Background
Breast cancer, the most common cancer in Chinese women and the second most common female cancer worldwide [
1], presents a serious threat to women’s health. The incidence of human epidermal growth factor receptor-2 (HER-2)-positive breast cancer comprises 23% of all breast cancer types and results in a poor prognosis. However, the development of an effective therapy for HER-2-positive breast cancer has proceeded slowly; no widely recognized treatment programmes have yet been accepted [
2]. The side effects of traditional therapeutic strategies have driven the development of natural compounds as new therapeutic drugs and promoted the urgent exploration of other anticancer strategies.
Delphinidin, an anthocyanidin monomer, exerts the strongest antioxidative efficiency of all anthocyanidins in the human diet [
3]. Our previous studies confirmed the anticancer activities of anthocyanidin and delphinidin-3-glucoside against breast cancer [
4‐
6]. However, although delphinidin is a major bioactive component of anthocyanidin, the mechanisms responsible for its effects against breast cancer remain poorly defined. Therefore, the present study aimed to provide further exploration of the anticancer effects of delphinidin.
Autophagy is shown to act in parallel with apoptosis and serves as an alternative mechanism to cell death when apoptosis is inhibited [
7]. However, a number of studies have suggested that autophagy induces protective effects against malignancy in the presence of cellular stress [
8,
9]. Delphinidin causes metabolic stress in breast cancer cells and acts a potential anticancer agent. Therefore, the exact role of autophagy induced by delphinidin needs further investigation. Many studies have indicated that autophagy can be activated by the monophosphate-activated protein kinase (AMPK) signalling pathway, which is associated closely with energy metabolism [
10,
11]; additionally, it is known that the classic AKT/protein kinase B-mammalian target of rapamycin (mTOR) signalling pathway initiates the vesicular double-membrane formation process of autophagy [
12]. Moreover, Atg5, a critical protein that mediates the expansion of autophagosomes, can be cleaved in response to metabolic stress and may even be the switch from autophagy to apoptosis [
13].
This study reported novel functions of delphinidin: the induction of apoptosis and protective autophagy in HER-2-positive MDA-MB-453 and BT474 breast cancer cells and the induction of autophagy through the modulation of the mTOR and AMPK signalling pathways. Our findings suggest potential clinical applications in which autophagic inhibition sensitizes cells to the anticancer effect of delphinidin. This research may support the future development of delphinidin or other phytochemicals, as a novel anticancer drug.
Methods
Cell culture and treatment
HER-2-positive breast cancer cells (MDA-MB-453 and BT474) were purchased from the Chinese Academy of Sciences (Shanghai, China). MDA-MB-453 cells were cultured in L-15 medium (Gibco, USA, 11415–064) supplemented with 10% foetal bovine serum (Millipore, USA, ES-009-B) at 37 °C in a humidified atmosphere containing 100% O2. BT474 cells were cultured in 1640 medium (Gibco, USA, A1049101) supplemented with 10% foetal bovine serum (Millipore, USA, ES-009-B), at 37 °C in a humidified atmosphere with 5% CO2. Delphinidin was dissolved in dimethyl sulphoxide (DMSO). The final concentration of DMSO (0.1% volume) was added to medium as a control. Where used, 3-methyladenine (3-MA) (5 mM) was dissolved in phosphate-buffered saline (PBS) and pretreated for 2.5 h before delphinidin administration. Bafilomycin A1 (BA1) (160 nM) was dissolved in DMSO and added 8 h before sample collection and observation.
Antibodies and reagents
Alexa Fluor 488 Phalloidin (8878), caspase-3 (9665 s), caspase-9 (9502), light chain 3 (LC3)-I/II (2775), AKT (4685), phospho-AKT (4051), mTOR (2972), phospho-mTOR (5536), eukaryotic initiation factor 4E (eIF4E, 2067), phospho-eukaryotic initiation factor 4E (p-eIF4E, 9741), ribosomal protein S6 kinase (p70s6k) (9202), phospho-ribosomal protein S6 kinase, (p-p70s6k, 9206), serine/threonine kinase LKB1/STK11 (3047), phospho-LKB1 (3482), forkhead box O3a (FOXO3a, 2497), AMPK (2532), phospho-forkhead box O 3a (p-FOXO3a, 9465), UNC-51-like kinase-1 (ULK1, 4776), phospho-UNC-51-like kinase-1 (ULK1, 12,753), anti-rabbit secondary antibody (7054), and anti-mouse secondary antibody (7056) were purchased from Cell Signaling Technology, USA. Atg5 (ab78073) and phospho-AMPK (ab195946) were purchased from Abcam, UK. β-Actin (TA-09) was purchased from ZSGB-BIO, China. Other reagents were obtained from the following sources as required: delphinidin (Sigma, USA, 43725); 3-MA (Sigma, USA, M9281); BA1 (Cayman, USA, 88899–55-2); DMSO (Sigma, USA, D2650); and cell counting kit-8 (CCK-8) (Dojindo, Japan, CK04–20).
Cell proliferation assay
MDA-MB-453 and BT474 cells were seeded into 96-well culture plates for 24 h and then treated with delphinidin for 48 h. CCK-8 assay was used to evaluate the viability of cells. The absorbance of the solutions in the wells was read at 450 nm in a microplate reader (BioTek, China, Powerwave XS).
Terminal deoxynucleotidyl transferase dUTP nick-end labelling assay
Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining was performed using the DeadEnd Fluorometric TUNEL system in accordance with the manufacturer’s instructions (Promega, USA, G3250). The samples were observed and recorded by using a fluorescence microscope (Olympus, Japan, IX71). The nuclei stained with bright green fluorescence were considered to be TUNEL-positive cells.
Immunoblotting
The adherent cells were collected in radio immunoprecipitation assay (RIPA) buffer by using a cell scraper. A protein assay kit (Thermo, USA, 23227) was used to quantify protein concentration. The samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the proteins were transferred to polyvinylidene fluoride membranes. Non-fat milk solution (5%) was used to block the membranes. After blocking, the membranes were treated with the primary antibodies at 4 °C overnight, washed three times in TBST, and then incubated with the secondary antibodies for 2 h at 37 °C. Finally, chemiluminescence reagents (Millipore, WBKLS0500) were used to visualize the stained proteins.
Transmission electron microscopy
The samples were fixed for 2 h with 2.5% glutaraldehyde in phosphate buffer, washed three times in 0.1 M phosphate buffer, fixed with 1% osmic acid for 2 h, and washed again. The samples were dehydrated with graded alcohol (50, 70, and 90%), dehydrated with 90% alcohol and 90% acetone at 4 °C, and finally dehydrated with 100% acetone at 18–21 °C (room temperature). The samples were embedded in a mixture of acetone and epoxy resin. The samples were solidified at different temperatures (37, 45, and 60 °C), sliced on an ultramicrotome (Leica, Germany, UC7), double stained with 3% uranyl acetate and lead citrate, and examined under a transmission electron microscope (JEOL, Japan, JEM-1011).
Immunofluorescence analysis
The cells were grown on coverslips in 6-well plates, administered delphinidin at different concentrations for 48 h, fixed in 4% formaldehyde, washed, and permeabilised with 0.1% Triton X-100. The samples were washed again and sequentially incubated with anti-LC3 primary antibody and Alexa Fluor 488 Phalloidin secondary antibody. The coverslips were then infiltrated with 60% glycerol and immediately examined under a fluorescence microscope (Olympus, IX71).
GFP-LC3 transient transfection
The cells were seeded on coverslips in 6-well plates for 24 h and then transfected with the apEX-GFP-hLC3WT plasmid (Addgene, USA, 24987) by using Lipofectamine 2000 (Invitrogen, USA, 11668019) based on the manufacturer’s protocol. After 24 h, the transfected cells were treated with different concentrations of delphinidin for 48 h and washed three times with PBS. The washed samples were fixed with 4% paraformaldehyde for 15 min and then washed again. A solution of 50% glycerol was used to infiltrate the coverslips and observe the distribution of green fluorescent protein (GFP)-LC3 punctate dots in cells. Finally, the images were photographed by using a fluorescence microscope (Olympus, Japan, IX71). The GFP-LC3 punctate dot assay was repeated three times.
Statistical analyses
All experimental data were presented as the mean ± standard error of the mean and each experiment was performed at least three times. The statistical analyses were performed by one-way ANOVA using SPSS version 21.0 (SPSS, IL, USA). Values of P < 0.05 were considered statistically significant.
Discussion
This study on delphinidin and HER-2 positive breast cancer demonstrated that delphinidin promoted antiproliferative effects and apoptosis in human HER-2 positive breast cancer MDA-MB-453 and BT474 cells. Furthermore, it was revealed that delphinidin simultaneously induced autophagy, which promoted cell viability and suppressed apoptotic cell death. The suppression of autophagy using 3-MA and BA1 in HER-2 positive breast cancer cells increased delphinidin-induced apoptosis and antiproliferation. Protective autophagy was induced over a range of treatment doses and was enhanced by an increase in the dose of delphinidin via suppression of the AKT/mTOR/eIF4e/p70s6k signalling pathway and activation of the LKB1/AMPK/ULK1/FOXO3a signalling pathway in HER-2 positive breast cancer cells.
Delphinidin, an anthocyanidin monomer, is prevalent in vegetables and fruits. Our previous research has demonstrated that anthocyanidin exerts an anticancer effect on HER-2 positive MDA-MB-453 breast cancer cells, but displays low cytotoxicity to normal breast cells [
4,
5]. As the monomer predominantly responsible for the activity of anthocyanidin, delphinidin has been shown to exert anticancer effects in various cancer models [
26,
27]. Consistent with previous reports [
26,
27], the present study showed that delphinidin induced antiproliferative effects and apoptosis in human HER-2 positive breast cancer MDA-MB-453 and BT474 cells. These findings are significant for the further development of delphinidin as a clinical chemopreventive agent.
Autophagy exerts contradictory effects in different stages of cancer cell formation. Certain phytochemicals, such as ampelopsin and baicalein, induce protective autophagy that counteract antiproliferative effects and apoptosis [
28,
29], whereas others, such as cyclovirobuxine D and
Zanthoxylum fruit, initiate autophagic cell death that sensitize cells to anticancer effects [
30,
31]. The present study demonstrated that delphinidin triggered autophagy and that autophagic inhibition by 3-MA and BA1 markedly enhanced the antiproliferative effects and apoptosis by delphinidin. It was suggested that the autophagy might exert protective efficiency in HER-2 positive breast cancer cells.
Caspase-dependent cell death, a method of apoptosis, is regulated in two main ways: the activation of exogenous death regulators and the endogenous release of cytochrome c. The activation of exogenous death regulators cleaves caspase-3, whereas cytochrome c release upregulates cleaved caspase-9 and cleaved caspase-3 [
32,
33]. Consequently, as delphinidin modulated the activation of caspase-3 and -9 in the present study, the cell death was believed to be mediated by the endogenous pathway. Several studies suggested that the endogenous pathway in cancer cells was related to activation of the endoplasmic reticulum stress pathway and the generation of reactive oxygen species [
28,
34]. To confirm that delphinidin-induced apoptosis was related to the endogenous pathway, further studies are necessary to detect the activity of the endoplasmic reticulum stress pathway and reactive oxygen species.
Many signalling pathways are involved in the induction of autophagy; however, the mTOR-related pathway, as a pivotal negative sensor of autophagy, is more significant. Many phytochemical compounds regulate autophagy through the mTOR pathway in MCF-7 breast cancer cell models [
28,
30]. To reveal the molecular mechanism of delphinidin-induced autophagy, the relationship between delphinidin, autophagy, and the mTOR pathway was explored. It was found that treatment with delphinidin specifically inhibited the AKT branch upstream of mTOR, affecting eIF4e and p70s6k downstream of mTOR phosphorylation, which suggested that delphinidin exerted a negative effect on mTOR activity. The result was similar to the effect of rapamycin, a natural inhibitor of mTOR and agonist of autophagy, on autophagy. It is well known that mTOR is a central pathway in the mediation of cell growth, protein synthesis, survival, and metabolism in response to hormones, nutrients, and other stimuli [
35]. The dysfunction of the mTOR pathway in mammary cells often leads to breast carcinogenesis [
36]. In the present study, the suppressive effect of delphinidin on HER-2 positive breast cancer cells was shown to occur through the mTOR pathway; thus, the proliferation inhibition and autophagy induced by delphinidin might be attributable to the same pathway.
AMPK, an energy sensor of cells, is associated with the activation of autophagy under the conditions of the catabolic processes of oxidative stress and energy starvation in eukaryotic cells. Previous studies have shown that AMPK activated autophagy through the direct activation of the downstream receptor ULK1, whereas others elucidated that AMPK activated autophagy through the inhibition of mTOR phosphorylation in pancreatic β cells [
24,
37]. The present study demonstrated that AMPK activated ULK1, a homolog of yeast ATG1, by phosphorylation at ser317 and a reduction in the activation of mTOR, which indicated the interaction between ULK1 and mTOR in delphinidin-induced autophagy. Shaw proposed that LKB1 and AMPK controlled mTOR signalling and cell growth. Hence, it was thought that the growth inhibition of MDA-MB-453 and BT474 cells induced by delphinidin was related to the activation of LKB1 and AMPK [
38]. FOXO3a, a member of forkhead box O (FoxO) family of transcription factors, has been reported to initiate the expression of autophagy-related genes [
38]. The present study showed that FOXO3a could be upregulated by the activation of AMPK, resulting in the induction of autophagy, which suggested that FOXO3a probably initiated autophagy-related genes. Several studies have found that the AMPK-FOXO3a axis plays an important role in the regulation of autophagy-related genes in different cell models [
39,
40].