A novel leptin antagonist peptide inhibits breast cancer growth in vitro and in vivo

The role of the obesity cytokine leptin in breast cancer progression has raised interest in interfering with leptin's actions as a valuable therapeutic strategy. Leptin interacts with its receptor through three different binding sites: I–III. Site I is crucial for the formation of an active leptin–leptin receptor complex and in its subsequent activation. Amino acids 39‐42 (Leu‐Asp‐Phe‐Ile‐ LDFI) were shown to contribute to leptin binding site I and their mutations in alanine resulted in muteins acting as typical antagonists. We synthesized a small peptide based on the wild‐type sequence of leptin binding site I (LDFI) and evaluated its efficacy in antagonizing leptin actions in breast cancer using in vitro and in vivo experimental models. The peptide LDFI abolished the leptin‐induced anchorage‐dependent and ‐independent growth as well as the migration of ERα‐positive (MCF‐7) and ‐negative (SKBR3) breast cancer cells. These results were well correlated with a reduction in the phosphorylation levels of leptin downstream effectors, as JAK2/STAT3/AKT/MAPK. Importantly, the peptide LDFI reversed the leptin‐mediated up‐regulation of its gene expression, as an additional mechanism able to enhance the peptide antagonistic activity. The described effects were specific for leptin signalling, since the developed peptide was not able to antagonize the other growth factors' actions on signalling activation, proliferation and migration. Finally, we showed that the LDFI pegylated peptide markedly reduced breast tumour growth in xenograft models. The unmodified peptide LDFI acting as a full leptin antagonist could become an attractive option for breast cancer treatment, especially in obese women.     

8-prenylnaringenin, a novel phytoestrogen, inhibits angiogenesis in vitro and in vivo

8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.     

A novel RGDS-analog inhibits angiogenesis in vitro and in vivo

In this study the anti-angiogenic action of a novel non-peptide RGDS-analog named RAM was tested in vitro and in vivo. RAM inhibited FGF-2-induced chemotaxis by 80% in an adhesion-independent way. Further, it induced HUVEC-apoptosis in collagen-seeded HUVEC, indicating that such pro-apoptotic effect was adhesion-independent. In vivo studies revealed that RAM inhibited FGF-2 induced angiogenesis by 60% in the mouse Matrigel-assay and in the chicken-egg chorion-allantoic membrane assay. Finally, RAM was markedly more stable in serum as compared to the template RGDS and after 24 h incubation in 100% serum was significantly more active than RGDS. Taken together these results show that RAM exerts anti-chemotactic and pro-apoptotic effects, by an unexpected adhesion-independent mechanism, as we have recently shown for the template RGDS molecule [Blood 103 (2004) 4180], and has in vivo relevant anti-angiogenic properties, with marked stability in serum; therefore, RAM represents a novel promising anti-angiogenic molecule.     

A novel anti-Cyr61 antibody inhibits breast cancer growth and metastasis in vivo

Cysteine-rich protein 61(CCN1/Cyr61) has been implicated as an important mediator in proliferation and metastasis of breast cancer, which indicated that blockage of Cyr61 might be a potent target for breast cancer treatment. However, the antitumor effect of anti-Cyr61 antibodies on breast cancer in vivo has not been reported so far. In this study, we reported the effect and likely mechanism of generated anti-human Cyr61 monoclonal antibodies (mAb) on Cyr61 high expression line MDA-MB-231, known as a highly malignant and invasive human breast cancer cell line, at aspects of proliferation and migration in vitro and in vivo. We found the mAb, denoted as 093G9, revealed inhibitory effects on MDA-MB-231 cell proliferation, migration, and invasion through downregulation of both AKT and ERK phosphorylation in vitro compared with its isotype control. 093G9 also showed significant efficacy on suppressing primary tumor growth and spontaneous lymph node metastasis in in vivo mouse model. The specific epitope recognized by 093G9 was identified to be ¹⁴⁰LPNLGCP¹⁴⁶, adjacent to the VWC domain of Cyr61 by Ph.D.-C7C phage library display system. Our study provides direct evidence that Cyr61 can be a potent therapeutic target for patients who bear high Cyr61 expression breast cancer. Furthermore, the mAb, 093G9 developed in our laboratory, has shown a promising therapeutic characteristic in breast cancer.     

A novel non-containing-nitrogen bisphosphonate inhibits both in vitro and in vivo angiogenesis

Bisphosphonates (BP) are powerful inhibitors of bone resorption and are widely used in the treatment of patients with metastasis-induced osteolysis. In the present study, we show that a novel non-nitrogen-containing BP (BP7033) that exhibits antitumor activity is a potent inhibitor of both in vivo and in vitro angiogenesis. When administered to mice, BP7033 inhibited tumoral angiogenesis (65% at 0.06mg/injection) as well as tumor growth (65% at 0.006mg/injection) in a tumor model of A431 cells xenografted in nude mice, with no sign of toxicity. Additionally, in vivo angiogenesis induced by vascular endothelial growth factor-containing Matrigel implants was reduced by 90% in the presence of BP7033 (0.6mg/plug). In vitro, BP7033 inhibited proliferation of human umbilical vein endothelial cells (HUVEC) (IC(50) value 3x10(-4) M) and completely prevented the formation of capillary-like tubules by HUVEC in Matrigel. Moreover, treatment of A431 cells by BP7033 induced an inhibition of Ras processing and a decrease in the secretion of both vascular endothelial growth factor and matrix metalloproteinase-2, two well-known stimulators of the proliferation and migration of endothelial cells. These findings indicate that this new BP compound has marked antiangiogenic properties and thus represents a promising candidate for treatment of malignant diseases with an angiogenic component.     

Honokiol,a small molecular weight natural product,inhibits angiogenesis in vitro and tumor growth in vivo

Natural products comprise a major source of small molecular weight angiogenesis inhibitors. We have used the transformed endothelial cell line SVR as an effective screen of natural product extracts to isolate anti-angiogenesis and anti-tumor compounds. Aqueous extracts of Magnolia grandiflora exhibit potent activity in our SVR proliferation assays. We found that the small molecular weight compound honokiol is the active principle of magnolia extract. Honokiol exhibited potent anti-proliferative activity against SVR cells in vitro. In addition, honokiol demonstrated preferential inhibition of primary human endothelial cells compared with fibroblasts and this inhibition was antagonized by antibodies against TNF alpha-related apoptosis-inducing ligand. In vivo, honokiol was highly effective against angiosarcoma in nude mice. Our preclinical data suggests that honokiol is a systemically available and non-toxic inhibitor of angiogenesis and should be further evaluated as a potential chemotherapeutic agent.     

Homocysteine inhibits angiogenesis in vitro and in vivo

Homocysteine has been reported to inhibit endothelial cell proliferation, which is closely related to angiogenesis. However, the relationship between homocysteine and angiogenesis is unknown. To clarify whether homocysteine would inhibit angiogenesis in vitro and in vivo, we examined the effect of homocysteine on tube formation by bovine aortic endothelial cells (BAECs) and by human microvessel endothelial cell-1 (HMEC-1) in vitro, and on angiogenesis in vivo using the chorioallantoic membrane (CAM) assay, as well as on BAEC proliferation and migration. Homocysteine, but not cysteine, inhibited BAEC proliferation, migration, and tube formation in a dose-dependent manner at concentrations from 0 to 10 mM. Homocysteine also inhibited tube formation by HMEC-1s. In these assay, 50% inhibition was induced by about 1 mM homocysteine. In the in vivo CAM assay, 0, 10, 100, 500, and 1000 microgram homocysteine induced an avascular zone by 0, 0, 16.7, 53.3 and 76.5%, respectively, also showing a dose-dependent effect. It was suggested that homocysteine inhibited angiogenesis by preventing proliferation and migration of endothelial cells.     

CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo

We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12 h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.     

Embellistatin, a microtubule polymerization inhibitor, inhibits angiogenesis both in vitro and in vivo

The efficient inhibition of angiogenesis is considered as a promising strategy for the treatment of angiogenesis-related diseases including cancer. Herein, we report that embellistatin, a bicyclic ketone compound known as a microtubule polymerization inhibitor, exhibits anti-angiogenic activity. Embellistatin inhibited in vitro angiogenesis of bovine aortic endothelial cells (BAECs) such as bFGF-induced invasion and tube formation as well as bFGF-induced mouse corneal angiogenesis in vivo. Notably, embellistatin exhibited stronger inhibition activity for the growth of BAECs than that of normal and cancer cell lines. Cell cycle analysis revealed that the compound arrests cell cycle at G2/M phase, which is associated with the increased expression of p21(WAF1) and p53 partly. These results demonstrate that embellistatin may serve the basis for the development of new anti-angiogenic agents.     

DTD, an anti-inflammatory ditriazine, inhibits angiogenesis in vitro and in vivo

The ditriazine derivative DTD (4,10-dichloropyrido[5,6:4,5]thieno[3,2-d':3,2-d]-1,2,3-ditriazine) has been previously reported to reduce the degree of granulomatous inflammation and vascular density in a murine air pouch granuloma model. The aim of this study was to test whether DTD affects angiogenesis. Our results show that DTD inhibits in vivo angiogenesis in the chorioallantoic membrane (CAM) assay at doses equal or lower than 0.3 nmol/egg. Different in vitro assays were used to study the potential effects of this compound on key steps of angiogenesis, namely, a colorimetric assay of cell proliferation/viability, a morphogenesis on Matrigel assay, zymographic assays for gelatinases and nuclear morphology and cell cycle analysis for apoptosis induction. Our data indicate that DTD inhibits proliferation but does not induce apoptosis in endothelial cells in vitro. DTD suppresses the endothelial capillary-like chord formation at concentrations lower than those required to inhibit proliferation. DTD treatment inhibits the matrix metalloproteinase-2 production in endothelial and fibrosarcoma cells, but does not affect the cyclooxygenase-2 expression in endothelial cells, as assessed by western blot analysis. Taken together, results here presented indicate that DTD exhibits an anti-angiogenic activity that is independent of inflammatory processes and make it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.     

Dihydrotanshinone I inhibits angiogenesis both in vitro and in vivo

Dihydrotanshinone I （DI）, a naturally occurring compound extracted from Salvia miltiorrhiza Bunge, has been reported to have cytotoxicity to a variety of tumor cells. In this study, we investigated its anti-angiogenic capacity in human umbilical vein endothelial cells. DI induced a potent cytotoxicity to human umbilical vein endothelial cells, with an IC50 value of approximately 1.28 μg/ml.At 0.25-1μg/ml, DI dose-dependently suppressed human umbilical vein endothelial cell migration, invasion, and tube formation detected by wound healing, Transwell invasion and Matrigel tube formation assays, respectively. Moreover, DI showed significant in vivo anti-angiogenic activity in chick embryo chorioailantoic membrane assay. DI induced a 61.1% inhibitory rate of microvessel density at 0.2 μg/egg. Taken together, our results showed that DI could inhibit angio-genesis through suppressing endothelial cell proliferation, migration, invasion and tube formation, indicating that DI has a potential to be developed as a novel anti-angiogenic agent.     

6]-Gingerol, a pungent ingredient of ginger, inhibits angiogenesis in vitro and in vivo

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has anti-bacterial, anti-inflammatory, and anti-tumor-promoting activities. Here, we describe its novel anti-angiogenic activity in vitro and in vivo. In vitro, [6]-gingerol inhibited both the VEGF- and bFGF-induced proliferation of human endothelial cells and caused cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells in response to VEGF, and strongly inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessel in the mouse cornea in response to VEGF. Moreover, i.p. administration, without reaching tumor cytotoxic blood levels, to mice receiving i.v. injection of B16F10 melanoma cells, reduced the number of lung metastasis, with preservation of apparently healthy behavior. Taken together, these results demonstrate that [6]-gingerol inhibits angiogenesis and may be useful in the treatment of tumors and other angiogenesis-dependent diseases.     

BRCC2 inhibits breast cancer cell growth and metastasis in vitro and in vivo via downregulating AKT pathway

In our previous study, we demonstrated that the BRCC2 (breast cancer cell 2) gene is a proapoptotic molecule that interacts with Bcl-X_L. BRCC2 downregulation is associated with poor disease-free and overall survival in breast cancer. In this study, we aimed to investigate the role of BRCC2 in tumor suppression in breast cancer. In clinical breast cancer samples, we found that BRCC2 expression was significantly downregulated in cancer lesions compared with paired normal breast tissues. By silencing or overexpressing BRCC2 in breast cancer cells, we found that BRCC2 could inhibit cell growth and metastasis in vitro. An in vivo assay showed that BRCC2 not only dramatically inhibited breast cancer cell xenograft formation and growth but also inhibited breast cancer cell metastasis in a lung metastasis model. Moreover, we demonstrated that BRCC2 inhibited breast cancer metastasis via regulation of the Akt pathway. Thus, our study provided evidence that BRCC2 functions as a novel tumor suppressor in breast cancer and may be a potential therapeutic target for breast cancer management.     

Lebectin, a Macrovipera lebetina venom-derived C-type lectin, inhibits angiogenesis both in vitro and in vivo

Integrins play an essential role in endothelial cell motility processes during angiogenesis and thus present interesting targets for the development of new anti-angiogenic agents. Snake venoms naturally contain a variety of proteins that can affect integrin-ligand interactions. Recently, the C-type lectin proteins (CLPs) have been characterized as efficient modulators of integrin functions. In this study, we investigated the anti-angiogenic activity of lebectin, a newly discovered CLP from Macrovipera lebetina venom. Human brain microvascular endothelial cells (HBMEC), used as an in vitro model, express alphavbeta3, alphavbeta5, and alpha5beta1 integrins, as well as the alpha2, alpha3, alpha6, and beta4 subunits. Our data show that lebectin acts as a very potent inhibitor (IC(50) approximately 0.5 nM) of HBMEC adhesion and migration on fibronectin by blocking the adhesive functions of both the alpha5beta1 and alphaV integrins. In addition, lebectin strongly inhibits both HBMEC in vitro tubulogenesis on Matrigel trade mark (IC(50) = 0.4 nM) and proliferation. Finally, using both a chicken CAM assay and a Matrigel trade mark Plug assay in nude mice, our results show that lebectin displays potent anti-angiogenic activity in vivo. Lebectin thus represents a new C-type lectin with anti-angiogenic properties with great potential for the treatment of angiogenesis-related diseases.     

A humanized anti-osteopontin antibody inhibits breast cancer growth and metastasis in vivo

Osteopontin (OPN) has been implicated as an important mediator of breast cancer progression and metastasis and has been investigated for use as a potential therapeutic target in the treatment of breast cancer. However, the in vivo antitumor effect of anti-OPN antibodies on breast cancer has not been reported. In this study, a mouse anti-human OPN antibody (1A12) was humanized by complementarity-determining region grafting method based on computer-assisted molecular modeling. A humanized version of 1A12, denoted as hu1A12, was shown to possess affinity comparable to that of its parental antibody. The ability of hu1A12 to inhibit cell migration, adhesion, invasion and colony formation was assessed in a highly metastatic human breast cancer cell line MDA-MB-435S. The results indicated that hu1A12 was effective in inhibiting the cell adhesion, migration, invasion and colony formation of MDA-MB-435S cells in vitro. hu1A12 also showed significant efficacy in suppressing primary tumor growth and spontaneous metastasis in a mouse lung metastasis model of human breast cancer. The specific epitope recognized by hu1A12 was identified to be ²¹²NAPSD²¹⁶, adjacent to the calcium binding domain of OPN. Our data strongly support that OPN is a potential target for the antibody-based therapies of breast cancer. The humanized anti-OPN antibody hu1A12 may be a promising therapeutic agent for the treatment of human breast cancer.     

A soluble form of GAS1 inhibits tumor growth and angiogenesis in a triple negative breast cancer model

We previously demonstrated the capacity of GAS1 (Growth Arrest Specific 1) to inhibit the growth of gliomas by blocking the GDNF–RET signaling pathway. Here, we show that a soluble form of GAS1 (tGAS1), decreases the number of viable MDA MB 231 human breast cancer cells, acting in both autocrine and paracrine manners when secreted from producing cells. Moreover, tGAS1 inhibits the growth of tumors implanted in female nu/nu mice through a RET-independent mechanism which involves interfering with the Artemin (ARTN)-GFRα3-(GDNF Family Receptor alpha 3) mediated intracellular signaling and the activation of ERK. In addition, we observed that the presence of tGAS1 reduces the vascularization of implanted tumors, by preventing the migration of endothelial cells. The present results support a potential adjuvant role for tGAS1 in the treatment of breast cancer, by detaining tumor growth and inhibiting angiogenesis.     

Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.