Ethanol extract of propolis and its constituent caffeic acid phenethyl ester inhibit breast cancer cells proliferation in inflammatory microenvironment by inhibiting TLR4 signal pathway and inducing apoptosis and autophagy

Abstract

Background: Propolis and its major constituent - caffeic acid phenethyl ester (CAPE) have good abilities on antitumor and anti-inflammation. However, little is known about the actions of propolis and CAPE on tumor in inflammatory microenvironment, and inflammatory responses play decisive roles at different stages of tumor development. To understand the effects and mechanisms of ethanol-extracted Chinese propolis (EECP) and its major constituent - CAPE in inflammation-stimulated tumor, we investigated their effects on Toll-like receptor 4 (TLR4) signaling pathway which plays a crucial role in breast cancer MDA-MB-231 cell line.

Methods: 80% confluent breast cancer MDA-MB-231 cells were stimulated with 1 μg/mL lipopolysaccaride (LPS). Then the cells were divided for treatment by CAPE (25 μg/mL) and EECP (25, 50 and 100 μg/mL), respectively. Cell viability, nitric oxide (NO) production and cell migration were measured by sulforhodamine B assay, chemical method and scratch assay. The levels of TLR4, MyD88, IRAK4, TRIF, caspase 3, PARP, LC3B and p62 were investigated through western blotting. The expression of TLR4, LC3B and nuclear factor-κB p65 (NF-κB p65) were tested by immunofluorescence microscopy assay.

Results: Treatment of different concentrations of EECP (25, 50 and 100 μg/mL) and CAPE (25 μg/mL) significantly inhibited LPS-stimulated MDA-MB-231 cell line proliferation, migration and NO production. Furthermore, EECP and CAPE activated caspase3 and PARP to induce cell apoptosis, and also upregulated LC3-II and decreased p62 level to induce autophagy during the process. TLR4 signaling pathway molecules such as TLR4, MyD88, IRAK4, TRIF and NF-κB p65 were all down-regulated after EECP and CAPE treatment in LPS-stimulated MDA-MB-231 cells.

Conclusions: These findings indicated that EECP and its major constituent - CAPE inhibited breast cancer MDA-MB-231 cells proliferation in inflammatory microenvironment through activating apoptosis, autophagy and inhibiting TLR4 signaling pathway. EECP and CAPE may hold promising prospects in treating inflammation-induced tumor.

Keywords: Anti-inflammatory; Antitumor; Caffeic acid phenethyl ester; Propolis; Toll like receptor 4.

Fig. 1

HPLC chromatograms of ethanol-extracted Chinese propolis (EECP) and caffeic acid phenethyl ester (CAPE)

Fig. 2

EECP and CAPE decreased LPS-stimulated MDA-MB-231 cells proliferation at 24 and 48 h. CAPE, cells treated with CAPE at 25 μg/mL. 25, 50 and 100 μg/mL, cells treated with EECP at 25, 50 and 100 μg/mL, respectively. (^* P < 0.05, ^** P < 0.01 vs control, n = 3). Data are means ± S.E.M

Fig. 3

EECP and CAPE inhibited LPS-stimulated MDA-MB-231 cells migration. a Cell migration micrographs obtained under a phase contrast microscope at 0, 24 and 48 h (×100). b Relative levels of cell migration. (^* P < 0.05, ^** P < 0.01 vs control, n = 3). Data are means ± S.E.M

Fig. 4

EECP and CAPE inhibited NO production at 48 h. (^* P < 0.05, ^** P < 0.01 vs control, n = 3). Data are means ± S.E.M

Fig. 5

EECP and CAPE induced apoptosis and autophagy in LPS-stimulated MDA-MB-231 cells. a The expression of caspase 3, procaspase 3 and PARP were detected by western blotting at 24 h. b Quantification of relative expression of procaspse 3, caspase 3 and PARP. c Cells were stained with anti-LC3B antibody for immunostaining. Immunofluorescence graphs showed an increase of endogenous punctuate LC3B. d The expression of LC3B and p62 were detected by western blotting at 24 h. e Quantification of relative expression of LC3-II, LC3-II/ LC3-Iand p62 in LPS-stimulated MDA-MB-231 cells. (^* P < 0.05, ^** P < 0.01 vs control, n = 3). Data are means ± S.E.M

Fig. 6

EECP and CAPE regulated the levels of TLR4, MyD88, IRAK4 and TRIF. a Cells were treated with EECP and CAPE for 24 h, respectively. Cells were stained with anti-TLR4 antibody. Immunofluorescence graphs showed a decrease of TLR4 level. b The expression of TLR4, MyD88, IRAK4 and TRIF in LPS-stimulated MDA-MB-231 cells were detected by western blotting at 48 h. c Quantification of relative expression of TLR4, MyD88, IRAK4 and TRIF in LPS-stimulated MDA-MB-231 cells. (^* P < 0.05, ^** P < 0.01 vs control, n = 3). Data are means ± S.E.M

Fig. 7

EECP and CAPE regulated the levels of NF- κB p65. a Fluorescent micrographs obtained at 48 h (×400). b The relative fluorescence intensity of NF-κB p65 in LPS-stimulated MDA-MB-231 cells. (^* P < 0.05, ^** P < 0.01 vs control, n = 3). Data are means ± S.E.M