Melittin from Apis florea Venom as a Promising Therapeutic Agent for Skin Cancer Treatment

Abstract

Melittin, a major component found in bee venom, is produced by the Apis species of the honey bee. In this study, the effect of melittin derived from Apis florea (Mel-AF), which is a wild honey bee species that is indigenous to Thailand, was investigated against human malignant melanoma (A375) cells. In this study, Mel-AF exhibited considerable potential in the anti-proliferative action of A375 cells. Subsequently, the cellular mechanism of Mel-AF that induced cell death was investigated in terms of apoptosis. As a result, gene and protein expression levels, which indicated the activation of cytochrome-c release and caspase-9 expression, eventually triggered the release of the caspase-3 executioner upon Mel-AF. We then determined that apoptosis-mediated cell death was carried out through the intrinsic mitochondrial pathway. Moreover, advanced abilities, including cell motility and invasion, were significantly suppressed. Mel-AF manipulated the actin arrangement via the trapping of stress fibers that were found underneath the membrane, which resulted in the defective actin cytoskeleton organization. Consequently, the expression of EGFR, a binding protein to F-actin, was also found to be suppressed. This outcome strongly supports the effects of Mel-AF in the inhibition of progressive malignant activity through the disruption of actin cytoskeleton-EGFR interaction and the EGFR signaling system. Thus, the findings of our current study indicate the potential usefulness of Mel-AF in cancer treatments as an apoptosis inducer and a potential actin-targeting agent.

Keywords: Apis florea; F-actin; apoptosis; epidermal growth factor receptor; malignant melanoma; melittin.

Figure 1

SDS-PAGE analysis of melittin peptide and bee venom extracts obtained from A. mellifera (AM) and A. florea (AF). Melittin peptide and venom extracts were separated on 15% SDS-PAGE and the patterns of the protein in all samples were visualized by 0.1% Coomassie Blue R250 staining. The major protein component was mainly found at ~3 kDa indicating melittin.

Figure 2

Effect of Mel-AF on A375 cell and NIH-3T3 cell viability. The cells were treated with the indicated concentration of Mel-AF (or Mel-AM) for 2 and 24 h. The viable cells after treatment were determined by MTT assay. (A,B) Anti-proliferative effects of Mel-AF compared to Mel-AM on A375 cells. (C) Non-cytotoxic effects of Mel-AF and Mel-AM on NIH-3T3 cells. Data are presented as mean ± SEM values of three independent experiments that were performed in triplicate. * p < 0.05 was determined by one-way analysis of variance.

Figure 3

Localization of Mel-AF on A375 cells was analyzed using a confocal laser scanning microscope. The cells were treated with FITC-conjugated Mel-AF for 2 h. (A) Confocal images show the localization of Mel-AF traced with FITC (green). The membrane compartment was indicated by staining with a specific antibody to CD46, a membrane cofactor protein followed by Alexa Fluor 568 rabbit anti-mouse IgG (red). The cellular nuclei were counterstained by DAPI (blue). (B) Orthogonal imaging analysis was performed to confirm the localization of Mel-AF on the cell membrane (yellow). Scale bar, 10 μm.

Figure 4

Effect of Mel-AF on apoptosis induction in A375 cells. Cells were treated with the indicated concentrations of Mel-AF (or Mel-AM) for 2 h. (A) Cells were then stained with FITC-Annexin V and PI to analyze early apoptotic (Annexin V+ PI−) and late apoptotic/death (Annexin V+ PI+) by flow cytometry. (B) Histogram shows the percentages of apoptotic cells for each treated sample at 2 h. (C) DNA fragmentation in treated cells were studied by TUNEL assay and analyzed by Flow cytometer. Data are presented as the mean ± SEM values of three independent experiments performed in triplicate. * p < 0.05 was determined by one-way analysis of variance.

Figure 5

Mel-AF induced apoptosis-related gene and protein expression through the intrinsic mitochondrial pathway in A375 cells. (A) Apoptosis-related genes, including Bcl-2, cytochrome-c, caspase-9, -8 and -3, were examined by quantitative real-time PCR. Relative mRNA expression was normalized to GAPDH internal control. (B) Western blot analysis of apoptotic protein expression was employed involving Bcl-2, cytochrome-c, caspase-9 and -3 with specific antibodies. GAPDH was used as an internal control to show equal protein loading. Data are presented as mean ± SEM values of three independent experiments performed in triplicate. * p < 0.05 was determined by one-way analysis of variance.

Figure 6

Mel-AF inhibited the migration and invasion activities of A375 cells. (A) Suppression of cell migration by Mel-AF was demonstrated using wound healing assay. Images depicting wounds at 0 and 24 h after treatment with Mel-AF (or Mel-AM) are shown (top). The gap distance was measured and the percentage of migration was calculated. (B) Decreases in cell invasion were determined by transwell invasion assay when compared with untreated cells (top). The percentage of cell invasion was calculated and compared with 100% cell invasion of the control. Data are presented as mean ± SEM values of three independent trials performed in triplicate. * p < 0.05 was determined by one-way analysis of variance.

Figure 7

Accumulation of actin stress fibers and suppression of EGFR expression upon Mel-AF treatment for 2 h. (A) The treated cells were fixed and the arrangement of actin cytoskeleton was investigated by F-actin staining (red). Images reveal that the cells that were treated with Mel-AF (or Mel-AM) formed abundant amounts of stress fibers beneath the cell membrane when compared with untreated cells. Scale bar, 50 µm. (B) F-actin levels were quantified by pixel intensity. (C) Expression of EGFR in Mel-AF (or Mel-AM) treated cells was investigated by western blot analysis with specific antibodies. GAPDH was used as an internal control to show equal protein loading. * p < 0.05 was determined by one-way analysis of variance.

Figure 8

Mechanisms of anticancer activity of Mel-AF in human malignant melanoma cells.