BB, a new EGFR inhibitor, exhibits prominent anti-angiogenesis and

[Cancer Biology & Therapy 8:17, 1640-1647; 1 September 2009]; ©2009 Landes Bioscience

Research Paper

BB, a new EGFR inhibitor, exhibits prominent anti-angiogenesis and antitumor activities Qi-Ming Sun,1 Ze-Hong Miao,1 Li-Ping Lin,1 Min Gui,1 Cai-Hua Zhu,1 Hua Xie,1 Wen-Hu Duan2 and Jian Ding1,* 1Division of Anti tumor Pharmacology; State Key Laboratory of Drug Research; 2Department of Medicinal Chemistry; Shanghai Institute of Materia Medica; Chinese Academy of Sciences; Shanghai, China

Abbreviations: BB, N-(3-bromophenyl)-7-methoxy-6-(3-(3-methoxypyrrolidin-1-yl) propoxy) quinazolin-4-amine; EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; NSCLC, non-small cell lung cancer; FBS, fetal bovine serum; ELISA, enzymelinked-immunosorbent assay; RTV, relative tumor volume; IC50, the concentration for 50% inhibition Key words: tyrosine kinase, EGFR, BB, antitumor, anti-angiogenesis

Aberrant activation of the epidermal growth factor receptor (EGFR) is closely associated with malignant progression of tumors. EGFR inhibitors have been used successfully in clinic in the treatment of solid tumors. In the present study, we revealed that BB, a new synthetic quinonazoline derivative, was a potent EGFR inhibitor. BB selectively inhibited EGFR with a IC50 value of 50 ± 37 nM, at least 32-fold more potent than suppressed all other ten tested receptor tyrosine kinases including the same family member ErbB2 (IC50 = 5.6 ± 3.2 μM). BB effectively abrogated autophosphorylation of the EGF-stimulated EGFR and phosphorylation of its key downstream signaling molecules ERK and AKT in A549 cells. BB was shown to suppress EGF-stimulated proliferation of A549 cells with an apparently lower IC50 value (0.33 ± 0.07 μM) than that (2.7 ± 0.4 μM) for the serum-stimulated cells. BB also inhibited the EGF-independent proliferation of a panel of tumor cells. In addition, BB exhibited anti-angiogenesis activity, as evidenced by antagonizing EGF-induced HMEC-1 migration in vitro, blocking HMEC-1 tube formation, and inhibiting microvessel sprouting from rat aortic rings. Most importantly, BB prominently inhibited in vivo tumorigenesis of NIH3T3 cells specifically driven by the activation-mutated EGFR genes. As reported, normal NIH3T3 cells lack tumorigenicity in nude mice. NIH3T3 cells transfected with the EGFR gene with activating mutation (A750P or L858R) produced rapidly growing xenografts in nude mice. BB, when given orally at 100 mg/ kg consecutively for 2 w, prominently inhibited the growth of the xenografts and reduced the number of microvessels. Taken *Correspondence to: Jian Ding; Division of Anti-tumor Pharmacology; State Key Laboratory of Drug Research; Shanghai Institute of Materia Medica; Chinese Academy of Sciences; Shanghai 201203 China; Tel.: +86.21.5080.6600 ext. 1305; Fax: +86.21.5080.6722; Email: [email protected] Submitted: 05/24/09; Accepted: 06/06/09 Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/article/9205

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together, the data indicate that BB is a new selective EGFR inhibitor with potent antitumor activity, revealing its potential as a promising anticancer candidate.

Introduction The human epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases, which consists of four members (ErbB1-4). All ErbB family members share a common structure organization that is composed of an extracellular ligandbinding domain, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. Except for ErbB2, all other three ErbB receptors have their own ligands. The binding of their respective ligands to the corresponding ErbB extracellular domains induces homodimerization or heterodimerization of the receptors and subsequent phosphorylation at the multiple tyrosine residues located in the intracellular region. These phosphorylated tyrosine residues serve as the docking sites for recruiting diverse effector proteins, which subsequently activate multiple signal transduction pathways, including the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI3K)/ AKT pathway.1-3 Aberrant EGFR activation, most frequently independent of its ligand stimulation, promotes multiple biological processes including survival, proliferation, invasion, metastasis, angiogenesis and decreased apoptosis, which play central roles in the progression of tumors derived from epidermal tissues. The most common mechanisms for the aberrant EGFR activation are overexpression resulting from EGFR gene amplification and activating mutations of the receptor.2 EGFR overexpression has been reported extensively in squamous-cell carcinomas of head and neck (SCCHN), non-small cell lung cancer (NSCLC), and ovarian and other tumor types. In contrast, the activating EGFR mutations are most frequently found in NSCLC though also found in SCLC, cholangiocarcinoma, ovarian, colorectal, head and neck, oesophageal and pancreatic cancer.4-9 Especially notably, such aberrant EGFR activation is closely correlated to the malignant progression and prognosis.

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Therefore, inhibitors targeting the EGFR receptor have been intensively investigated as molecularly-targeted antitumor agents. Several EGFR inhibitors such as gefitinib (Iressa) and Erlotinib, have been used to treat cancers especially NSCLCs in clinic. However, their limited therapeutic spectrum against cancer and inevitable acquired drug resistance require continuous efforts in developing new EGFR inhibitors. In the current study, we identified and characterized BB as a new orally available EGFR inhibitor. BB potently inhibited EGFR phosphorylation and downstream signal transduction, resulting in significant inhibition on tumor growth and angiogenesis.

Results BB selectively inhibits the tyrosine kinase activity of EGFR. BB was a new synthesized compound targeting EGFR. To determine the selectivity of BB against EGFR, we used ELISA assays to measure the effects of BB on the enzymatic activity of a panel of 11 tyrosine kinases consisting of EGFR, EphB2, SRC, KIT, ErbB2, KDR, PDGFRβ, FLT-1, FGFR1, FGFR2 and EphA2. BB exhibited potent inhibition against the kinase activity of EGFR (Table 1); the suppression of BB against EGFR was 32 times more potent than that against EphB2 and above 100 times stronger than that against all other tested tyrosine kinases (Table 1). The result indicates an apparent selectivity of BB against EGFR. BB suppresses the biochemical pathways in which cellular EGFR operates. MAPK and PI3K/AKT pathways are critical for tumor proliferation, survival and response to exterior stimuli.16 It is well established that EGFR, activated via its self-phosphorylation, regulates both these pathways by phosphorylating their components such as ERK in the MAPK pathway and AKT in the PI3K/AKT pathway.17 To further prove BB targeting EGFR, we chose the non-small cell lung cancer (NSCLC) cell line A549 as a cellular model based on the fact that the famous EGFR inhibitor Iressa has been approved for the treatment of patients with advanced NSCLC.18 Exposure of A549 cells to the growth factor EGF rapidly increased the level of cellular EGFR autophosphorylation and subsequently enhanced the phosphorylation levels of its downstream targets ERK and AKT, which is reflective of the activation of the biochemical pathways operated by EGFR. BB as well as the reference drug Iressa prominently reduced, or even almost completely prevented the phosphorylation of EGFR, ERK and AKT stimulated by EGF (Fig. 2A). At the same time, the total protein levels of EGFR, ERK and AKT kept unchanged in A549 cells exposed either to EGF or to the tested compounds (Fig. 2A). The data indicate that BB inhibits the biochemical pathways in which the cellular EGFR operates. BB inhibits in vitro proliferation of tumor cells in both EGF-dependent and -independent manners. As revealed in Figure 2A, the EGFR-related pathways were maintained under a status of low activation in A549 cells; the addition of EGF activated those pathways and could make the cells addict to EGF. To detect the consequence of the inhibition of the EGFR-related pathways by BB, we used SRB assays to examine the effects of BB on the proliferation of A549 cells in the absence and presence of EGF, respectively. The result showed that A549 cells in the absence of EGF were pretty resistant to BB, with an IC50 of 15.3 μM; in www.landesbioscience.com

Table 1 Differential inhibitory activities of BB against a panel of tyrosine kinases Kinases EGFR

IC50 ± SD (µM)* 0.05 ± 0.04

ErbB2

5.64 ± 3.20

KIT

5.45 ± 1.54

EphB2

1.61 ± 0.13

SRC

5.29 ± 3.48

KDR

9.95 ± 1.32

PDGFR

>10

FLT-1

>10

FGFR1

>10

FGFR2

>10

EPHa2

>10

*The ability of BB to inhibit the enzymatic activities of a panel of recombinant tyrosine kinases was examined by ELISA assays. IC50 values were expressed as mean ± SD of at least three independent determinations. IC50 values quoted as “greater than” denoted the inability to reach an IC50 value with the highest concentration tested (10 µM).

contrast, EGF significantly sensitized the cells to BB, with a 66.5 times lower IC50 (0.23 μM) (Fig. 2B). The data indicate that BB, on one hand, exerts the EGF-dependent proliferative inhibition, and may have some relatively weak EGF-independent activities on the other. To further confirm such EGF-independency, we examined the proliferative inhibition of BB in additional 13 tumor cell lines that possess distinct levels of EGFR protein19,20 (Fig. 2D). The result showed that under routine culture conditions, the proliferative inhibition of BB against those tumor cell lines was, though relatively weak, quite certain (Fig. 2C), strengthening the conclusion from A549 cells that BB also possesses EGF-independent activities to certain degree (Fig. 2B). BB represses neoangiogenesis in in vitro and ex vivo models. The EGFR signaling has been demonstrated to drive neoangiogenesis, including endothelial cell migration and microvessels restructuring.21 To further examine the effects of BB on the EGFR signaling, we evaluated whether BB affected neoangiogenesis using several in vitro and ex vivo models. Boyden chamber assays showed that BB reduced the migration of endothelial HMEC-1 cells driven by EGF (20 ng/mL, for 6 h) in a concentrationdependent manner (Fig. 3A). Consistently, the HMEC-1 tube formation assay revealed that BB disrupted the ability of the endothelial cells to form capillary-like tubule structures (Fig. 3B). The aorta sprout outgrowth assay provides an ex vivo approach to comprehensively investigating the process of new blood vessels formation and anti-angiogenic effect.22 This assay showed that BB notably suppressed the sprout outgrowth from the rat aorta rings (Fig. 3C). These data collectively indicate the anti-angiogenic activity of BB. BB inhibits the proliferation and tumorigenesis of the NIH3T3 cells transfected with EGFR carrying activating mutations and reduces in vivo angiogenesis. Activating mutations of EGFR persistently activate the receptor independently of its

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Figure 1. Chemical structure of BB.

ligand EGF, which has been demonstrated to be attributable to tumorigenesis and to be highly correlated with tumor progression and prognosis.23 L858R and A750P, occurring in exons 21 and 19, respectively, are two most common EGFR activating mutations, characteristic of their good drug sensitivity to EGFRtargeted agents.24,25 To test whether BB combats those activating mutations, we transfected the EGFR expression plasmids with the mutant EGFR gene of L858R or A750P into NIH3T3 cells. Both two mutated EGFRs, designated as L858R-EGFR/NIH3T3 and A750P-EGFR/NIH3T3 cells, respectively, were stably expressed very well and the mutations resulted in constitutive receptor activation (EGFR phosphorylation) in the absence of exogenous EGF (Fig. 4A). As expected, BB inhibited phosphorylation of mutant EGFR and its downstream molecule ERK in L858R-EGFR/ NIH3T3 and A750P-EGFR/NIH3T3 cells, and both cell lines were consistently sensitive to BB as well as the reference Iressa (Fig. 4B and C). To further detect whether BB reduces the in vivo proliferation and growth of the NIH3T3 cells transfected with EGFR of activating mutation, we seeded the cells into nude mice. Although the parent NIH3T3 cells did not form detectable tumors as reported,15 both two transfected cells produced tumors that grew very rapidly (Fig. 4D and E). Both BB and the reference Iressa, administered orally once daily at a dose of 100 mg/kg, dramatically suppressed the proliferation and growth of the tumors formed by L858REGFR/NIH3T3 and A750P-EGFR/NIH3T3 cells, respectively (Fig. 4D and E). To examine whether BB reduces in vivo angiogenesis, we conducted immunohistochemical staining of the above tumor tissues with the antibody against CD31, a specific endothelial marker reflective of angiogenesis. The result showed that the number of endothelial cells in the tumors treated with BB or Iressa was remarkably decreased (Fig. 5), indicating that BB also inhibits in vivo angiogenesis.

Discussion EGFR has been an established molecular target, the inhibitors of which are well-tolerated and effective in cancer therapy, especially against subgroups of NSCLC tumors with aberrant EGFR activation. In current report, we presented a new compound BB that was found to elicit prominent EGFR-targeted inhibition. BB revealed a good selectivity against EGFR; even between EGFR and the same family member ErbB2, there was an enormous difference of more than 100 fold potency at the molecular level. The EGFR-targeted inhibition of BB was conceptually validated by using the NSCLC 1642

A549 cell line because BB disrupted the signaling transduction via the EGFR-operated biochemical pathways, as evidenced by the fact that BB dramatically reduced the EGF-driven phosphorylation of both AKT and ERK. The potent inhibition of BB on EGFR was further reflected in its apparent antiproliferation activity in A549 cells and its notable anti-angiogenesis shown by its inhibiting HMEC-1 migration and tube formation and the sprout outgrowth from rat aorta rings. To continue the proof of concept, we transfected the EGFR plasmids with sensitive activating mutations (L858R or A750P) into NIH3T3 cells. BB was found, either in vitro or in vivo, to inhibit the proliferation and growth of the cells carrying those mutations. BB also suppressed the angiogenesis of the tumors developed by those cells in nude mice. Taken together, all the data indicate that BB is a new potent EGFR inhibitor. A noteworthy result in our study is that BB could inhibit EGF-independent, persistent EGFR activation, manifested by its in vitro anti-proliferation against the tested tumor cell lines with distinct EGFR expression. Moreover, the effects of BB on other tyrosine kinases such as EphB2, SRC, KIT, ErbB2 and KDR, though relatively weak, are likely to collectively contribute to its anti-proliferation as well as anti-angiogenesis. This implies a potential relatively wide spectrum against tumors. In conclusion, the current study demonstrates that BB is a potent EGFR inhibitor with good selectivity, prominent antiproliferation and anti-angiogenic property. Its new structure, availability of oral administration and good tolerance in animals further show its promising druggability.

Materials and Methods Agents. BB (N-(3-bromophenyl)-7-methoxy-6-(3-(3-metho xypyrrolidin-1-yl)propoxy)-quinazolin-4-amine, Fig. 1) was designed by introducing the common pharmacore pyrrolidine into the core structure of quinazoline to obtain optimal activity and favored pharmacokinetics profile. Iressa were provided by the Department of Medicinal Chemistry, Shanghai Institute of Materia medica, Chinese Academy of Sciences. The chemical identity of BB was supported by nuclear magnetic resonance and mass spectroscopy. The purity of BB determined by HPLC was 99%. A stock solution of 10 mM BB was prepared in 100% dimethyl sulfoxide, kept in aliquot at -20°C and thawed immediately prior to each experiment. Cell lines and culture. Human hepatocellular carcinoma BEL-7402 and SMMC-7721 cell lines were obtained from the cell bank of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Human promyelocytic leukemia HL-60, chronic myelogenous leukemia K562, lung adenocarcinoma A549, colorectal carcinoma HT-29, skin malignant melanoma A-375, breast carcinoma BT-474, and cervical carcinoma HeLa cell lines were purchased from the American Type Culture Collection (Manassas, VA). Human colorectal carcinoma HCT-116, breast carcinoma MCF-7, renal carcinoma 786-O, and ovarian carcinoma SK-OV-3 cell lines were from the Japanese Foundation of Cancer Research (Tokyo, Japan). Most of cell lines were maintained in RPMI-1640 medium (GIBCO, Grand Island, NE). In addition, HCT-116, HeLa, A-375 and A549 cells were maintained in 5A,

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Figure 2. BB inhibits EGFR-downstream signaling pathways and tumor cell proliferation. (A) BB suppressed EGFR-downstream signaling pathways in A549 cells. Starved A549 cells were treated as described in Material and Methods. Lysates were initially probed using antibodies against phosphoEGFR, phospho-ERK, phospho-AKT and subsequently reprobed using antibodies against the respective total protein to visualize the protein levels. (B) BB combated EGF-stimulated cell proliferation. Proliferation assays based on sulforhodamin B staining were performed in 96-well plates using 10% FBS or 20 ng/mL EGF (in the presence of 1% FBS) to stimulate the cells in the absence or presence of BB. The data given from three independent experiments were expressed as mean ± SD. (C) Proliferation inhibition of BB against a panel of human tumor cell lines. Cells were treated with the tested compounds for 72 h. Cell viability was determined with 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide or sulforhodamin B assays. IC50s were expressed as mean ± SD from three independent experiments. (D) Expression levels of EGFR in different cell lines. EGFR expression was determined by western blotting.

MEM, DMEM and F12 medium (GIBCO, Grand Island, NE), respectively. All other cell lines were maintained in RPMI-1640 medium (GIBCO, Grand Island, NE). All the above medium was supplemented with 10% heat-inactivated fetal bovine serum www.landesbioscience.com

(GIBCO, Grand Island, NE), L-glutamine (2 mM), penicillin (100 IU/mL), streptomycin (100 μg/mL) and HEPES (10 mM) (For MCF-7 cells, the medium was additionally supplemented with 1 mM sodium pyruvate and with 0.01 mg/mL bovine insulin). All

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to the institutional ethical guidelines on animal care. Recombinant protein production. The catalytic moieties of EGFR, ErbB2, VEGFR2, KIT, SRC, FGFR1, FGFR2, EphA2, EphB2 and PDGFR were expressed as His-tagged proteins following infection of high five (T. ni.) cells with engineered baculoviruses as described elsewhere previously.10 The purified FLT-1 was purchased from Sigma (St. Louis, MO). Biochemical protein kinase inhibiton assays. ELISA assays were used to determine the selectivity profile of BB to inhibit kinase activity of protein kinases. In briefly, the kinase assays were performed in 96-well plates that were coated overnight with 2.5 μg of a poly(Glu, Tyr)4:1 (Sigma, Saint Louis) in 0.125 mL of PBS per well. The purified kinases were diluted in the kinase assay buffer (100 mM Hepes pH 7.5, 40 mM Figure 3. BB inhibits angiogenesis in vitro. (A) BB reduced HMEC-1 migration. HMEC-1 cells were seeded and MgCl2, 0.2 mM MnCl2, 0.4 mM sodium orthovanadate and treated as described in the section of Material and Methods. (left) Representative images from three independent experiments with similar results (magnification 100x). (right) The inhibition rate of HMEC-1 migration by BB. The 2 mM DL-dithiothreitol) and data were expressed as mean ± SD, n = 3. (B) BB disrupted tube formation of HMEC-1 cells. Left, representative added to all test wells at 1 images from three independent experiments with similar results (magnification 100x). (right) The inhibition rate ng protein per 0.1 mL volume of tube formation. The values were expressed as mean ± SD, n = 3. (C) The inhibitory effect of BB on microvesbuffer. Gradient concentrations sel sprouts arising from rat aortic rings. Images given were representative of two independent experiments with of the tested compounds were similar results (magnification 40x). added to tested wells (10 μL/ well). The kinase reaction was the cells were cultured in a humidified atmosphere of 95% air plus initiated by the addition of 5 μM ATP and the plates were incubated for 60 min at 37°C. After reaction, 100 μL/well of 5% CO2 at 37°C. Human microvascular endothelium HMEC-1 cells were a kind the anti-phosphotyrosine antibody (PY99) diluted in T-PBS gift from Prof. He Lu (Shanghai Institute of Materia Medica, containing 5 mg/mL bovine serum albumin was added and incuChinese Academy of Sciences) and propagated in MCDB131 bated at 37°C for 30 min. Then the plate was incubated with 100 medium (GIBCO, Grand Island, NE) with 10% heat-inactivated μL/well secondary antibody diluted in T-PBS containing 5 mg/ fetal bovine serum (GIBCO, Grand Island, NE), 10 ng/ml EGF mL bovine serum albumin at 37°C for 30 min. Finally, 100 μl (Sigma, Saint Louis, MO) and 1 μg/mL hydrocortisone (Sigma, of the color development solution (0.03% H2O2 and 2 mg/mL o-phenylenediamine in 0.1 M citrate buffer, pH 5.5) was added Saint Louis, MO). Mouse embryonic fibroblast NIH3T3 cells were obtained and the plate was incubated at room temperature until color from the American Type Culture Collection (Manassas, VA) emerged. The reaction was terminated by the addition of 50 μL of and cultured in DMEM medium supplemented with 10% heat- 2 M H2SO4, and A492 absorbency was measured using a multiwell spectrophotometer (Sunnyvale, CA). The ­inhibition rate (%) inactivated calf serum. Animals. BALB/cA nu/nu female mice aged 4–5 w were bred in was calculated as [1 - (A492/A492control)] x 100%. The IC50 was the Shanghai Institute of Materia Medica. The animals were housed fitted with the 4-parameter curve. The data were expressed as mean in sterile cages under laminar airflow hoods in a specific pathogen-free ± SD from three independent experiments. Western blotting analyses. A549 cells were seeded in six-well room with a 12 h light and 12 h dark schedule, and fed autoclaved chow and water ad libitum. All experiments were performed according plates and incubated to 80% confluence, then incubated for 2 h 1644

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Figure 4. BB inhibits in vitro proliferation and in vivo tumorigenesis of NIH3T3 cells carrying activating EGFR mutations. (A) Ligand-independent activation of the Mutant EGFR. NIH3T3 cells expressing mutant EGFR were lysed and immunoblotted with the antibody to total EGFR or the antibody specific to phospho-EGFR. Both EGFR mutants exhibited constitutive phosphorylation. (B) BB inhibited the phosphorylation of mutant EGFR and its downstream signal molecule ERK in transfected NIH3T3 cells. Cells expressing A750P-EGFR or L858R-EGFR were treated for 2 h at indicated concentrations of BB and then subjected to immunoblotting for phopho-EGFR, EGFR, phopho-ERK and ERK. (C) BB inhibited the in vitro proliferation of NIH3T3 cells carrying activating EGFR mutations. Cells seeded in 96-well plates were treated with various concentrations of drugs for 72 h. Cell viability was determined by sulforhodamin B assays. IC50s were shown as mean ± SD of three independent experiments. (D and E) BB suppressed the in vivo tumorigenesis of NIH3T3 cells carrying activating EGFR mutations. Tumors were initiated by subcutaneous injection of 1 x 106 L858R-EGFR/NIH3T3 or A750P-EGFR/ NIH3T3 cells to nude mice. The animals were randomly divided into groups when tumor volume reached 100 to 200 mm3 and received BB or Iressa orally at a dose of 100 mg/kg/day. Data points represented the means for six mice, with SD bars shown in two directions. **p < 0.01, versus the normal saline treatment (control).

in serum-free medium in the presence of different concentrations of BB or solvent control, and subsequently stimulated by the addition of EGF (20 ng/mL) for 10 min. After stimulation, cells were washed twice with ice-cold PBS and lysed in the lysis buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 137 mM NaCl, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol, and 1% Triton X-100). Lysates were clarified by centrifugation at 15,000 xg for 10 min, resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk for 1 h at room temperature and then probed with primary antibody overnight at 4°C. Immunoreactive bands were visualized by incubation with horseradish peroxidaseconjugated secondary antibodies and application of an enhanced chemiluminescent system. www.landesbioscience.com

Cell proliferation assays. The effects of BB on cell proliferation were examined using a panel of human tumor cell lines. Cells were seeded into 96-well plates and incubated overnight for attachment, then treated in triplicate with gradient concentrations of the tested compounds at 37°C for 72 h. For suspension cell lines, proliferative activity was assessed by measuring the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (Sigma, Saint Louis, MO) to a colored product.11 Sulforhodamin B (Sigma, Saint Louis, MO) assays, as previously described,12 were applied to solid tumor cell lines. The IC50 was fitted with 4-parameter curves. The data were expressed as mean ± SD from three independent experiments. The EGF-stimulated A549 growth inhibition of BB was measured as described. Briefly, A549 cells were seeded in 96-well

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Figure 5. BB represses tumor angiogenesis in vivo. Mice were killed two weeks after treatment with BB for histological examinations. Slides were stained with anti-CD31 antibody using diaminobenzidine as chromagen, and then counterstained with hematoxylin. The brown color showed the CD31-positive cells (endothelium cells, pointed with arrows). (A) Typical photographs of primary tumor sections with immunohistochemistry staining (magnification, 200x). (B) The histogram represented the number of microvessels in (A). Similar results were obtained from three separate experiments. *p < 0.05; **p < 0.01 versus control.

plates and incubated overnight, then incubated for 72 h in medium containing 1% bovine serum albumin and 20 ng/mL EGF in the presence of different concentrations of BB or the solvent control. HMEC-1 migration assays. HMEC-1 migration assays were performed in a transwell Boyden Chamber13 (8 μm pore size, Costar, MA), which was coated with 1% gelatin. The lower chamber contained 600 μL MCDB131 medium supplemented with 0.5% serum and 20 ng/mL EGFR. One hundred microlitres

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cell suspensions (1 x 105 cells/well) with various concentrations of BB or the solvent control diluted in serum-free MCDB131 medium was added to the upper chamber. After 6 h incubation at 37°C in a CO2 incubator, all non-migrant cells were removed from the upper face of the transwell membrane with a cotton swab; the migrated cells were fixed with 90% ethanol and then stained with 0.1% crystal violet in 0.1 M borate and 2% ethanol (pH 9.0). The stained cells were photographed under a microscope and subsequently extracted with 10% acetic acid. The OD value was determined at 600 nm, and the inhibition rate of migration was calculated as [1 - (ODBB - ODblank)/(ODcontrol - ODblank)] x 100%. Matrigel endothelial cell tube formation assays. The tube formation of HMEC-1 cells was conducted for the assay of in vitro angiogenesis.14 Briefly, a 96-well plate was coated with 50 μL of matrigel (Becton Dickinson Labware, MA USA), which was allowed to solidify at 37°C for 1 h. HMEC-1 (1.5 x 104 cells/ well) were seeded on the matrigel and cultured in MCDB131 medium (1% fetal bovine serum, 20 ng/mL EGF) containing different concentrations of BB or the solvent control for 8 h, and then photographed under a microscope (Olympus, DP70, Japan). The quantity of tube formation was valued by the total length of the tube-like structures in each tested well using Image-Pro Express (Media Cybernetics, Inc., Bethesda, MD). The inhibition rate of tube formation was calculated as [1 - (tubes BB/tubes control)] x 100%. Rat aortic ring assays. The aortas were harvested from six-weekold Sprague-Dawley rats. Each aorta was cut into 1-mm slices and embedded in 30 μL Matrigel in 24-well plates. The aortic rings were then fed with 500 μL of M199 medium (10% FCS) with different concentrations of BB or the solvent control, and photographed after 6 d incubation at 37°C in a CO2 incubator. Transfection and infection. The pBabe-Puro vectors containing human mutant EGFRs were obtained from Addgene Inc., (Cambridge, MA; www.addgene.org/pgvec1). And then replication incompetent retroviruses were produced from these vectors by transfection into Phoenix 293T packaging cells (Orbigen, San Diego) using Lipofectamine 2000 (Invitrogen, CA).15 NIH3T3 cells were infected with these retroviruses in the presence of polybrene. Forty-eight hours after infection, 2 μg/mL puremycin (Sigma, St. Louis, MO) was added and pooled stable cell lines were selected, from which clonal cell lines were derived. Antitumor activity against EGFR-transfected-NIH3T3 xenografts in nude mice. Nude mice, aged 4–5 weeks, were subcutaneously injected with different mutant EGFR transfected NIH3T3 cells (2 x 106). When the tumor reached a volume of 100–200 mm3, the mice were randomly assigned into control and treatment groups. Control groups were given vehicle alone, and treatment groups received BB or Iressa (p.o. 100 mg/kg/day). The sizes of tumor volume (V) were calculated as follows: V = (length x width2)/2. The individual relative tumor volume (RTV) was calculated as follows: RTV = Vt/V0, where VT is the volume on a given day, and V0 is the volume at the beginning of the treatment. The therapeutic effect of the compounds was expressed as the volume

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ratio of treatment to control (T/C): T/C (%) = 100% x (mean RTV of the treated group/mean RTV of the control group). Immunohistochemistry. Immunohistochemistry assays were used to examine whether tumor growth inhibited by BB was associated with its inhibition of tumor vessel formation. Tumor specimens were fixed in 4% paraformaldehyde for 1 week before transferred to 70% ethanol. Tumor samples were subsequently paraffin-embedded, and sections were cut and baked onto microscope slides. Slides were incubated with anti-CD31 antibody (Santa Cruz Inc., CA) and then secondary antibodies and visualized using a colorimetric method. All sections were countstained with hematoxylin. The microvessels from five randomly chosen fields were counted and photographed under a microscope (Olympus, DP70, Japan). Statistical analyses. The significance of differences between means assessed by Student’s t test, p < 0.05 was regarded to be statistically significant. Acknowledgements

This work was supported by grants from the National Natural Sciences Foundation of China (No. 30721005) the Ministry of Science and Technology of the China (No. 2004CB518903) and the Science and Technology Commission of Shanghai Municipality (STCSM) (No. 08DZ1980200).

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