Recent advances in targeted anti-vasculature therapy: the neuroblastoma model.

Novel anti-vasculature strategies that are emerging for the treatment of cancer and for the inhibition of angiogenesis may be a promising new tool for the adjuvant therapy of malignant tumours. Over the last fifteen years, several reports have been published concerning the relationship between tumour progression and angiogenesis in experimental models of neuroblastoma in vitro and in vivo. Moreover, a high vascular index in neuroblastoma correlates with poor prognosis, suggesting dependence of aggressive tumour growth on active angiogenesis. Here, we present an overview of the most recent advances in anti-vasculature therapy of neuroblastoma, and describe some preclinical results as well as future perspectives.

Improvement of aqueous solubility of fenretinide and other hydrophobic anti-tumor drugs by complexation with amphiphilic dextrins.

This study relates to the preparation of a series of amphiphilic dextrins and their evaluation as complexing agents for anti-tumor hydrophobic drugs such as fenretinide, paclitaxel, etoposide, and camptothecin. The amphiphilic dextrins were obtained by conjugation of low molecular weight dextrin (average molecular weight 1670, average polymerization degree 9.33 glucose monomer) with hydrocarbon chains at substitution degree of about 0.1 mole hydrocarbon chain per mole of glucose monomer, as confirmed by 1H-NMR spectra. The conjugates were highly soluble in water and dissolved with formation of nano-aggregates endowed with hydrophobic inner cores able to host hydrophobic drugs by complexation. Complexation raised hydrophobic drugs aqueous solubility; the best results were obtained with fenretinide. Solid complexes with fenretinide were prepared by using three different approaches: the kneading method, the co-solubilisation method, and the co-precipitation method. Kneading method provided the complexes endowed with the best functional properties. Thermogravimetric analysis on solid samples suggested a notable thermal stability up to 300 degrees C for both the conjugated dextrins and the solid complexes. In differential scanning calorimetry profiles no significant differences were observed among amphiphilic dextrins and complexed drug, indicating that the guest molecule exists in an amorphous state in the solid matrices. Particle size analysis confirmed the dimensional suitability of the complexes for parenteral administration. Moreover, sustained drug release, in vitro, has been observed from all the complexes analyzed. Regarding the biological effects, the cytotoxicity of complexed fenretinide towards HTLA-230 neuroblastoma cell line was always higher than the free drug, suggesting that complexation increased drug bioavailability. These findings, taken together, indicated that these biodegradable, self-assembling dextrin conjugates may be regarded as new potential complexing agents for hydrophobic drugs and, in particular, for fenretinide, to increase drug solubility, bioavailability, and thus therapeutic efficacy.

Micellar complexes of all-trans retinoic acid with polyvinylalcohol-nicotinoyl esters as new parenteral formulations in neuroblastoma.

All-trans-retinoic acid (ATRA) is now included in many antitumor therapeutic schemes for the treatment of acute promyelocytic leukemia, Kaposi's sarcoma, head and neck squamous cell carcinoma, ovarian carcinoma, bladder cancer, and neuroblastoma. Unfortunately, its poor aqueous solubility hampers its parenteral formulation, whereas oral administration of ATRA is associated with progressively diminishing drug levels in plasma, which is related to induction of retinoic acid-binding proteins and increased drug catabolism by cytochrome P450-mediated reactions. An ATRA formulation, obtained by complexation of the drug into polymeric micelles, might be suitable for parenteral administration overcoming these unwanted effects. To this purpose, amphiphilic polymers were prepared by polyvinylalcohol (PVA) partial esterification with nicotinoyl moieties and their functional properties evaluated with regard to ATRA complexation. The physicochemical characteristics of the polymers and the complexes were analyzed by 1H-NMR, Dynamic Light Scattering (DLS), Capillary Electophoresis (CE), and were correlated with the complex ability to improve the drug solubilization and release the free drug in an aqueous environment. Subsequently, the best complex, providing the highest ATRA solubilization and release, was evaluated in vitro to test its cytotoxicity towards neuroblastoma cell lines. The PVA substitution degree calculated from 1H-NMR was found to be 5.0%, 8.2%, 15.3% (nicotinoyl moiety:PVA monomer molar ratio), while capillary electrophoresis analysis on the complexes revealed that the drug loadings were 0.95%, 1.20%, 4.76% (ATRA:polymer w:w) for PVA substitution degrees of 5.0%, 8.2%, and 15.3%, respectively. Complexation strongly increased ATRA aqueous solubility, which reached 1.20 +/- 0.25 mg/mL. The DLS measurements of the polymers and the complexes in aqueous solutions revealed mean sizes always below 400 nm, low polydispersity (min 0.202 +/- 0.013, max 0.450 +/- 0.032), and size almost unaffected by concentration. Drug fractional release did not exceed 8% after 48 h. The cytotoxicity studies against neuroblastoma cell lines outlined a significant growth inhibition effect of complexed ATRA with respect to free ATRA. These data suggest that the systems analyzed may be suitable carriers for parenteral administration of ATRA and other hydrophobic antitumor drugs, where the carriers are required to improve drug aqueous solubility and delay drug release almost after their accumulation in solid tumors.

Fenretinide-polyvinylalcohol conjugates: new systems allowing fenretinide intravenous administration.

N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) has been shown to be active toward many tumors without appreciable side effects. However its in vitro activity does not match a correspondent efficacy in vivo. The main reason is that the drug's hydrophobicity hinders its bioavailability in the body fluids. Even if the drug is previously dissolved in organic solvents, such as ethanol or DMSO, the subsequent dilution in body fluids trigger its precipitation in fine aggregates characterized by very low dissolution efficiency, never reaching amounts suitable for therapeutic response. To date no intravenous formulation of 4-HPR exists on the market. The 4-HPR linkage to a hydrophilic polymer by a covalent bond easily hydrolyzable in aqueous environment is expected to increase the drug's aqueous solubility, providing the free drug after hydrolysis of the covalent bond. This may be a useful tool for the preparation of aqueous intravenous formulations of 4-HPR. For this purpose, we linked 4-HPR to polyvinylalcohol (PVA) by a carbonate bond at different drug/hydroxy vinyl monomer molar ratios. We demonstrated that conjugation increased 4-HPR aqueous solubility and strongly inhibited neuroblastoma cell proliferation. In addition, in an in vivo neuroblastoma metastatic model, we obtained a significant antitumor effect as a consequence of the improved drug bioavailability.

Enhancement of oleyl alcohol anti tumor activity through complexation in polyvinylalcohol amphiphilic derivatives.

Oleyl alcohol was complexed with new amphiphilic polyvinylalcohol derivatives with the aim of increasing its aqueous solubility, thus improving bioavailability and favoring its antitumor activity. Water-soluble amphiphilic polymers were prepared by polyvinyl alcohol (PVA) substitution with oleyl chains through a succinyl spacer at 2% and 3% substitution degree. The complexes were obtained by spray-drying hydroalcoholic solutions of the substituted polymers and free oleyl alcohol at different weight ratios (3:1; 5:1; 10:1 w/w). The main physicochemical characteristics of the complexes were analyzed and correlated to the cytotoxic activity of oleyl alcohol toward tumor cell lines. The complexes strongly increased the aqueous solubility of oleyl alcohol and provided oleyl alcohol release in the presence of extractive conditions (simulating in vivo absorption). The complexes obtained by 10:1 polymer:fatty alcohol weight ratio offered higher release rates than the 5:1 and 3:1 ratios, respectively. Complexation also increased oleyl alcohol cytotoxicity toward tumor cells due to increased availability of the active molecule in the aqueous phase. Pure polymers were found to be biocompatible and no toxic effect was detected up to the highest concentration used in the present study (500 mu g/ml). The complexation of oleyl alcohol with the polymers analyzed here efficiently increased the availability of the fatty alcohol in aqueous environment. The enhanced cytotoxicity toward tumor cells of the complexed oleyl alcohol and the polymer biocompatibility make these amphiphilic PVA derivatives interesting candidates for soluble pharmaceutical formulations containing hydrophobic drugs whose therapeutic potential is often underestimated due to unsuitable levels of their aqueous solubilization.

Amphiphilic poly(vinyl alcohol) derivatives as complexing agents for fenretinide.

Poly(vinyl alcohol) (PVA) substituted with oleyl chains and tetraethyleneglycol monoethyl ether chains (TEGMEE) at 1.5% and 1% degrees of substitution respectively (mol of substituent to mol of hydroxyvinyl monomer) has previously been shown to self-assemble in water, providing aggregates selectively cytotoxic toward tumor cells vs normal cells. These polymers have also been shown to increase the long-term survival of nude mice injected with both human and murine neuroblastoma cell lines. In the present work, we changed the substitution degree of the oleyl chains on the poly(vinyl alcohol) backbone and maintained constant at 1% the degree of TEGMEE substitution. We evaluated the main physicochemical characteristics of the final polymers, their cytotoxicity toward tumor cells, and their complexing ability for hydrophobic molecules. The aim was to investigate the possibility of improving intrinsic antitumor efficacy of the polymer by changing the degree of oleyl chain substitution and further increase activity by complexation with antitumor drugs. The polymers were prepared at oleyl chain substitution degrees ranging from 0.5 to 3% (mol of substituent to mol of hydroxyvinyl monomer). The most active was again the 1.5% substituted polymer. It was further characterized by exhibiting the highest complexing ability toward hydrophobic molecules allowing the formation of a complex with fenretinide (HPR). The polymer-HPR complex was stable in aqueous environment and released the free drug prevalently in the presence of fluid hydrophobic phases. It was cytotoxic toward tumor cells with minimal activity toward normal cells. Antitumor activity exceeded that of the separate complex components resulting from the concomitant effect of the polymer and the HPR solubilized by complexation.

Preparation and evaluation of polyvinyl alcohol-co-oleylvinyl ether derivatives as tumor-specific cytotoxic systems.

A series of poly(vinyl alcohol) amphiphilic derivatives have been prepared to obtain polymeric aggregates in aqueous phase holding thermodynamic instability. The aim was to evaluate their ability to interact with tumor cells eliciting selective cytotoxicity. The poly(vinyl alcohol) derivatives were prepared by partial substitution of poly(vinyl alcohol) (MW 10 kDa) with both oleyl chains and poly(ethylene glycol) monoethyl ethers (PEGMEE) of different molecular weights. The substitution degree was 1.5% for the oleyl chains and 1% for the PEGMEE chains (moles of substituent per 100 mol of hydroxyvinyl monomer). The polyvinyl derivatives obtained easily dissolved in water. Dynamic and static light scattering measurements on the polymer aqueous solutions indicated the formation of polymeric aggregates characterized by low polydispersity (0.232-0.299) and mean size (218-382 nm) in the range suitable for intravenous administration. Moreover, they were characterized by different packing densities and thermodynamic instabilities driving the polymers to interact with hydrophobic membranes. Among the analyzed polymers, the poly(vinyl alcohol)-co-oleylvinyl ether substituted with triethylene glycol monoethyl ether (P10(4)) provided in solution the highest affinity for hydrophobic membranes. P10(4), moreover, was the most cytotoxic toward the tumor cell lines analyzed (neuroblastoma: SH-SY5Y, IMR-32, HTLA-230. melanoma: MZ2-MEL, RPMI7932.), while it did not appreciably alter the viability of the normal resting lymphocytes. The peculiar behavior of the P10(4) aggregates has been correlated to their high thermodynamic instability in solution due to the high packing density that triggers the polymeric aggregates to interact with hydrophobic membranes such as the tumor cell membranes, thus eliciting cytotoxicity.

Modified doxorubicin for improved encapsulation in PVA polymeric micelles.

Polyvinylalcohol, partially substituted with lipophilic acyl chains, generates polymeric micelles in aqueous phase, containing a hydrophobic core able to encapsulate lipophilic drugs. Two types of polymers were obtained by conjugation of polyvinylalcohol with oleoyl or linoleoyl chains as pendant groups. The polymers, at a substitution degree of approximately 1%, are soluble in water and form polymeric micelles whose size increases with polymer concentration. Doxorubicin was hydrophobized, by linking an oleoyl chain via amide bond, to make the drug more similar to the substituted polymers and promote its encapsulation into the inner core of the micelles. The properties of the drug-polymer systems were evaluated in solution by dynamic light scattering technique and correlated to the physicochemical characteristics of the drug and the substituted polymers. Solubilization tests revealed that the similarity of the chain, in both the polymer and the drug, promotes better drug encapsulation in the oleoyl than linoleoyl derivative. The drug-polymer systems are stable in phosphate buffer saline (pH 7.4) at 37 degrees C, and the release of the drug is activated by the presence of the proteolytic enzyme pronase-E. The enzyme activated drug release and the size of the polymeric micelles, compatible with the pore dimensions of the tumor vessels, make these systems interesting for targeting lipophilic drugs to solid tumors, where the proteolytic enzyme concentration strongly raises with respect to the other body compartments.

Modified polyvinylalcohol for encapsulation of all-trans-retinoic acid in polymeric micelles.

All-trans-retinoic acid (ATRA) is now included in many antitumor therapeutic schemes for the treatment of acute promyelocytic leukaemia, Kaposi's sarcoma, head and neck squamous cell carcinoma, ovarian carcinoma, bladder cancer and neuroblastoma. Unfortunately its poor aqueous solubility hampers its parenteral formulation. To date, there is no parenteral formulation of ATRA commercially available and oral administration of ATRA is associated with progressively diminishing ATRA levels in plasma, which is related to induction of retinoic acid-binding protein and increased drug catabolism by cytochrome P-450-mediated reaction. An ATRA formulation, obtained by complexation of the drug into polymeric micelles, might be suitable for parenteral administration overcoming these unwanted effects. To this purpose we prepared an amphiphilic polymer by polyvinylalcohol (PVA) substitution with oleyl amine at 1.5% substitution degree (mol substituent per 100 mol hydroxyvinylmonomer) and evaluated its functional properties with regard to ATRA complexation. The substituted polymer displayed ability to interact with ATRA both in aqueous solution and in the solid state following spray-drying of drug-polymer hydro-alcoholic solutions. The spray-dried complexes rapidly dissolved in water providing high levels of ATRA solubilization as a function of the drug-polymer weight ratio. The complexes characterized by 1:5 drug-polymer weight ratio provided higher levels of ATRA solubilization than 1:3 and 1:10 drug-polymer weight ratios respectively. Pre-formed polymeric micelles in water equilibrated in the presence of excess solid ATRA provided the lowest levels of solubilization. The drug release from the complexes was very slow in PBS, indicating their suitability in antitumor drug targeting where a fundamental requirement is stability towards drug release for at least 24 h, corresponding to the average circulation time period of macromolecular carriers. The cytotoxicity studies against neuroblastoma cell lines outlined increased cytotoxicity of complexed ATRA with respect to free ATRA, likely due to the increased bioavailability of the hydrophobic drug from the complex. We conclude that ATRA entrapped into self-assembling polymer micelles may be a useful parenteral ATRA formulation overcoming the unwanted pharmacological mechanism that lead to acquired retinoid resistance.

Immunoliposomal fenretinide: a novel antitumoral drug for human neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). Here, we investigated the antitumor activity of anti-GD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-immunoliposomes (anti-GD2-SIL) showed specific, competitive binding to, and uptake by, various NB cell lines. Moreover, anti-GD2-SIL-HPR presented increased selectivity and efficacy in inhibiting NB cell proliferation through the induction of apoptosis, compared to free drug and SL-HPR. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. However, similar, but significantly less potent antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 mAb alone (P=0.0297 and P=0.0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that, although anti-GD2 mAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only following treatment with anti-GD2-SIL-HPR (P<0.0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.

Anti-GD2 monoclonal antibody immunotherapy: a promising strategy in the prevention of neuroblastoma relapse.

In spite of the satisfactory frequency of clinical response to first-line therapy in neuroblastoma (NB), complete eradication of NB cells is rarely achieved. As a consequence, the majority of patients with advanced stage NB undergo relapse, which is often resistant to conventional treatment and rapidly overwhelming. Thus, after induction of the apparent remission, new therapeutic strategies are needed to completely eradicate the small number of surviving NB cells and to prevent relapse. We explored the potential of different doses of the anti-GD2 monoclonal antibody (mAb) 14G2a in an experimental metastatic model where a limited number of HTLA-230 human NB cells are injected i.v. into nude mice, leading to extensive metastases and death of animals within 7-8 weeks. Treatment with 14G2a mAb (1-4 mg/kg cumulative dose given as five i.v. daily administrations) dramatically reduced the metastatic spread of NB cells and prolonged the long-term survival of treated mice in a dose-dependent manner. Neither macrophages nor NK cells appeared to contribute to the protective effect of antibody treatment in vivo, suggesting either an involvement of granulocytes or a complement-mediated cytotoxicity towards NB cells. Whatever the effecting mechanism(s) involved, these results strongly support the clinical use of anti-GD2 mAbs after first-line induction regimens.

Inhibition of neuroblastoma-induced angiogenesis by fenretinide.

Retinoids are a class of natural or synthetic compounds that participate in the control of cell proliferation, differentiation and fetal development. The synthetic retinoid fenretinide (HPR) inhibits carcinogenesis in various animal models. Retinoids have also been suggested to be effective inhibitors of angiogenesis. The effects of HPR on certain endothelial cell functions were investigated in vitro, and its effects on angiogenesis was studied in vivo, by using the chorioallantoic membrane (CAM) assay. HPR inhibited vascular endothelial growth factor- (VEGF-) and fibroblast growth factor-2- (FGF-2)-induced endothelial cell proliferation without affecting endothelial motility; moreover, HPR inhibited growth factor-induced angiogenesis in the CAM assay. Furthermore, a significant antiangiogenic potential of HPR has also been observed in neuroblastoma (NB) biopsy-induced angiogenesis in vivo. We previously demonstrated that supernatants derived from NB cell lines stimulated endothelial cell proliferation. In the present study, we found that this effect was abolished when NB cells were incubated in the presence of HPR. VEGF- and FGF-2-specific ELISA assays, performed on both NB cells derived from conditioned medium and cellular extracts, indicated no consistent effect of HPR on the level of these angiogenic cytokines. Moreover, RT-PCR analysis of VEGF and FGF-2 gene expression confirmed the above lack of effect. HPR was also able to significantly repress the spontaneous growth of endothelial cells, requiring at least 48-72 hr of treatment with HPR, followed by a progressive accumulation of cells in G(1) at subsequent time points. Finally, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGF receptor 2 and FGF-2 receptor-2 was reduced after implantation of HPR-loaded sponges, as compared to control CAMs. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors.

Angiogenesis extent and expression of matrix metalloproteinase-2 and -9 correlate with progression in human neuroblastoma.

In human tumors changes in angiogenesis and expression of extracellular matrix-degrading enzymes occur simultaneously during invasion and metastasis. Tissues from 20 biopsies of human neuroblastoma (NB) were investigated immunohistochemically by using an antibody against factor VIII to determine their microvessel number, and by in situ hybridisation to determine the expression of mRNA of the matrix metalloproteinase-2 (MMP-2) and MMP-9. The extent of angiogenesis and the expression of the MMP-2 and MMP-9 mRNA were upregulated in advancing stages. These in situ data suggest that angiogenesis and degradation of extracellular matrix occur simultaneously with NB tumor progression.

Delivery of c-myb antisense oligodeoxynucleotides to human neuroblastoma cells via disialoganglioside GD(2)-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.

Autocrine regulation of volume-sensitive anion channels in airway epithelial cells by adenosine.

The activity of volume-sensitive Cl- channels was studied in human tracheal epithelial cells (9HTEo-) by taurine efflux experiments. The efflux elicited by a hypotonic shock was partially inhibited by adenosine receptor antagonists, by alpha,beta-methyleneadenosine 5'-diphosphate (alphabetaMeADP), an inhibitor of the 5'-ectonucleotidase, and by adenosine deaminase. On the other hand, dipyridamole, a nucleoside transporter inhibitor, increased the swelling-induced taurine efflux. Extracellular ATP and adenosine increased taurine efflux by potentiating the effect of hypotonic shock. alphabetaMeADP strongly inhibited the effect of extracellular ATP but not that of adenosine. These results suggest that anion channel activation involves the release of intracellular ATP, which is then degraded to adenosine by specific ectoenzymes. Adenosine then binds to purinergic receptors, causing the activation of the channels. To directly demonstrate ATP efflux, cells were loaded with [3H]AMP, and the release of radiolabeled molecules was analyzed by high performance liquid chromatography. During hypotonic shock, cell supernatants showed the presence of ATP, ADP, and adenosine. alphabetaMeADP inhibited adenosine formation and caused the appearance of AMP. Under hypotonic conditions, elevation of intracellular Ca2+ by ionomycin caused an increase of ATP and adenosine in the extracellular solution. Our results demonstrate that volume-sensitive anion channels are regulated with an autocrine mechanism involving swelling-induced ATP release and then hydrolysis to adenosine.

N-(4-hydroxyphenyl) retinamide is cytotoxic to melanoma cells in vitro through induction of programmed cell death.

Melanoma is a highly malignant and increasingly common tumour. Since metastatic melanoma remains incurable, new treatment approaches are needed. Previously, we reported that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (fenretinide, HPR) induces apoptosis in neuroblastoma cells, sharing a neuroectodermal origin with melanoma cells. Since no data exist thus far on the effects of HPR on human melanoma tumours, our purpose was to investigate the in vitro modulation of cell growth and apoptosis by HPR in melanoma cells. Ten human melanoma cell lines were exposed in vitro to increasing concentrations of HPR. Dose-dependent growth inhibition and cytotoxicity were observed. According to cytofluorimetric analysis, propidium iodide staining and TUNEL assay, HPR-treated melanoma cells were shown to undergo apoptosis. However, IC50 values ranged from 5 to 28 microM, while IC90 values were between 10 and 45 microM. These last concentrations are approximately 10-fold higher than those achievable in patients given oral HPR. To explore the potential of new delivery strategies, HPR was loaded at high concentrations into immunoliposomes directed to disialoganglioside GD2, a tumour-specific antigen extensively expressed by neuroectoderma-derived tumours. Treatment of melanoma cells for a short time (2 hr) with HPR-containing immunoliposomes followed by culture in drug-free medium gave rise to apoptosis of target cells, whereas cells treated for 2 hr with equivalent concentrations of the free drug survived. The efficacy of immunoliposomal HPR was strongly dependent on the density of GD2 expression in the different cell lines.

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 microM 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.

Induction of differentiation and apoptosis by interferon-gamma in human neuroblastoma cells in vitro as a dual and alternative early biological response.

Interferon-gamma (IFN-gamma) has a well known differentiation-promoting activity on several neuroblastoma (NB) cell lines and has also been reported to induce apoptosis in different cellular models. We have investigated the potential of IFN-gamma to trigger, besides differentiation, programmed cell death in NB cells and the relationship between these processes. Nine NB cell lines, characterized by different phenotypic and maturational features, were cultured in the presence of IFN-gamma (1000 IU/ml) for up to 5 days with either only one treatment at the start of the culture or renewing the culture medium (with or without IFN-gamma) every other day. Neuronal differentiation was assessed by evaluation of morphological changes and expression of mature cytoskeletal proteins, while apoptosis was evaluated at the desired times by fluorescent and electronic microscopy, DNA content analysis and DNA fragmentation assay. Our findings show that apoptosis is an early (mainly non post-differentiative) event and is much more evident following a single IFN-gamma administration. Moreover, IFN-gamma-triggered apoptosis is independent of the cellular phenotype (schwannian or neuronal) and appears to be mutually exclusive with respect to differentiation at the single cell level. Our results strengthen the potential of IFN-gamma as a promising therapeutic agent for NB.

Increase of metaiodobenzylguanidine uptake and intracellular half-life during differentiation of human neuroblastoma cells.

Iodine-labelled metaiodobenzylguanidine (MIBG) is a radiopharmaceutical used for diagnostic imaging and targeted radiotherapy of neuroendocrine tumors. We previously reported that the ability of a neuroblastoma (NB) cell line, LAN-5, to accumulate MIBG was powerfully stimulated by interferon-gamma (IFN-gamma), a well-known NB differentiation-promoting agent. To extend the above findings, we have investigated 5 NB cell lines for their ability to accumulate 125I-MIBG in basal conditions or after various combinations of differentiative stimuli. Our results show that association of IFN-gamma and tumor necrosis factor-alpha boosts MIBG uptake in the early times of incubation in LAN-5 and GI-LI-N cells, while both SK-N-SH and SK-N-BE(2)c cells are strongly stimulated by co-treatment with IFN-gamma and all-trans retinoic acid. Moreover, although only LAN-5 and GI-LI-N cells are sensitive to IFN-gamma alone, the combination of IFN-gamma and IFN-alpha causes a synergistic increase in MIBG uptake in all the NB cell lines tested. From experiments on MIBG release we conclude that no intracellular storage within specialized structures took place during differentiation. The observed enhancement in MIBG accumulation results from an increased uptake of the drug only. This conclusion was confirmed by analyzing MIBG-transporter gene expression, which was increased in cells subjected differentiative regimens. According to these findings, inducing differentiation of NB cells in vitro appears to improve their MIBG incorporation ability powerfully.

Differential effects of N-(4-hydroxyphenyl)retinamide and retinoic acid on neuroblastoma cells: apoptosis versus differentiation.

Retinoids exert various important biological effects in the control of normal growth, differentiation, and fetal development. While retinoic acid (RA) has entered clinical trials as a differentiation-promoting agent, it is only recently that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has been shown to be of potential clinical interest in cancer chemoprevention and treatment. Since thus far no data exist on the effects of HPR on neural crest cell-derived tumors, we have examined its in vitro effects on neuroblastoma (NB) cell lines and found that at relevant pharmacological concentrations it induces a dose-dependent growth inhibition. The antiproliferative effects of HPR were, in six of six cell lines tested, drastically more potent that those induced by an equimolar dose of RA. Time course growth analysis showed that HPR at 3 x 10(-6) M induces a very rapid (24-72 h) fall in thymidine uptake (> 90%), whereas at 3 x 10(-7) M it exhibits cytostatic effects. In contrast to RA, HPR did not show morphological changes typical of NB cell maturation nor did it induce the expression of any cytoskeletal protein associated with neuronal differentiation. DNA flow cytofluorimetric analysis revealed that HPR did not induce an arrest in a specific phase of the cell cycle while triggering apoptosis. This phenomenon was evidenced both by the visualization of "DNA ladders" on gel electrophoresis and by a quantitative assay for evaluating programmed cell death based upon the labeling of DNA breaks with tritiated thymidine. With the latter method, apoptotic cells were detectable as early as 3-6 h after treatment of NB cells with 10(-5) M HPR, while more than 50% of cells were apoptotic by 24-72 h following exposure to 3 x 10(-6) M HPR. In contrast, RA induced a low rate of apoptosis in NB cells only after 3-5 days. Time lapse photomicroscopy showed that NB cells treated with HPR underwent a death process highly reminiscent of apoptosis, with progressive condensation of the cytoplasm around the nucleus and intense cell shrinkage. The cells then rounded up and detached from the plate. Furthermore, propidium iodide staining of the DNA showed that a high proportion of cells treated with HPR displayed a small and brightly staining nucleus; chromatin appeared aggregated into dense masses in the nuclear periphery, a typical feature of apoptotic cells. In conclusion, our study demonstrates that contrary to the differentiation-promoting activity of RA, HPR dramatically suppresses NB cell growth by inducing programmed cell death.(ABSTRACT TRUNCATED AT 400 WORDS)