The Effect of Bee Venom and Melittin on Glioblastoma Cells in Zebrafish Model

Abstract

Previous in vitro studies have shown the therapeutic potential of bee venom (BV) against different types of glioblastoma cells. Our aim was to evaluate the cytotoxic effect of BV on glioma in the zebrafish model. First, safe concentrations of BV and melittin were determined by determining the LD₅₀ for each substance. Two human glioma cell lines, 8MGBA and LN-229, were used in this study. After staining the tested cells for visualization under UV light, they were then implanted into 2-day-old zebrafish embryos. Zebrafish were incubated for 3 days with crude BV and melittin at concentrations of 1.5 and 2.5 µg/mL vs. control group. Tumor growth was assessed with a stereo microscope. We found differential proliferative responses of two human glioma lines in a zebrafish model. The 8MGBA cell line, but not LN-229, showed proliferative potential when implanted into 2-day-old zebrafish embryos. This study showed a dose-dependent cytotoxic effect only for BV against 8MGBA cells. The observed cytotoxic effect is not dependent on the presence of the peptide melittin-the main BV component with the greatest cytotoxic potential. Simultaneously, a slight increase in LN-229 cell proliferation was observed after 3 days of incubation with melittin at a concentration of 2.5 µg/mL. This indicates that any consideration of bee venom as a therapeutic substance must take into account the type of glioblastoma.

Keywords: bee venom; glioblastoma; melittin; zebrafish model.

Figure 1

Structural formula of melittin (PubChem CID 16129627).

Figure 2

The mortality of zebrafish exposed to different concentrations of BV (A) and melittin (C). The percentage of zebrafish mortality (y-axis) as a function of the logarithm of the drug concentration (x-axis). The red dashed line represents the 50% viability, which is used to determine the LC₅₀ (Lethal Concentration 50) value for each compound. The LC₅₀ value is the concentration at which 50% of the zebrafish larvae are expected to die. The right side of the figure indicates the lack of cardiotoxicity of the bee venom (B) and melittin (D) concentrations used. The mean heart rate did not change significantly.

Figure 3

Representative images of glioblastoma cells’ xenotransplantation into zebrafish embryos. Microscopic observation of the 8MGBA cells (A) and LN-229 cells (B) at 5 dpf zebrafish xenograft upon treatment with BV and melittin (Mel) (1.5 µg/mL and 2.5 µg/mL) vs. control zebrafish embryos xenotransplanted with glioblastoma cells and incubated without BV or Mel.

Figure 4

Bar graphs representing the percentage of 8MGBA cells xenotransplanted into zebrafish embryos treated for 72 h with BV (A) or melittin (B) at the concentrations of 1.5 µg/mL and 2.5 µg/mL for each compound, or without any treatment (cont 3d). Initial measurement of cell percentage before treatment was set to 100% (cont). Mean (SEM), * p  <  0.05, **** p  <  0.0001 compared to control.

Figure 5

Bar graphs representing the percentage of LN-229 cells xenotransplanted into zebrafish embryos treated for 72 h with BV (A) or melittin (B) at the concentrations of 1.5 µg/mL and 2.5 µg/mL for each compound, or without any treatment (cont 3d). Initial measurement of cell percentage before treatment was set to 100% (cont). Mean (SEM), ** p < 0.001 compared to control.