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The excitatory amino acids (EAA) L-glutamate (L-Glu), L-aspartate (L-Asp) and D-aspartate (D-Asp) are thought to play a neurotransmitter/neuromodulator role in neuronal communications. Recently, a high level of EAA L-Glu, D- and L-Asp isomers has been found in the forebrain of Naples high-excitability (NHE) rat line that models the mesocortical variant of Attention-Deficit Hyperactivity Disorder (ADHD). The aim of this study was to assess the functions of D-Asp using two forms, i.e. free D-Asp or D-Asp diethyl ester (DEE) as prodrug, on brain and behaviour. Thus, prepuberal rats were given, for two weeks daily, an i.p. injection of D-Asp or DEE or vehicle. Then rats were exposed to two spatial novelties i.e. Làt and radial Olton maze. Behaviour was monitored for indices of activity, non-selective attention (NSA), selective spatial attention (SSA) and emotional reactivity. L-Glu and D- and L-Asp were detected by HPLC in cognitive and non-cognitive brain areas such as prefrontal cortex, striatum, hippocampus and hypothalamus. Results indicate that subchronic D-Asp or DEE (i) reduced EAA levels in the NHE and increased it in the random-bred controls (NRB) rats, (ii) in the Làt-maze D-Asp increased horizontal activity in NHE but DEE decreased it in NRB rats, (iii) in the Olton maze D-Asp and DEE decreased vertical activity in NHE and NRB rats respectively, (iv) D-Asp impaired attention only in NRB decreasing number of arms visited before first repetition. Therefore, data demonstrate differential effects of prepuberal subchronic D-Asp and DEE that may be related to different basal EAA levels in NHE and NRB rats.

The Naples high-excitability (NHE) rats are thought to model the mesocortical variant of attention-deficit hyperactivity disorder (ADHD). The aim of this study was to investigate forebrain level of L-glutamate, L-aspartate and D-aspartate, in NHE vs. Naples random bred (NRB) control rats. Thus, prepuberal NHE and NRB rats were daily handled in the 5th and 6th week of postnatal life. Then rats were exposed to two spatial novelties i.e. a Lat and a Olton maze for 10 min. Amino acids were detected by HPLC in the prefrontal cortex (PFC), striatum (STR), hippocampus (HPC) and hypothalamus (HYP). Results indicate that all amino acids were higher in NHE than in NRB rats. This, in turn, may explain the behavioural hyperactivity and attention deficit of this animal model of ADHD.

Naples High-Excitability (NHE) rats model the mesocortical variant of Attention-Deficit Hyperactivity Disorder (ADHD). Recently, a high level of excitatory amino acids (EAA) has been found in the forebrain of NHE rats. The aim of this study was to verify the effect of postnatal stimulation in prepuberal rats on forebrain EAA. Thus, prepuberal NHE and Naples Random Bred (NRB) control rats were daily handled (PS) or they were left undisturbed throughout (NO-PS). One hour after the last stimulation, PS and NO-PS rats were exposed to a spatial novelty in a Lat-maze and one day later to a non-reinforced Olton maze. In both tests the horizontal (HA) and vertical (frequency – VA and duration of rearing – RD) components of behaviour indexed activity and non-selective attention (NSA). Moreover, in the Olton maze the position of the number of arms visited before first repetition (FE) and to criterion (NVTC), indexed selective spatial attention (SSA). Amino acids were detected by HPLC in prefrontal cortex (PFC), striatum (STR), hippocampus (HPC) and hypothalamus (HYP). Results indicate that (i) in the Lat-maze, only for HA, NO-PS NHE rats were more active than PS, (ii) in the Olton maze NO-PS rats of both lines showed shorter rearing durations than PS, (iii) EAA level was higher in NHE than in NRB rats and (iv) NO-PS vs. PS treatment increased level of EAA across the forebrain in both rat lines. In contrast in NHE NO-PS rats l -glutamate ( l -Glu) decreased in HYP and l -aspartate ( l -Asp) decreased in HPC. In conclusion, postnatal stimulation in prepuberal rats significantly affects forebrain excitatory amino acids and behaviour in NHE line. Thus EAA are modulated by genetic determinants and environmental (epigenetic) factors.

We know that the Yin Yang 1 protein (YY1) overexpression is positively and strongly correlated with the degree of malignancy of bone tumors. Therefore, we questioned whether we could influence cell invasiveness by deleting YY1 in human osteosarcoma cells (SaOs-2), as tested in Matrigel-coated filters and metastasis implantation of such osteosarcoma cells in vivo, by serial analysis with nuclear magnetic resonance. Moreover, we focused our work on the chemokine receptor CXCR4 and its inhibition by T22 antibody, as well as on systemic (direct in vivo assay) and computer-assisted imaging of angiogenesis-related metastasis. Results showed that cell invasiveness and metastasis implantation by wild-type SaOs-2 cells, as evaluated by histology and immunohistochemistry, are associated with up-regulation of CXCR4 expression, which in turn was significantly reduced by T22. In addition, deletion of YY1 (siRNAYY1-SaOs-2) induced a significant decrease of cell invasion and metastasis growth. This phenomenon was associated with decreased vascular endothelial growth factor (VEGF)/angiogenesis and a complex rearrangement of the gene expression profile as evaluated by microarray analysis. In conclusion, YY1 and VEGF/CXCR4 seem to intervene in the pathogenesis of the malignant phenotype of osteosarcoma by acting on cell invasiveness and metastasis growth. [Cancer Res 2008;68(6):1797–808]

New nanocarriers are obtained by assembling two amphiphilic monomers: one containing the bioactive peptide CCK8 spaced, by a polydisperse poly(ethylene glycol), from two hydrophobic tails ((C18)2PEG2000CCK8), and the other containing a chelating agent able to give stable radiolabeled indium-111 complexes linked to the same hydrophobic moiety ((C18)2DTPAGlu). The size and shape of the supramolecular aggregates were structurally characterized by dynamic light scattering, small-angle neutron scattering, and cryogenic transmission electronic microscopy. Under the experimental conditions we investigated (pH 7.4 and molar ratio between monomers 30:70), there is the presence of high polydisperse aggregates: rod-like micelles with a radius of approximately 40 A and length >700 A, open bilayer fragments with thickness approximately 65 A, and probably vesicles. The presence of the bioactive peptide well exposed on the external surface of the aggregate allows selective targeting of nanocarriers towards the cholecystokinin receptors overexpressed by the cancerous cells. In vitro binding assays and in vivo biodistribution studies by nuclear medicine experiments using indium-111 are reported. Moreover, preliminary data concerning the drug loading capability of the aggregates and their drug efficiency on the target cells is reported by using the cytotoxic drug doxorubicin. Incubation of receptor-positive and control cells with peptide-containing aggregates filled with doxorubicin shows significantly lower cell survival in receptor-expressing cells relative to the control, for samples incubated in the presence of doxorubicin.

The development of receptor targeting radiolabeled ligands has gained much interest in recent years for diagnostic and therapeutic applications in nuclear medicine. Cholecystokinin (CCK) receptors have been shown to be overexpressed in a subset of neuroendocrine and other tumors. We are evaluating binding and biodistribution properties of a CCK8 peptide derivative labeled with (99m)Tc(I)-tricarbonyl. The CCK8 peptide was modified at its N-terminus by adding to its N-terminus two lysine-histidine modules (KH), where histidine is coupled to the side chain of the lysine ((KH)(2)-CCK8). (99m)Tc(I)-tricarbonyl was generated with the IsoLinktrade mark kit. A431 cells stably transfected with a cDNA encoding for the human CCK2 receptor were utilized to determine binding affinity, internalization, and retention of the labeled peptide, in comparison with wild-type A431 cells. A nude mouse tumor model was obtained by generating A431-CCK2R and A431-control tumors in opposite flanks of the animals. High specific activity labeling with (99m)Tc was achieved. In A431-CCK2R cells, specific saturable binding was observed as well as evident internalization of the radiolabeled peptide after binding. Biodistribution experiments showed rapid, specific localization of (KH)(2)-CCK8 on A431-CCK2R xenografts compared with control tumors, although absolute uptake values were not markedly higher compared with background activity. Clearance of unbound radioactivity was both urinary and hepatobiliary. In imaging experiments, while targeting to CCK2R positive tumors could be appreciated, there was poor contrast between target and nontarget areas. (KH)(2)-CCK8 shows adequate in vitro and in vivo properties for CCK2R targeting although improvement of biodistribution warrant further development.

The development of receptor targeting radiolabeled ligands has gained much interest in recent years for diagnostic and therapeutic applications in nuclear medicine. Cholecystokinin (CCK) receptors have been shown to be overexpressed in a subset of neuroendocrine and other tumors. We are evaluating binding and biodistribution properties of a CCK8 peptide derivative labeled with Tc-99m(I)-tricarbonyl. The CCK8 peptide was modified at its N-terminus by adding to its N-terminus two lysine-histidine modules (KH), where histidine is coupled to the side chain of the lysine ((KH)(2)-CCK8). Tc-99m(I)-tricarbonyl was generated with the IsoLink (TM) kit. A431 cells stably transfected with a cDNA encoding for the human CCK2 receptor were utilized to determine binding affinity, internalization, and retention of the labeled peptide, in comparison with wild-type A431 cells. A nude mouse tumor model was obtained by generating A431-CCK2R and A431-control tumors in opposite flanks of the animals. High specific activity labeling with 99(m)Tc was achieved. In A431-CCK2R cells, specific saturable binding was observed as well as evident internalization of the radiolabeled peptide after binding. Biodistribution experiments showed rapid, specific localization of (KH)(2)-CCK8 on A431-CCK2R xenografts compared with control tun-tors, although absolute uptake values were not markedly higher compared with background activity. Clearance of unbound radioactivity was both urinary and hepatobiliary. In imaging experiments, while targeting to CCK2R positive tumors could be appreciated, there was poor contrast between target and nontarget areas. (KH)2-CCK8 shows adequate in vitro and in vivo properties for CCK2R targeting although improvement of biodistribution warrant further development. (C) 2008 Wiley Periodicals, Inc.

Interferon alpha (IFNalpha) induces an EGF-Ras-->Raf-1-->Erk dependent survival pathway counteracting apoptosis induced by the cytokine. In this paper we have evaluated the effects of the combination between farnesyl-transferase inhibitor (FTI) R115777 and IFNalpha on the growth inhibition and apoptosis of cancer cells. Simultaneous exposure to R115777 and IFNalpha produced synergistic both antiproliferative and proapoptotic effects. In these experimental conditions, IFNalpha and R115777 completely antagonized the increased activity of both Ras and Erk-1/2 induced by IFNalpha and strongly reduced Akt activity. Furthermore, treatment with R115777 in combination with IFNalpha regimen induced tumor growth delay on established KB cell xenografts in nude mice, while the single agents were almost inactive. R115777 was again able to antagonize the Ras-dependent survival pathway induced by IFNalpha also in vivo. Raf-1, one of the downstream targets of Ras, has been reported to activate bcl-2 through displacement and/or phosphorylation of Bad. We have found that IFNalpha induced mitochondrial localization of Raf-1 that was antagonized by R115777. Moreover, IFNalpha increased Raf-1/bcl-2 immuno-conjugate formation and intracellular co-localization and enhanced phosphorylation of Bad at Ser 112 and again R115777 counteracted all these effects. Moreover, the use of plasmids encoding for dominant negative or dominant positive Raf-1 antagonized and potentiated, respectively, the co-immunoprecipitation between Raf-1 and bcl-2. In conclusion, FTI R115777 strongly potentiates the antitumor activity of IFNalpha both in vitro and in vivo through the inhibition of different survival pathways that are dependent from isoprenylation of intracellular proteins such as ras.

In human mammary and prostate cancer cells, steroid hormones or epidermal growth factor (EGF) trigger association of the androgen receptor (AR)-estradiol receptor (ER) (α or β) complex with Src. This interaction activates Src and affects the G1 to S cell cycle progression. In this report, we identify the sequence responsible for the AR/Src interaction and describe a 10 amino-acid peptide that inhibits this interaction. Treatment of the human prostate or mammary cancer cells (LNCaP or MCF-7, respectively) with nanomolar concentrations of this peptide inhibits the androgen- or estradiol-induced association between the AR or the ER and Src the Src/Erk pathway activation, cyclin D1 expression and DNA synthesis, without interfering in the receptor-dependent transcriptional activity. Similarly, the peptide prevents the S phase entry of LNCaP and MCF-7 cells treated with EGF as well as mouse embryo fibroblasts stimulated with androgen or EGF. Interestingly, the peptide does not inhibit the S phase entry and cytoskeletal changes induced by EGF or serum treatment of AR-negative prostate cancer cell lines. The peptide is the first example of a specific inhibitor of steroid receptor-dependent signal transducing activity. The importance of these results is highlighted by the finding that the peptide strongly inhibits the growth of LNCaP xenografts established in nude mice. © 2007 Nature Publishing Group All rights reserved.

Context: We previously showed that BAG3 protein, a member of the BAG (Bcl-2-associated athanogene) co-chaperone family, modulates apoptosis in human leukemias. The expression of BAG3 in other tumor types has not been extensively investigated so far. Objective: The objective of this study was to analyze BAG3 expression in thyroid neoplastic cells and investigate its influence in cell apoptotic response to TNF-related apoptosis-inducing ligand (TRAIL). Design, Setting, and Patients: We investigated BAG3 expression in human thyroid carcinoma cell lines, including NPA, and the effect of BAG3-specific small interfering RNA on TRAIL-induced apoptosis in NPA cells. Subsequently, we analyzed BAG3 expression in 30 benign lesions and 56 carcinomas from patients of the Naples Tumor Institute Fondazione Senatore Pascale. Main Outcome Measures: The main outcome measures were: analysis of BAG3 protein in NPA cells by Western blot and immunocytochemistry; analysis of apoptosis in TRAIL-stimulated NPA cells by flow cytometry; and evaluation of BAG3 expression in specimens from thyroid lesions by immunohistochemistry. Results: BAG3 was expressed in human thyroid carcinoma cell lines; small interfering RNA-mediated downmodulation of its levels significantly (P < 0.0195) enhanced NPA cell apoptotic response to TRAIL. The protein was not detectable in 19 of 20 specimens of normal thyroid or goiters, whereas 54 of 56 analyzed carcinomas (15 follicular, 28 papillary, and 13 anaplastic) were clearly positive for BAG3 expression. Conclusions: BAG3 downmodulates the apoptotic response to TRAIL in human neoplastic thyroid cells. The protein is specifically expressed in thyroid carcinomas and not in normal thyroid tissue or goiter.

Interactions between cancer cells and stromal cells are critically involved in tumor formation, progression and development of metastasis. Here we report for the first time that human pancreatic adenocarcinoma (PDAC) cells secrete BAG3 that binds and activates macrophages, inducing their activation and the secretion of pancreatic cancer- supporting factors. We also identify IFITM-2 as a BAG3 receptor, by its co-precipitation with BAG3 from macrophage cell membrane and by impairment of BAG3- induced activation in IFITM-2- silenced macrophages. Finally we show the importance of this pathway in vivo since in nu/nu mice carrying orthotopic xenografts of human PDAC (MIA PaCa-2) cells, BAG3 sequestration by an anti-BAG3 antibody results in reduced tumor growth and prevents metastastasis formation. In conclusion we have identified a novel paracrine loop involved in PDAC growth and metastatic spreading and shown that its pharmacological blockage with an anti-BAG3 antibody has therapeutic potential. Citation Format: Alessandra Rosati, Anna Basile, Raffaella D'Auria, Morena d'Avenia, Margot De Marco, Antonia Falco, Michelina Festa, Luana Guerriero, Vittoria Iorio, Maria Pascale, Renato Franco, Claudio Arra, Antonio Barbieri, Domenica Rea, Giulio Menichini, Michael Hahne, Marteen Bijlsma, Daniela Barcaroli, David A. Tuveson, Jelena Todoric, Michael Karin, Vincenzo De Laurenzi, Maria Caterina Turco. PDAC cells release BAG3 that activates a paracrine loop with stromal macrophages. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2393. doi:10.1158/1538-7445.AM2015-2393

597 Background: Trastuzumab (T), a monoclonal antibody against ErbB2, has improved the prognosis of breast cancer patients, but ErbB2 blockade in cardiomyocytes can produce asymptomatic left ventri...

and on the EndoPredict as well as on the MammaPrint test results were compared. In comparison to the clinical-pathological prognostic markers the EndoPredict would lead to a change of therapy in 36.4% (16/44). Conclusion: It was noticeable that the amount of recommendations for chemotherapy grew by one-third, which is in contrast to all studies published so far. One reason could be a selection bias of the sample cohort because of the high amount of tumours with a grading of 3. If the choice of therapy would base on the MammaPrint result the EndoPredict would lead to a change of therapy in 37.5% (15/40). Both tests lead to different risk classification in 34.2% of patients. Our results shed doubt on the reliability of the current gene expression tests. Follow-up of our patients for extended time periods will be necessary to properly assess the value of the assays. Disclosure of Interest: No significant relationships.

Purpose: Epidermal growth factor receptor (EGFR) overexpression has been implicated in the development of head and neck squamous cell carcinomas (HNSCC) and represents a potential therapeutic target for this disease. We have reported previously that growth inhibitory concentrations of IFN-α enhance the expression and activity of EGFR and that this effect could represent an escape mechanism to the growth inhibition and apoptotic cell death induced by IFN-α. In this study, we investigate whether the combination of IFN-α and gefitinib (Iressa, AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom), a selective EGFR tyrosine kinase inhibitor, might have a cooperative antitumor effect on HNSCC-derived cell lines. Experimental Design: The interaction of IFN-α and gefitinib was evaluated in vitro on HNSCC-derived cell lines by median drug effect analysis calculating a combination index with CalcuSyn software and in vivo by using HNSCC xenografts in nude mice. The mechanism of gefitinib and IFN-α interactions was also studied by analysis of cell cycle kinetics, apoptosis assays, and Western blotting of EGFR signal transduction components. Results: Simultaneous exposure to gefitinib and IFN-α produced synergistic antiproliferative and proapoptotic effects compared with single drug treatment. Furthermore, daily treatment of gefitinib (50 mg/kg p.o.) in combination with an IFN-α regimen (50,000 units s.c. three times weekly) induced tumor growth delay and increased survival rate on established HNSCC xenografts in nude mice. Moreover, the concomitant treatment with gefitinib suppressed the stimulation of extracellular signal-regulated kinase phosphorylation/activity induced by IFN-α both in vitro and in vivo. Conclusion: The observed cooperative antitumor effects could be, at least in part, explained by the inhibition exerted by gefitinib of an IFN-α-induced EGF-dependent survival pathway, which involves extracellular signal-regulated kinase activation. These results provide a rationale for the clinical evaluation of gefitinib in combination with IFN-α in HNSCC.

SummaryHMGA2 gene amplification and overexpression in human prolactinomas and the development of pituitary adenomas in HMGA2 transgenic mice showed that HMGA2 plays a crucial role in pituitary tumorigenesis. We have explored the pRB/E2F1 pathway to investigate the mechanism by which HMGA2 acts. Here we show that HMGA2 interacts with pRB and induces E2F1 activity in mouse pituitary adenomas by displacing HDAC1 from the pRB/E2F1 complex—a process that results in E2F1 acetylation. We found that loss of E2F1 function (obtained by mating HMGA2 and E2F1−/− mice) suppressed pituitary tumorigenesis in HMGA2 mice. Thus, HMGA2-mediated E2F1 activation is a crucial event in the onset of these tumors in transgenic mice and probably also in human prolactinomas.

The HMGA1 protein is a major factor in chromatin architecture and gene control. It plays a critical role in neoplastic transformation. In fact, blockage of HMGA1 synthesis prevents rat thyroid cell transformation by murine transforming retroviruses, and an adenovirus carrying the HMGA1 gene in the antisense orientation induces apoptotic cell death in anaplastic human thyroid carcinoma cell lines, but not in normal thyroid cells. Moreover, both in vitro and in vivo studies have established the oncogenic role of the HMGA1 gene. In this study, to define HMGA1 function in vivo, we examined the consequences of disrupting the Hmga1 gene in mice. Both heterozygous and homozygous mice for the Hmga1-null allele show cardiac hypertrophy due to the direct role of HMGA1 on cardiomyocytic cell growth regulation. These mice also developed hematologic malignancies, including B cell lymphoma and myeloid granuloerythroblastic leukemia. The B cell expansion and the increased expression of the RAG1/2 endonuclease, observed in HMGA1-knockout spleen tissues, might be responsible for the high rate of abnormal IgH rearrangements observed in these neoplasias. Therefore, the data reported here indicate the critical role of HMGA1 in heart development and growth, and reveal an unsuspected antioncogenic potential for this gene in hematologic malignancies. (Cancer Res 2006; 66(5): 2536-43)

Evidence suggests that CRIPTO-1 (CR-1) might be involved in the pathogenesis of human carcinoma. In the present study, we have screened the expression of CR-1 mRNA and protein in a wide panel of human cancer cell lines by using reverse transcriptase (RT)-PCR, real-time PCR and immunocytochemistry. Results of these experiments demonstrate that CR-1 is expressed in several, different carcinoma types. The anchorage-independent growth of colon, ovarian, lung and breast carcinoma cells was significantly inhibited by treatment with anti-CR-1 second generation antisense oligonucleotides. Similar results were obtained with anti-transforming growth factor alpha (TGF-alpha) and anti-amphiregulin (AR) antisense oligonucleotides. Treatment of carcinoma cells with CR-1 antisense oligonucleotides resulted in a significant reduction in the levels of expression of CR-1 mRNA and protein, and in the levels of activation of Akt. Finally, oral administration of either CR-1, AR or TGF-alpha antisense oligonucleotides produced a significant reduction in the growth of GEO colon carcinoma xenografts in nude mice that was associated with a reduction in the levels of expression of the target proteins. Taken together, these data strongly suggest that CR-1 might represent a novel target for therapeutic intervention in different carcinoma types.

Regulatory peptides and their analogs are being extensively investigated as radiopharmaceuticals for cancer imaging and therapy. Receptors of the cholecystokinin family have been shown to be overexpressed in different types of neuroendocrine tumors. The purposes of this study were to evaluate the chole- cystokinin octapeptide amide (CCK8) peptide tagged with a diethylenetriaminepentaacetic acid derivative (DTPAGlu) and to test whether a 111In-labeled conjugate (111In-DTPAGlu-G-CCK8, a derivative containing the chelating agent DTPAGlu bound through a glycine linker at the N-terminal end of the bioactive peptide CCK8) is suitable for cholecystokinin-B receptor (CCKBR) imaging. Methods: CCK8 was synthesized by solid- phase techniques and covalently coupled to DTPAGlu through a glycine linker at its amino terminus. The compound was labeled with 111In. The radiochemical purity and stability of the com- pound were assessed by chromatographic methods. NIH-3T3 and A431 cells overexpressing CCKBR were used to charac- terize the in vitro properties of the compound. Nude mice bear- ing control and CCKBR-overexpressing A431 xenografts were used as an in vivo model. Results: DTPAGlu-G-CCK8 showed rapid and efficient labeling with 111In. The radiolabeled conju- gate showed specific binding to both cell lines overexpressing CCKBR. Binding was saturable, with a dissociation constant of ?20 nmol/L in both cell systems. Both cell lines showed inter- nalization of the ligand after interaction with the receptor. Bio- distribution studies showed rapid localization of 111In-DTPAGlu- G-CCK8 on CCKBR-overexpressing A431 xenografts that was severalfold higher than that on control tumors at all time points tested. Unbound activity showed rapid clearance of over 80% through the kidneys by 30 min after injection. The labeled pep- tide conjugate was very stable in serum but showed a rapid breakdown after injection. Incubation with kidney homogenates suggested that most breakdown occurred in the kidneys, favor- ing the clearance of unbound activity. Conclusion: Our findings indicate that the in vitro and in vivo characteristics of 111In- DTPAGlu-G-CCK8 are favorable for CCKBR imaging, as the peptide shows high-affinity binding to the receptor, is internal- ized in CCKBR-expressing cells, and shows avid uptake in CCKBR-overexpressing xenografts, with rapid clearance of un- bound radioactivity through the kidneys. Furthermore, the ease of synthesis, high labeling efficiency, and chemical stability of DTPAGlu make this chelating moiety an ideal candidate for widespread use in peptide radiolabeling for nuclear medicine applications.

Cholecystokinin (CCK) receptors of the subtype B (CCK-BR) have been shown to be overexpressed in certain neuroendocrine tumors including medullary thyroid cancer. Our recent work has focused on new methods to radiolabel the CCK8 peptide with In-111 or Tc-99m for CCK-B receptor imaging. Derivatives of CCK8 were obtained by addition at the N-terminus in solid phase of a DTPA derivative (DTPAGlu) linked through a glycine spacer (DTPAGlu-G-CCK8) or cysteine, glycine and a diphenylphosphinopropionyl moiety (PhosGC-CCK8) for labeling with In-111 and 99mTc, respectively. CCK-BR overexpressing A431 cancer cell lines were utilized to characterize in vitro Properties of the two compounds as well as for generating xenografts in nude mice for in vivo characterization. Both In-111-DTPAGlu-G-CCK8 and (99m)TcPhosGC-CCK8 showed similar binding affinities for CCK-BR with dissociation constants of 20-40 nM, were internalized after interaction with the receptor and displayed prolonged cellular retention times. Specific in vivo interaction with the receptor of both CCK8 analogs was observed in our animal model In-111-DTPAGlu-G-CCK8 showed better target to non-target ratios, although it appeared to be rapidly metabolized after injection and activity cleared through the kidneys. Tc-99m-PhosGC-CCK8 was more stable in vivo but showed marked hepatobiliary clearance with resulting high background activity in the bowel. The rapid clearance and lower background obtained with 111In-DTPAGlu-G-CCK8 make this a better candidate for further development.

We have recently developed a candidate HIV-1 vaccine based on virus-like particles (VLPs) expressing a gp120 from an Ugandan HIV-1 isolate of the clade A (HIV-VLP(A)s). In vivo immunogenicity experiments were performed in Balb/c mice, with an immunization schedule based on a multiple-dose regimen of HIV-VLP(A)s without adjuvants, showing a significant induction of both humoral and cellular immunity. The Env-specific cellular response was investigated in vitro, scoring for both the proliferative response of T helper cells and the cytolytic activity of cytotoxic T lymphocytes (CTLs). Furthermore, immune sera showed >50% neutralization activity against both the autologous field isolate and the heterologous T cell adapted B-clade HIV-1(IIIB) viral strain. This is one of the first examples of HIV-1 vaccines based on antigens derived from the A clade, which represents >25% of all isolates identified world wide. In particular, the A clade is predominant in sub-Saharan countries, where 70% of the global HIV-1 infections occur, and where vaccination is the only rational strategy for an affordable prevention against HIV-1 infection.

Cardiac monitoring and managing of patients receiving antineoplastic therapy is an actual issue. We agree with the interesting article by Fallah-Rad et al. ([1][1]): new, sensitive indexes of cardiac function are needed to predict cardiac dysfunction before ejection fraction (EF) is compromised.

Aim: Anthracyclines are first line drugs against cancer, but produce a well-known cardiomyopathy through multiple mechanisms, which also include, among many, Ca2+ overload due to reduced SERCA2a activity and inappropriate opening of the RyR2, and impaired myocardial energetics. Anthracyclines generate Reactive Oxigen and Nitrogen Species (ROS and RNS), posing the heart at increased demand for oxygen, thus setting the stage for a metabolic ischemia that also activates late INa, the target of Ranolazine (RAN). Here, we aim at assessing whether RAN, diminishing intracellular Ca2+ through its inhibition of late INa blunts anthracyclines cardiotoxicity. Methods: To evaluate cardiac function in vivo, fractional shortening (FS) and ejection fraction (EF) were measured by M/B mode echocardiography and radial and longitudinal strain (RS and LS) were measured using 2D speckle-tracking ecocardiography, in C57/BL6 mice, 2-4 mo old, at day 0, and after 2 and 7 days of daily administration of Doxorubicin (Doxo, 2.17 mg/kg/day, ip). These measurements were repeated after 5 days of RAN treatment (750 mg/kg/day, a dose comparable to the one used in humans) iniziated at the end of Doxo treatment. Results: In our in vivo studies, after 7 days with Doxo, FS decreased to 50.5 ± 8.4%, p Conclusions: RAN post-treatment blunts cardiotoxic effects due to anthracyclines, as demonstrated by the normalization of the values of FS, EF and RS. It remains to explain the persistent abnormality of the LS. Disclosure: All authors have declared no conflicts of interest.

Aim: ErbB2 is overexpressed in about 25% of breast cancers. It modulates myocardial development and function in the heart. Trastuzumab (T), an anti-ErbB2 inhibitor, has improved the prognosis of patients with breast cancer, but is related to an increased risk of asymptomatic left ventricular (LV) dysfunction (3–34%) and heart failure (2–4%). The mechanisms of T cardiotoxicity are not entirely known and can include changes in Ca2+ regulation related to blockade of ErbB2. Here, we aim at assessing whether ranolazine (RAN), diminishing intracellular Ca2+ through its inhibition of late Ina, blunts T cardiotoxicity in vivo. Methods: To evaluate cardiac function in vivo, fractional shortening (FS) and ejection fraction (EF) were measured by echocardiography M-Mode in C57BL6 mice, 2-4 mo old, pretreated with RAN (750mg/kg/day, a dose comparable to the one used in humans) per os for 3 days. RAN was then administered for additional 7 days, alone and together with T (2.25 mg/kg/day ip), according to our well established protocol. Results: In our in vivo studies, after 7 days with T, FS decreased to 49 ± 1.5%, p < 0.01 vs 60 ± 0.5% (sham), and EF to 81 + 2%, p < 0.01 vs 91 ± 1% (sham). RAN alone did not change FS (59 ± 2%) nor EF 89 ± 1%. Interestingly, in mice treated with RAN and T, RAN prevents the reduction of EF and FS vs T alone (FS was 58 ± 1%, EF was 90 ± 1%, p = 0.01 and p < 0.01 respectively). Conclusions: In our mouse model, T produces LV dysfunction and RAN blunts T cardiotoxic effects. We plan to test RAN as a cardioprotective agent with other target-therapy drugs in our experimental models and to define the mechanisms of cardioprotection.

e13508 Background: ErbB2 is overexpressed in about 25% of breast cancers. It modulates myocardial development and function in the heart. Trastuzumab (T), an anti-ErbB2 inhibitor, has improved the p...

A majority of human colon carcinomas coexpress the epidermal growth factor (EGF)-related peptides transforming growth factor alpha (TGFalpha), amphiregulin (AR) and CRIPTO-1 (CR). We have synthesized novel, antisense mixed backbone oligonucleotides (AS MBOs) directed against TGFalpha, AR and CR. We screened the EGF-related AS MBOs for their ability to inhibit the anchorage independent growth of GEO human colon carcinoma cells. The MBOs that showed a high in vitro efficacy were then used for in vivo experiments. TGFalpha, AR and CR AS MBOs were able to inhibit the growth of GEO tumor xenografts in nude mice in a dose-dependent manner. Furthermore, the AS MBOs were able to specifically inhibit the expression of the target mRNAs and proteins in the tumor xenografts. A more significant tumor growth inhibition was observed when mice were treated with a combination of the three AS MBOs as compared to treatment with a single AS MBO. Finally, tumors from mice treated with TGFalpha, AR and CR AS MBOs showed a significant reduction of microvessel count, as compared with tumors from untreated mice or from mice treated with a single AS MBO. These data suggest that combinations of AS oligonucleotides directed against different growth factors might represent a novel, experimental therapy approach of colon carcinomas.

Vascular endothelial growth factor A (VEGF) is a potent mitogen for endothelial cells in vitro and promotes neo-angiogenesis in vivo. VEGF overexpression occurs in most human malignancies including thyroid carcinomas in which elevated VEGF expression is associated with a high tumorigenic potential. To investigate the role of VEGF in angiogenesis associated with development of thyroid carcinomas, we constitutively expressed VEGF121 into a poorly tumorigenic cell line (NPA) expressing minimal levels of endogenous VEGF. Here we report that VEGF overexpressing NPA cells showed the same growth potential as untransfected NPA in vitro but formed well-vascularized tumors when injected subcutaneously into nude mice with markedly reduced latency compared to parental cells. A complementary approach was to suppress VEGF expression in a highly tumorigenic anaplastic cell line (ARO) by the transfection of an antisense construct. Antisense-transfected ARO cells expressed reduced constitutive levels of VEGF, showed the same growth potential as untransfected ARO cells in vitro and formed small tumors characterized by minimal vascularization, extensive necrosis and longer latency compared to parental or vector-transfected ARO cells in vivo. Finally, we investigated the expression of both VEGF tyrosine kinase receptors (Flt-1 and Flk-1/KDR) in tumor specimens by RT - PCR. Expression of (Flt-1 and Flk-1/KDR) was low in tissue specimens derived from NPA tumors, but was found enhanced in NPA VEGF tumors; conversely, the expression of VEGF receptors was high in tissue specimens derived from ARO tumors but was decreased in tumors derived from VEGF depleted ARO cells. These results clearly demonstrate that VEGF indirectly promotes the growth of thyroid tumors by stimulating angiogenesis.

Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue1. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described2. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathke's pouch3. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2 –/– results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.

169 Background: ErbB2 is overexpressed in about 25% of breast cancers; in the heart, it modulates myocardial development and function. Trastuzumab (T), an anti-ErbB2 inhibitor, has improved the prognosis of patients with breast cancer, but is related to an increased risk of asymptomatic left ventricular (LV) dysfunction (3-34%) and heart failure (2-4%). Conventional measures of ventricular function, such as fractional shortening (FS) and ejection fraction (FE) are insensitive in detecting early cardiomyopathy induced by antineoplastic therapy. Here, we aim at assessing whether myocardial strain by 2D-speckle tracking (ST) is able to identify early LV dysfunction in mice treated with doxorubicin (D) and T, alone or in combination (D+T) and to relate data of cardiac function with tissue alterations. Methods: Cardiac function was measured with FS, by M-mode echocardiography, and with radial myocardial strain with ST in sedated C57BL/6 mice (8-10 wk old) at time 0, 2 and 6 days of daily administration of D (2.17 mg/kg/day), T (2.25 mg/kg/day), D+T (2.17 mg/kg/day + 2.25 mg/kg/day) and in a control group. In excised hearts, we evaluated TNFα and CD68 by immunohistochemistry; interstizial fibrosis was analyzed with picrosirius red staining. Results: FS was reduced in group D and D+T at 2 days (52+0.2% and 49+2%), both p2at 6 days in group D (16.46% and 155), in group T+D (12.35% and 74.16) and in group T (5.65% and 72.32) p

631 Background: ErbB2 is overexpressed in about 25% of breast cancers; in the heart, it modulates myocardial development and function. Trastuzumab (T), an anti-ErbB2 inhibitor, has improved the prognosis of patients with breast cancer, but is related to an increased risk of asymptomatic left ventricular (LV) dysfunction (3-34%) and heart failure (2-4%). Conventional measures of ventricular function, such as fractional shortening (FS) and ejection fraction (FE) are insensitive in detecting early cardiomyopathy induced by antineoplastic therapy. Aim of this study is to evaluate whether myocardial strain by 2D-speckle tracking (ST) is able to identify early LV dysfunction in mice treated with doxorubicin (D) and T, alone or in combination (D+T) and to relate data of cardiac function with tissue alterations. Methods: Cardiac function was measured with FS, by M-mode echocardiography, and with radial myocardial strain with ST in sedated C57BL/6 mice (8-10 wk old) at time 0, 2 and 6 days of daily administration of D, T, D+T and in a control group. In excised hearts, we evaluated TNFα and CD68 by immunohistochemistry; interstizial fibrosis was analyzed with picrosirius red staining. Results: FS was reduced in group D and D+T at 2 days (52+0.2% and 49+2% respectively), both p2at 6 days in group D (16.46% and 155 respectively), in group T+D (12.35% and 74.16) and in group T (5.65% and 72.32) p

Background Doxorubicin (DOX) produces a well-known cardiomyopathy through multiple mechanisms, which include, among many, Ca2+ overload due to reduced SERCA2a activity and inappropriate opening of the RyR2, and impaired myocardial energetics. DOX generates Reactive Oxygen and Nitrogen Species (ROS and RNS), posing the heart at increased demand for oxygen, setting the stage for metabolic ischemia that also activates late INa, target of ranolazine (RAN). Here, we aim at assessing whether RAN, diminishing intracellular Ca2+ through inhibition of late INa, and enhancing myocardial glucose utilization (and/or reverting impairment of glucose utilization caused by chemotherapy) prevents DOX cardiotoxicity. Methods We measured left ventricular (LV) function with fractional shortening (FS) by echocardiography in C57BL6 mice, 2-4 mo old, pretreated with RAN (370mg/kg/day, a dose comparable to the one used in humans) per os for 3 days. RAN was then administered for additional 7 days, alone and together with DOX (2.17mg/kg/day ip), according to our well established protocol. Hearts were then excised, mRNA expression was analyzed by qRT- PCR, interstitial fibrosis with picrosirius red staining. By Western blotting, we measured the activation of the apoptotic pathway. Results After 7 days with DOX, FS decreased to 50 ± 2%, p = .002 vs 60 ± 1% (sham). RAN alone did not change FS (59 ± 2%). Interestingly, in mice treated with RAN + DOX, the reduction in FS was milder: 57 ± 1%, p = 0.01 vs DOX alone. DOX-cardiotoxicity was accompanied by significant elevations in ANP (1000 folds), BNP (500 folds), CTGF (26 folds) and MMP2 (81 folds) mRNAs, while co-treatment with RAN significantly lowered these same genes compared to DOX. The alterations in extracellular matrix remodeling were confirmed by an increase of interstitial collagen with DOX (3.66%), p = .004 vs 2.19% (sham), which was normal in hearts co-treated with RAN (2.02%, p = .0002 vs DOX). Finally, the levels of PARP and pro-Caspase 3 were significantly decreased in DOX (indicating activation of apoptosis) but not in RAN + DOX. Conclusions In mice, RAN prevents DOX cardiotoxic effects. We plan to test RAN as a cardioprotective agent with other antineoplastic cardiotoxic drugs in mice, and to better characterize the cardioprotective mechanisms of RAN in these settings. Disclosure All authors have declared no conflicts of interest.

e11052 Background: Lapatinib (L) inhibits ErbB2-tyrosine kinase domain and has been reported (in the few existing trials) to be associated with a low risk of cardiac dysfunction (2%), while the prototypical Trastuzumab (T) increases the frequency of asymptomatic ejection fraction decrease by 3-18%, and the risk of heart failure by 2-4%. Here, we characterize our L murine cardiotoxicity model. Methods: Cardiac function was measured with fractional shortening (FS) by M-mode echocardiography, and with radial myocardial strain (%) with speckle tracking (ST) in sedated C57BL6 mice (2-4 mo. old) at day 0, and after 2 and 7 days of daily administration of L (100mg/kg/day orally), and in control mice. For comparison we used our well-established models with T (2.25 mg/kg/day, ip), and Doxorubicin (D, 2.17 mg/kg/day ip) as positive control. On excised hearts, we evaluated TNFα and CD68 by immunohistochemistry. Results: FS was reduced with D at 2 days: 52±0.2%, p2 (72±6) compared to L (2±.2% and 27±4, respectively, both p

e13539 Background: Anthracyclines produce a well-known cardiomyopathy through multiple mechanisms, which also include, among many, Ca2+ overload due to reduced SERCA2a activity and inappropriate opening of the RyR2, and impaired myocardial energetics. Anthracyclines generate Reactive Oxigen and Nitrogen Species (ROS and RNS), posing the heart at increased demand for oxygen, thus setting the stage for a metabolic ischemia that also activates late INa, the target of ranolazine (RAN). Here, we aim at assessing whether RAN, diminishing intracellular Ca2+ through its inhibition of late INa, and enhancing myocardial glucose utilization (and/or reverting impairment of glucose utilization caused by chemotherapy) blunts anthracyclines cardiotoxicity. Methods: To assess for toxicity in vitro, rat H9C2 cardiomyoblasts were pretreated with RAN (0.1-1mM) for 72 hours and then treated with doxorubicin (DOX, 0.1 mM) for additional 24 hours. Cells counts were assessed by Trypan exclusion test. To evaluate cardiac function in vivo, fractional shortening (FS) and ejection fraction (EF) were measured by echocardiography in C57BL6 mice, 2-4 mo old, pretreated with RAN (370mg/kg/day, a dose comparable to the one used in humans) per os for 3 days. RAN was then administered for additional 7 days, together with DOX (2.17mg/kg/day ip), according to our well established protocol. Results: After DOX, only 68% of the cells were viable. RAN alone did not affect cell survival, but blunted DOX toxicity, rescuing % cell survival to 87% (p=.01 vs DOX alone). In our in vivo studies, after 7 days with DOX, FS decreased to 50±2%, p=.002 vs 60±1% (sham), and EF to 81±2%, p=0.0001 vs 91±1% (sham). RAN alone did not change FS (59±2%) nor EF (89±1%). Interestingly, in mice treated with RAN and DOX, the reduction in cardiac function was milder: FS was 57±1%, EF was 89±1%, p=0.01 and 0.0009 respectively, vs DOX alone. Conclusions: RAN blunts DOX cardiotoxic effects in 2 different models, in vitro and in vivo. We plan to test RAN as a cardioprotective agent with other antineoplastic cardiotoxic drugs in our experimental models, and to better characterize the cardioprotective mechanisms of RAN in all these settings.

The interaction between the chemokine receptor CXCR4 and its ligand, CXCL12, has been shown in pre-clinical models to facilitate tumour metastasis and to be involved in tissue repair and in recruitment of hematopoietic precursors. AMD3100, a potent CXCR4 antagonist, has been approved by FDA for stem cell mobilization in patients with multiple myeloma and non-Hodgkin lymphoma. Several other CXCR4 inhibitors are in clinical development (BTK140, CTCE-9908, MDX-1338 and POL6326), with the aim of inhibiting the development and progression of metastasis. In the rational design of CXCR4 inhibitors, a new class of cyclic peptide CXCL12-based CXCR4 inhibitors was discovered (IT Patent M12010A 000096; PCT WO2011/092575 A1) and validated for efficacy in in vitro and in vivo models. Among the tested peptides RFF-1 showed potent and specific inhibitory effects on CXCR4-mediated functions in multiple in vitro assays (P-ERK induction, migration and calcium mobilization CXCL12-induced and binding to CXCR4). C57/BL mice were injected with B16 melanoma cells transfected with human CXCR4. A 4,5 fold reduction in lung metastases was observed after 10 days treatment with 2 mg/kg intraperitoneally (i.p.) RFF-1 compared to the CXCR4 inhibitor AMD3100 (1.25mg/kg i.p), the powerful CXCR4 inhibitor not suitable for prolonged administration, that reduced lung metastases of about 4 fold. In another model of mouse osteosarcoma, K7M2 murine cells, were injected in Balb/C mice. A 3 fold reduction in lung metastases was observed after 15 days treatment with 10 mg/kg i.p. RFF-1 whereas AMD3100 treatment (2.5mg/kg i.p.) lead to 1.8 fold increase in lung metastases. Additionally, a 30% reduction in primary cell growth was achieved in a subcutaneous model of human renal cancer SN12C cells when i.p. treated with 2 mg/kg RFF-1. With the aim to evaluate RFF-1 efficacy in hematopoietic stem cell (HSC) mobilization DBA/2 healthy mice were injected with a single s.c. RFF-1 dose and peripheral blood HSC mobilization was evaluated by colony forming unit assay. Treatment with 30 mg/kg RFF-1 results in rapid, potent and durable mobilization of HSC reaching faster the peak compared to 20 mg/kg AMD3100. These results have prompted us to pursue the clinical development of peptide RFF-1. A first in human Phase I trial is planned to determine the maximal tolerated dose and recommended Phase 2 dose, toxicity profile, pharmacokinetics and antitumor activity of RFF-1 in patients with refractory solid tumors. The Phase I-II study of a novel CXCR4 antagonist provides the opportunity to develop settings in diseases where there is increasing evidence of a role for CXCR4 in the development and progression of metastases such as relapsed glioblastoma, neoadjuvant colorectal and breast cancer, and advanced prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4645. doi:1538-7445.AM2012-4645

e11040 Background: Anti-ErbB2 therapies are associated with increased risk of left ventricular (LV) dysfunction. Trastuzumab increases the frequency of asymptomatic decrease in LV ejection fraction (LVEF) by 3-18%, and the risk of heart failure (HF) by 2-4%. The newer ErbB2 antibody rhuMAb 2C4 (pertuzumab) seems to affect ligand induced ErbB signaling in a more direct fashion, affecting EGFR/ErbB2 dimerization; yet, its cardiac side effects are only beginning to emerge. Here, we test whether the murine 2C4 induces cardiac dysfunction in normal mice. Methods: In vivo cardiac function was measured with LV fractional shortening (FS) by M-mode echocardiography in sedated C57BL/6 mice (2-4 mo. old) at day 0, and after 2 and 6 days of daily i.p. administration of 2C4 (2.25 μg/g/day) or doxorubicin (Doxo, 2.17 μg/g/day) as a positive control, and in sham animals. With Speckle Tracking echocardiography (ST) we also evaluated radial myocardial strain (%), a very sensitive parameter which can predict LV dysfunction...

e13612 Background: The mTOR inhibitor temsirolimus is being evaluated for anticancer efficacy in hundreds of clinical trials and is approved for treatment of advanced renal cell carcinoma. The PI3K...

Introduction: The CXCR4 inhibitor AMD3100 is FDA-approved to provide stem cell mobilization for autologous transplantation in NHL and Multiple Myeloma patients. Newly designed CXCR4 inhibitory molecules, named Hys-7 inv, Phe-7 inv and Hys-OH, were recently discovered and validated in in vitro and in vivo studies (patent MI2010A-000093). Aim of the study was to evaluate the role of new CXCR4 inhibitors in hematopoietic stem cell and neutrophil mobilization. Experimental procedures: 60 DBA/2 female were injected subcutaneously with 5 or 30 mg/kg of CXCR4 antagonist in comparison with AMD3100 (3 or 20 mg/kg). Blood was collected at 1, 2, 6 and 24 hours from the treatment. Neutrophilis were evaluated trough flow cytometry (Ly-6G+ cells) and colony forming unit assay (CFU) of the mobilized stem cell were performed. Bone marrow was also evaluated through flow cytometry and cytological analysis. Results: Hys-7 inv, Phe-7 inv and Hys-OH, cyclic peptides CXCR4 antagonists, induced potent stem cell and neutrophils mobilization compared with AMD3100. Treatment resulted in a rapid and dose related increment in neutrophils count as well as hematopoietic stem and progenitor cells increase in peripheral blood (2 to 5 fold increase). Although Hys-7inv and Hys-OH induced neutrophils increment similar to AMD3100 the kinetic was different. AMD3100 (20 mg/kg) mobilized Ly-6G cells within 1 hour but transiently (no increase at 24 hours); instead Hys-7inv and Hys-OH mobilized Ly-6G within 2 hours lasting up to 24 hours. Interestingly, Phe-7 inv increased neutrophils mobilization within 1 hour lasting up to 24 hours. AMD3100 treated bone marrow showed an increase in progenitor cells myelo-committed (from 1.5 to 3 fold increase) and polimorphonuclear cells (PMN) (1.5 to 2.5 fold increase) and a reduction in mature-lymphocyte (0.4 to 0.28 fold increase); CXCR4 inhibitor peptides increased the immature myelo-committed progenitor cells (from 2 to 3 fold increase VS control) compared with AMD3100 (1.5 fold increase VS control) reducing PMN (1.5 to 1.65 fold increase) compared to AMD3100 (2.5 fold increase). CFU showed increased colonies in peripheral blood from mice treated with cyclic-peptides (from 2.5 to 4 fold increase) compared to AMD3100 (3.5 fold increase); the highest number of CFU was reached earlier in mice treated with Hys-7inv, Phe-7 inv and Hys-OH rapidly (within 1h) versus AMD3100 (6 hours). Conclusions: In vivo studies demonstrated that three newly synthesized CXCR4 inhibitors increased neutrophil and hematopoietic stem cells release from the bone marrow with different kinetic and duration compared to the FDA approved stem cell mobilizer AMD3100. Ongoing experiments are evaluating advantages in mobilizing precursors cells with CXCR4 cyclic-peptides antagonists compared to AMD3100. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 394. doi:10.1158/1538-7445.AM2011-394

e11096 Background: New targeted therapies have improved the prognosis of patients with breast cancer but cause several off-target effects, such as left ventricular dysfunction and heart failure tha...

Modulation of the axis CXCR4/CXCL12 affected primary tumor growth and secondary lesions development. Moreover, CXCR4 inhibition increases hematopoietic stem cell mobility in patients undergoing autologous transplantation for myeloma and non-Hodgkin lymphoma. Although several CXCR4 inhibitors were described neither one reached satisfactory efficacy, bioavailability and acceptable toxicity. To develop new CXCR4 inhibitors through a rationale design approach, comparative studies were undertaken evaluating the N-terminal structure of the ligand CXCL12 and v-MIP II, an inhibitory chemokine like produced by the herpes virus 8-Kaposi associated. A common amino acidic motif (RFF) was identified in both structures although in inverted orientation. Having as a core this small domain, a new library (20 units) of cycle-peptide molecules was generated. 20 units of cycle-peptide molecules were synthesized that consists of 5 and 7 amino-acid residues cycled by a S-S bridge designed to interact with the receptor CXCR4. The peptides were characterized for functionally CXCR4 interaction through: 1. inhibition of CXCR4 binding; 2. inhibition of migration CXCL12-induced; 3. inhibition of calcium release CXCL12-induced; 4. Inhibition of P-Erk CXCL12-induced. Four peptides were identified as CXCR4 inhibitors. These four CXCR4 inhibitory peptides were further evaluated for the in vivo efficacy through inhibition of metastasis formation. B16 mouse melanoma cells transfected with CXCR4 were injected in the vein tail in C57 Bl mice and peptide treatment followed for 10 days. Dramatic reduction in number and size of lung metastases were registered in mice peptides-treated. Moreover, xenograft of human renal cancer cells SN12C-EGFP were subcutaneously injected and systemic peptides treatment followed for 10 days. Statistically significant decrease in tumor growth was showed in peptides treated mice. Taken together the data define four new CXCR4 inhibitory peptides, designed through innovative strategy, effective in “in vivo” systems deserving further development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2270.

The oral fluoropyrimidine capecitabine is converted to the active compound 5-florouracil (5-FU) in several steps, the last of which is the conversion of 5′-deoxy-5-fluorouridine (5′DFUR) to 5-FU by thymidine phosphorylase (TP). Capecitabine was designed to take advantage of the increased levels of TP observed in tumors as opposed to normal tissues, potentially allowing for selective toxicity in tumors. In this study we found that the antiproliferative effect induced by the histone deacetylase inhibitor vorinostat in colorectal cancer cells in vitro and in vivo xenograft models, was paralleled by downregulation of thymidilate synthase, an essential enzyme for DNA synthesis and the target of 5-FU, and by upregulation of TP. These effects were evident at the mRNA and at the protein level and were not observed in peripheral blood lymphocyte (PBL) from healthy donors treated ex vivo by vorinostat. On the basis of these results, we have evaluated in vitro the effects of the combination between vorinostat and the capecitabine metabolite 5′-DFUR on the growth of human LoVo, LS174T and SW620 colon cancer cell lines, showing that simultaneous exposure of equipotent doses of the two agents, for 96 hours, resulted in synergistic antiproliferative effect in all cell lines as demonstrated by calculating combination indexes. We also demonstrated that vorinostat and 5′-DFUR in combination, compared to single agents treatment, increased apoptotic cell death, demonstrated by flow cytometry analysis of DNA content after propidium iodide staining, by the induction of BAX protein expression and by the cleavage of PARP. The observed apoptotic effect could be, at least in part, related to the increase in the ROS content induced by vorinostat and 5′-DFUR combination compared to single agents treatment. In order to verify in vivo the synergistic effects on tumor cell growth demonstrated in vitro, we evaluated vorinostat in combination with capecitabine in SW620 colon cancer cells xenograft model. A marked inhibition of tumor growth was observed in mice group treated with vorinostat (100 mg/kg p.o. daily) plus capecitabine (359 mg/Kg p.o. daily) as compared to single agents treatment, with a consequent increase by 2.4-fold of tumor growth delay and a substantial increase in survival. Analysis of xenografts tumor sections by immunohistochemistry showed a significant increase in apoptotic cells in vorinostat plus capecitabine treated group compared with control, or single agent treated groups, as well as the down-regulation TS and upregulation TP proteins expression in mice treated with vorinostat alone or in combination with capecitabine. Overall these data suggested that the association of vorinostat plus capecitabine could be clinically explored in colon cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5442.

Inhibition of the axis CXCR4/CXCL12 can affect tumor growth and metastases. Moreover, CXCR4 inhibition increases hematopoietic stem cell mobility in patients undergoing autologous transplantation for myeloma and non-Hodgkin lymphoma. Although several inhibitors were described neither one reached satisfactory efficacy, bioavailability and acceptable toxicity. To develop new CXCR4 inhibitors through a rationale design approach, comparative studies were undertaken evaluating the N-terminal structure of the ligand CXCL12 and v-MIP II, an inhibitory chemokine like produced by the herpes virus 8-Kaposi associated. A common amino acidic motif (RFF) was identified in both structures although in inverted orientation. Having as a core this small domain, a new library (20 units) of cycle-peptide molecules was generated. 20 units of cycle-peptide molecules were synthesized that consists of 5 and 7 amino-acid residues cycled by a S-S bridge designed to interact with the receptor CXCR4. The peptides were characterized for functionally CXCR4 interaction through: 1. inhibition of CXCR4 binding; 2. inhibition of migration CXCL12-induced; 3. inhibition of calcium CXCL12-induced; 4. Inhibition of P-Erk CXCL12-induced. Four peptides were identified as possible CXCR4 inhibitors. These four CXCR4 inhibitory peptides were further evaluated for the in vivo efficacy through inhibition of metastasis formation. B16 mouse melanoma cells transfected with CXCR4 were injected in the vein tail in C57 Bl mice and peptide treatment followed for 10 days. Dramatic reduction in number and size of lung metastases were registered in mice peptides-treated. Moreover, xenograft of human renal cancer cells SN12C-EGFP were subcutaneously injected and systemic peptides treatment followed for 10 days. Statistically significant decrease in tumor growth was showed in peptides treated mice. Taken together the data define three new CXCR4 inhibitory peptides, designed through innovative strategy, effective in “in vivo” deserving further development. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B50