Gabriella Pagnan

Targeting Liposomal Chemotherapy via Both Tumor Cell–Specific and Tumor Vasculature–Specific Ligands Potentiates Therapeutic Efficacy

Neuroblastoma, the most common solid tumor of infancy derived from the sympathetic nervous system, continues to present a formidable clinical challenge. Sterically stabilized immunoliposomes (SIL) have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. We showed that SIL loaded with doxorubicin (DXR) and targeted to the disialoganglioside receptor GD(2) [aGD(2)-SIL(DXR)] led to a selective inhibition of the metastatic growth of experimental models of human neuroblastoma. By coupling NGR peptides that target the angiogenic endothelial cell marker aminopeptidase N to the surface of DXR-loaded liposomes [NGR-SL(DXR)], we obtained tumor regression, pronounced destruction of the tumor vasculature, and prolonged survival of orthotopic neuroblastoma xenografts. Here, we showed good liposome stability, long circulation times, and enhanced time-dependent tumor accumulation of both the carrier and the drug. Antivascular effects against animal models of lung and ovarian cancer were shown for formulations of NGR-SL(DXR). In the chick embryo chorioallantoic assay, NGR-SL(DXR) substantially reduced the angiogenic potential of various neuroblastoma xenografts, with synergistic inhibition observed for the combination of NGR-SL(DXR) with aGD(2)-SIL(DXR). A significant improvement in antitumor effects was seen in neuroblastoma-bearing animal models when treated with the combined formulations compared with control mice or mice treated with either tumor- or vascular-targeted liposomal formulations, administered separately. The combined treatment resulted in a dramatic inhibition of tumor endothelial cell density. Long-term survivors were obtained only in animals treated with the combined tumor- and vascular-targeted formulations, confirming the pivotal role of combination therapies in treating aggressive metastatic neuroblastoma.

Expression of ΔNp73 is a molecular marker for adverse outcome in neuroblastoma patients

The p73 gene is a p53 homologue which induces apoptosis and inhibits cell proliferation. Although p73 maps at 1p36.3 and is frequently deleted in neuroblastoma (NB), it does not act as a classic oncosuppressor gene. In developing sympathetic neurons of mice, p73 is predominantly expressed as a truncated anti-apoptotic isoform (ΔNp73), which antagonizes both p53 and the full-length p73 protein (TAp73). This suggests that p73 may be part of a complex tumor-control mechanism. To determine the role of ΔNp73 in NB we analyzed the pattern of expression of this gene in vivo and evaluated the prognostic significance of its expression. Our results indicate that ΔNp73 expression is associated with reduced apoptosis in a NB tumor tissue. Expression of this variant in NB patients significantly correlates with age at diagnosis and VMA urinary excretion. Moreover it is strongly associated with reduced survival (HR=7.93; P<0.001) and progression-free survival (HR=5.3; P<0.001) and its role in predicting a poorer outcome is independent from age, primary tumor site, stage and MYCN amplification (OS: HR=5.24, P=0.012; PFS: HR=4.36, P=0.005). In conclusion our data seem to indicate that ΔNp73 is a crucial gene in neuroblastoma pathogenesis.

Delivery of c-myb Antisense Oligodeoxynucleotides to Human Neuroblastoma Cells Via Disialoganglioside GD2-Targeted Immunoliposomes: Antitumor Effects

Background Advanced-stage neuroblastoma resists conventional treatment; hence, novel therapeutic approaches are required. We evaluated the use of c-myb antisense oligodeoxynucleotides (asODNs) delivered to cells via targeted immunoliposomes to inhibit c-Myb protein expression and neuroblastoma cell proliferation in vitro. Methods Phosphorothioate asODNs and control sequences were encapsulated in cationic lipid, and the resulting particles were coated with neutral lipids to produce coated cationic liposomes (CCLs). Monoclonal antibodies directed against the disialoganglioside GD(2) were covalently coupled to the CCLs. (3)H-labeled liposomes were used to measure cellular binding, and cellular uptake of asODNs was evaluated by dot-blot analysis. Growth inhibition was quantified by counting trypan blue dye-stained cells. Expression of c-Myb protein was examined by western blot analysis. Results Our methods produced GD(2)-targeted liposomes that stably entrapped 80%-90% of added c-myb asODNs. These liposomes showed concentration-dependent binding to GD(2)-positive neuroblastoma cells that could be blocked by soluble anti-GD(2) monoclonal antibodies. GD(2)-targeted liposomes increased the uptake of asODNs by neuroblastoma cells by a factor of fourfold to 10-fold over that obtained with free asODNs. Neuroblastoma cell proliferation was inhibited to a greater extent by GD(2)-targeted liposomes containing c-myb asODNs than by nontargeted liposomes or free asODNs. GD(2)-targeted liposomes containing c-myb asODNs specifically reduced expression of c-Myb protein by neuroblastoma cells. Enhanced liposome binding and asODN uptake, as well as the antiproliferative effect, were not evident in GD(2)-negative cells. Conclusions Encapsulation of asODNs into immunoliposomes appears to enhance their toxicity toward targeted cells while shielding nontargeted cells from antisense effects and may be efficacious for the delivery of drugs with broad therapeutic applications to tumor cells.

Antiproliferative and pro-apoptotic activity of eugenol-related biphenyls on malignant melanoma cells

BackgroundMalignant melanoma is one of the most aggressive skin cancer and chemotherapeutic agents currently in use are still unsatisfactory. Prevention and early diagnosis are the only effective tools against this tumour whose incidence and mortality rates are highly increased during the last decades in fair skin populations. Therefore the search for novel therapeutic approaches is warranted. Aim of this work was to identify and test new compounds with antiproliferative and cytotoxic activity on melanoma cells. We tested eugenol together with six natural and synthetic eugenol-related compounds for their capability to inhibit cell growth on primary melanoma cell lines established from patients tissue samples.ResultsEugenol and isoeugenol monomers and their respective O-methylated forms did not show to inhibit melanoma cells proliferation. Conversely, the dimeric forms (biphenyls) showed some antiproliferative activity which was mild for dehydrodieugenol, higher for its O,O- methylated form (O,O-dimethyl-dehydrodieugenol), and markedly pronounced for the racemic mixture of the brominated biphenyl (6,6-dibromo-dehydrodieugenol) (S7), being its enantiomeric form (S) the most effective compared to the other compounds. Such activity resulted to be selective against tumour cells, without affecting cultured normal human skin fibroblasts. Dose and time dependence curves have been obtained for the enantiomeric form S7-(S). Then IC50 and minimal effective doses and times have been established for the melanoma cell lines tested. TUNEL and phosphatidylserine exposure assays demonstrated the occurrence of apoptotic events associated with the antiproliferative activity of S7-(S). Cytotoxic activity and apoptosis induced by treating melanoma cells with eugenol-related biphenyls was partially dependent by caspase activation.ConclusionOur findings demonstrate that the eugenol related biphenyl (S)-6,6-dibromo-dehydrodieugenol elicits specific antiproliferative activity on neuroectodermal tumour cells partially triggering apoptosis and its activity should be further investigated on in vivo melanoma models in order to evaluate the real anticancer effectiveness on such tumour.

GD2‐mediated melanoma cell targeting and cytotoxicity of liposome‐entrapped fenretinide

Melanoma is a highly malignant and increasingly common neoplasm. Because metastatic melanoma remains incurable, new treatment approaches are needed. Immunoliposomes have been previously shown to enhance the selective localization of immunoliposome‐entrapped drugs to solid tumors with improvements in the therapeutic index of the drugs. Previously, we reported that the synthetic retinoid fenretinide (HPR) is an inducer of apoptosis in neuroblastoma (NB) cells, sharing the neuroectodermal origin with melanoma cells. HPR is a strong inducer of apoptosis also in melanoma cells, although at doses 10‐fold higher than those achievable clinically. Thus, our purpose was to investigate the in vitro potentiation of its cytotoxic effect on melanoma cells in combination with long‐circulating GD2‐targeted immunoliposomes. GD2 is a disialoganglioside extensively expressed on tumors of neuroectodermal origin, including melanoma. Murine anti‐GD2 antibody (Ab) 14.G2a and its human/mouse chimeric variant ch14.18 have been ligated to sterically stabilized liposomes by covalent coupling of Ab to the polyethylene glycol (PEG) terminus. Ab‐bearing liposomes showed specific, competitive binding to and uptake by various melanoma cell lines compared with liposomes bearing non‐specific isotype‐matched Abs or Ab‐free liposomes. Cytotoxicity was evaluated after 2 hr treatment, followed by extensive washing and 72 hr incubation. This treatment protocol was designed to minimize non‐specific adsorption of liposomes to the cells, while allowing for maximum Ab‐mediated binding. When melanoma cells were incubated with 30 μM HPR entrapped in anti‐GD2 liposomes, a significant reduction in cellular growth was observed compared to free HPR, entrapped HPR in Ab‐free liposomes or empty liposomes. Cytotoxicity was not evident in tumor cell lines of other origins that did not express GD2. Growth of NB cells was also inhibited by immunoliposomes with entrapped HPR. Int. J. Cancer 81:268–274, 1999.

Expression and methylation of CASP8 in neuroblastoma: identification of a promoter region.

Neuroblastoma (NB) is a tumor of infancy that presents a high rate of spontaneous regression, a phenomenon that likely reflects the activation of an apoptotic and/or differentiation program. An attractive hypothesis suggested that the caspase-8 gene (CASP8), which encodes a key enzyme at the top of the apoptotic cascade, is an anti-oncogene that can be inactivated by methylation or deletion in MYCN-amplified neuroblastoma1. However, subsequent reports have not fully confirmed this model of NB development2, 3. To clarify this question, we have studied CASP8 expression in primary tumors and NB cell lines and its relationship to CpG methylation at the putative 5 regulatory region of the gene.

Induction of apoptosis in human neuroblastoma cells by abrogation of integrin-mediated cell adhesion

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extra‐cellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN‐5 cells it acted as a differentiation‐promoting agent, while it had an anti‐proliferative effect on GI‐LI‐N cells, driving them driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a “DNA ladder” on gel electrophoresis, by propidium iodine staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI‐LI‐N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti‐sense oligonucleotides to β, integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell‐matrix interations by means of perturbing antibody against β, subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings incidate that abrogation of cell adhesion through down‐modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death. Int. J. Cancer 70:688–698, 1997.

Enhanced Antitumor Efficacy of Clinical-Grade Vasculature-Targeted Liposomal Doxorubicin

Purpose:In vivo evaluation of good manufacturing practice-grade targeted liposomal doxorubicin (TVT-DOX), bound to a CD13 isoform expressed on the vasculature of solid tumors, in human tumor xenografts of neuroblastoma, ovarian cancer, and lung cancer. Experimental Design: Mice were implanted with lung, ovarian, or neuroblastoma tumor cells via the pulmonary, peritoneal, or orthotopic (adrenal gland) routes, respectively, and treated, at different days post inoculation, with multiple doses of doxorubicin, administered either free or encapsulated in untargeted liposomes (Caelyx) or in TVT-DOX. The effect of TVT-DOX treatment on tumor cell proliferation, viability, apoptosis, and angiogenesis was studied by immunohistochemical analyses of neoplastic tissues and using the chick embryo chorioallantoic membrane assay. Results: Compared with the three control groups (no doxorubicin, free doxorubicin, or Caelyx), statistically significant improvements in survival was seen in all three animal models following treatment with 5 mg/kg (maximum tolerated dose) of TVT-DOX, with long-term survivors occurring in the neuroblastoma group; increased survival was also seen at a dose of 1.7 mg/kg in mice bearing neuroblastoma or ovarian cancer. Minimal residual disease after surgical removal of neuroblastoma primary mass, and the enhanced response to TVT-DOX, was visualized and quantified by bioluminescence imaging and with magnetic resonance imaging. When treated with TVT-DOX, compared with Caelyx, all three tumor models, as assayed by immunohistochemistry and chorioallantoic membrane, showed statistically significant reductions in cell proliferation, blood vessel density, and microvessel area, showing increased cell apoptosis. Conclusion: TVT-DOX should be evaluated as a novel angiostatic strategy for adjuvant therapy of solid tumors.

Combined targeting of perivascular and endothelial tumor cells enhances anti-tumor efficacy of liposomal chemotherapy in neuroblastoma.

The therapeutic index of anti-cancer drugs is increased when encapsulating them in tumor-targeted liposomes. Liposome-entrapped doxorubicin (DXR), targeting the tumor vasculature marker, aminopeptidase N (APN), displayed enhanced anti-tumor effects and prolonged survival in human neuroblastoma (NB)-bearing mice. Here we exploited a peptide ligand of aminopeptidase A (APA), discovered by phage display technology for delivery of liposomal DXR to perivascular tumor cells. Immunohistochemistry, performed in NB-bearing mice, showed APA expression in the vascular wall of NB primary and metastatic lesions. APA-targeted peptides displayed specific binding to APA-transfected cells in vitro, and also accumulation in the tumor of NB-bearing mice. Consequently, novel, APA-targeted, DXR-liposomes were developed and in vivo proof-of-principle was established, alone and in combination with APN-targeted DXR-loaded liposomes, in NB-bearing mice. Mice receiving APA-targeted liposomal DXR exhibited an increased life span in comparison to control mice, but to a lesser extent relative to that in mice treated with APN-targeted formulation, moreover the greatest increase in TUNEL-positive tumor cells was observed in animals treated with APN-targeted formulations. Mice treated with a combination of APA- and APN-targeted, liposomal DXR had a significant increase in life span compared to each treatment administered separately. There was a significant increase in the level of apoptosis in the tumors of mice on the combination therapy, and a pronounced destruction of the tumor vasculature with nearly total ablation of endothelial cells and pericytes. The availability of novel ligands binding to additional tumor vasculature-associated antigens will allow the design of sophisticated combinations of ligand-targeted liposomal anti-cancer drugs.

Combined Therapeutic Effects of Vinblastine and Rapamycin on Human Neuroblastoma Growth, Apoptosis, and Angiogenesis

Purpose: Vinblastine and rapamycin displayed synergistic inhibition of human neuroblastoma-related angiogenesis. Here, we studied the antitumor activity of vinblastine and rapamycin against human neuroblastoma. Experimental Design: Cell proliferation, cell cycle progression, and apoptosis were evaluated by measuring 3H-thymidine incorporation, bromodeoxyuridine uptake, and phosphatidylserine exposure, respectively. The in vivo sensitivity of neuroblastoma cells to vinblastine and rapamycin was determined in orthotopic neuroblastoma-engrafted mice. Angiogenesis was assessed by the chick embryo chorioallantoic membrane assay. Results: Each compound alone was able to induce a dose-dependent significant inhibition of cell proliferation, with a dramatically enhanced antiproliferative effect for the drugs used in combination. A marked G2-M cell cycle arrest with a nearly complete depletion of S phase was associated. The combined treatment triggered an increased apoptosis compared with either drug tested alone. A significant inhibition of tumor growth and microvessel area was obtained in neuroblastoma-bearing mice when treated with vinblastine or rapamycin alone, and a more dramatic effect with the combined treatment, compared with control mice. The therapeutic effectiveness, expressed as increased life span, was statistically improved by the combined therapy, compared with mice treated with either drug tested separately. Histologic evaluation of primary tumors showed that the combined treatment inhibited proliferation and angiogenesis and induced apoptosis. Combined treatment of neuroblastoma cells and neuroblastoma-bearing mice with vinblastine and rapamycin induced the down-modulation of both vascular endothelial growth factor production and vascular endothelial growth factor receptor 2 expression. In the chorioallantoic membrane assay, angiogenesis induced by human neuroblastoma biopsy specimens was significantly inhibited by vinblastine and rapamycin. Conclusions: These results may be relevant to design new therapeutic strategies against neuroblastoma.