Ethanolic extract of propolis (EEP) enhances the apoptosis- inducing potential of TRAIL in cancer cells

Abstract

Ethanolic extract of propolis (EEP) is one of the richest sources of phenolic acids and flavonoids. EEP and its phenolic compounds have been known for various biological activities including immunopotentiation, chemopreventive and antitumor effects. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is a naturally occurring anticancer agent that preferentially induces apoptosis in cancer cells and is not toxic toward normal cells. We examined the cytotoxic and apoptotic effect of EEP and phenolic compounds identified in propolis in combination with TRAIL on HeLa cancer cells. HeLa cells were resistant to TRAIL-induced apoptosis. Our study demonstrated that EEP and its components significantly sensitize to TRAIL induced death in cancer cells. The percentage of the apoptotic cell after exposure to 50 microg/mL EEP and 100 ng/mL TRAIL increased to 71.10 +/- 1.16%. The strongest cytotoxic effect in combination with TRAIL on HeLa cells exhibited apigenin and CAPE at the concentration of 50 microM (58.87 +/- 0.75% and 49.59 +/- 0.39%, respectively). In this report, we show for the first time that EEP markedly augmented TRAIL mediated apoptosis in cancer cells and confirmed the importance of propolis in chemoprevention of malignant tumors.

Figure 1

Chemical structures of the phenolic compounds used in this study.

Figure 2

EEP induced apoptosis in HeLa cells. The cancer cells were incubated for 48 hours with EEP at the concentrations of 5 – 50 μg/mL. Detection of apoptotic cell death by annexin V-FITC staining using flow cytometry.

Figure 3

Cytotoxic activity of EEP phenolic components in HeLa cells. The cancer cells were incubated for 48 hours with the compounds at the concentrations of 50 μM. The percentage of death cells was measured by MTT cytotoxicity assay.

Figure 4

TRAIL induced apoptosis in HeLa cells. The cancer cells were incubated for 48 hours with TRAIL at the concentrations of 50 – 200 ng/mL. Detection of apoptotic cell death by annexin V-FITC staining using flow cytometry.

Figure 5

TRAIL induced apoptosis in combination with EEP in HeLa cells. Detection of apoptotic cell death by annexin V-FITC staining using flow cytometry. (A) The cancer cells were incubated for 48 hours with TRAIL at the concentration of 50 – 200 ng/mL and EEP at the concentration of 5 – 50 μg/mL and the cancer cells were incubated for 48 hours with TRAIL at the concentration of 100 ng/mL and EEP at the concentration of 5 – 50 μg/mL. (B) The same data was presented in 3-D figure.

Figure 6

Cytotoxic activity of EEP phenolic components in combination with TRAIL in HeLa cells. The cancer cells were incubated for 48 hours with the compounds at the concentrations of 50 μM and TRAIL at the concentrations of 100 ng/mL. The percentage of cell death was measured by MTT cytotoxicity assay.

Figure 7

TRAIL induced apoptosis in combination with EEP, after and before exposure to EEP in HeLa cells. Detection of apoptotic cell death by annexin V-FITC staining using flow cytometry. HeLa cancer cells were: 1) treated with EEP in combination with TRAIL for 48 hours, 2) pretreated with EEP for 24 hours, followed by TRAIL for another 24 hours and 3) pretreated with TRAIL for 24 hours, followed by EEP for another 24 hours.