Nutr Res Pract.  2010 Dec;4(6):455-461.

The effects of nutrient depleted microenvironments and delta-like 1 homologue (DLK1) on apoptosis in neuroblastoma

• Kim Y

Abstract

The tumor microenvironment, particularly sufficient nutrition and oxygen supply, is important for tumor cell survival. Nutrition deprivation causes cancer cell death. Since apoptosis is a major mechanism of neuronal loss, we explored neuronal apoptosis in various microenvironment conditions employing neuroblastoma (NB) cells. To investigate the effects of tumor malignancy and differentiation on apoptosis, the cells were exposed to poor microenvironments characterized as serum-free, low-glucose, and hypoxia. Incubation of the cells in serum-free and low-glucose environments significantly increased apoptosis in less malignant and more differentiated N-type IMR32 cells, whereas more malignant and less differentiated I-type BE(2)C cells were not affected by those treatments. In contrast, hypoxia (1% O2) did not affect apoptosis despite cell malignancy. It is suggested that DLK1 constitutes an important stem cell pathway for regulating self-renewal, clonogenicity, and tumorigenicity. This raises questions about the role of DLK1 in the cellular resistance of cancer cells under poor microenvironments, which cancer cells normally encounter. In the present study, DLK1 overexpression resulted in marked protection from apoptosis induced by nutrient deprivation. This in vitro model demonstrated that increasing severity of nutrition deprivation and knock-down of DLK1 caused greater apoptotic death, which could be a useful strategy for targeted therapies in fighting NB as well as for evaluating how nutrient deprived cells respond to therapeutic manipulation.

Keyword

Neuroblastoma; nutrient deprivation; apoptosis; DLK1

MeSH Terms

Anoxia
Apoptosis
Cell Death
Cell Survival
Neuroblastoma
Neurons
Oxygen
Stem Cells
Tumor Microenvironment
Oxygen

Figure

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  Fig. 1 Propidium iodide (PI) and Hoechst 33342 (Hoechst) staining demonstrate the appearance of nuclei with condensed chromatin in NB cells. BE(2)C cells (A) and IMR32 cells (B) were maintained with (left panels) or without (right panels) serum in media for 48 hr and then stained with PI (red) and Hoechst (blue) to visualize nuclear morphological changes (arrow). Both PI-and Hoechst-positive cells were counted as apoptotic cells. Images were detected by a fluorescence microscope. Magnification = ×100.

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  Fig. 2 More malignant I-type cells showed less apoptosis induced by serum deprivation. More advanced and less differentiated I-type BE(2)C cells and less advanced and more differentiated IMR32 cells were incubated at different concentrations of serum (10%, 1%, or 0%). After 48 hours, the cells were fixed and double stained with PI and Hoechst to evaluate apoptosis. Apoptotic cells with compromised membranes were positive for PI staining. Both PI- and Hoechst-positive cells were counted as apoptotic cells. A total of ≥ 300 cells were counted in 3-4 random fields. Bars are means ± SE. Different letters for given bars indicate the values are significantly different from each other (P < 0.05) (Tukey's Post Hoc test after one-way ANOVA).

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  Fig. 3 The effect of tumor microenvironment (nutrient deprivation and hypoxia) on apoptosis. (A) BE(2)C and IMR32 cells were incubated at different combinations of nutrient deprivation (S+G, S+LG, SF+G, and SF+LG) for 48 hr. (B) BE(2)C and IMR32 cells were incubated under combination of hypoxia and serum deprivation (N+S, N+SF, H+S, and H+SF) for 48 hr. Determination of apotosis was performed as described in Fig. 2. A total of ≥ 300 cells were counted in 3-4 random fields. Bars are means ± SE. Different letters for given bars indicate the values are significant different from each other (P < 0.05) (Tukey's Post Hoc test after one-way ANOVA). S, 10% FBS (v.v); SF, serum-free; G, 25 mM of glucose; LG, low-glucose (25 µM); N, normoxia (21% O2); H, hypoxia (1% O2)

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  Fig. 4 DLK1 ameliorated nutrient depletion-induced apoptosis. (A) IMR32 cells were infected with full length DLK1 (DLK1-FL). Western blotting was performed to verify the over-expression of DLK1. β-actin was used as a loading control. (B) DLK1 over-expressed IMR32 cells were shifted to various conditions of media and maintained for 48h. The determination of apotosis was performed as described in Fig. 2. A total of ≥ 300 cells were counted in 3-4 random fields. Bars are means ± SE. *Statistically different, as compared with control (Student's t-test) (P < 0.05). 10%, 10% of serum (v.v); 0.2%, 0.2% of serum (v.v); G, 25 mM of glucose; LG, low-glucose (25 µM)

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  Fig. 5 DLK1 knock-down increased nutrient depletion-induced apoptosis. (A) BE(2)C cells were transfected with siDLK1 oligonucleotides (siDLK1-05 or siDLK1-07). Knockdown of DLK1 expression by siDLK1-05 or -07 was confirmed by Western blotting. β-actin was used as a loading control. (B) After transfection with siDLK1 oligonucletides, cells were incubated with nutrient depletion conditions, either serum-free or low-glucose (25 µM of glucose). The determination of apotosis was performed as described in Fig. 2. A total of ≥ 300 cells were counted in 3-4 random fields. Bars are the mean ± SE. Different letters for given bars indicate values are significantly different from each other (P < 0.05) (Tukey's Post Hoc test after one-way ANOVA)

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