hTERT Transduction Extends the Lifespan of Primary Pediatric Low-Grade Glioma Cells While Preserving the Biological Response to NGF.

The neurotrophin nerve growth factor (NGF) modulates the growth of human gliomas and is able to induce cell differentiation through the engagement of tropomyosin receptor kinase A (TrkA) receptor, although the role played in controlling glioma survival has proved controversial. Unfortunately, the slow growth rate of low-grade gliomas (LGG) has made it difficult to investigate NGF effects on these tumors in preclinical models. In fact, patient-derived low-grade human astrocytoma cells duplicate only a limited number of times in culture before undergoing senescence. Nevertheless, replicative senescence can be counteracted by overexpression of hTERT, the catalytic subunit of telomerase, which potentially increases the proliferative potential of human cells without inducing cancer-associated changes. We have extended, by hTERT transduction, the proliferative in vitro potential of a human LGG cell line derived from a pediatric pilocytic astrocytoma (PA) surgical sample. Remarkably, the hTERT-transduced LGG cells showed a behavior similar to that of the parental line in terms of biological responses to NGF treatment, including molecular events associated with induction of NGF-related differentiation. Therefore, transduction of LGG cells with hTERT can provide a valid approach to increase the in vitro life-span of patient-derived astrocytoma primary cultures, characterized by a finite proliferative potential.

Preclinical evaluation of the novel 7-substituted camptothecin Namitecan (ST1968) in paediatric tumour models.

PURPOSE: The present study aimed to evaluate the new water soluble camptothecin analogue Namitecan (ST1968) in preclinical paediatric tumour models of the nervous system comprehensive of neuroblastoma, primitive neuroectodermal tumours/PNET and medulloblastoma where the drug was compared to Irinotecan. METHODS: Cellular sensitivity to the drug was assessed by MTT and clonogenic assays. Propidium iodide staining was used for cell cycle perturbation studies. The genotoxic effects were quantified by Comet assay, whereas apoptosis was assessed by PARP cleavage and sub-G1 accumulation. Tumour response was investigated in xenograft models in nude mice. RESULTS: The cellular response to Namitecan was heterogeneous with IC(50) (2 h) ranging between 0.14 and 13.26 µM, whereas SN38 (the active metabolite of Irinotecan) appeared more effective (IC(50): 0.03-11.7 µM). Interestingly, prolonged drug incubation times up to 72 h enhanced Namitecan cytotoxicity, with similar colony inhibition curves between the two analogues (IC(50), nM-SN38: 0.9 ± 0.2; Namitecan: 0.7 ± 0.4). DNA damage, accumulation in late-S/G2 phases and induction of apoptosis appeared important players of Namitecan cytotoxicity in our models. In vivo, Namitecan was superior to Irinotecan in three out of five xenograft models, with reversible weight loss (10 %). In the sensitive SK-N-AS xenograft, Namitecan showed a high retention in tumours consistently with: high antitumour response, rapid drug-mediated DNA damage (60 % mean TailDNA after 1 h from drug inoculation), persistent cell cycle perturbation (60-40 % G2 accumulation after 48-72 h, respectively) and apoptosis. Studies with Namitecan and platinum agents in this model showed a significant enhancement of antitumour activity of the drugs combination versus single agents. CONCLUSIONS: Our preclinical data strongly support the interest of further investigations on the well-tolerated Namitecan either as a single agent or in combination in paediatric oncology.

Enhanced cell cycle perturbation and apoptosis mediate the synergistic effects of ST1926 and ATRA in neuroblastoma preclinical models.

Retinoic acid therapy is nowadays an important component of treatment for residual disease of stage IV neuroblastoma after multimodal therapy. Nevertheless, arising resistance and treatment toxicity could represent relevant limiting factors. In the present study, we show that retinoic acid enhances the cytostatic and apoptogenic properties of the novel adamantyl retinoid ST1926 in a panel of neuroblastoma cells with different p53 status and caspase 8 expression, resulting in synergistic effects as assessed by Combination Index and Isobologram analysis. Under conditions where the two drugs alone produced no toxic effects, their combination resulted in enhanced G2-M arrest and sub-G1 population as shown by BrdU pulse-chase and labeling experiments. PARP cleavage, caspase 3, 8 and 9 activation and modulation of DR4 and FAS were indicative of enhanced apoptosis triggered by the co-incubation of the two drugs whereas neither ST1926-mediated genotoxic damage nor ATRA-differentiating effects were affected by the combined treatment. Caspase-3 and 8-mediated apoptosis appeared to play an important role in the drugs synergism. In fact, the addition of a pan-caspase inhibitor ZVAD-FMK reverted this effect in SK-N-DZ cells, and synergism was confined to limited drugs doses in HTLA cells not expressing caspase-8. Although not modulated, p53 appeared to enhance cells responsiveness to retinoid/ATRA combination. In vivo studies in the most sensitive neuroblastoma model SK-N-DZ, confirmed enhanced activity of the drugs combination vs single treatments. The study provides important lines of evidence that such a drugs combination could represent a less toxic and more effective approach for maintenance treatment in children with neuroblastoma.

Antitumor activity and pharmacokinetics of oral gimatecan on pediatric cancer xenografts.

PURPOSE: This study compared the antitumor activity and the pharmacological profile of gimatecan given orally and irinotecan (CPT-11) on pediatric tumor xenografts. EXPERIMENTAL DESIGN: Gimatecan was tested in two neuroblastoma cell lines (SK-N-DZ and SK-N-(BE)2c) and on TE-671 rhabdomyosarcoma cells using two different schedules. We characterized its pharmacokinetic profile in nude mice bearing human SK-N-DZ and TE-671 cell lines. RESULTS: Gimatecan appears to have high plasma disposition. The drug was present in plasma almost completely as the intact lactone form and showed substantial activity in all tumor models. Prolonged daily treatment with low doses of gimatecan produced significant tumor regression in all tumor xenografts. CONCLUSION: The antitumor activity and the promising pharmacological profile indicate gimatecan as an excellent candidate for clinical treatment of pediatric tumors.

Antitumor effect in medulloblastoma cells by gefitinib: Ectopic HER2 overexpression enhances gefitinib effects in vivo.

The effects of the epidermal growth factor receptor (EGFR) inhibitor gefitinib on cell growth and signaling were evaluated in three medulloblastoma (MB) cell lines (D283, D341, Daoy), one supratentorial primitive neuroectodermal tumor cell line (PFSK), and four MB primary cultures. Cell lines showed diverse expression of EGFR and human epidermal receptor 2 (HER2), with high levels of constitutively activated HER2 in the HER2-overexpressing D341 and D283 cells. Gefitinib sensitivity varied across lines and was not related to expression of HER receptors or receptor baseline activation. Gefitinib induced G(0)/G(1) arrest in all lines, whereas apoptosis was dose-dependently induced only in D283 and D341 cells. The molecular response to gefitinib was investigated in Daoy and D341 lines, which showed a higher (half-maximal inhibitory concentration [IC(50)], 3.8 microM) and lower (IC(50), 6.6 microM) sensitivity to the agent, respectively. Gefitinib inhibited constitutive and EGF-triggered EGFR phosphorylation in both lines but was ineffective in constitutive activation of HER2 in D341 cells. Phosphorylated AKT inhibition paralleled that of phosphorylated EGFR, suggesting the presence of an autocrine gefitinib-sensitive EGFR/AKT pathway. On the whole, EGF-dependent signaling was less responsive to ligand stimulation and gefitinib inhibition in D341 cells, which correlated with the lower sensitivity to gefitinib's antiproliferative effect of this line. In vivo, the growth of D341 and Daoy xenografts treated with gefitinib at 150 mg/kg per day was inhibited by approximately 50%. Ectopically overexpressed HER2 in Daoy cells significantly increased sensitivity to gefitinib's antitumor effects in vivo (tumor volume inhibition = 78%). Our data indicate that gefitinib might be a molecularly targeted agent for the treatment of MB.

The cellular response to PPARgamma ligands is related to the phenotype of neuroblastoma cell lines.

Neuroblastoma (NB) is a phenotypically heterogeneous tumor, displaying cells of neuronal, melanocytic, or glial/schwannian lineage. This cellular heterogeneity is also present in vitro, where cells of neuroblastic (N)- or stromal (S)-type may be identified. Ligands of peroxisome proliferator-activated receptor gamma (PPARgamma) have been shown to inhibit growth in different tumor cell lines. The purpose of this study was to determine PPARgamma expression and the response to its ligands in NB cell lines with different phenotypes. We used eight NB cell lines with N-, mixed, and S-phenotype. PPARgamma expression was found in all NB cell lines, regardless of their phenotype. Mutational analysis and transactivation assays showed that PPARgamma is not mutated and remains functional in NB cells. Two PPARgamma ligands, 15-deoxy-delta12,14-prostaglandin J2 (PGJ2) and rosiglitazone, inhibited growth of all cell lines, with PGJ, being the most potent agent. PGJ2, but not rosiglitazone, induced arrest of the cells in the G2/M phase as well as apoptosis. The sensitivity to the two ligands appeared to be more related to the phenotype than PPARgamma expression, with the S-type cells being less sensitive than the N-type, partly because of their lower capability of undergoing apoptosis. No synergistic effect on growth inhibition was observed when all cell lines were co-treated with 9-cis retinoic acid (9-cis RA) and rosiglitazone. Our data indicate that PPARgamma expression and function are maintained in phenotipically different NB cell lines. Activation of PPARgamma by its synthetic ligands might have a therapeutic role in advanced NB.