Targeting survivin via PI3K but not c-akt/PKB by anticancer drugs in immature neutrophils

Myelosuppression is the most common unwanted side effect associated with the administration of anticancer drugs, and infections remain a common cause of death in chemotherapy-treated patients. Several mechanisms of the cytotoxicity of these drugs have been proposed and may synergistically operate in a given cell. Survivin expression has been associated with cancer, but recent reports suggest that this molecule is also expressed in several immature and mature hematopoietic cells. Here, we provide evidence that treatment of immature neutrophils with anticancer drugs reduced endogenous survivin levels causing apoptosis. The anticancer drugs did not directly target survivin, instead they blocked the activity of phosphatidylinositol-3-OH kinase, which regulated survivin expression and apoptosis in these cells. Strikingly, and in contrast to other cells, this pathway did not involve the serine/threonine kinase c-akt/PKB. Moreover, in combination with anticancer drug therapy, rapamycin did not induce increased myelosuppression in an experimental lymphoma mouse model. These data suggest that drugs that block either c-akt/PKB or signaling molecules located distal to c-akt/PKB may preferentially induce apoptosis of cancer cells as they exhibit no cytotoxicity for immature neutrophils.

Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase

Eph receptor tyrosine kinases are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Caveolae are flask-shaped invaginations of the cell membrane; their major structural protein, caveolin-1, has been shown to regulate signaling molecules localized in these micro-domains. The interaction of caveolin-1 with several of these proteins is mediated by the binding of its scaffolding domain to a region containing hydrophobic amino acids within these proteins. The presence of such a motif within the EphB1 kinase domain prompted us to investigate the caveolar localization and regulation of EphB1 by caveolin-1. We report that EphB1 receptors are localized in caveolae, and directly interact with caveolin-1 upon ligand stimulation. This interaction, as well as EphB1-mediated activation of extracellular-signal-regulated kinase (ERK), was abrogated by overexpression of a caveolin-1 mutant lacking a functional scaffolding domain. Interaction between Ephs and caveolin-1 is not restricted to the B-subclass of receptors, since we show that EphA2 also interacts with caveolin-1. Furthermore, we demonstrate that the caveolin-binding motif within the kinase domain of EphB1 is primordial for its correct membrane targeting. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of EphB1.

Selective in vitro targeting of GRP and NMB receptors in human tumours with the new bombesin tracer 177Lu-AMBA

PURPOSE: To investigate the in vitro binding properties of a novel radiolabelled bombesin analogue, (177)Lu-AMBA, in human neoplastic and non-neoplastic tissues selected for their expression of the bombesin receptor subtypes GRP-R, NMB-R and BRS-3. METHODS: In vitro receptor autoradiography was performed in cancers expressing the various bombesin receptor subtypes. The novel radioligand (177)Lu-AMBA was used and compared with established bombesin radioligands such as (125)I-Tyr(4)-bombesin and (125)I-[D: -Tyr(6),beta-Ala(11),Phe(13),Nle(14)]-bombesin(6-14). In vitro incidence of detection of each of the three bombesin receptor subtypes was evaluated in each tumour. RESULTS: (177)Lu-AMBA identified all GRP-R-expressing tumours, such as prostatic, mammary and renal cell carcinomas as well as gastrointestinal stromal tumours. (177)Lu-AMBA also identified all NMB-expressing tumours, but did not detect BRS-3-expressing tumours or BRS-3-expressing pancreatic islets. GRP-R-expressing peritumoural vessels were heavily labelled with (177)Lu-AMBA. In contrast to the strongly GRP-R-positive mouse pancreas, the human pancreas was not labelled with (177)Lu-AMBA unless chronic pancreatitis was diagnosed. In general, the sensitivity was slightly better with (177)Lu-AMBA than with the conventional bombesin radioligands. CONCLUSION: The present in vitro study suggests that (177)Lu-AMBA may be a very useful in vivo targeting agent for GRP-R-expressing tumours, NMB-R-expressing tumours and GRP-R-expressing neoangiogenic vessels.

PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl-2 and Bcl-xL genes

OBJECTIVE: To determine whether a specifically designed bispecific (Bcl-2/Bcl-xL) antisense oligonucleotide (ASO) induces apoptosis and enhances chemosensitivity in human prostate cancer LNCaP cells, as Bcl-2 and Bcl-xL are both anti-apoptotic genes associated with treatment resistance and tumour progression in many malignancies, including prostate cancer. MATERIALS AND METHODS: Inhibition of Bcl-2 and Bcl-xL expression by the bispecific ASO was evaluated using real-time reverse transcription-polymerase chain reaction and Western blotting, while growth inhibition and induction of apoptosis were analysed by a crystal violet assay, flow cytometry and Western blotting of apoptosis-relevant proteins. The effect of combined treatment with bispecific ASO and chemotherapy or small-interference RNA (siRNA) targeting the clusterin gene was also investigated. RESULTS: Bispecific ASO reduced Bcl-2 and Bcl-xL expression in LNCaP cells in a dose-dependent manner. There was cell growth inhibition, increases in the sub-G0-G1 fraction, and cleavage of caspase-3 and poly(ADP-Ribose) polymerase proteins in LNCaP cells after bispecific ASO treatment. Interestingly, Bcl-2/Bcl-xL bispecific ASO treatment also resulted in the down-regulation of Mcl-1 and up-regulation of Bax. The sensitivity of LNCaP cells to mitoxantrone, docetaxel or paclitaxel was significantly increased, reducing the 50% inhibitory concentration by 45%, 80% or 90%, respectively. Furthermore, the apoptotic induction by Bcl-2/Bcl-xL bispecific ASO was synergistically enhanced by siRNA-mediated inhibition of clusterin, a cytoprotective chaperone that interacts with and inhibits activated Bax. CONCLUSIONS: These findings support the concept of the targeted suppression of Bcl-2 anti-apoptotic family members using multitarget inhibition strategies for prostate cancer, through the effective induction of apoptosis.

[Lys40(Ahx-DTPA-111In)NH2]exendin-4, a very promising ligand for glucagon-like peptide-1 (GLP-1) receptor targeting

High levels of glucagon-like peptide-1 (GLP-1) receptor expression in human insulinomas and gastrinomas provide an attractive target for imaging, therapy, and intraoperative tumor localization, using receptor-avid radioligands. The goal of this study was to establish a tumor model for GLP-1 receptor targeting and to use a newly designed exendin-4-DTPA (DTPA is diethylenetriaminepentaacetic acid) conjugate for GLP-1 receptor targeting. METHODS: Exendin-4 was modified C-terminally with Lys(40)-NH(2), whereby the lysine side chain was conjugated with Ahx-DTPA (Ahx is aminohexanoic acid). The GLP-1 receptor affinity (50% inhibitory concentration [IC(50)] value) of [Lys(40)(Ahx-DTPA)NH(2)]exendin-4 as well as the GLP-1 receptor density in tumors and different organs of Rip1Tag2 mice were determined. Rip1Tag2 mice are transgenic mice that develop insulinomas in a well-defined multistage tumorigenesis pathway. This animal model was used for biodistribution studies, pinhole SPECT/MRI, and SPECT/CT. Peptide stability, internalization, and efflux studies were performed in cultured beta-tumor cells established from tumors of Rip1Tag2 mice. RESULTS: The GLP-1 receptor affinity of [Lys(40)(Ahx-DTPA)NH(2)]exendin-4 was found to be 2.1 +/- 1.1 nmol/L (mean +/- SEM). Because the GLP-1 receptor density in tumors of Rip1Tag2 mice was very high, a remarkably high tumor uptake of 287 +/- 62 %IA/g (% injected activity per gram tissue) was found 4 h after injection. This resulted in excellent tumor visualization by pinhole SPECT/MRI and SPECT/CT. In accordance with in vitro data, [Lys(40)(Ahx-DTPA-(111)In)NH(2)]exendin-4 uptake in Rip1Tag2 mice was also found in nonneoplastic tissues such as pancreas and lung. However, lung and pancreas uptake was distinctly lower compared with that of tumors, resulting in a tumor-to-pancreas ratio of 13.6 and in a tumor-to-lung ratio of 4.4 at 4 h after injection. Furthermore, in vitro studies in cultured beta-tumor cells demonstrated a specific internalization of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]exendin-4, whereas peptide stability studies indicated a high metabolic stability of the radiopeptide in beta-tumor cells and human blood serum. CONCLUSION: The high density of GLP-1 receptors in insulinomas as well as the high specific uptake of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]exendin-4 in the tumor of Rip1Tag2 mice indicate that targeting of GLP-1 receptors in insulinomas may become a useful imaging method to localize insulinomas in patients, either preoperatively or intraoperatively. In addition, Rip1Tag2 transgenic mice represent a suitable animal tumor model for GLP-1 receptor targeting.

Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors

Targeting neuroendocrine tumors expressing somatostatin receptor subtypes (sst) with radiolabeled somatostatin agonists is an established diagnostic and therapeutic approach in oncology. While agonists readily internalize into tumor cells, permitting accumulation of radioactivity, radiolabeled antagonists do not, and they have not been considered for tumor targeting. The macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to two potent somatostatin receptor-selective peptide antagonists [NH(2)-CO-c(DCys-Phe-Tyr-DAgl(8)(Me,2-naphthoyl)-Lys-Thr-Phe-Cys)-OH (sst(3)-ODN-8) and a sst(2)-selective antagonist (sst(2)-ANT)], for labeling with (111/nat)In. (111/nat)In-DOTA-sst(3)-ODN-8 and (111/nat)In-DOTA-[4-NO(2)-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH(2)] ((111/nat)In-DOTA-sst(2)-ANT) showed high sst(3)- and sst(2)-binding affinity, respectively. They did not trigger sst(3) or sst(2) internalization but prevented agonist-stimulated internalization. (111)In-DOTA-sst(3)-ODN-8 and (111)In-DOTA-sst(2)-ANT were injected intravenously into mice bearing sst(3)- and sst(2)-expressing tumors, and their biodistribution was monitored. In the sst(3)-expressing tumors, strong accumulation of (111)In-DOTA-sst(3)-ODN-8 was observed, peaking at 1 h with 60% injected radioactivity per gram of tissue and remaining at a high level for >72 h. Excess of sst(3)-ODN-8 blocked uptake. As a control, the potent agonist (111)In-DOTA-[1-Nal(3)]-octreotide, with strong sst(3)-binding and internalization properties showed a much lower and shorter-lasting uptake in sst(3)-expressing tumors. Similarly, (111)In-DOTA-sst(2)-ANT was injected into mice bearing sst(2)-expressing tumors. Tumor uptake was considerably higher than with the highly potent sst(2)-selective agonist (111)In-diethylenetriaminepentaacetic acid-[Tyr(3),Thr(8)]-octreotide ((111)In-DTPA-TATE). Scatchard plots showed that antagonists labeled many more sites than agonists. Somatostatin antagonist radiotracers therefore are preferable over agonists for the in vivo targeting of sst(3)- or sst(2)-expressing tumors. Antagonist radioligands for other peptide receptors need to be evaluated in nuclear oncology as a result of this paradigm shift.

Local targeting of malignant gliomas by the diffusible peptidic vector 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid-substance p

PURPOSE: Malignant glial brain tumors consistently overexpress neurokinin type 1 receptors. In classic seed-based brachytherapy, one to several rigid (125)I seeds are inserted, mainly for the treatment of small low-grade gliomas. The complex geometry of rapidly proliferating high-grade gliomas requires a diffusible system targeting tumor-associated surface structures to saturate the tumor, including its margins. EXPERIMENTAL DESIGN: We developed a new targeting vector by conjugating the chelator 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid to Arg(1) of substance P, generating a radiopharmaceutical with a molecular weight of 1,806 Da and an IC(50) of 0.88 +/- 0.34 nmol/L. Cell biological studies were done with glioblastoma cell lines. neurokinin type-1 receptor (NK1R) autoradiography was done with 58 tumor biopsies. For labeling, (90)Y was mostly used. To reduce the "cross-fire effect" in critically located tumors, (177)Lut and (213)Bi were used instead. In a pilot study, we assessed feasibility, biodistribution, and early and long-term toxicity following i.t. injection of radiolabeled 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid substance P in 14 glioblastoma and six glioma patients of WHO grades 2 to 3. RESULTS: Autoradiography disclosed overexpression of NK1R in 55 of 58 gliomas of WHO grades 2 to 4. Internalization of the peptidic vector was found to be specific. Clinically, the radiopharmeutical was distributed according to tumor geometry. Only transient toxicity was seen as symptomatic radiogenic edema in one patient (observation period, 7-66 months). Disease stabilization and/or improved neurologic status was observed in 13 of 20 patients. Secondary resection disclosed widespread radiation necrosis with improved demarcation. CONCLUSIONS: Targeted radiotherapy using diffusible peptidic vectors represents an innovative strategy for local control of malignant gliomas, which will be further assessed as a neoadjuvant approach.

Immune cell migration across the blood–brain barrier: molecular mechanisms and therapeutic targeting

The endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier protect the CNS from the constantly changing milieu within the bloodstream. The BBB strictly controls immune cell entry into the CNS, which is rare under physiological conditions. During a variety of pathological conditions of the CNS, such as viral or bacterial infections, or during inflammatory diseases, such as multiple sclerosis, immunocompetent cells readily traverse the BBB and subsequently enter the CNS parenchyma. Most of the available information on immune cell entry into the CNS is derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Consequently, our current knowledge on traffic signals mediating immune cell entry across the BBB during immunosurveillance and disease results mainly from experimental data in the EAE model. Therefore, a large part of this review summarizes these findings. Similarly, the potential benefits and risks associated with therapeutic targeting of immune cell trafficking across the BBB will be discussed in the context of multiple sclerosis, since elucidation of the molecular mechanisms relevant to this disease have largely relied on the use of its in vivo model, EAE.