R. Carosio

Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake

BackgroundNeuroblastoma (NB) is an extra-cranial solid tumour of childhood. In spite of the good clinical response to first-line therapy, complete eradication of NB cells is rarely achieved. Thus, new therapeutic strategies are needed to eradicate surviving NB cells and prevent relapse. Sodium ascorbate has been recently reported to induce apoptosis of B16 melanoma cells through down-regulation of the transferrin receptor, CD71. Since NB and melanoma share the same embryologic neuroectodermal origin, we used different human NB cell lines to assess whether the same findings occurred.ResultsWe could observe dose- and time-dependent induction of apoptosis in all NB cell lines. Sodium ascorbate decreased the expression of CD71 and caused cell death within 24 h. An increase in the global and specific caspase activity took place, as well as an early loss of the mitochondrial transmembrane potential. Moreover, intracellular iron was significantly decreased after exposure to sodium ascorbate. Apoptotic markers were reverted when the cells were pretreated with the iron donor ferric ammonium citrate (FAC), further confirming that iron depletion is responsible for the ascorbate-induced cell death in NB cells.ConclusionSodium ascorbate is highly toxic to neuroblastoma cell lines and the specific mechanism of vitamin C-induced apoptosis is due to a perturbation of intracellular iron levels ensuing TfR-downregulation.

Neuroblastoma-targeted Nanoparticles Entrapping siRNA Specifically Knockdown ALK

RNA interference molecules have some advantages as cancer therapeutics, including a proved efficacy on both wild-type (WT) and mutated transcripts and an extremely high sequence-specificity. The most significant hurdle to be overcome if exogenous small interfering RNAs (siRNA) is to be used therapeutically is the specific, effective, nontoxic delivery of siRNA to its intracellular site of action. At present, human applications are confined almost exclusively to targets within the liver, where the delivery systems naturally accumulate, and extra-hepatic targets remain a challenge. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that has recently been shown to contribute to the cell growth and progression of human neuroblastoma (NB). We investigated its potential as a therapeutic target in NB by generating anti-GD₂-targeted nanoparticles that carry ALK-directed siRNA, which are specifically and efficiently delivered to GD₂-expressing NB cells. Relative to free ALK-siRNA, anti-GD₂-targeted liposomal formulations of ALK-siRNA had low plasma clearance, increased siRNA stability, and improved binding, uptake, silencing and induction of cell death, and specificity for NB cells. In NB xenografts, intravenous (i.v.) injection of the targeted ALK-siRNA liposomes showed gene-specific antitumor activity with no side effects. ALK-selective siRNA entrapped in anti-GD₂-targeted nanoparticles is a promising new modality for NB treatment.

The Combined Therapeutic Effects of Bortezomib and Fenretinide on Neuroblastoma Cells Involve Endoplasmic Reticulum Stress Response

Purpose: The proteasome inhibitor bortezomib inhibited cell growth and angiogenesis in neuroblastoma. Bortezomib has been shown to induce synergistic activity when combined with other antineoplastic agents. Here we have investigated the antitumor activity of bortezomib in combination with fenretinide, a synthetic retinoid, against neuroblastoma cells. Experimental Design: Different neuroblastoma cell lines were tested for sensitivity to bortezomib and fenretinide, given alone or in different dose-dependent and time-dependent combination schedules. Cell proliferation, cell viability, and apoptosis were evaluated by measuring 3H-thymidine incorporation, trypan blue staining, DNA fragmentation, and western blot analysis. Angiogenesis was assessed by the chick embryo chorioallantoic membrane assay. An orthotopic neuroblastoma mouse model was used to examine in vivo sensitivity. Results: Each compound alone was able to induce a dose-dependent inhibition of cell proliferation, with a significant enhanced antiproliferative effect for the drugs used in combination. This inhibition was characterized by marked G2-M and G1 cell cycle arrest with nearly complete depletion of S phase. Bortezomib and fenretinide in association triggered an increased apoptosis through activation of specific genes of the endoplasmic reticulum stress compared with either drug tested alone. Tumor-bearing mice treated with bortezomib plus fenretinide lived statistically significantly longer than mice treated with each drug alone. Histologic evaluation and chorioallantoic membrane analysis of primary tumors showed that the combined therapeutic activity of bortezomib and fenretinide rested upon antitumor and antiangiogenic mechanisms. Conclusions: These findings provide the rationale for the development of a new therapeutic strategy for neuroblastoma based on this pharmacologic combination.

Selective Therapeutic Targeting of the Anaplastic Lymphoma Kinase With Liposomal siRNA Induces Apoptosis and Inhibits Angiogenesis in Neuroblastoma.

The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is involved in the pathogenesis of different types of human cancers, including neuroblastoma (NB). In NB, ALK overexpression, or point mutations, are associated with poor prognosis and advanced stage disease. Inhibition of ALK kinase activity by small-molecule inhibitors in lung cancers carrying ALK translocations has shown therapeutic potential. However, secondary mutations may occur that, generate tumor resistance to ALK inhibitors. To overcome resistance to ALK inhibitors in NB, we adopted an alternative RNA interference (RNAi)-based therapeutic strategy that is able to knockdown ALK, regardless of its genetic status [mutated, amplified, wild-type (WT)]. NB cell lines, transduced by lentiviral short hairpin RNA (shRNA), showed reduced proliferation and increased apoptosis when ALK was knocked down. In mice, a nanodelivery system for ALK-specific small interfering RNA (siRNA), based on the conjugation of antibodies directed against the NB-selective marker GD(2) to liposomes, showed strong ALK knockdown in vivo in NB cells, which resulted in cell growth arrest, apoptosis, and prolonged survival. ALK knockdown was associated with marked reductions in vascular endothelial growth factor (VEGF) secretion, blood vessel density, and matrix metalloproteinases (MMPs) expression in vivo, suggesting a role for ALK in NB-induced neoangiogenesis and tumor invasion, confirming this gene as a fundamental oncogene in NB.

The use of the orthotopic model to validate antivascular therapies for cancer

The orthotopic model reproduces aspects of the tumour microenvironment and emulates a number of important biological features of cancer progression, angiogenesis, metastasis and resistance. Due to its parallels with human cancer, the model can be used to evaluate therapeutic responses to various therapies. This review outlines the importance of using the orthotopic implantation of tumour cells in mice models for evaluating the effectiveness of antivascular therapies.

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.

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 ∼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°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.

In vitro and In vivo Antitumor Activity of the Novel Derivatized Polyvinyl Alcohol-Based Polymer P10(4)

Purpose: The major limitation to successful chemotherapy of neuroblastoma is the toxicity of traditional antitumor drugs. Hence, less toxic and more effective drugs are to be found, and novel formulations of conventional compounds allowing a more favorable biodistribution should be sought for. In an attempt to pursue this task, we recently synthesized an amphiphilic polymer based on a polyvinyl alcohol backbone [P10(4)]. Experimental Design: The cytotoxic activity of P10(4) was evaluated both in vitro on neuroblastoma and melanoma cell lines and in vivo in pseudometastatic neuroblastoma models. Apoptosis was assessed by morphology, cytofluorimetric analysis of DNA content, and DNA fragmentation assay. Caspases activation was investigated by kits specific for caspase-1, caspase-2, caspase-3, caspase-4, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, and caspase-13. Colony formation was evaluated by soft agar assay. Results: P10(4) exerted a potent cytotoxic activity on different neuroblastoma and melanoma cell lines through induction of both extrinsic and intrinsic caspase cascades and subsequent apoptosis. Moreover, the clonogenic potential of cells that survived P10(4) treatment was strongly reduced. Next, we tested the effects of P10(4) in nude mice injected with both a human and a murine neuroblastoma cell lines i.v. P10(4) significantly increased the life span and the long-term survival of treated mice over controls. No side effects were observed, even at doses higher than those used for therapeutic purposes. Conclusions: Our data suggest that P10(4) holds promise as an anticancer compound and, because of its lack of interaction with DNA, is unlikely to give rise to drug resistance.

Enhanced anti-neuroblastoma activity of a fenretinide complexed form after intravenous administration

Objectives  The major limitation to successful chemotherapy of neuroblastoma (NB) is the toxicity and the poor bioavailability of traditional drugs.

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 μ 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.