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

<p>Abstract</p> <p>Background</p> <p>Neuroblastoma (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.</p> <p>Results</p> <p>We 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.</p> <p>Conclusion</p> <p>Sodium 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.</p

Membrane expression of TfR (CD71) by neuroblastoma cell lines and its modulation by sodium ascorbate

<p><b>Copyright information:</b></p><p>Taken from "Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake"</p><p>http://www.molecular-cancer.com/content/6/1/55</p><p>Molecular Cancer 2007;6():55-55.</p><p>Published online 30 Aug 2007</p><p>PMCID:PMC2000471.</p><p></p> Cells were seeded in six well plates and treated for 6 and 24 hours with increasing concentrations of sodium ascorbate. For each bar group, the concentration was: 0 (control), 0.5 mM, 1 mM, 2 mM and 3 mM. Cells were than washed and incubated with a FITC-coniugated mouse monoclonal antibody specific to human CD71 and analyzed by flow cytometry. The data are the mean ± S.D. from three independent experiments, each in triplicate

Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake-2

<p><b>Copyright information:</b></p><p>Taken from "Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake"</p><p>http://www.molecular-cancer.com/content/6/1/55</p><p>Molecular Cancer 2007;6():55-55.</p><p>Published online 30 Aug 2007</p><p>PMCID:PMC2000471.</p><p></p> for 24 hours and iron levels were measured in cell lysates. After terminating the incubation, cells were collected by scraping and washed three times with PBS and then lysed in specific buffer. Iron levels were analyzed with a Cobas Integra 800 system as described under "Material and Method". Values indicated are the mean ± S.D. of three separate experiments

Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake-4

<p><b>Copyright information:</b></p><p>Taken from "Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake"</p><p>http://www.molecular-cancer.com/content/6/1/55</p><p>Molecular Cancer 2007;6():55-55.</p><p>Published online 30 Aug 2007</p><p>PMCID:PMC2000471.</p><p></p>of sodium ascorbate (in the presence or absence of FAC 70 μg/ml). After 16 hours, cells detached in the medium were collected by centrifugation, resuspended and incubated with AnnexinV-FITC. Apoptosis was quantified as increased green fluorescence by flow cytometry. The data are the mean ± S.D. from four independent experiments. Statistical analysis was done by Student's test (*, p < 0.005)

Representative example of cytofluorimetric analysis showing the effect of vitamin C alone or in combination with FAC, on the mitochondrial membrane potential of SH-SY5Y (panel A) and HTLA-230 (panel B) stained with JC-1 probe

<p><b>Copyright information:</b></p><p>Taken from "Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake"</p><p>Molecular Cancer 2007;6():55-55.</p><p>Published online 30 Aug 2007</p><p>PMCID:PMC2000471.</p><p></p> In abscissa FL-1 (green fluorescence); in ordinate FL-2 (red fluorescence). Numbers represent the percentage of non-apoptotic red fluorescent cells (R1, R3) and that of apoptotic green fluorescent cells (R2, R4). Sodium ascorbate induces a marked increase in apoptosis which is fully prevented by treatment with FAC. SH-SY5Y and HTLA-230 were incubate in complete medium in the absence (A1, B1) or in presence of 1 mM sodium ascorbate (A2, B2) and 2 mM sodium ascorbate (A4, B4) for 16 hours. Cells were pretreated (A3, A5, B3, B5) with 70 μg/ml of FAC for 3 hours. Comparable results were obtained in three independent experiments

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

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

Dual-Targeting Nanoparticles for In Vivo

Ovarian cancer is the most lethal gynecologic cancer. Claudin-3 and-4, the receptors for Clostridium Perfringens Enterotoxin (CPE), are overexpressed in over 70% of these tumors. Here we synthesized and characterized poly(lactic-co-glycolic-acid) (PLGA) nanoparticles (NP) modified with the carboxi-terminal binding domain of CPE (c-CPE-NP) for the delivery of suicide gene therapy to chemotherapy-resistant ovarian cancer cells. As a therapeutic payload we generated a plasmid encoding for the Diphteria Toxin subunit-A (DT-A) under the transcriptional control of the p16 promoter, a gene highly differentially expressed in ovarian cancer cells. Flow cytometry and immunofluorescence demonstrated that c-CPE-NPs encapsulating the CMV GFP plasmid (CMV GFP c-CPE-NP) were significantly more efficient than control NP modified with a scrambled peptide (CMV GFP scr-NP) in transfecting primary chemotherapy-resistant ovarian tumor cell lines in vitro (p=0.03). Importantly, c-CPE-NPs encapsulating the p16 DT-A vector (p16 DT-A c-CPE-NP) were significantly more effective than control p16 DT-A scr-NP in inducing ovarian cancer cell death in vitro (% cytotoxicity: mean ± STDV = 32.9 ± 0.15 and 7.45 ± 7.93, respectively, p=0.03). In vivo bio-distribution studies demonstrated efficient transfection of tumor cells within 12 hours after intraperitoneal (IP) injection of CMV GFP c-CPE-NP in mice harboring chemotherapy-resistant ovarian cancer xenografts. Finally, multiple IP injections of p16 DT-A c-CPE-NP resulted in a significant inhibition of tumor growth compared to control NP in chemotherapy-resistant tumor-bearing mice (p=0.041). p16 DT-A c-CPE-NP may represent a novel dual-targeting therapeutic approach for the selective delivery of gene therapy to chemotherapy-resistant ovarian cancer cells