1. Yu-Fen Wang1, Ya-Chi Chan1, Dar-Ren Chen1, 2, Hui-Yi Lin3
P1-169
Inhibition of ADAM17 and subsequent EGFR-PI3K-mTOR activation by aloe-emodin may promote cytotoxicity of tamoxifen
Yu-Fen Wang1, Ya-Chi Chan1, Dar-Ren Chen1, 2, Hui-Yi Lin3
1Cancer Research Center, Changhua Christian Hospital, 2Comprehensive Breast Cancer Center, Changhua Christian Hospital, Changhua, Taiwan, 3School of Pharmacy, China Medical University, Taichung, Taiwan
Background
A disintegrin and metalloproteases (ADAMs), the zinc-dependent transmembrane metalloproteases, shed the extracellular domains of membrane-bound growth factors, cytokines and receptors. In breast cancer, numerous members of the ADAM family including tumor necrosis factor-α converting enzyme (TACE/ADAM17) have been associated with tumorigenesis and cancer progression. ADAM17 promotes cancer progression by releasing HER/EGFR ligands. Aloe-emodin (AE), derived from rhubarb (Rheum palmatum) and aloe vera, exhibits anticancer activity through multiple action mechanisms. In estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-over-expressed breast cancer cells, suppression of ER and HER2 pathways by AE leads to cell death. According to our past study, AE may enhance tamoxifen-induced cytotoxicity in breast cancer. This study aimed to investigate effects of AE supplement on ADAM17-related pathways in tamoxifen-treated breast cancer cells.
Figure 2. Effects of AE on the tamoxifen-induced apoptosis in MCF-7 cells. Cells were treated as indicated treatment for 72 hours, and subsequent analyses of apoptosis was performed. * and ** stand for P < 0.05 and P < respectively when compared with the tamoxifen alone treatment group.
24 hrs
72 hrs
Tamoxifen (μg/mL) 9
AE (μg/mL) 4
HIF-1α
ADAM17
EGFR
Total PI3K
Phospho-PI3K
Total mTOR
Phospho-mTOR
Vimentin
Materials and Methods
MCF-7 cells were treated with combined AE and tamoxifen (Tam-AE) for indicated time. MTT assay and Muse viability assay were used for detecting cell viability. Flow cytometry was performed to determine apoptosis and autophagy. Protein expression was analyzed by using Western blot.
Figure 3. Representative data of protein expression of HIF-1α, ADAM17, EGFR, PI3K, mTOR, and vimentin after Tam-AE treatment in MCF-7 cells. Three independent experiments were performed. The staining of Coomassie dye in each lane was used as internal control.
Results
Compared to tamoxifen or AE treatment alone, combination of tamoxifen and AE resulted in more apparent apoptosis in MCF-7 cells. When combined with tamoxifen, AE significantly reduced ADAM17 expression as well as HIF-1α, EGFR and vimentin, and inhibited activation of PI3K and mTOR. In addition, wound healing assay showed Tam-AE treatment inhibited more cell migration when compared with tamoxifen or AE alone group. 0h
24h
Control AE
Tam 9
Tam-AE
(A)
(B)
Control AE
Figure 4. Results of wound healing assay after Tam-AE treatment in MCF-7 cells. Three independent experiments were performed.
Tamoxifen
Tamoxifen + AE
Conclusions
The results suggested that AE may enhance anticancer activity of tamoxifen through inhibiting ADAM17 expression, and the subsequent EGFR-PI3K-mTOR pathway is thus blocked. Reduction of ADAM17 expression may be resulted from down-regulated of the transcription factor HIF-1α by AE supplement.
Furthermore, the lowered ADAM17 protein expression may also hinder epithelial to mesenchymal transition in breast cancer cells.
Figure 1. MCF-7 cells after treatment with AE and tamoxifen. (A) Cell viability. (B) Cell morphology. AE was used to treat cells in the presence or absence of tamoxifen for 24 and 72 hours, respectively. Cell morphology was photographed using a phase contrast microscope. Data are expressed as mean ± SD of four independent experiments. * and ** stand for P < 0.05 and P < respectively when compared with the tamoxifen alone treatment group.