Epigallocatechin gallate inhibits sphere formation of neuroblastoma BE(2)-C cells

Abstract

Objectives: A growing number of epidemiological studies have demonstrated that the consumption of green tea inhibits the growth of a variety of cancers. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, has been shown to have an anti-cancer effect against many cancers. Most cancers are believed to be initiated from and maintained by a small population of tumor-initiating cells (TICs) that are responsible for chemotherapeutic resistance and tumor relapse. In neuroblastoma, an aggressive pediatric tumor that often relapses and has a poor prognosis, TICs were recently identified as spheres grown in a serum-free non-adherent culture used for neural crest stem cell growth. Although EGCG has been reported to induce growth arrest and apoptosis in neuroblastoma cells, its effect on neuroblastoma TICs remains to be defined.

Methods: Gene expression was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). The effects of EGCG on cell proliferation, apoptosis, and sphere formation were determined by cell counting, propidium iodide staining, and sphere (>100 μm in diameter) counting, respectively.

Results: Neuroblastoma BE(2)-C cells showed increased expression of stem cell markers (nanog homeobox [NANOG] and octamer-binding transcription factor 4 [OCT4]), as well as decreased expression of neuronal differentiation markers (Cu(2+)-transporting ATPase alpha polypeptide [ATP7A] and dickkopf homolog 2 [DKK2]) in spheres grown in serum-free non-adherent culture, compared to parental cells grown in conventional culture. Although EGCG induced growth arrest and apoptosis in the parental cells in a dose-dependent manner, it was not effective against spheres. However, EGCG potently inhibited sphere formation in the BE(2)-C cells.

Conclusions: The present results suggest that EGCG may inhibit the development of TICs in BE(2)-C cells.

Fig. 1

Spheres from BE(2)-C cells. a BE(2)-C cells were cultured either in a 10% serum adherent condition for 3 days (parental cells) or in a serum-free non-adherent condition over 4 weeks (spheres) and examined by phase-contrast microscopy. The images shown are representative of three independent experiments. Scale bars show 100 μm. b Total RNA was prepared from parental cells and spheres. The relative mRNA expression of nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT4), Cu²⁺-transporting ATPase alpha polypeptide (ATP7A), and dickkopf homolog 2 (DKK2) to phosphoglycerate kinase 1 (PGK1) was analyzed by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). The mean expression in parental cells was set at 1. The data shown are means ± SD of three independent experiments

Fig. 2

Effect of epigallocatechin gallate (EGCG) on cell proliferation and apoptosis of BE(2)-C cells. a Parental cells and spheres were seeded into a 24-well plate and treated with the indicated concentrations of EGCG. After 48 h, the number of cells was counted manually. Cell proliferation was defined as the percentage of cells in each sample in relation to that in a control sample (without EGCG). The data shown are means ± SD of three independent experiments. b Parental cells and spheres were seeded into a 6-well plate, treated with the indicated concentrations of EGCG for 48 h, and stained with propidium iodide (PI). Apoptosis was defined as the percentage of PI-positive cells in each sample analyzed by flow cytometry. The data shown are means ± SD of three independent experiments

Fig. 3

Effect of EGCG on sphere formation in BE(2)-C cells. Parental cells were seeded into a 96-well plate, cultured in sphere medium (SM) containing the indicated concentrations of EGCG for 14–17 days, and examined by bright-field microscopy. The number of spheres (>100 μM in diameter) was counted manually. Sphere formation was defined as the percentage of spheres in each sample in relation to that in a control sample (without EGCG). The data shown are means ± SD of three independent experiments