BW1023U90: A new GRP receptor antagonist for small-cell lung cancer cells

Gastrin-releasing peptide (GRP) receptor antagonists were synthesized and their ability to interact with small-cell lung cancer (SCLC) cells determined. [¹²⁵I]BW1023U90, bound with high affinity (K_d = 2 nM) to a single class of sites (Bmax = 55 fmol/mg protein) using SCLC cell line NCI-H345. [¹²⁵I]BW1023U90 binding was time dependent and reversible even at 37°C as the ligand was minimally internalized. Specific [¹²⁵I]BW1023U90 binding was inhibited with high affinity by GRP as well as bombesin (BB) but not neuromedin B (NMB). BW1023U90 inhibited the ability of BB to elevate cytosolic Ca²⁺ and increase the growth of SCLC cells. A BW1023U90 analogue, BW2258U89 (10 μg/day, SC) slowed SCLC xenograft formation in nude mice and [¹²⁵I]BW1023U90 localized to SCLC tumors 1 h after injection into nude mice. BW2258U89 (4% by weight) was placed in microspheres and slowly released over a 3-week period in nude mice bearing SCLC xenografts. The microspheres containing BW2258U89 strongly inhibited SCLC growth in vivo. A radioimmunoassay was developed for the GRP receptor antagonists and the rabbit antiserum cross-reacted totally with BW2258U89 or BW1023U90. BW2258U89 immunoreactivity (5 nM) was detected in the plasma of nude mice containing the microspheres after 1 week. These data suggest that GRP receptor antagonists bind to receptors on SCLC tumors.     

促吞噬肽衍生物 T 肽对裸鼠术后残瘤 VEGF 表达的影响

目的：探讨促吞噬肽衍生物T肽抑制裸鼠术后残瘤生长的作用机理。方法：建立MCF-7乳腺癌裸鼠皮下移植瘤术后残瘤模型,观察8mg／kg剂量T肽对残余肿瘤组织的生长情况,并采用免疫组化和Western印迹检测给药后残瘤组织血管内皮生长因子（VEGF）的表达,采用RT-PCR检测不同T肽浓度对血管内皮细胞VEGF基因转录的影响。结果：T肽对MCF-7乳腺癌裸鼠皮下移植瘤术后残瘤生长表现出良好的抑制作用,按照瘤重得出的抑制率为68．2％,按照肿瘤体积得出的抑制率为67．6％,T肽给药组裸鼠残瘤组织中VEGF的表达量较空白对照组明显减少。血管内皮细胞中T肽呈剂量相关性抑制VEGF基因的转录。结论：T肽的抗癌作用与其抑制肿瘤微环境肿瘤组织和血管内皮细胞中VEGF的表达有密切联系,预示着T肽有潜力成为预防术后残瘤生长的抗癌新药。     

Human T cells targeted with anti-T3 cross-linked to antitumor antibody prevent tumor growth in nude mice

Human peripheral blood T cells were tested for the ability to prevent tumor growth in nude mice when targeted with anti-T3 cross-linked to antitumor antibodies. LS174T human colon adenocarcinoma cells were mixed with human PBL coated either with anti-T3 (Fab) cross-linked to 315F6 (Fab) (an antitumor monoclonal antibody) or with no antibody, and were injected subcutaneously into nude mice. Tumor growth was totally inhibited at effector to target (E:T) ratios of 7.0:1 and 2.1:1, and was partially inhibited at 0.7:1 with antibody-coated PBL, but was not inhibited by uncoated PBL. T cell-mediated protection against tumor growth occurred when an antitumor was physically cross-linked to anti-T3. Neither a mixture of unlinked anti-T3 and antitumor antibodies nor anti-human MHC class I cross-linked to antitumor antibody prevented tumor growth. Whereas in vitro cytotoxicity was mediated exclusively by T8+ cells and was augmented by brief exposure of effector cells to IL 2, tumor neutralization in vivo was mediated by both T4+ and T8+ cells and was not significantly stimulated by prior exposure of the cells to IL 2. We conclude that human T cells, when targeted with appropriate antibody heteroaggregates, can specifically inhibit tumor growth at low E:T ratios, and that cells mediating tumor neutralization in vivo may differ from those mediating cytotoxicity in vitro.     

Down-regulation of nucleophosmin/B23 mRNA delays the entry of cells into mitosis

In investigating the regulation of nucleophosmin/B23 mRNA expression at the entry of mitosis, the results of Northern gel blot analysis showed that the nucleophosmin/B23 mRNA levels significantly increased in prometaphase (nocodazole-arrested) or metaphase (colchicine-arrested) cells collected by mitotic shake-off. A higher level of nucleophosmin/B23 mRNA was detected in all the collected mitotic cells arrested by treatment with nocodazole for 10-18h as compared to that in G2 cells. An attempt was then made to determine whether the regulation of nucleophosmin/B23 mRNA plays a role in the control of entry into mitosis. Down-regulation of nucleophosmin/B23 mRNA by transfection of its antisense construct resulted in the delay of cells entering mitosis. The demonstration of the characteristic changes in the mRNA level of nucleophosmin/B23 during the entry of cells into mitosis implicates the importance of nucleophosmin/B23 in the control of the mitotic fate of nucleoli and cell growth.     

Characterization and cellular localization of nucleophosmin/B23 in HeLa cells treated with selected cytotoxic agents (studies of B23-translocation mechanism).

Previous studies indicated that nucleophosmin/B23, an abundant nucleolar phosphoprotein, accumulated in the nucleoplasm (B23-translocation) of cells after exposure to selected cytotoxic drugs. Attempts were made to understand the B23-translocation mechanism. This paper reports that: (1) B23-translocation is a reversible process. Upon removal of camptothecin, which induced B23-translocation in HeLa cells, nucleophosmin/B23 relocalized into nucleoli within 2 h. Relocation occurs in the presence of cycloheximide which inhibits new protein synthesis. There is no reduction or degradation of nucleophosmin/B23 detected during drug treatments. Nucleophosmin/B23 has a half-life of 18-20 h. Taken together, these results indicate that B23-translocation is a reversible process. Drug treatment causes redistribution of nucleophosmin/B23 in nucleoplasm. (2) Inhibition of RNA synthesis does not cause the B23-translocation. Over 80% of RNA synthesis was inhibited in HeLa cells by treatment with actinomycin D, camptothecin, and methotrexate. While actinomycin D and camptothecin cause B23-translocation in all cells, 40% of methotrexate-treated cells remain untranslocated. (3) There is no significant change of phosphorylation in nucleophosmin/B23 during drug treatment. An identical oligomeric cross-linkage pattern was obtained in drug-treated cells. (4) HeLa cells treated with B23-translocation effective drugs have small and round nucleoli while control cells have large and irregular-shaped nucleoli.     

Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function

Many tumor cells shed specialized membrane vesicles known as exosomes. In this study, we show that pretreatment of mice with exosomes produced by TS/A or 4T.1 murine mammary tumor cells resulted in accelerated growth of implanted tumor cells in both syngeneic BALB/c mice and nude mice. As implanted TS/A tumor cells grew more rapidly in mice that had been depleted of NK cells, we analyzed the effects of the tumor-derived exosomes on NK cells. The tumor-derived exosomes inhibit NK cell cytotoxic activity ex vivo and in vitro as demonstrated by chromium release assays. The treatment of mice with TS/A tumor exosomes also led to a reduction in the percentages of NK cells, as determined by FACS analysis, in the lungs and spleens. Key features of NK cell activity were inhibited, including release of perforin but not granzyme B, as well as the expression of cyclin D3 and activation of the Jak3-mediated pathways. Human tumor cell lines also were found to produce exosomes that were capable of inhibiting IL-2-stimulated NK cell proliferation. Exosomes produced by dendritic cells or B cells did not. The presentation of tumor Ags by exosomes is under consideration as a cancer vaccine strategy; however, we found that pretreatment of mice with tumor exosomes blunted the protective effect of syngeneic dendritic cells pulsed ex vivo with tumor exosomes. We propose that tumor exosomes contribute to the growth of tumors by blocking IL-2-mediated activation of NK cells and their cytotoxic response to tumor cells.     

High-affinity urokinase-derived cyclic peptides inhibiting urokinase/urokinase receptor-interaction: effects on tumor growth and spread

Urokinase-type plasminogen activator (uPA) binds with high affinity to its specific cell surface receptor (uPAR) (CD87) via a well-defined sequence within the N-terminal region of uPA (uPA(19-31)). Since this uPA/uPAR-interaction plays a significant role in tumor cell invasion and metastasis, it has become an attractive therapeutic target. Two small peptidic cyclic competitive antagonists of uPA/uPAR-interaction have been developed, based on the uPAR binding site in uPA: WX-360 (cyclo(21,29)[D-Cys21]-uPA(21-30)[S21C;H29C]) and its norleucine (Nle) derivative WX-360-Nle (cyclo(21,29)[D-Cys21]-uPA(21-30)[S21C;K23Nle;H29C]). These peptides display an only five to 10-fold lower affinity to uPAR as compared to the naturally occurring uPAR-ligand uPA. In this study, WX-360 and WX-360-Nle were tested in nude mice for their potency to inhibit tumor growth and intraperitoneal spread of lacZ-tagged human ovarian cancer cells. Intraperitoneal administration of either cyclic peptide (20 mg peptide/kg; 1x daily for 37 days) into the tumor-bearing nude mice resulted in a significant reduction of tumor weight and spread within the peritoneum as compared to the untreated control group. This is the first report demonstrating effective reduction of tumor growth and spread of human ovarian cancer cells in vivo by small synthetic uPA-derived cyclic peptides competitively interfering with uPA/uPAR-interaction. Thus, both WX-360 and WX-360-Nle are promising novel compounds to reduce dissemination of human ovarian carcinoma.     

Induction of T-cell-mediated immunity against MethA fibrosarcoma by intratumoral injections of a bacillus Calmette-Guérin nucleic acid fraction

Summary MY-1, which consists of DNA and RNA extracted and purified from bacillus Calmette-Guérin (BCG), has been shown to have strong antitumor activity against various experimental tumors. To examine the role of T cells in the antitumor mechanism of MY-1, the effect of MY-1 injection on the development of tumor-specific immunity against MethA fibrosarcoma was investigated. MY-1 injections inhibited tumor growth less effectively in T-cell-deficient nude mice than in normal BALB/c mice. MethA tumor growth was suppressed after inoculation with L3T4-positive lymphocytes from tumor-bearing mice treated with MY-1. MethA-specific delayed-type hypersensitivity was also detected in tumor-bearing mice treated with MY-1. Immunohistochemical analyses showed that many L3T4-positive and a few Lyt2-positive cells infiltrated the regressing tumors. These results indicate that intratumoral MY-1 injections induce a MethA-specific, L3T4-positive cell-mediated, delayed-type hypersensitivity, which is necessary for the tumor regression.     

Antitumor activity of IL-32β through the activation of lymphocytes,and the inactivation of NF-κB and STAT3 signals

Cytokine and activation of lymphocytes are critical for tumor growth. We investigated whether interleukin (IL)-32β overexpression changes other cytokine levels and activates cytotoxic lymphocyte, and thus modify tumor growth. Herein, IL-32β inhibited B16 melanoma growth in IL-32β-overexpressing transgenic mice (IL-32β mice), and downregulated the expressions of anti-apoptotic proteins (bcl-2, IAP, and XIAP) and cell growth regulatory proteins (Ki-67 antigen (Ki-67) and proliferating cell nuclear antigen (PCNA)), but upregulated the expressions of pro-apoptotic proteins (bax, cleaved caspase-3, and cleaved caspase-9). IL-32β also inhibited colon and prostate tumor growth in athymic nude mice inoculated with IL-32β-transfected SW620 colon or PC3 prostate cancer cells. The forced expression of IL-32β also inhibited cell growth in cultured colon and prostate cancer cells, and these inhibitory effects were abolished by IL-32 small interfering RNA (siRNA). IL-10 levels were elevated, but IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were reduced in the tumor tissues and spleens of IL-32β mice, and athymic nude mice. The number of cytotoxic T (CD8⁺) and natural killer (NK) cells in tumor tissues, spleen, and blood was significantly elevated in IL-32β mice and athymic nude mice inoculated with IL-32β-transfected cancer cells. Constituted activated NF-κB and STAT3 levels were reduced in the tumor tissues of IL-32β mice and athymic nude mice, as well as in IL-32β-transfected cultured cancer cells. These findings suggest that IL-32β inhibits tumor growth by increasing cytotoxic lymphocyte numbers, and by inactivating the NF-κB and STAT3 pathways through changing of cytokine levels in tumor tissues.     

Cytolytic T cells recognize the two amino-terminal domains of H-2 K antigens in tandem in influenza A infected cells

Simian sarcoma virus-transformed NIH 3T3 (SSV-NIH 3T3) and SSV-NRK cells secrete a potent growth-promoting activity identical with the platelet-derived growth factor (PDGF) in mitogenic assays. The secreted activity is blocked by anti-PDGF antisera and competes with 125I-PDGF for receptor binding, suggesting that the secreted protein is the transforming protein of SSV, p28v-sis, or its processed product. Secreted p28v-sis appears to stimulate autocrine cell growth of SSV-transformed cells because anti-PDGF antisera block 3H-thymidine incorporation into growing SSV-NIH 3T3 and SSV-NRK cells. SSV-transformed cells have reduced numbers of high-affinity 125I-PDGF receptors; PDGF/p28v-sis receptor was purified from SSV-NIH 3T3 cells and retained active protein tyrosine kinase activity stimulated by PDGF. The rate of tumor growth in athymic nude mice injected with SSV-transformed cells was compared with levels of secreted growth factor activity. The rate of tumor growth in nude mice correlated directly with levels of p28v-sis secreted by SSV-transformed cells.     

5-烯丙基-7-二氟亚甲基白杨素对人肺癌A549细胞裸鼠移植瘤生长的影响

目的研究5-烯丙基-7-二氟亚甲基白杨素（ADFMChR）对人肺癌A549细胞裸鼠移植瘤生长的影响。方法建立人肺癌A549细胞裸鼠移植瘤模型,测定荷人肺癌裸鼠移植瘤的大小和重量,应用免疫组化SP法检测移植瘤组织中PCNA、VEGF、CD31的表达。结果ADFMChR对肺癌移植瘤生长有显著抑制作用（P〈0.01）,5.0、10.0、20.0 mg/（kg.bw）的ADFMChR对移植瘤的瘤重抑制率分别为42.98%,82.31%和89.91%。免疫组化检测结果表明：ADFMChR具有抑制肺腺癌裸鼠移植瘤细胞PCNA、VEGF及CD31蛋白表达作用。结论ADFMChR抑制肺腺癌裸鼠移植瘤的生长作用与其抑制移植瘤细胞PCNA、VEGF以及CD31的蛋白表达相关。     

Human immunodeficiency virus (HIV)-infected tumor xenografts as an in vivo model for antiviral therapy: role of alpha/beta interferon in restriction of tumor growth in nude mice injected with HIV-infected U937 tumor cells.

The host factors involved in the restriction of tumor growth were studied in nude mice transplanted with a cloned line of chronically human immunodeficiency virus (HIV)-infected U937 cells. HIV-infected and uninfected U937 cells exhibited the same growth patterns in culture. However, HIV-infected cells were not tumorigenic when injected subcutaneously in nude mice, whereas large solid tumors were observed in mice injected with uninfected U937 cells. Injection of nude mice with antibody to alpha/beta interferon (IFN-alpha/beta) enabled HIV-infected U937 cells to grow progressively in approximately 90 to 100% of mice. HIV-infected U937 cells formed solid tumors in the majority (60 to 90%) of either immunosuppressed (splenectomized, irradiated, and anti-asialo-GM1-treated) or genetically immunodeficient (bg/nu/xid) nude mice. In mice treated with antibodies to IFN-alpha/beta with established HIV-positive tumors, a direct correlation was found between p24 antigenemia and tumor size. Treatment of established HIV-positive U937 cell tumors with human IFN-alpha or mouse IFN-alpha/beta resulted in a clear-cut inhibition of both tumor growth and p24 HIV antigenemia. In contrast, treatment with tumor necrosis factor alpha markedly inhibited tumor growth but did not significantly decrease serum p24 levels. 3'-Azido-3'-deoxythymidine treatment did not affect either tumor growth or the levels of serum p24 antigen. These data indicate that endogenous IFN-alpha/beta is a crucial factor in the restriction of both tumor growth and p24 antigenemia in mice injected with HIV-infected tumor cells. Moreover, the results suggest that the development of HIV-1 p24 antigenemia in athymic immunosuppressed mice may represent an interesting in vivo model for anti-HIV therapy.     

CD40 ligation in vivo can induce T cell independent antitumor effects even against immunogenic tumors

Antitumor effects of CD40 ligation appear to involve distinct antitumor effector cells in different experimental models. In this study, we tested whether T cells were required for antitumor effects of agonistic anti-CD40 mAb (alphaCD40) against immunogenic versus poorly immunogenic tumors. Treatment of mice bearing poorly immunogenic B16 melanoma and its more immunogenic variant, B16-hsp72.1, with alphaCD40 resulted in a similar level of tumor growth suppression. Depletion of T cells did not reduce the antitumor effects in these 2 tumor models. To generate antitumor T cell responses, C57BL/6 mice were immunized with irradiated B16-hsp72.1. Treatment of these vaccinated mice challenged with a high dose of B16-hsp72.1 tumor cells with alphaCD40 induced tumor growth suppression, which was reduced by T-cell depletion, demonstrating that T cells were involved in the antitumor effect of alphaCD40. However, immunized mice depleted of T cells and treated with alphaCD40 were still able to suppress tumor growth as compared to tumor growth in immunized, T cell-depleted mice not treated with alphaCD40, suggesting that T cells were not required for the antitumor effect of alphaCD40. To confirm a lack of correlation between tumor immunogenicity and T-cell requirement in antitumor effects of CD40 ligation, we found that alphaCD40 induced tumor growth suppression in nude and SCID/beige mice bearing highly immunogenic tumors such as Meth A sarcoma, suggesting that macrophages may play a role. Indeed, both poorly immunogenic and highly immunogenic tumors were sensitive to in vitro growth inhibition by macrophages from alphaCD40-treated mice. Taken together, our results indicate that antitumor effects induced by alphaCD40, even against immunogenic tumors, can be observed in the absence of T cells and may involve macrophages.     

Sensitivity of tumor cells towards CIGB‐300 anticancer peptide relies on its nucleolar localization

CIGB‐300 is a novel anticancer peptide that impairs the casein kinase 2‐mediated phosphorylation by direct binding to the conserved phosphoacceptor site on their substrates. Previous findings indicated that CIGB‐300 inhibits tumor cell proliferation in vitro and induces tumor growth delay in vivo in cancer animal models. Interestingly, we had previously demonstrated that the putative oncogene B23/nucleophosmin (NPM) is the major intracellular target for CIGB‐300 in a sensitive human lung cancer cell line. However, the ability of this peptide to target B23/NPM in cancer cells with differential CIGB‐300 response phenotype remained to be determined. Interestingly, in this work, we evidenced that CIGB‐300's antiproliferative activity on tumor cells strongly correlates with its nucleolar localization, the main subcellular localization of the previously identified B23/NPM target. Likewise, using CIGB‐300 equipotent doses (concentration that inhibits 50% of proliferation), we demonstrated that this peptide interacts and inhibits B23/NPM phosphorylation in different cancer cell lines as evidenced by in vivo pull‐down and metabolic labeling experiments. Moreover, such inhibition was followed by a fast apoptosis on CIGB‐300‐treated cells and also an impairment of cell cycle progression mainly after 5 h of treatment. Altogether, our data not only validates B23/NPM as a main target for CIGB‐300 in cancer cells but also provides the first experimental clues to explain their differential antiproliferative response. Importantly, our findings suggest that further improvements to this cell penetrating peptide‐based drug should entail its more efficient intracellular delivery at such subcellular localization. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Essential requirement of I-A region-identical host bone marrow or bone marrow-derived cells for tumor neutralization by primed L3T4+ T cells

The antitumor activity of Meth A-hyperimmunized BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by the Winn test in H-2 incompatible bone marrow chimeras in closed colony CD-1 (nu/nu), inbred DDD/1(nu/nu) (H-2s), or inbred BALB/c(nu/nu) (H-2d) mice as recipients. We found that Meth A-Im-SPL suppressed Meth A growth in the chimera nude mice which were reconstituted with bone marrow cells of the H-2d haplotype (i.e., BALB/c, DBA/2 and B10.D2), but not in the chimeras which were reconstituted with bone marrow cells of the H-2a, H-2b, or H-2k haplotype (i.e., B10.A, B10, and B10.BR). These results suggested that H-2 restriction occurred between Meth A-Im-SPL and bone marrow or bone marrow-derived cells in tumor neutralization. Furthermore, Meth A-Im-SPL did not suppress Meth 1 tumors (antigenically distinct from Meth A tumors) in the presence or absence of mitomycin C-treated Meth A in a Winn assay. These results suggested that there is tumor specificity in the "effector phase" as well as in the "induction phase". The phenotype of the effectors in the Meth A-Im-SPL was Thy-1.2+ and L3T4+, because Meth A-Im-SPL lost their antitumor activity with pretreatment with anti-Thy-1.2 monoclonal antibody (mAb) and complement or anti-L3T4 mAb and complement, but not with anti-Lyt-2.2 mAb and complement or complement alone. Positively purified L3T4+ T cells from Meth A-Im-SPL (Meth A-Im-L3T4), obtained by the panning method, suppressed the tumor growth in the chimera nude mice which were reconstituted with bone marrow cells of B10.KEA2 mice (that were I-A region-identical with Meth A-Im-L3T4 cells but not others in H-2) as well as B10.D2 cells (that were fully identical with Meth A-Im-L3T4 cells in H-2). We conclude that Meth A-Im-SPL (L3T4+) neutralized the tumors in collaboration with I-A region-identical host bone marrow or bone marrow-derived cells, and the neutralization was not accompanied by the "bystander effect."     

Lymphotactin expression by engineered myeloma cells drives tumor regression: mediation by CD4+ and CD8+ T cells and neutrophils expressing XCR1 receptor.

The C chemokine lymphotactin has been characterized as a T cell chemoattractant both in vitro and in vivo. To determine whether lymphotactin expression within tumors could influence tumor growth, we transfected an expression vector for lymphotactin into SP2/0 myeloma cells and tested their ability to form tumors in BALB/c and nude mice. Transfection did not alter cell growth in vitro. Whereas SP2/0 cells gave rise to a 100% tumor incidence, lymphotactin-expressing SP2/0-Lptn tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells and neutrophils. Regression of the SP2/0-Lptn tumors was associated with a type 1 cytokine response and dependent on both CD4(+) and CD8(+) T cells, but not NK cells. Both SP2/0 and SP2/0-Lptn tumors grew in nude mice, but growth of the latter tumors was retarded and associated with heavy neutrophil responses; this retardation of SP2/0-Lptn tumor growth was reversed by neutrophil depletion of the mice. Our data also indicate that mouse neutrophils express the lymphotactin receptor XCR1 and that lymphotactin specifically chemoattracts these cells in vitro. Thus, lymphotactin has natural adjuvant activities that may augment antitumor responses via effects on both T cells and neutrophils and thereby could be important in gene transfer immunotherapies for some cancers.     

Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1

Neuropilin-1 (NRP-1), a non-tyrosine kinase receptor of vascular endothelial growth factor-165 (VEGF165), was found expressed on endothelial and some tumor cells. Since its overexpression is correlated with tumor angiogenesis and progression, the targeting of NRP-1 could be a potential anti-cancer strategy. To explore this hypothesis, we identified a peptide inhibiting the VEGF165 binding to NRP-1 and we tested whether it was able to inhibit tumor growth and angiogenesis. To prove the target of peptide action, we assessed its effects on binding of radiolabeled VEGF165 to recombinant receptors and to cultured cells expressing only VEGFR-2 (KDR) or NRP-1. Antiangiogenic activity of the peptide was tested in vitro in tubulogenesis assays and in vivo in nude mice xenotransplanted in fat-pad with breast cancer MDA-MB-231 cells. Tumor volumes, vascularity and proliferation indices were determined. The selected peptide, ATWLPPR, inhibited the VEGF165 binding to NRP-1 but not to tyrosine kinase receptors, VEGFR-1 (flt-1) and KDR; nor did it bind to heparin. It diminished the VEGF-induced human umbilical vein endothelial cell proliferation and tubular formation on Matrigel and in co-culture with fibroblasts. Administration of ATWLPPR to nude mice inhibited the growth of MDA-MB-231 xenografts, and reduced blood vessel density and endothelial cell area but did not alter the proliferation indices of the tumor. In conclusion, ATWLPPR, a previously identified KDR-interacting peptide, was shown to inhibit the VEGF165 interactions with NRP-1 but not with KDR and to decrease the tumor angiogenesis and growth, thus validating, in vivo, NRP-1 as a possible target for antiangiogenic and antitumor agents.