibition of apoptosis. This implies that the inhibition of the HER2/HER3 heterodimer function provided an especially effective strategy for blocking the HER2/neu-mediated transformation of breast cancer cells. Our results also demonstrated that apigenin dissociated the complex of HER2/neu and GRP94 that preceded the depletion of HER2/neu. Apigenin-induced degradation of mature HER2/neu involves polyubiquitination of HER2/neu and subsequent hydrolysis by the proteasome. Several cancers, including breast cancer, have a lower incidence in Asia than in Western countries (1Rose D.P. Boyar A.P. Wynder E.L. Cancer. 1986; 58: 2363-2371Crossref PubMed Scopus (518) Google Scholar). This may be attributed to the Asian dietary regimen rich in flavonoid-containing plants, which are thought to be anti-tumorigenic. Among the plant flavonoids, apigenin (4′,5,7,-trihydroxyflavone) is a chemopreventive compound (2Kuo M.L. Lee K.C. Lin J.K. Mutat. Res. 1992; 270: 87-95Crossref PubMed Scopus (96) Google Scholar, 3Chaumontet C. Bex V. Gaillard-Sanchez I. Seillan-Heberden C. Suschetet M. Martel P. 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Cancer. 1997; 28: 236-247Crossref PubMed Scopus (232) Google Scholar), and it possesses growth inhibitory properties against many human cancer cell lines, including breast (8Yin F. Giuliano A.E. Law R.E. Van Herle A.J. Anticancer Res. 2001; 21: 413-420PubMed Google Scholar), colon (9Wang W. Heideman L. Chung C.S. Pelling J.C. Koehler K.J. Birt D.F. Mol. Carcinog. 2000; 28: 102-110Crossref PubMed Scopus (264) Google Scholar), skin (10Caltagirone S. Rossi C. Poggi A. Ranelletti F.O. Natali P.G. Brunetti M. Aiello F.B. Piantelli M. Int. J. Cancer. 2000; 87: 595-600Crossref PubMed Scopus (430) Google Scholar), thyroid (11Yin F. Giuliano A.E. Van Herle A.J. Thyroid. 1999; 9: 369-376Crossref PubMed Scopus (103) Google Scholar), leukemia (12Wang I.K. Lin-Shiau S.Y. Lin J.K. Eur. J. Cancer. 1999; 35: 1517-1525Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar), and prostate carcinomas cells (13Sanjay G. Farrukh A. Hasan M. Oncogene. 2002; 21: 3727-3738Crossref PubMed Scopus (265) Google Scholar). Apigenin has been demonstrated to be a protein kinase inhibitor, and it achieves this inhibitory effect by competing with ATP (14Geahlen R.L. Koonchanok N.M. McLaughlin J.L. J. Nat. Prod. 1989; 52: 982-986Crossref PubMed Scopus (125) Google Scholar). Our previous report (15Huang Y.T. Kuo M.L. Liu J.Y. Huang S.Y. Lin J.K. Eur. J. Cancer. 1996; 32A: 146-151Abstract Full Text PDF PubMed Scopus (60) Google Scholar) showed that apigenin inhibited TPA 1The abbreviations used are: TPA12-O-tetradecanoylphorbol-13-acetatePI3Kphosphatidylinositol 3-kinasePKBprotein kinase BGSK-3glycogen synthase kinase-3PDK3-phosphoinositide-dependent protein kinaseDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumPBSphosphate-buffered salineMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideLLnLN-acetyl-Leu-Leu-norleu-alCQchloroquineHsp90heat shock protein 90.-induced c-jun and c-fos expression and TPA-mediated tumor promotion of mouse skin. 12-O-tetradecanoylphorbol-13-acetate phosphatidylinositol 3-kinase protein kinase B glycogen synthase kinase-3 3-phosphoinositide-dependent protein kinase Dulbecco's modified Eagle's medium fetal bovine serum phosphate-buffered saline 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide N-acetyl-Leu-Leu-norleu-al chloroquine heat shock protein 90. Breast cancer is the most common cancer among women in the Western world and the second leading cause of cancer-related deaths in women (16Greenle R.T. Hill-Harmon M.B. Thun M. CA Cancer J. Clin. 2001; 51: 15-36Crossref PubMed Scopus (3332) Google Scholar). Gene amplification and/or overexpression of some oncogenes have been implicated in breast cancers. HER2/neu (also known as ErbB2) is among the most characterized oncogenes linked with poor prognosis in breast cancer (17Slamon D. Clark M. Wong S. Levin W. Ullrich A. McAuire W. Science. 1987; 235: 177-181Crossref PubMed Scopus (10014) Google Scholar). Overexpression of HER2/neu is found in ∼30% of human breast cancers and correlates with more aggressive tumors and more resistance to cancer chemotherapy (18Menard S. Tagliabue E. Campiglio M. Pupa S.M. J. Cell Physiol. 2000; 182: 150-162Crossref PubMed Scopus (251) Google Scholar). An increase in HER2/neu expression also enhances malignant phenotypes of cancer cells, including those with metastatic potential (19Yu D.H. Hung M.C. Oncogene. 1991; 6: 1991-1996PubMed Google Scholar, 20Yu D.H. Wang S.S. Dulski K.M. Nicolson G.L. Hung M.C. Cancer Res. 1994; 54: 3150-3156Google Scholar, 21Yusa K. Sugimot Y. Yamori T. Yamamoto T. Toyoshima K. Tsuruo T. J. Natl. Cancer Inst. 1990; 82: 1632-1635Crossref Scopus (35) Google Scholar). The association of HER2/neu overexpression in cancer cells with chemoresistance and metastasis provides a plausible interpretation for the poor clinical outcome of patients with HER2/neu-overexpressing cancers; it suggests that the enhanced tyrosine kinase activity of HER2/neu might play a critical role in the initiation, progression, and outcome of human tumors. HER2/neu is a member of the class II receptor (ErbB) tyrosine kinase family, which in human includes the HER1 (epidermal growth factor receptor, ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). ErbB receptors are essential mediators of cell proliferation and differentiation. Their aberrant activation is associated with the development and severity of many cancers. Homo- and hetero-dimerization of ErbB receptors result in a wide variety of cellular signal transduction. Dimerization of HER2/neu and HER3 occurs frequently and is a preferred heterodimer (22Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar). The HER2/HER3 dimer constitutes a high affinity co-receptor for heregullin, which is capable of potent mitogenic signaling (23Karunagaran D. Tzahar E. Beerli R.R. Chen X. Graus-Porta D. Ratzkin B.J. Seger R. Hynes N.E. Yarden Y. EMBO J. 1996; 15: 254-264Crossref PubMed Scopus (589) Google Scholar). HER3 is a kinase-defective protein that is phosphorylated by HER2/neu. Tyrosine-phosphorylated HER3 is able to directly couple to PI3K (phosphatidylinositol 3-kinase), a lipid kinase involved in the proliferation, survival, adhesion, and motility of tumor cells (24Fedi P. Pierce J.H. di Fiore P.P. Kraus M.H. Mol. Cell. Biol. 1994; 14: 492-500Crossref PubMed Google Scholar, 25Prigent S.A. Gullick W.J. EMBO J. 1994; 13: 2831-2841Crossref PubMed Scopus (320) Google Scholar, 26Duronio V. Scheid M.P. Ettinger S. Cell Signal. 1998; 10: 233-239Crossref PubMed Scopus (186) Google Scholar, 27Hellyer N.J. Kim M.S. Koland J.G. J. Biol. Chem. 2001; 276: 42153-42161Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). 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Matin A. Yu D. Xia W. Sorgi F. Huang L. Hung M.C. Cancer Gene Ther. 1996; 3: 168-174PubMed Google Scholar, 37Yu D. Matin A. Xia W. Sorgi F. Huang L. Hung M.C. Oncogene. 1995; 11: 1383-1388PubMed Google Scholar, 38Zhang Y. Yu D. Xia W. Hung M.C. Oncogene. 1995; 10: 1947-1954PubMed Google Scholar) or by using anti-HER2/neu antibodies (39Drebin J.A. Link V.C. Weinberg R.A. Greene M.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9129-9133Crossref PubMed Scopus (197) Google Scholar, 40Drebin J.A. Link V.C. Greene M.I. Oncogene. 1988; 2: 273-277PubMed Google Scholar, 41Katsumata M. Okudaira T. Samanta A. Clark D.P. Drebin J.A. Jolicoeur P. Greene M.I. Nat. Med. 1995; 1: 644-648Crossref PubMed Scopus (103) Google Scholar, 42Park J.W. Hong K. Carter P. Asgari H. Guo L.Y. Keller G.A. Wirth C. Shalaby R. Kotts C. Wood W.I. Papahadjopoulos D. Benz C.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1327-1331Crossref PubMed Scopus (255) Google Scholar) can suppress tumor growth and dissemination. One therapeutic approach that has already reached clinical application is the use of an unarmed monoclonal antibody called Trastuzumab (Herceptin™) (43Pegram M. Slamon D. Semin. Oncol. 2000; 27: 13-19PubMed Google Scholar). Studies have attributed the therapeutic potential of anti-HER2/neu antibodies to their ability to enhance intracellular degradation of the cell surface-localized oncoprotein (44Kasprzyk P.G. Song S.U. Di Fiore P.P. King C.R. Cancer Res. 1992; 52: 2771-2776PubMed Google Scholar). These findings suggest that manipulating HER2/neu may be of substantial value in the treatment of breast cancer. Apigenin has been shown to efficiently inhibit the growth of MCF-7 and MDA-MB-468 breast carcinoma cell lines, and its growth inhibitory effects are mediated by targeting different signal transduction pathways (8Yin F. Giuliano A.E. Law R.E. Van Herle A.J. Anticancer Res. 2001; 21: 413-420PubMed Google Scholar). However, MCF-7 and MDAMB-468 express only basal levels of HER2/neu. Here, we investigated the effectiveness of apigenin against a series of breast cancer cells having different levels of HER2/neu expression. We showed that apigenin efficiently inhibited the growth of MDAMB-453 HER2/neu-overexpressing breast cancer cells. Induction of apoptosis was also observed in these HER2/neu-overexpressing breast cancer cells. In addition, to elucidate the molecular mechanism of apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells, the apoptotic machinery and the expression of several cell survival genes were investigated. We demonstrated that HER2/neu was degraded in MDA-MB-453 HER2/neu-overexpressing breast cancer cells by proteasomal degradation, and that the inhibition of cell growth and induction of apoptosis by apigenin may be through suppression of HER2/HER3 signaling and disruption of the PI3K/Akt-dependent pathway. Cell Culture—The human breast cancer cell lines used in this study were MDA-MB-453, BT-474, and SKBr-3, all of which overexpress HER2/neu, and MCF-7, which expresses the basal level of HER2/neu. We also used HBL-100 cell line, which is derived from a normal human breast tissue transformed by SV40 large T antigen and expresses a basal level of HER2/neu. All of the cells were grown in DMEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and gentamicin (50 mg/ml). Cells were grown in a humidified incubator at 37 °C under 5% CO2 in air. Cell Transfection—The plasmid pSV2-erbB2, a constitutive expression vector, carries the 4.4-kb full-length human HER2/neu cDNA under the control of the SV40 promoter/enhancer sequence. Two million cells were transfected with 2 μg of DNA mediated by 10 μl of Lipofectin reagent (Invitrogen). Experiments were performed 24 h after transfection. Western Blot Analysis—The cells (1.5 × 106) were seeded onto a 100-mm tissue culture dish in 10% FBS DMEM/F-12 and cultured for 24 h. The cells were then incubated in 1% FBS DMEM/F-12 treating with various dose of apigenin for various time periods. Cells were washed three times with PBS and then lysed in gold lysis buffer (10% glycerol, 1% Triton X-100, 137 mm NaCl, 10 mm NaF, 1 mm EGTA, 5 mm EDTA, 1 mm sodium pyrophosphate, 20 mm Tris-HCl, pH 7.9, 100 mm β-glycerophosphate, 1 mm sodium orthovanadate, 0.1% SDS, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin). Protein content was determined against a standardized control, using the Bio-Rad protein assay kit (Bio-Rad Laboratories). A total of 50 μg of protein was separated by SDS-PAGE and transferred to nitrocellulose filter paper (Schleicher & Schuell, Inc., Keene, NH). Nonspecific binding on the nitrocellulose filter paper was minimized with a blocking buffer containing nonfat dry milk (5%) and Tween 20 (0.1%, v/v) in PBS (PBS/Tween 20). Then the filter paper was incubated with primary antibodies followed by incubation with horseradish peroxidase-conjugated goat anti-mouse antibody (1:2500 dilution, Roche Applied Science, Indianapolis, IN). Reactive bands were visualized with an enhanced chemiluminescence system (Amersham Biosciences, Arlington Heights, IL). The intensity of the bands was scanned and quantified with National Institutes of Health Image software. In Vitro HER2/neu Tyrosine Kinase Assay—Immunocomplex was precipitated from lysate of MDA-MB-453 cells with monoclonal anti-HER2/neu antibody c-neu (Ab-3) on protein-A-conjugated agarose beads (40 μl) (Roche Applied Science) and then washed three times with 50 mm Tris-HCl buffer containing 0.5 m LiCl (pH 7.5) and once in assay buffer (50 mm Tris-HCl (pH 7.5) and 10 mm MnCl2). Radiolabeled ATP (10 μCi of [γ-32P]ATP, Amersham Biosciences) and 10 μl of enolase (2.5 mg/ml, Sigma Chemical Co., St. Louis, MO) were added to the beads, followed by incubation for 20 min at room temperature. The reaction products were separated by 8% SDS-PAGE. The gel was dried and visualized by autoradiography. In Vitro PI3K Assay—MDA-MB-453 cell extracts (500 μg) were immunoprecipitated with anti-PI3K(p85) antibody (Upstate Biotechnology, Inc.) and protein A-Sepharose beads (Repligen). Immunoprecipitation complexes were washed three times with 1% Triton X-100 in PBS, twice with a buffer containing 0.5 m LiCl, 0.1 m Tris, pH 7.5, and twice with 10 mm Tris, pH 7.5, 100 mm NaCl, and 1 mm EDTA. Complexes were incubated for 20 min at room temperature with 1 μm ATP, 5 μCi of [γ-32P]ATP (Amersham Biosciences), and a 0.5 μm/ml lipid mix of phosphatidylinositol and phosphatidylserine (1:1) in 10 mm HEPES (pH 7.0), and 1 mm EGTA. The reaction was quenched by 1 m HCl, and lipids were extracted with CHCl3:CH3OH (1:1). The organic layer was analyzed by thin-layer chromatography developed with 1-propanol:methanol:glacial acetic acid (50:15:35) and detected by autoradiography. In Vitro Akt Kinase Assay—Kinase activity was assayed using a New England Biolabs Akt Kinase Kit. Akt was immunoprecipitated, washed twice with lysis buffer, then twice with kinase buffer (25 mm Tris, pH 7.5, 5 mm β-glycerolphosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4, 10 mm MgCl2). 200 μm ATP and 1 μg of substrate (paramyosin fused to a GSK-3 crosstide) were added, and assays were performed at 30 °C for 30 min. Reaction mixtures were separated by 10% SDS-PAGE, and the p-GSK-3 reaction product was detected by immunoblotting. In Vitro grp94 Autophosphorylation Assay—MDA-MB-453 cell extracts (500 μg) were immunoprecipitated with GRP94 (Upstate Biotechnology, Inc.). Mixtures were incubated for 3 h at 4 °C, and then 40 μl of protein-A-conjugated agarose beads was added. After rotation at 4 °C for overnight, immunocomplexes were washed 5 times with 800 μl each of a washing buffer (50 mm Tris, 100 mm NaF, 50 mm NaCl, 2 mm EDTA, 2 mm sodium orthovanadate, 10 mm sodium pyrophosphate, 10% glycerol, 1% Nonidet P-40, pH 8.0) and finally resuspended in 50 mm Tris, pH 7.4. Ten μl of the immunocomplex beads was incubated in a buffer containing 30 mm Hepes, 400 kBq of 0.2 mm [γ-32P]ATP, pH 7.5, in the presence of 5 mm CaCl2 or MgCl2 at 37 °C for 30 min with occasional mixings. The reaction was terminated by adding 5× SDS sample buffer and boiling for 5 min. The proteins eluted from the immunoaffinity resins were analyzed by SDS-PAGE and autoradiography. Pulse-chase Labeling Assay—MDA-MB-453 cells were grown to 70% confluence in 100-mm dishes in DMEM/F-12 supplemented with 10% fetal calf serum. Plates were washed and then incubated in DMEM lacking methionine and cysteine for 20 min and then pulsed for 30 min in 1 ml of deficient media containing 10% dialyzed fetal calf serum and 0.1 mCi of [35S]methionine (Trans-Label, ICN). After pulsing, plates were washed once in complete media and then incubated in complete media containing either 40 μm apigenin or vehicle control (0.l% Me2SO). After incubation, plates were washed three times in PBS, and the cells were lysed in gold lysis buffer. Cell lysates were cleared by a 10-min spin at 12,000 × g, and then an equal amount of protein (500 μg) was immunoprecipitated with monoclonal antibody Ab-3 as described above. Immunocomplexes were separated by 8% SDS-polyacrylamide gel. The gel was dried and visualized by fluorography. Immunofluorescence Assay—MDA-MB-453 cells were plated on coverslips placed in six-well plates. Experiments were performed 24 h after cell attachment. Cells were fixed in PBS containing 4% paraformaldehyde for 10–15 min at room temperature. Cells were rinsed with PBS for 2–3 times followed by blocking with 1% normal goat serum for 30 min. Incubations were performed with primary antibodies diluted in blocking buffer at 4 °C for overnight, after which coverslips were washed and incubated for 30 min with the fluorescein isothiocyanate-conjugated secondary antibodies diluted in blocking buffer. Coverslips were washed and mounted in Vectashield (Vector Laboratories, Burlingame, CA) and viewed under a Leica TCS SP2 confocal laser-scanning microscope (Leica Microsystems, Heidelberg, Germany). MTT Assay—Cells were seeded in a 96-well microtiter plate (1 × 104 cells/well) overnight, then treated with varying concentrations of apigenin, and incubated for an additional 72 h. The effect of apigenin on cell growth was examined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Briefly, 20 μl of MTT solution (5 mg/ml, Sigma Chemical Co.) was added to each well and incubated for 4 h at 37 °C. The supernatant was aspirated, and the MTT-formazan crystals formed by metabolically viable cells were dissolved in 200 μl of Me2SO. Finally, the absorbance was monitored by a microplate reader at a wavelength of 595 nm. DNA Extraction and Electrophoretic Analysis—Cells (4 × 105 cells/ml) were harvested, washed in PBS, and then lysed by digestion buffer containing 0.5% Sarkosyl, 0.5% mg/ml proteinase K, 50 mm Tris (pH 8.0), and 10 mm EDTA at 55 °C for 3 h. RNase A (0.5 mg/ml) was added, and the mixture was incubated at 55 °C for 24 h, after which the DNA was extracted by phenol/chloroform/isoamyl alcohol (25:24:1). Approximately 20 μg of DNA was loaded in each well, and electrophoresed into a 1.8% agarose gel (containing ethidium bromide) at 50 V for 120 min. The gel was then visualized under a UV light and photographed. Flow Cytometry—Cells (2 × 105) were cultured in 60-mm Petri dishes and incubated for various times. Then cells were harvested, washed with PBS, resuspended in 200 μl of PBS, and fixed in 800 μl of iced 100% ethanol at -20 °C. After being left to stand overnight, cell pellets were collected by centrifugation, resuspended in 1 ml of hypotonic buffer (0.5% Triton X-100 in PBS and 0.5 μg/ml RNase), and incubated at 37 °C for 30 min. Then 1 ml of propidium iodide solution (50 μg/ml) was added, and the mixture was allowed to stand on ice for 30 min. Fluorescence emitted from the propidium iodide-DNA complex was quantitated after excitation of the fluorescent dye by FAC-Scan cytometry (BD Biosciences, San Jose, CA). Apigenin Preferentially Inhibited the Proliferation of HER2/neu-overexpressing Breast Cancer Cells—The growth of the tested cell lines was inhibited by apigenin in a dose-dependent manner but to varying extents (Fig. 1). At a 40 μm concentration, apigenin blocked 48% of growth in HER2/neu-overexpressing MDA-MB-453 cells. However, under the same conditions, it inhibited only 31%of growth in MCF7 (basal HER2/neu levels). Apigenin had little effect on the immortalized non-cancerous HBL-100 breast cell line even at 70 μm. These results suggest that apigenin preferentially suppresses growth of the HER2/neu-overexpressing breast cancer cell lines. Apigenin Induced Apoptosis in the HER2/neu-overexpressing Breast Cancer Cells—Apigenin-treated MDA-MB-453 cell lines underwent apoptosis in a dose- and time-dependent manner as measured by flow cytometry using propidium-iodide staining (Fig. 2A). A significant number of the cells (55.37%) started to undergo apoptosis as early as 36 h after treatment with 40 μm apigenin. A lower concentration of apigenin (20 μm) resulted in apoptosis in fewer cells (∼20% at 36 h). As shown in Fig. 2B,by comparing with vehicle control, apigenin treatment (20 and 40 μm for 48 h) resulted in DNA fragmentations in MDA-MB-453 cells. Similarly, treatment with apigenin at 40 μm for 24 and 48 h resulted in the formation of a DNA ladder. Apigenin Inhibited PI3K Activity and Akt Kinase in HER2/neu-overexpressing Breast Cancer Cells—A key mechanism by which HER2/neu overexpression stimulates tumor cell growth and renders cells chemoresistant is through the HER2/neu receptor. This mechanism involves the PI3K/Akt signaling pathway, and human breast cancer cells with overexpression and amplification of HER2/neu have been shown to make increased use of the signaling pathway mediated by PI3K/Akt (17Slamon D. Clark M. Wong S. Levin W. Ullrich A. McAuire W. Science. 1987; 235: 177-181Crossref PubMed Scopus (10014) Google Scholar, 45Yokota J. Yamamoto T. Toyoshima K. Terada M. Sugimura T. Battifora H. Cline M.J. Lancet. 1986; 1: 765-767Abstract PubMed Scopus (341) Google Scholar). Activated Akt is considered the focal point of a survival pathway known to protect cells from apoptosis by several stimuli, whereas in a recent report, apigenin displayed potent inhibitory effects on PI3K activity (46Agullo G. Gamet-Payrastre L. Manenti S. Viala C. Remesy C. Chap H. Payrastre B. Biochem. Pharmacol. 1997; 53: 1649-1657Crossref PubMed Scopus (519) Google Scholar). As shown in Fig. 3A, our results also indicated that apigenin possessed inhibitory effects on PI3K activity in the HER2/neu-overexpressing breast cancer cells. Furthermore, we found that in the HER2/neu-overexpressing breast cancer cell lines MDA-MB-453, BT-474, and SKBr-3, treatment with apigenin had no effect on steady-state levels of total PI3K protein, whereas its downstream effector of phosphorylated Akt was inhibited in a dose- and time-dependent manner (Fig. 3B). Wortmannin and LY294002 are known to be irreversible PI3K inhibitors and were used here as positive controls (Fig. 3B). Treatment of the HER2/neu-overexpressing breast cancer cell lines with wortmannin almost completely inhibited Akt phosphorylation at 2 h, whereas the reduced inhibition at 16 h post-treatment (Fig. 3B) is presumably attributable to wortmannin having a relatively short half-life. Akt kinase has been shown to phosphorylate several key substrates that regulate protein translation, apoptosis, and cellular proliferation (47Marte B.M. Downward J. Trends. Biochem. Sci. 1997; 22: 355-358Abstract Full Text PDF PubMed Scopus (649) Google Scholar, 48Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1400) Google Scholar), and phosphorylation of its substrate, glycogen synthase kinase-3 (GSK-3), was demonstrated here in MDA-MB-453 cells (Fig. 3C). Apigenin caused dephosphorylation of GSK-3 at concentrations associated with inhibition of Akt activation, but at the same time, treatment with apigenin had no effect on steady-state levels of total Akt kinase protein (Fig. 3C). To test whether apigenin directly inhibited the Akt kinase, Akt was immunoprecipitated from untreated MDAMB-453 cells. After treatment of the precipitates with various concentrations of apigenin, measurement of the Akt kinase activity showed that apigenin have

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