Nutr Res Pract.  2010 Aug;4(4):276-282.

Effect of retinoic acid and delta-like 1 homologue (DLK1) on differentiation in neuroblastoma

• Kim Y

Abstract

The principal objective of this study was to evaluate the chemopreventive and therapeutic effects of a combination of all-trans-retinoic acid (RA) and knockdown of delta-like 1 homologue (Drosophila) (DLK1) on neuroblastoma, the most common malignant disease in children. As unfavorable neuroblastoma is poorly differentiated, neuroblastoma cell was induced differentiation by RA or DLK1 knockdown. Neuroblastoma cells showed elongated neurite growth, a hallmark of neuronal differentiation at various doses of RA, as well as by DLK1 knockdown. In order to determine whether or not a combination of RA and DLK1 knockdown exerts a greater chemotherapeutic effect on neuroblastoma, cells were incubated at 10 nM RA after being transfected with SiRNA-DLK1. Neuronal differentiation was increased more by a combination of RA and DLK1 knockdown than by single treatment. Additionally, in order to assess the signal pathway of neuroblastoma differentiation induced by RA and DLK1 knockdown, treatment with the specific MEK/ERK inhibitors, U0126 and PD 98059, was applied to differentiated neuroblastoma cells. Differentiation induced by RA and DLK1 knockdown increased ERK phosphorylation. The MEK/ERK inhibitor U0126 completely inhibited neuronal differentiation induced by both RA and DLK1 knockdown, whereas PD98059 partially blocked neuronal differentiation. After the withdrawal of inhibitors, cellular differentiation was fully recovered. This study is, to the best of our knowledge, the first to demonstrate that the specific inhibitors of the MEK/ERK pathway, U0126 and PD98059, exert differential effects on the ERK phosphorylation induced by RA or DLK1 knockdown. Based on the observations of this study, it can be concluded that a combination of RA and DLK1 knockdown increases neuronal differentiation for the control of the malignant growth of human neuroblastomas, and also that both MEK1 and MEK2 are required for the differentiation induced by RA and DLK1 knockdown.

Keyword

Retinoic acid; DLK1; differentiation; neuroblastoma

MeSH Terms

Butadienes
Child
Flavonoids
Humans
Neurites
Neuroblastoma
Neurons
Nitriles
Phosphorylation
Signal Transduction
Tretinoin
Butadienes
Flavonoids
Nitriles
Tretinoin

Figure

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  Fig. 1 All-trans-retinoic acid (RA) induces neuronal differentiation in neuroblastoma and down-regulates stem cell markers (CD133, SOX2, and DLK1). A. BE(2)C cells were treated without (w/o) or with RA (10 nM, 100 nM, or 1 µM) for 5 d. β-Tubulin III expression was detected by immunofluorescence. Magnification = ×100. B. BE(2)C cells were treated without (-) or with 1µM RA (+) for 5 d. CD133, SOX2, and DLK1 expression were detected by immunofluorescence. Magnification = ×100

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  Fig. 2 Neuronal differentiation was increased by a combination of RA and DLK1 knockdown. BE(2)C cells were transfected with DLK1 SiRNA (05), SiRNA (07), or a nontargetting SiRNA (Ctrl). Cells were treated without (w/o) or with 10 nM RA for 3 d. A. β-Tubulin III expression was detected by immunofluorescence. B. DLK1 expression was detected by immunofluorescence.

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  Fig. 3 MAPK inhibitors block ERK phosphorylation induced by RA. BE(2)C cells were treated at various concentrations (0, 1 µM, 10 µM) of RA. Cells were treated with MEK/ERK inhibitors, U0126 (5 µM) and PD98059 (PD, 5 µM) on the next day, and then maintained for 3 d. ERK phosphorylation was analyzed by Western Blotting. β-actin was used as a loading control.

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  Fig. 4 MAPK inhibitors, U0126 or PD 98059 (PD) blocks neuronal differentiation induced by RA. Cells were treated without (w/o) or with RA (1 nM, 10 nM) and then treated with 5 µM of U0126 (A) or PD (B) on the next day. The cells were maintained for 3 d. Cell morphology was examined by phase contrast microscopy. Magnification = 100×

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  Fig. 5 MAPK inhibitors, U0126 or PD 98059 (PD) blocks neuronal differentiation induced by DLK1 knockdown. BE(2)C cells were infected with control or ShRNA-DLK1 and then treated with various concentrations (0, 2.5 µM, 5 µM, 10 µM) of U0126 (A) or PD (B) next day. Cells were maintained for 3 d. Cell morphology was examined by phase contrast microscopy. Magnification = 100×

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  Fig. 6 Neuronal differentiation induced by DLK1 knockdown was recovered after withdrawal of MAPK inhibitors. BE(2)C cells were infected with control or ShRNA-DLK1. Cells were treated with 5 µM U0126 (A) or PD (B) next day and maintained for 5 d (middle panel). After 3 days of treatment, U0126 or PD was withdrawn and cells were maintained for 2 d. Cell morphology was examined by phase contrast microscopy. Magnification = 100×

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