
Amygdalin, D-mandelonitrile-β-D-glucoside-6-β-glucoside, belongs to aromatic cyanogenic glycoside group derived from rosaceous plant seed. Mounting evidence has supported the anti-cancer effects of amygdalin. However, whether amygdalin indeed acts as an anti-tumor agent against breast cancer cells is not clear. The present study aimed to investigate the effect of amygdalin on the proliferation of human breast cancer cells. Here, we show that amygdalin exerted cytotoxic activities on estrogen receptors (ER)-positive MCF7 cells, and MDA-MB-231 and Hs578T triple-negative breast cancer (TNBC) cells. Amygdalin induced apoptosis of Hs578T TNBC cells. Amygdalin downregulated B-cell lymphoma 2 (Bcl-2), upregulated Bcl-2-associated X protein (Bax), activated of caspase-3 and cleaved poly ADP-ribose polymerase (PARP). Amygdalin activated a pro-apoptotic signaling molecule p38 mitogen-activated protein kinases (p38 MAPK) in Hs578T cells. Treatment of amygdalin significantly inhibited the adhesion of Hs578T cells, in which integrin α5 may be involved. Taken together, this study demonstrates that amygdalin induces apoptosis and inhibits adhesion of breast cancer cells. The results suggest a potential application of amygdalin as a chemopreventive agent to prevent or alleviate progression of breast cancer, especially TNBC.
Breast cancer has been estimated as one of most commonly diagnosed types of cancer among women (Siegel
Many natural chemopreventive compounds are shown to reduce the risk of cancer through induction of apoptosis, programmed cell death (Tanaka, 2013). Mitochondrial-mediated apoptosis is induced by multiple molecular events including decrease in B-cell lymphoma 2 (Bcl-2), increases in Bcl-2-associated X protein (Bax), and activation of caspase-3 (Chiarugi
Tumor metastasis is a complex process involving extensive interactions between the tumor cells and host tissues including tumor cell dissociation, intravasation, extravasation, adhesion, and angiogenesis (Alizadeh
Amygdalin, D-mandelonitrile-β-D-glucoside-6-β-glucoside, belongs to the aromatic cyanogenic glycoside group and is widely distributed in the rosaceous plant seed, for example, apricot, peach, apple, cherry, plum,
Mounting evidence has supported that amygdalin induces apoptotic cell death of various cancer cells such as promyelocytic leukemia, prostate cancer, cervical and liver cancer cells (Kwon
Despite many studies have demonstrated the anti-cancer effects of amygdalin on various cancer cells, the chemopreventive potential of amygdalin in breast cancer is poorly understood at present. In this study, we aimed to investigate the chemopreventive potential of amygdalin against breast cancer. To this end, we examined the anti-proliferative effect of amygdalin on ER-positive MCF7 human breast carcinoma cells and MDA-MB-231 and Hs578T TNBC cells. In addition, we investigated the effect of amygdalin on apoptosis and adhesion of Hs578T TNBC cells.
Hs578T and MDA-MB-231 breast cancer cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). MCF7 cells were from American Type Culture Collection (ATCC, Manassas, USA). Hs578T and MCF7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 μg/mL penicillin-streptomycin as previously described (Koh
Amygdalin (depicted in Fig. 1) was purchased from Sigma Aldrich (St. Louis, MO, USA) and dissolved in serum-free media. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) was purchased from Sigma Aldrich (St. Louis, MO, USA). Z-VAD-FMK was purchased from R&D system, Inc. (Minneapolis, MN, USA).
Cells (1×104 cells) cultured in a 96-well plate were treated with amygdalin for 24 hr. After incubation, 25 μL of 5 mg/mL of MTT was added and incubated for 3 hr. Then the formazan was dissolved with 100 μL of DMSO and the optical density was measured at 540 nm using ELISA reader (Synergy 2; BioTek Instruments, Inc., Winooski, VT, USA).
The cells were cultured to 70% confluency and incubated in serum-free media containing various concentrations of amygdalin (0, 10, 20 and 40 mg/mL) for 24 hr. Immunoblot analysis was performed as described previously (Moon
Adhesion assays was performed on 96-well plates coated with 5 μg/mL collagen as previously described (Miao
We investigated the cytotoxic effects of amygdalin on human breast carcinoma cells. We performed MTT assay upon treatment with various concentrations of amygdalin for 24 hr. As shown in Fig. 2, amygdalin inhibited proliferation of MCF7, MDA-MB-231 and Hs578T cells in a dose-dependent manner. The IC50 values of amygdalin in MCF7, MDA-MB-231 and Hs578T cells were 30.8, 48.5 and 52.9 mg/mL, respectively. The data demonstrate that amygdalin exerted cytotoxic effect on ER-positive MCF7 as well as MDA-MB-231 and Hs578T TNBC cells.
To evaluate if cytotoxic effect of amygdalin on breast cancer cells involves apoptosis, we determined the expression levels of apoptosis-related proteins in Hs578T TNBC cells. Cells were treated with amygdalin at various concentrations for 24 hr. The levels of Bcl-2, Bax, PARP and pro-caspase-3 were determined by immunoblot analysis. As shown in Fig. 3A, amygdalin increased the expression level of pro-apoptotic protein Bax and decreased that of anti-apoptotic Bcl-2. The level of pro-caspase-3 was decreased by amygdalin treatment. PARP cleavage was observed in Hs578T cells treated with amygdalin.
A kinetic study was performed to examine the effect of amygdalin on the activation of p38 MAPK which is known as a pro-apoptotic signaling molecule (Xia
To examine whether a caspase-3 inhibitor might attenuate amygdalin-induced cytotoxic effect, we treated the Hs578T cells with a caspase-3 inhibitor, 25 μM Z-VAD-FMK for 24 hr. Amygdalin-induced decrease in pro-caspase-3 level was recovered by Z-VAD-FMK. Increase in cleaved PARP by amygdalin was also recovered by Z-VAD-FMK (Fig. 3C). As shown in Fig. 3D, amygdalin-induced cytotoxicity was significantly attenuated by treatment with Z-VAD-FMK. These results suggest that amygdalin may induce apoptosis in Hs578T cells via caspase-3 pathway.
We next examined the effect of amygdalin on adhesive phenotype of Hs578T cells. The adhesion of Hs578T cells was significantly inhibited by treatment of amygdalin in a dose-dependent manner (Fig. 4A). The level of integrin α5 was decreased by amygdalin treatment (Fig. 4B). The results demonstrate that amygdalin effectively inhibited the adhesive phenotype of Hs578T breast cancer cells.
Compounds naturally derived from plants have provided a number of useful cancer chemotherapeutic drugs by wide variety of anti-tumor effects. The anti-tumor activity of amygdalin was reported
Reduction or resistance of apoptosis often leads to malignant progression of cancer (Evan and Vousden, 2001). Various signaling pathways may trigger the apoptotic process in human cancers (Townson
Integrins regulate cell adhesion to the extracellular matrix, a cellular process that mediates cell differentiation, metastasis and angiogenesis (Howe
Recent studies reported that various natural products inhibit tumor cell growth and metastasis, and induce apoptosis of cancer cells (Mantena
The present study was supported by the Duksung Women’s University Research Grant 2013. The authors thank Dr. Eun-Sook Kim and Dr. Minsoo Koh at Duksung Women’s University (Seoul, Korea) for helpful discussion.
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