Evelyne Zeira

Role of high expression levels of CXCR4 in tumor growth, vascularization, and metastasis

Hormone refractory metastatic prostate cancer remains an incurable disease. We found that high expression levels of the chemokine receptor CXCR4 correlated with the presence of metastatic disease in prostate cancer patients. Positive staining for CXCL12, the ligand for CXCR4, was mainly present in the tumor‐associated blood vessels and basal cell hyperplasia. Subcutaneous xenografts of PC3 and 22Rv1 prostate tumors that overexpressed CXCR4 in NOD/SCID mice were two‐ to threefold larger in volume and weight vs. controls. Moreover, blood vessel density, functionality, invasiveness of tumors into the surrounding tissues, and metastasis to the lymph node and lung were significantly increased in these tumors. Neutralizing the interactions of CXCL12/CXCR4 in vivo with CXCR4 specific antibodies inhibited the CXCR4‐dependent tumor growth and vascularization. In vitro, CXCL12 induced the proliferation and VEGF secretion but not migration of PC3 and 22Rv1 cells overexpressing CXCR4. Similar effects of CXCR4 overexpression on tumor growth in vivo were also noted in two breast cancer lines, suggesting that the observed effect of CXCR4 is not unique to prostate tumor cells. Thus high levels of the chemokine receptor CXCR4 induce a more aggressive phenotype in prostate cancer cells and identify CXCR4 as a potential therapeutic target in advanced cases of metastatic prostate cancer.

A Pivotal Role of Cyclic AMP-Responsive Element Binding Protein in Tumor Progression

Tumor microenvironment controls the selection of malignant cells capable of surviving in stressful and hypoxic conditions. The transcription factor, cyclic AMP-responsive element binding (CREB) protein, activated by multiple extracellular signals, modulates cellular response by regulating the expression of a multitude of genes. Previously, we have demonstrated that two cystein residues, at the DNA binding domain of CREB, mediate activation of CREB-dependent gene expression at normoxia and hypoxia. The construction of a dominant-positive CREB mutant, insensitive to hypoxia cue (substitution of two cystein residues at position 300 and 310 with serine in the DNA binding domain) and of a dominant negative CREB mutant (addition of a mutation in serine133), enabled a direct assessment, in vitro and in vivo, of the role of CREB in tumor progression. In this work, we demonstrate both in vitro and in vivo that CREB controls hepatocellular carcinoma growth, supports angiogenesis, and renders resistance to apoptosis. Along with the identification, by DNA microarray, of the CREB-regulated genes in normoxia and hypoxia, this work demonstrates for the first time that in parallel to other hypoxia responsive mechanisms, CREB plays an important role in hepatocellular carcinoma tumor progression.

Liposomal immunostimulatory DNA sequence (ISS-ODN): an efficient parenteral and mucosal adjuvant for influenza and hepatitis B vaccines.

Synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory sequences (ISS-ODN, also known as CpG-ODNs) have been shown to display in experimental models potent Th1-biassed immunoadjuvant activity upon parenteral or mucosal co-administration with a variety of antigens. In an attempt to potentiate adjuvant activity, and to reduce dose and number of administrations, ISS-ODN was entrapped (up to 90% efficiency) in large (1.5 microm) multilamellar liposomes using a simple and fast (5 min) procedure. Mice were vaccinated once or twice intramuscularly (i.m.) or intranasally (i.n.) with subunit influenza vaccines (consisting of the viral hemagglutinin and neuraminidase, HN) or with hepatitis B surface antigen particles (HBsAg), either non-encapsulated or liposome-encapsulated, together with free or liposomal ISS-ODN (5-25 microg per dose). At 3-12 weeks post-vaccination, the humoral (systemic, mucosal) and cellular responses and protective immunity were assessed. Vaccine formulations containing liposomal ISS-ODN co-administered with either soluble antigen or liposomal antigen (in the same vesicles or in separate vesicles) were up to 30 times more effective than formulations containing un-encapsulated ISS-ODN in inducing: (a) antigen-specific serum and mucosal IgG2a and IgA antibodies; (b) splenocyte proliferative response, cytotoxic activity and IFNgamma production; (c) a DTH response; and (d) protection against virus challenge. The response was Th1-dominant in the influenza model and a mixed Th1+Th2 response in the hepatitis B model. No adverse reactions were noted. Thus, liposomal encapsulation of ISS-ODN further enhances its inherent adjuvant activity.

CXCR4 antagonist 4F-benzoyl-TN14003 inhibits leukemia and multiple myeloma tumor growth

OBJECTIVE The chemokine receptor CXCR4 and its ligand CXCL12 are involved in the progression and dissemination of a diverse number of solid and hematological malignancies. Binding CXCL12 to CXCR4 activates a variety of intracellular signal transduction pathways that regulate cell chemotaxis, adhesion, survival, proliferation, and apoptosis. MATERIALS AND METHODS Here, we demonstrate that the CXCR4 antagonist, 4F-benzoyl-TN14003 (BKT140), but not AMD3100, exhibits a CXCR4-dependent preferential cytotoxicity toward malignant cells of hematopoietic origin. BKT140 significantly and preferentially stimulated multiple myeloma apoptotic cell death. BKT140 treatment induced morphological changes, phosphatidylserine externalization, decreased mitochondrial membrane potential, caspase-3 activation, sub-G1 arrest, and DNA double-stranded breaks. RESULTS In vivo, subcutaneous injections of BKT140 significantly reduced, in a dose-dependent manner, the growth of human acute myeloid leukemia and multiple myeloma xenografts. Tumors from animals treated with BKT140 were smaller in size and weights, had larger necrotic areas and high apoptotic scores. CONCLUSIONS Taken together, these results suggest a potential therapeutic use for BKT140 in multiple myeloma and leukemia patients.

A3 adenosine receptors and mitogen-activated protein kinases in lung injury following in vivo reperfusion

IntroductionAlthough activation of A3 adenosine receptors attenuates reperfusion lung injury and associated apoptosis, the signaling pathway that mediates this protection remains unclear. Adenosine agonists activate mitogen-activated protein kinases, and these kinases have been implicated in ischemia/reperfusion injury; the purpose of this study was therefore to determine whether A3 adenosine receptor stimulation with reperfusion modulates expression of the different mitogen-activated protein kinases. In addition, we compared the effect of the A3 adenosine agonist IB-MECA with the newly synthesized, highly selective A3 adenosine receptor agonist MRS3558 on injury in reperfused lung.MethodStudies were performed in an in vivo spontaneously breathing cat model, in which the left lower lobe of the lung was isolated and subjected to 2 hours of ischemia and 3 hours of reperfusion. The selective A3 adenosine receptor agonists IB-MECA (0.05 mg/kg, 0.1 mg/kg, or 0.3 mg/kg) and MRS3558 (0.05 mg/kg or 0.1 mg/kg) were administered before reperfusion.ResultsBoth A3 adenosine receptor agonists administered before reperfusion markedly (P < 0.01) attenuated indices of injury and apoptosis, including the percentage of injured alveoli, wet/dry weight ratio, myeloperoxidase activity, TUNEL (in situ TdT-mediated dUTP nick end labeling)-positive cells, and caspase 3 activity and expression. The more pronounced effects at low doses were observed with MRS3558. Increases in phosphorylated c-Jun amino-terminal protein kinase (JNK), p38, and extracellular signal-regulated kinase (ERK)1/2 levels were observed by the end of reperfusion compared with controls. Pretreatment with the A3 agonists upregulated phosphorylated ERK1/2 levels but did not modify phosphorylated JNK and p38 levels.ConclusionThe protective effects of A3 adenosine receptor activation are mediated in part through upregulation of phosphorylated ERK. Also, MRS3558 was found to be more potent than IB-MECA in attenuating reperfusion lung injury. The results suggest not only that enhancement of the ERK pathway may shift the balance between cell death and survival toward cell survival, but also that A3 agonists have potential as an effective therapy for ischemia/reperfusion-induced lung injury.

Interaction between CXCR4 and CCL20 pathways regulates tumor growth

The chemokine receptor CXCR4 and its ligand CXCL12 is overexpressed in the majority of tumors and is critically involved in the development and metastasis of these tumors. CXCR4 is expressed in malignant tumor cells whereas its ligand SDF-1 (CXCL12) is expressed mainly by cancer associated fibroblasts (CAF). Similarly to CXCR4, the chemokine CCL20 is overexpressed in variety of tumors; however its role and regulation in tumors is not fully clear. Here, we show that the chemokine receptor CXCR4 stimulates the production of the chemokine CCL20 and that CCL20 stimulates the proliferation and adhesion to collagen of various tumor cells. Furthermore, overexpression of CCL20 in tumor cells promotes growth and adhesion in vitro and increased tumor growth and invasiveness in vivo. Moreover, neutralizing antibodies to CCL20 inhibit the in vivo growth of tumors that either overexpress CXCR4 or CCL20 or naturally express CCL20. These results reveal a role for CCL20 in CXCR4-dependent and -independent tumor growth and suggest a therapeutic potential for CCL20 and CCR6 antagonists in the treatment of CXCR4- and CCL20-dependent malignancies.

Immunogenicity and safety of a novel IL-2-supplemented liposomal influenza vaccine (INFLUSOME-VAC) in nursing-home residents.

Influenza and its complications account for substantial morbidity and mortality, especially among the elderly. In young adults, immunization provides 70-90% protection, while among the elderly the vaccine may be only </=50% effective; hence, the need for new, more immunogenic vaccines. We compared the safety and immunogenicity of a novel, interleukin-2 (IL-2) -supplemented trivalent liposomal influenza vaccine (designated INFLUSOME-VAC) with that of a commercial trivalent split virion vaccine in community-residing elderly volunteers (mean age 81 years) in winter of 2000/2001. Eighty-one individuals were randomly assigned to be vaccinated intramuscularly, either with the standard vaccine (n=33) or with INFLUSOME-VAC (n=48) prepared from the former. The two vaccines contained equal amounts of hemagglutinin (HA) ( approximately 15 microgram of each viral strain); INFLUSOME-VAC consisted of liposomal antigens admixed with liposomal human IL-2 (Lip IL-2) (33 microgram = 6x10(5) IU/dose). At 1 month post-vaccination, seroconversion rates (tested by hemagglutination inhibition) for the A/New Caledonia (H1N1) and A/Moscow (H3N2) strains were significantly higher (P=0.04) in the INFLUSOME-VAC group (65 versus 45%, 44 versus 24%, respectively). Moreover, INFLUSOME-VAC induced a greater anti-neuraminidase (NA-N2) response (P<0.05). Anti-IL-2 antibodies were undetected, and no increase in anti-phospholipid IgG antibodies was found in the INFLUSOME-VAC group. Adverse reactions were similar in both groups. Thus, INFLUSOME-VAC appears to be both safe and more immunogenic than the currently used vaccine in the elderly.

Attenuation of reperfusion lung injury and apoptosis by A2A adenosine receptor activation is associated with modulation of Bcl-2 and Bax expression and activation of extracellular signal-regulated kinases.

Adenosine receptors (AR) and extracellular signal-regulated kinases (ERK) have been implicated in tissue protection and apoptosis regulation during ischemia/reperfusion (I/R) injury. This study tests the hypothesis that reduction of reperfusion lung injury after A2AAR activation is associated with attenuation of apoptosis, modulation of ERK activation, and alterations in antiapoptotic and proapoptotic protein expression (Bcl-2 and Bax, respectively). Experiments were performed in intact-chest, spontaneously breathing cats in which the arterial branch of the left lower lung lobe was occluded for 2 h and reperfused for 3 h (I/R group). Animals were treated with the selective A2AAR agonist ATL313 given 5 min before reperfusion alone or in combination with the selective A2AAR antagonist ZM241385. Western blot analysis showed significant reduction in expression of Bcl-2 and increase in expression of Bax after reperfusion, compared with control lungs. Phosphorylated ERK1/2 levels were also increased after reperfusion. Compared with the I/R group, ATL313 markedly (P < 0.01) attenuated indices of injury and apoptosis including the percentage of injured alveoli, wet-dry weight ratio, myeloperoxidase activity, in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive cells, and caspase 3 activity and expression. Furthermore, compared with reperfused lungs, in ATL313-pretreated lungs, Western blot analysis demonstrated substantial ERK1/2 activation, increased expression of Bcl-2, and attenuated expression of Bax. The protective effects of ATL313 were blocked by pretreatment with ZM241385. In summary, the present study shows that in vivo activation of A2AAR confers protection against reperfusion lung injury. This protection is associated with decreased apoptosis and involves ERK1/2 activation and alterations in antiapoptotic Bcl-2 and proapoptotic Bax proteins.

Femtosecond laser: a new intradermal DNA delivery method for efficient, long-term gene expression and genetic immunization

A femtosecond laser beam gene transduction (SG‐LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 μg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg‐specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNγ, and IL‐4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG‐LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV‐infected replicating cells. Tumor growth retardation was induced in vacci‐nated mice challenged with an HBsAg‐expressing syn‐geneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.—Zeira, E., Manevitch, A., Manevitch, Z., Kedar, E., Gropp, M., Daudi, N., Barsuk, R., Harati, M., Yotvat, H., Troilo, P. J., Griffiths, T. G., II, Pacchione, S. J., Roden, D. F., Niu, Z., Nussbaum, O., Zamir, G., Papo, O., Hemo, I., Lewis, A., Galun, E. Femtosecond laser: a new intradermal DNA delivery method for efficient, long‐term gene expression and genetic immunization. FASEB J. 21, 3522–3533 (2007)

Activation of A3 Adenosine Receptor Provides Lung Protection Against Ischemia-Reperfusion Injury Associated with Reduction in Apoptosis

Apoptosis has been described in various models of ischemia‐reperfusion (IR) injury, including lung transplantation. A3 adenosine receptor (AR) has been linked to a variety of apoptotic processes. The effect of A3AR activation on lung injury and apoptosis, following IR, has not been reported to date. In a spontaneously breathing cat model, in which the left lower lobe of the lung was isolated and subjected to 2 h of ischemia and 3 h of reperfusion, we tested the effect of IB‐MECA, a selective A3AR agonist, on lung apoptosis and injury. Significant increase in the extent of apoptosis was observed following lung reperfusion. IB‐MECA, administered before IR, and before or with reperfusion, markedly (p < 0.01) attenuated indices of injury and apoptosis including the percentage of injured alveoli, wet/dry weight ratio, myeloperoxidase activity, in situ terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end‐labeling (TUNEL) positive cells, and caspase 3 activity and expression. The protective effects of IB‐MECA were completely blocked by pretreatment with the selective A3AR antagonist MRS‐1191. In summary, even when given after the onset of ischemia, the A3AR agonist IB‐MECA conferred a powerful protection against reperfusion lung injury, which was associated with decreased apoptosis. This suggests a potentially important role for A3AR in lung IR injury.