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• W1994061224 abstract "Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. A synthetic library was screened to identify inhibitors of tumor cell migration. From this, we discovered that CAC-1098 (aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC50 = 5 and 50 nm, respectively. We synthesized KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the bromide group of CBI-0997 with a methoxyl group. Like CBI-0997, KRIBB3 has anti-migratory and anti-invasive activities in MDA-MB-231 cells. Because KRIBB3 has a better drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding proteins. Using affinity chromatography, we identified Hsp27 as a target protein of KRIBB3 in vitro. Treatment of MDA-MB-231 cells with phorbol 12-myristate 13-acetate induced protein kinase C-dependent phosphorylation of Hsp27 and tumor cell migration. In contrast, treatment of MDA-MB-231 cells with KRIBB3 blocked phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of KRIBB3 on tumor cell invasion, and knockdown of Hsp27 using small interfering RNA inhibited tumor cell migration. Overall, our results demonstrate that KRIBB3 inhibits tumor cell migration and invasion by blocking protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27. Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. A synthetic library was screened to identify inhibitors of tumor cell migration. From this, we discovered that CAC-1098 (aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC50 = 5 and 50 nm, respectively. We synthesized KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the bromide group of CBI-0997 with a methoxyl group. Like CBI-0997, KRIBB3 has anti-migratory and anti-invasive activities in MDA-MB-231 cells. Because KRIBB3 has a better drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding proteins. Using affinity chromatography, we identified Hsp27 as a target protein of KRIBB3 in vitro. Treatment of MDA-MB-231 cells with phorbol 12-myristate 13-acetate induced protein kinase C-dependent phosphorylation of Hsp27 and tumor cell migration. In contrast, treatment of MDA-MB-231 cells with KRIBB3 blocked phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of KRIBB3 on tumor cell invasion, and knockdown of Hsp27 using small interfering RNA inhibited tumor cell migration. Overall, our results demonstrate that KRIBB3 inhibits tumor cell migration and invasion by blocking protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27. Traditionally, genetic mutagenesis has proven to be a useful tool in solving the function of a wide range of genes in biological process. Recently, a chemical genetic approach has been developed to elucidate the principles of a wide range of biological processes (for review, see Refs. 1Schreiber S.L. Bioorg. Med. Chem. 1998; 6: 1127-1152Crossref PubMed Scopus (365) Google Scholar, 2Stockwell B.R. Nature. 2004; 432: 846-854Crossref PubMed Scopus (390) Google Scholar, 3Mayer T.U. Trends Cell Biol. 2003; 13: 270-277Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In chemical genetics, instead of site-specific mutation as in traditional genetics, the function of a protein is inhibited or activated using small chemicals. Therefore, chemical genetics seeks to identify novel small molecules that afford functional dissection of cell biological pathways. Such chemicals are useful as bioactive molecular probes and allow further analysis of the relationship between target processes or proteins within cells and their cellular function. Metastasis plays a major role in morbidity and mortality from breast cancer (4Mundy G.R. Nat. Rev. Cancer. 2002; 2: 584-593Crossref PubMed Scopus (2290) Google Scholar). The metastatic potential of cancer cells is related to the ability to digest the extracellular matrix, migrate, cross blood vessel walls, and reach the blood circulation (for review, see Refs. 5Pantel K. Brakenhoff R.H. Nat. Rev. Cancer. 2004; 4: 448-456Crossref PubMed Scopus (1063) Google Scholar, 6Friedl P. Wolf K. Nat. Rev. Cancer. 2003; 3: 362-374Crossref PubMed Scopus (2469) Google Scholar, 7Chambers A.F. Groom A.C. MacDonald I.C. Nat. Rev. Cancer. 2002; 2: 563-572Crossref PubMed Scopus (3069) Google Scholar, 8Hood J.D. Cheresh D.A. Nat. Rev. Cancer. 2002; 2: 91-100Crossref PubMed Scopus (1484) Google Scholar). Cell movement is a complex process involving a number of steps, including the disruption of cell-cell junctions, cytoskeletal rearrangements, and the constant remodeling of adhesive contacts with the extracellular matrix (for review, see Refs. 9Ridley A.J. Schwartz M.A. Burridge K. Firtel R.A. Ginsberg M.H. Borisy G. Parsons J.T. Horwitz A.R. Science. 2003; 302: 1704-1709Crossref PubMed Scopus (3831) Google Scholar, 10Franz C.M. Jones G.E. Ridley A.J. Dev. Cell. 2002; 2: 153-158Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 11Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3277) Google Scholar). Cell migration contributes to several other important pathological processes, including vascular disease, osteoporosis, chronic inflammatory disease such as rheumatoid arthritis and multiple sclerosis, cancer, and mental retardation. Anticancer drug development strategy has traditionally focused on inhibition of cancer cell growth. However, other processes in tumor progression can be potential targets for cancer treatment. Among these, migration is one of the interesting processes in tumor progression. Expression of different growth factor receptors in malignant cells has been reported and can participate not only in transducing growth signals, but also in contributing to the motile properties of malignant cancer cells. Multiple growth factors and their receptors are involved in the malignant progression (12van der Valk P. Lindeman J. Kamphorst W. Ann. Oncol. 1997; 8: 1023-1029Abstract Full Text PDF PubMed Scopus (50) Google Scholar). The number of cells expressing multiple growth factors and their receptors tends to increase with increasing malignancy grade. High metastatic cells exhibit higher spontaneous rates of migration compared with their low metastatic counterparts (13Clark E.A. Golub T.R. Lander E.S. Hynes R.O. Nature. 2000; 406: 532-535Crossref PubMed Scopus (1308) Google Scholar, 14Volk T. Geiger B. Raz A. Cancer Res. 1984; 44: 811-824PubMed Google Scholar). Hsp27 (heat shock protein of 27 kDa) belongs to a family of abundant and ubiquitous stress proteins that are detectable in virtually all organisms from prokaryotes to mammals (15Concannon C.G. Gorman A.M. Samali A. Apoptosis. 2003; 8: 61-70Crossref PubMed Scopus (454) Google Scholar). In unstressed cells, Hsp27 levels are generally low, and it exists predominantly as a large oligomeric unit of up to 800 kDa. The size of this oligomeric unit is dependent on a number of parameters, including temperature and degree of phosphorylation of Hsp27 (16Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar, 17Zantema A. Verlaan-De Vries M. Maasdam D. Bol S. van der Eb A. J. Biol. Chem. 1992; 267: 12936-12941Abstract Full Text PDF PubMed Google Scholar). Under stress conditions, an increase in the level of Hsp27 expression is preceded by phosphorylation-induced reorganization of the multimeric status of Hsp27. Phosphorylation occurs at four different residues, Ser-15, Ser-78, Ser-82, and Thr-143, and this induces redistribution of the large oligomers to small tetrameric units (16Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar, 17Zantema A. Verlaan-De Vries M. Maasdam D. Bol S. van der Eb A. J. Biol. Chem. 1992; 267: 12936-12941Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of Hsp27 is catalyzed by MAPKAPK-2 3The abbreviations used are: MAPKAPKmitogen-activated protein kinase-activated protein kinasePKCprotein kinase CFAKfocal adhesion kinaseFBSfetal bovine serumsiRNAsmall interfering RNAMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinasePI3Kphosphatidylinositol 3-kinasePMAphorbol 12-myristate 13-acetate. and MAPKAPK-3 (18Landry J. Lambert H. Zhou M. Lavoie J.N. Hickey E. Weber L.A. Anderson C.W. J. Biol. Chem. 1992; 267: 794-803Abstract Full Text PDF PubMed Google Scholar, 19Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (154) Google Scholar, 20Stokoe D. Engel K. Campbell D.G. Cohen P. Gaestel M. FEBS Lett. 1992; 313: 307-313Crossref PubMed Scopus (472) Google Scholar), protein kinase C (PKC) (21Maizels E.T. Peters C.A. Kline M. Cutler R.E. Shanmugam M. Hunzicker-Dunn M. Biochem. J. 1998; 332 (Jr.): 703-712Crossref PubMed Scopus (101) Google Scholar), cGMP-dependent protein kinase (22Butt E. Immler D. Meyer H.E. Kotlyarov A. Laass K. Gaestel M. J. Biol. Chem. 2001; 276: 7108-7113Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and protein kinase D (23Doppler H. Storz P. Li J. Comb M.J. Toker A. J. Biol. Chem. 2005; 280: 15013-15019Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The increased phosphorylation of Hsp27 is detectable several minutes after exposure to stress, and a subsequent increase in the expression level of the protein is detectable within several hours (24Landry J. Chretien P. Laszlo A. Lambert H. J. Cell. Physiol. 1991; 147: 93-101Crossref PubMed Scopus (125) Google Scholar). Hsp27 has been recognized as a potent regulator of cytoskeleton dynamics (actin microfilaments). The actin cytoskeleton is modulated by both the spatial arrangement as well as the polymerization dynamics of its different elements (25Liang P. MacRae T.H. J. Cell Sci. 1997; 110: 1431-1440Crossref PubMed Google Scholar). Overexpression of Hsp27 increases the stability of F-actin microfilaments during exposure to stress (26Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar, 27Huot J. Lambert H. Lavoie J.N. Guimond A. Houle F. Landry J. Eur. J. Biochem. 1995; 227: 416-427Crossref PubMed Scopus (173) Google Scholar, 28Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. J. Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar). The exact mechanism by which Hsp27 stabilizes F-actin is poorly characterized. Actin microfilaments are not the only components of the cytoskeleton that have been reported to interact with Hsp27. Hsp27 colocalizes with tubulin/microtubles (29Hino M. Kurogi K. Okubo M.A. Murata-Hori M. Hosoya H. Biochem. Biophys. Res. Commun. 2000; 271: 164-169Crossref PubMed Scopus (87) Google Scholar). Higher Hsp27 expression levels are commonly detected in a variety of different cancers, including breast (30Love S. King R.J. Br. J. Cancer. 1994; 69: 743-748Crossref PubMed Scopus (81) Google Scholar, 31Oesterreich S. Weng C.N. Qiu M. Hilsenbeck S.G. Osborne C.K. Fuqua S.A. Cancer Res. 1993; 53: 4443-4448PubMed Google Scholar), prostate (32Cornford P.A. Dodson A.R. Parsons K.F. Desmond A.D. Woolfenden A. Fordham M. Neoptolemos J.P. Ke Y. Foster C.S. Cancer Res. 2000; 60: 7099-7105PubMed Google Scholar), gastric (33Chen J. Kahne T. Rocken C. Gotze T. Yu J. Sung J.J. Chen M. Hu P. Malfertheiner P. Ebert M.P. J. Proteome Res. 2004; 3: 1009-1016Crossref PubMed Scopus (70) Google Scholar, 34Ehrenfried J.A. Herron B.E. Townsend C.M. Evers B.M. Surg. Oncol. 1995; 4 (Jr.): 197-203Crossref PubMed Scopus (49) Google Scholar), and ovarian (35Langdon S.P. Rabiasz G.J. Hirst G.L. King R.J. Hawkins R.A. Smyth J.F. Miller W.R. Clin. Cancer Res. 1995; 1: 1603-1609PubMed Google Scholar) cancers. Several studies point to the ability of Hsp27 to increase the metastatic potential of tumor cells in nude mice as well as to enhance their resistance to therapy (36Blackburn R.V. Galoforo S.S. Berns C.M. Armour E.P. McEachern D. Corry P.M. Lee Y.J. Int. J. Cancer. 1997; 72: 871-877Crossref PubMed Scopus (26) Google Scholar, 37Katoh M. Koninkx J. Schumacher U. Cancer Lett. 2000; 161: 113-120Crossref PubMed Scopus (19) Google Scholar). mitogen-activated protein kinase-activated protein kinase protein kinase C focal adhesion kinase fetal bovine serum small interfering RNA mitogen-activated protein kinase/extracellular signal-regulated kinase kinase phosphatidylinositol 3-kinase phorbol 12-myristate 13-acetate. We now report that we have identified anti-migratory compounds by chemical library screening. One of the inhibitors, KRIBB3, inhibited both tumor cell migration and invasion. In addition, we found that KRIBB3 bound to Hsp27 and inhibited cell migration by blocking PKC-dependent Hsp27 phosphorylation. Materials—Anti-phospho-Ser-78 Hsp27 monoclonal antibody was purchased from Upstate Biotechnology. Antibodies against Hsp27, AKT, phospho-AKT, phospho-PKCμ, and phosphorylated phospholipase Cβ3 were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against focal adhesion kinase (FAK) and p130cas and antibody PY-20 were purchased from Transduction Laboratories (Lexington, KY). Chemicals used in these experiments were purchased from Sigma and Calbiochem. CAC-1098 (aurintricarboxylic acid) was purchased from Sigma, and CBI-1098 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) and KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) were synthesized in our laboratory. Cell Culture—The cell lines used were obtained originally from American Type Culture Collection. The human breast cancer cell line MDA-MB-231 was maintained in RPMI 1640 medium (Invitrogen) supplement with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen) and 25 mm HEPES. Cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 in an incubator. Construction of an Hsp27-overexpressing MDA-MB-231 Cell Line— Full-length Hsp27 was obtained by PCR using oligonucleotides 5′-CGCGGATCCATGACCGAGCGCCGCGTCC-3′ (sense) and 5′-GGAATTCGTGGGCATCCGGGCTAAGG-3′ (antisense) with an expressed sequence tag clone containing Hsp27 (Korean UniGene Clone ID hMU001508, cDNA clone MGC:21487) as a template. The PCR product was digested with BamHI and EcoRI and then inserted into the pcDNA3.1 mammalian expression vector that had been digested with BamHI and EcoRI. To generate cells stably expressing Hsp27, MDA-MB-231 cells were transfected using Lipofectamine and 1 μg of pcDNA3.1-Hsp27. Clones were selected in growth medium containing 0.8 mg/ml G418 for 21 days. To avoid colony-specific variation, we used whole populations of selected colonies for further study. Knockdown of the Hsp27 Protein Using Hsp27 Small Interfering RNA (siRNA)—The Hsp27 and control siRNAs were purchased from Cell Signaling Technology. Cells plated at a density of 8 × 104 cells/well in 6-well plates were transfected with 100 nm Hsp27 and control siRNA oligoduplexes after preincubation for 20 min with Oligofectamine in serum-free Opti-MEM I (Invitrogen). 4 h after the beginning of the incubation, 0.5 ml of RPMI 1640 containing 30% FBS was added (without antibiotics). 48 h after transfection, cells were collected and used for migration assay or for preparation of whole cell lysates. Cell Proliferation Assays—Proliferation assays were performed as described previously (38Han D.C. Lee M-Y. Shin K.D. Jeon S.B. Kim J.M. Son K.-H. Kim H.C. Kim H.M. Kwon B.-M. J. Biol. Chem. 2004; 279: 6911-6920Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Briefly, 5000 cells were seeded onto 96-well plates in RPMI 1640 medium containing 10% FBS. After 20-24 h, cells were replenished with fresh complete medium containing either a test compound or 0.1% Me2SO. After incubation for 48 h, the cell proliferation reagent WST-1 (Roche Applied Science) was added to each well. The amount of WST-1 formazan produced was measured at 450 nm using an enzyme-linked immunosorbent assay reader (Bio-Rad). Cell Migration Assays—Migration was measured using a 48-well Boyden chamber (Neuro Probe, Inc., Gaithersburg, MD). Various concentrations of chemicals in RPMI 1640 medium with 10% FBS were placed into the base wells separated from the top wells by polycarbonate filters (8-μm pore size, 25 × 80 mm, polyvinylpyrrolidone-free; Neuro Probe, Inc.). Cells were harvested by trypsinization, washed once with serum-free RPMI 1640 medium containing 0.5 mg/ml soybean trypsin inhibitor (Sigma), and washed twice with serum-free RPMI 1640 medium. Cells were resuspended in serum-free RPMI 1640 medium and added to the upper chamber at 8 × 103 cells/well. Cells were incubated for 16 h at 37 °C in a humidified atmosphere of 5% CO2. At the end of the experiment, cells were fixed with methanol for 10 min and stained with modified Giemsa stain (Sigma) for 1 h. Cells on the upper side of the membrane were then removed using a cotton swab. The migrated cells were counted under a light microscope at ×100 magnifications. The following kinase inhibitors were used at the concentrations indicated: rottlerin (PKCδ inhibitor; 5 μm), PD98059 (MEK inhibitor; 40 μm), bisindolylmaleimide I (broad PKC inhibitor; 5 μm), Y27632 (ROCK inhibitor; 10 μm), wortmannin (phosphatidylinositol 3-kinase (PI3K) inhibitor; 10 μm), SB203580 (p38 mitogen-activated protein kinase inhibitor; 10 μm), AG1478 (epidermal growth factor receptor inhibitor; 10 μm), and PP2 (Src inhibitor; 50 μm). Cell Invasion Assay—MDA-MB-231 cells were starved overnight in serum-free RPMI 1640 medium. Thawed Matrigel (BD Biosciences) was diluted 1:20 with 1× RPMI 1640 medium, and 100 μl was used to coat each invasion chamber (Transwell, BD Biosciences) equipped with an 8-μm pore size Micropore filter. After the chambers were incubated at 37 °C for 1 h, unbound Matrigel was aspirated and rinsed gently using serum-free RPMI 1640 medium. The Matrigel barrier was reconstituted with 100 μlof serum-free medium for 2 h at 37 °C. In the meantime, MDA-MB-231 cells were harvested after trypsinization, washed once with serum-free RPMI 1640 medium containing 0.5 mg/ml soybean trypsin inhibitor, and washed twice with serum-free RPMI 1640 medium. Cells were diluted to 1 × 105 cells/ml, and 200 μl was inoculated into the upper chamber. 10% FBS as chemoattractant was added to 24-well culture dishes. After incubation at 37 °C for 24 h, the Matrigel on the filter was removed with a cotton swab. The filter was then stained with crystal violet (300 μl of 5 mg/ml crystal violet dissolved in 20% methanol) and incubated for 30 min. The membrane was washed several times with 1× phosphate-buffered saline, and the cells that had penetrated the filter were counted under a microscope. Western Blotting and Immunoprecipitation—Lysates were prepared using radioimmune precipitation assay buffer as described previously (38Han D.C. Lee M-Y. Shin K.D. Jeon S.B. Kim J.M. Son K.-H. Kim H.C. Kim H.M. Kwon B.-M. J. Biol. Chem. 2004; 279: 6911-6920Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). 40 μg of protein was resolved by 7.5 or 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Roche Applied Science). The membrane was blocked with 1% Western blocking reagent (Roche Applied Science) in Tris-buffered saline/Tween (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, and 0.05% Tween 20). The primary antibodies were used as recommended by the manufacturers. The secondary antibodies used were horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.). The membrane was incubated with primary antibody for 2 h at room temperature, washed three times with Tris-buffered saline/Tween, and visualized with chemiluminescent β-peroxidase reagents (Roche Applied Science). For immunoprecipitation, 400 μg of lysates was incubated with primary antibody for 2 h at 4°Cin a rotary shaker, and 40 μl of protein G-agarose beads was then added. After 1 h, bead-containing lysates were centrifuged and washed three times with lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 5 mm EDTA, 30 mm Na2HPC4, 50 mm NaF, 0.5 mm NaVO4, 2 mm PMSF, and 1% aprotinin). Bead-bound proteins were resolved by SDS-PAGE and immunoblotted using specific antibody. Detection of KRIBB3-binding Proteins—MDA-MB-231 cells were washed with phosphate-buffered saline and then homogenized using a 27-gauge syringe in binding buffer (10 mm Tris-HCl (pH 7.4), 50 mm KCl, 5 mm MgCl2, 1 mm EDTA, and 0.1 mm Na3VO4). The cell lysate was centrifuged at 13,000 rpm for 30 min at 4 °C, and the supernatant was collected. After the supernatant of MDA-MB-231 cells had been precleared by incubation with immobilized streptavidin (Sigma) for 60 min at 4 °C, followed by centrifugation at 500 × g for 5 min, the cleared supernatants were incubated with biotinyl-KRIBB3 compound. After incubation for 2 h at 4°C, proteins associated with the biotinyl-KRIBB3 compound were precipitated with UltraLink-immobilized NeutrAvidin-agarose (Pierce). Precipitated samples were applied to the column. Samples were washed with 10 bed volumes of wash buffer containing 50 mm HEPES (pH 7.5), 30 mm NaCl, 1 mm EDTA, 2.5 mm EGTA, 0.1% Tween 20, 10% (v/v) glycerol, 1 mm NaF, 0.1 mm Na3VO4, and protease inhibitor mixture (1 tablet/10 ml; Roche Applied Science). Samples were eluted from the column with 5 bed volumes of elution buffer (0.1 m glycine HCl (pH 2.8)). Samples were boiled in SDS-PAGE sample buffer, separated on a 10% polyacrylamide gel, and visualized by Coomassie Brilliant Blue staining. Peptide Analysis of the KRIBB3-binding Protein—In-gel specific KRIBB3-binding protein was cut out and sent to the Korea Basic Science Institute (Daejeon, Korea) for peptide analysis. The sample was digested with trypsin. Peptide tandem mass spectrometry analysis of the digested peptides was performed using an electrospray ionization quadrupole time-of-flight mass spectrometer as described previously (39Nam M.H. Heo E.J. Kim J.Y. Kim S.I. Kwon K.H. Seo J.B. Kwon O. Yoo J.S. Park Y.M. Proteomics. 2003; 3: 2351-2367Crossref PubMed Scopus (50) Google Scholar). Screening of Anti-migratory Chemicals—We searched for anti-migratory compounds using cell-based screening. A whole cell-based assay was preferred because of its ability to simultaneously assess multiple targets. In addition, this approach can avoid problems with drug permeability by identifying active compounds that freely enter the cell. The effect of chemicals on the cell migration of MDA-MB-231 cells was measured using a modified Boyden chamber assay (Fig. 1A). Cell migration was performed in the presence of 10 μm chemical, and 10% FBS was used as a chemoattractant. The components provided by serum are certain highly specific proteins called growth factors. Serum-containing growth factors are insulin-like growth factor-1, epidermal growth factor, and platelet-derived growth factor. Upon screening ∼12,000 synthetic chemicals for “cell stoppers” that inhibit migration of the human breast cancer cell line MDA-MB-231, we identified CAC-1098 and CBI-0997 (Fig. 1B). CAC-1098 and CBI-0997 inhibited migration of MDA-MB-231 cells with IC50 = 5 and 50 nm, respectively (Fig. 1C). To clarify whether the inhibitory action on motile functions could be due to a cytotoxic effect, the cell stoppers were tested for their effects on MDA-MB-231 cell proliferation. The cell stoppers CAC-1098 and CBI-0997 inhibited proliferation of MDA-MB-231 cells with GI50 > 100 and 25 μm, respectively (Fig. 1D), where GI50 is the concentration of an inhibition at which 50% inhibition of the cell growth is seen. These results indicate that the cell stoppers significantly inhibit cell migration at 0.1-1 μm without cytotoxic problems. We synthesized several derivatives of CBI-0997 and analyzed their anti-migratory activities. 4K. D. Shin, M.-Y. Lee, D.-S. Shin, S. Lee, K.-H. Son, S. Koh, Y.-K. Paik, B.-M. Kwon, and D. C. Han, unpublished data. In this study, we found that replacement of the bromide group with a methoxyl group did not change its anti-migratory activity, and we decided to study the methoxyl derivative further. We named the methoxyl derivative (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) KRIBB3 (Fig. 1B). KRIBB2 (4-ethyl-5-methoxy-2-(3-methyl-4-phenylisoxazole-5-yl)phenol) is an inactive analog of KRIBB3 and was used as a negative control. CAC-1098, another identified cell stopper, is a polymeric carboxylated triphenylmethane derivative (Fig. 1B). Aurintricarboxylic acid has been reported to be an angiogenesis inhibitor (40Gagliardi A. Collins D.C. Cancer Res. 1993; 53: 533-535PubMed Google Scholar) and to have anti-proliferative activity in vascular smooth muscle cells (41Benezra M. Ben-Sasson S.A. Regan J. Chang M. Bar-Shavit R. Vlodavsky I. Arterioscler. Thromb. 1994; 14: 1992-1999Crossref PubMed Scopus (43) Google Scholar). However, when MDA-MB-231 cells were treated with 0.01 μm CAC-1098, >80% of cell migration was inhibited. In addition, we could not detect any anti-proliferative activity of CAC-1098 up to 10 μm (Fig. 1, C and D). This result implies the possibility that CAC-1098 may inhibit angiogenesis by inhibiting migration of vascular endothelial cells. It will be interesting to test this possibility. Successful identification of previously known anti-angiogenesis compounds validated our approach to identify novel compounds that may inhibit cell migration. Inhibition of Tumor Invasion by Cell Stoppers—The invasive behavior of cancer cells is accompanied by increased cell movement. This suggests that cell stoppers can block tumor invasion. The effect of cell stoppers on MDA-MB-231 cell invasion through Matrigel-coated porous filters in response to a chemotactic stimulus was examined in an 8-μm pore size invasion chamber assay. Cells were treated with cell stoppers at different concentrations (0-100 μm) for 24 h, and 10% FBS was used as a chemoattractant. CAC-1098 and KRIBB3 inhibited invasion of MDA-MB-231 cells in a dose-dependent manner, with half-maximal inhibition at 2.8 and 0.15 μm, respectively (Fig. 1, E and F). In particular, invasion was almost completely blocked by 1 μm KRIBB3 (by ∼90%). Analysis of Phosphorylation of FAK, p130cas, and AKT—When cells were treated with KRIBB3, they became rounded within 5 min (Fig. 2A). Cells treated with vehicle (Me2SO) did not induce any detectable morphological changes (data not shown). Cell spreading is regulated by integrin receptors and ligands (extracellular matrix). Activation of integrins induces phosphorylation of many focal adhesion proteins, including FAK (42Guan J.L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (724) Google Scholar) and p130cas (43Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Cells lacking the tyrosine kinase FAK or Src have more and larger adhesions and migrate poorly (44Webb D.J. Parsons J.T. Horwitz A.F. Nat. Cell Biol. 2002; 4 (-E100): E97Crossref PubMed Scopus (604) Google Scholar, 45Alahari S.K. Reddig P.J. Juliano R.L. Int. Rev. Cytol. 2002; 220: 145-184Crossref PubMed Scopus (64) Google Scholar). The interaction of FAK with Src and the adaptor proteins p130cas and Crk appears to regulate adhesion turnover. Therefore, we analyzed tyrosine phosphorylation of whole cell lysates or specific focal adhesion proteins after immunoprecipitation. In general, tyrosine phosphorylation of whole cell lysates increased upon KRIBB3 treatment, reached a maximum after 3 h, and then gradually decreased (Fig. 2B). However, the profile of tyrosine phosphorylation of whole cell lysates was not altered by treatment with Me2SO. To test whether FAK is tyrosine-phosphorylated by KRIBB3, FAK was immunoprecipitated with anti-FAK antibody and blotted with phosphotyrosine-specific antibody. As shown in Fig. 2C, we could not detect any decrease in FAK phosphorylation. In contrast, we could see a significant increase in FAK phosphorylation 3 h after KRIBB3 treatment. Similarly, p130cas was immunoprecipitated with anti-p130cas antibody and analyzed with phosphotyrosine-specific antibody. However, we could not detect any decrease in p130cas phosphorylation (Fig. 2D). These results suggest that FAK and p130cas are not involved in KRIBB3-induced anti-migratory activity. Directed cell migration toward a soluble growth factor is a general phenotype of motile cells and requires highly polarized intracellular signaling events promoting the recruitment of pleckstrin homology domain-containing proteins to the leading edge (46Parent C.A. Devreotes P.N. Science. 1999; 284: 765-770Crossref PubMed Scopus (729) Google Scholar, 47Firtel R.A. Chung C.Y. BioEssays. 2000; 22: 603-615Crossref PubMed Scopus (156) Google Scholar). This localized increase in phosphatidylinositol 3-phosphates is the result of spatial activation of PI3K (48Funamoto S. Meili R. Lee S. Parry L. Firtel R.A. Cell. 2002; 109:" @default.
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