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

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Sodium ascorbate induces apoptosis in neuroblastoma cell lines. Cells were seeded in six well plates and treated for 6 or 24 hours with increasing concentrations of sodium ascorbate. For each bar group, the concentration was: 0 mM (control), 0.5 mM, 1 mM, 2 mM, 3 mM. Cells were then processed and stained with propidium iodide as described in "Materials and Methods". The percent of cells in the sub-G₁ phase of the cell cycle were considered apoptotic. The data are the mean ± S.D. from three independent experiments.

Figure 2

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

Figure 3

Reduction of intracellular iron levels by sodium ascorbate. 30 × 10⁶ of either HTLA-230 or SH-SY5Y were treated with the indicated doses of sodium ascorbate 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.

Figure 4

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. 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.

Figure 5

Effect of FAC on sodium ascorbate-induced apoptosis. HTLA-230 were grown in either complete medium alone (control cells), or medium containing 2 mM 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).

Figure 6

Effect of vitamin C on caspases activation. HTLA-230 and SH-SY5Y were treated with the indicated concentrations for 12 and 24 hours. After detachment, cells were stained with the pan-caspase-fluorescent-probe z-VAD-FMK according to the manufactures instructions. Relative increases in fluorescence emission were detected by single color flow cytometry. The data are the mean ± S.D. from three independent experiments.

Figure 7

Effect of sodium ascorbate on single caspases activation. HTLA-230 and SH-SY5Y were incubated in the absence (0 mM) or presence of the indicated concentrations for 16 hours and then analyzed for single caspases by flow cytometry as described in Material and Method. Coloums, mean of three different experiments, each done in triplicate; bars, SD.