Propolis Inhibits Neurite Outgrowth in Differentiating SH-SY5Y Human Neuroblastoma Cells

Abstract

Propolis is a multicomponent, active, complex resinous substance collected by honeybees from a variety of plant sources. We have studied the effect of propolis on neurite outgrowth of SH-SY5Y human neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). Propolis, at a concentration of 3 μg/mL, had no significant effect on the viability of differentiating SH-SY5Y cells. However, the neurite outgrowth of the differentiating SH-SY5Y cells treated with propolis (0.3~3 μg/mL) for 48 hr was significantly inhibited in a dose-dependent manner. Treatment of RA-stimulated differentiating SH-SY5Y cells with 0.3 to 3 μg/mL propolis resulted in decreased level of transglutaminase and 43-kDa growth-associated protein (GAP-43) in a dose-dependent manner. The results indicate that propolis is able to inhibit neurite outgrowth of differentiating SH-SY5Y cells.

Keywords: GAP-43; Neurite outgrowth; Propolis; SH-SY5Y neuroblastoma cell; TGase.

Fig. 1

Effect of propolis on the cell viability of differentiating SH-SY5Y cells. The cells were treated with propolis for 48 hr in the presence of RA (10 μM). Then, the viability of SH-SY5Y cells was determined by MTT assay. The percentage of viable cells was calculated by defining the viability of cells without RA and propolis treatment as 100%. Data are expressed as the mean ± standard error obtained from four independent experiments.

Fig. 2

Effects of propolis on neurite outgrowth in differentiating SH-SY5Y neuroblastoma cells. (A) The cells were induced to differentiate for 48 hr with RA (10 μM) in the presence or absence of propolis (0.3~3 μg/mL). Cells were fixed in 4% paraformaldehyde and stained with Coomassie Brilliant Blue (CBB). Shown are images of typical fields of cells viewed by inverted light microscope. (B) Neurite outgrowth was quantified by counting the number of cells exhibiting neurites that were two times longer than the cell body diameter in length. The proportion of cells with neurites was expressed as a percentage of the total number of cells. Approximately 300 cells were counted in each sample. The data are expressed as the mean ± standard error of four independent experiments. *p<0.05 when compared to RA-treated cells. Scale bar = 100 μm.

Fig. 3

Effect of propolis on the expression of TGase in differentiating SH-SY5Y cells. The cells were treated with various concentrations of RA (10 μM) and propolis for 48 hr. The Western blot data represents one of three experiments. Densitometric analysis was performed to determine the intensity of TGase bands. Values are normalized to β-actin band and expressed as the percentage of cells treated with retinoic acid (RA) only. Data are mean ± standard error of three independent experiments. *p< 0.05 when compared to RA-treated cells.

Fig. 4

Effect of propolis on the expression of GAP-43 in differentiating SH-SY5Y cells. The cells were treated with various concentrations of propolis in the presence of 10 μM RA for 48 hr. The Western blot data represents one of three experiments. Densitometric analysis was performed to determine the intensity of GAP-43 bands. Values are normalized to β-actin band and expressed as the percentage of cells treated with retinoic acid (RA) only. Data are mean standard error of three independent experiments. *p< 0.05 when compared to RA-treated cells.