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Abstract

Glycosylation of cell surface proteins plays an important role in the regulation of apoptosis. It has been demonstrated that knockdown of epithelial cell adhesion molecule promoted apoptosis, inhibited cell proliferation, and caused cell-cycle arrest. In this study, we investigated whether and how N-glycosylation of epithelial cell adhesion molecule influenced the apoptosis in breast cancer cells. We applied the N-glycosylation mutation epithelial cell adhesion molecule plasmid to express deglycosylation of epithelial cell adhesion molecule and then to study its function. Our results showed that deglycosylation of epithelial cell adhesion molecule promoted apoptosis and inhibited cell proliferation. Deglycosylation of epithelial cell adhesion molecule enhanced the cytotoxic effect of 5-fluorouracil, promoting apoptosis by downregulating the expression of the anti-apoptotic protein Bcl-2 and upregulating the expression of the pro-apoptotic proteins Bax and Caspase 3 via the extracellular-signal-regulated kinase 1/2 and c-Jun N-terminal kinase mitogen-activated protein kinase signaling pathways in MCF-7 and MDA-MB-231 cells. These findings are important for a better understanding of epithelial cell adhesion molecule apoptosis regulation and suggest epithelial cell adhesion molecule as a potential target for the treatment of breast cancer.

Keywords

Epithelial cell adhesion molecule N-glycosylation cell apoptosis breast cancer signaling pathway

Introduction

Breast cancer is the most common cancer and is the primary cause of death among women globally.^1,2 In 2012, an estimated 1.7 million cases and 521,900 deaths occurred.³ Incidence rates continue to increase despite more efforts during the past two decades. Therefore, it is imminent to explore the mechanism of disease progression and find a more effective therapy treatment.

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that is highly expressed on most carcinomas including breast cancer.^4–6 Human EpCAM is a polypeptide of 314 amino acids (aa), consisting of a large extracellular domain (N-terminal) of 242 aa which contains three N-glycosylation sites, a single-spanning transmembrane domain of 23 aa, and a short cytoplasmic domain of 26 aa (C-terminal). Increasing clinical evidence has confirmed that EpCAM is involved in cancer progression and is associated with a poor prognosis.^7–9 More evidence suggested that EpCAM played a critical factor in tumor development, progression, and metastasis.^10,11 For example, EpCAM is frequently overexpressed in patients with acute myeloid leukemia (AML), with EpCAM⁺ leukemic cells exhibiting enhanced chemoresistance and oncogenesis.¹² Breast cancer cells overexpressing EpCAM could be resistant to neoadjuvant chemotherapy (NAC), contributing to a poor prognosis.¹³

Glycosylation is one of the most common post-translational modification reactions. Glycosylation modification of cell surface proteins regulates cellular functions including migration, growth, proliferation, adhesion, and apoptosis. Tumorigenic cells possess the change of glycosylation modification, which may lead to metastasis, uncontrolled proliferation, and the inhibition of apoptosis.^14–16 It has been reported that protein glycosylation was involved in potentially all aspects of human growth and development. Altered glycans on the tumor- and host-cell surface and in the tumor microenvironment have been identified to mediate critical events at the onset and/or during tumor progression.^17,18 EpCAM is N-glycosylated at N74, N111, and N198.⁸ EpCAM is differentially glycosylated in healthy tissue and tumor cells of the head and neck area.¹⁹ In our previous study, we have suggested that knockdown of EpCAM promoted apoptosis, inhibited cell proliferation, and caused cell-cycle arrest.²⁰ Little is known about the contribution of glycosylation modifications of EpCAM to the adaptive response to apoptosis inducers. In this study, we aimed to analyze the influence of N-glycosylation of EpCAM on apoptosis in breast cancer cells.

Materials and methods

Materials

MCF-7 and MDA-MB-231 cells were obtained from American Type Culture Collection. Dulbecco’s Modified Eagle Medium (DMEM)/F12 (1:1), fetal bovine serum (FBS), Lipofectamine™ Reagent, and Plus™ Reagent were purchased from Invitrogen. Extracellular-signal-regulated kinase (ERK) inhibitor (PD98059) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125) were purchased from Sigma. The anti Bcl-2, Bax, Caspase 3, pJNK, JNK, pERK, ERK, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) were purchased from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody and anti-rabbit secondary antibody were purchased from Santa Cruz Biotechnology. An enhanced chemiluminescence (ECL) assay kit was purchased from Amersham. EpCAM expression plasmid and si-EpCAM sequence were purchased from the ProteinTech Company. All the other reagents were of the highest purity commercially available.

Cell culture

MCF-7 and MDA-MB-231 cells were maintained in medium DMEM supplemented with 10% calf serum, 1% penicillin/streptomycin, 1-mM sodium pyruvate, 1.5 g/L sodium bicarbonate, and 10-mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Cells were incubated in a 5% CO₂ humidified atmosphere at 37°C.

Mutation of N-glycosylation sites by site-directed mutagenesis

We produced N-glycosylation mutant by substituting asparagines (N) with glutamine (Q) in all the three N-glycosylation sites of EpCAM. EpCAM harboring substitutions at the N-glycosylation sites were all generated by the TAKALA Company. All mutations were verified by DNA sequencing to ensure the presence of the correct mutation and the absence of any other randomly introduced mutations.

Cell viability assay

Cells (2 × 10³/100 mL) were seeded in 96-well plates and treated on the following day with 5-fluorouracil (5-FU) alone, EpCAM expression plasmid, si-EpCAM sequence, and N-glycosylation mutation EpCAM plasmid in combination with 5-FU. Cell viability was analyzed using the Cell Counting Kit-8 (CCK-8) according to the manufacturer’s instructions. In brief, the supernatant of each group was removed after treated separately, and cells were incubated in DMEM medium containing CCK8 for another 2 h at 37°C. Optical density (OD) was read at 450 nm on a microplate reader (Bio-Rad). The viability (%) was calculated according to the following equation: Viability (%) = (OD_treated / OD_medium) × 100.

Detection of apoptosis by 4′,6-diamidino-2-phenylindole staining

Morphological evaluation of cell apoptosis was performed using 4′,6-diamidino-2-phenylindole (DAPI) staining which detected the nuclei of both apoptotic and living cells. Cells grown on the glass coverslips were fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) for 30 min, washed for 15 min in 0.1% Triton X-100/PBS, and incubated in dark with DAPI (10 mg/mL) for 15 min. The stained cells were studied using a fluorescence microscope. The rate of apoptotic cells was recorded in 10 random nonoverlapping fields by two blinded observers. Stained nuclei were visualized under ultraviolet (UV) excitation and photographed using an Olympus fluorescence microscopy (Olympus).

Western blot

To prepare whole cell extracts, cells at 90% confluent were washed in PBS before incubation with lysis buffer (1% Triton X-100; 150-mM NaCl; 10-mM Tris, pH 7.4; 1-mM ethylenediaminetetraacetic acid (EDTA); 1-mM ethylene glycol tetraacetic acid (EGTA), pH 8.0; 0.2-mM Na₃VO₄; 0.2-mM phenylmethylsulfonyl fluoride; and 0.5% Nonidet P-40) on ice for 10 min. The cell lysates were clarified by centrifugation at 9000g for 10 min, and the supernatants were collected. Protein concentration was determined with the Coomassie Protein Assay Reagent using bovine serum albumin (BSA) as a standard. Cell lysates (50 µg) were separated by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) mini-gel. Samples were transferred electrophoretically to nitrocellulose membranes, blocked with Tween 20/Tris-HCl buffer solution (TTBS) containing 5% fat-free dry milk for 2 h, and incubated for 3 h with the appropriate primary antibodies at the dilutions recommended by the suppliers. After incubation with an HRP-conjugated anti-goat secondary antibody, immunoreactive proteins were visualized with ECL detection system. Western blots shown are representative of at least three independent experiments. Densitometry of each band for the target protein was quantified by densitometry analysis with Labworks 4.6. The protein band intensity was quantified by the mean ± standard error of the mean (SEM) of three experiments for each group as determined from densitometry relative to GAPDH.

Statistical analysis

The quantitative data derived from three independent experiments are expressed as mean (±standard deviation (SD)). Unpaired Student’s t tests were used to analyze between-group differences that is repeated, and p < 0.05 was considered statistically significant.

Results

N-glycosylation of EpCAM played an essential role in 5-FU-induced apoptosis in breast cancer cells

The effect of EpCAM and 5-FU on the morphology of MCF-7 and MDA-MB-231 cells was assessed by microscopic observation. The cells were transfected with EpCAM expression plasmid, si-EpCAM sequence, N-glycosylation mutation EpCAM plasmid, and 7.5 µg/mL 5-FU for 48 h, and were observed under a fluorescence microscope after staining with DAPI. Observations under the microscope showed that apoptotic cells in 5-FU-treated groups exhibited nuclear condensation, nuclear fragmentation, and chromatin margination. Cell morphology in response to EpCAM expression plasmid in combination with 5-FU treatment had no significant alterations. Treatment with 7.5 µg/mL 5-FU combined with si-EpCAM and N-glycosylation mutation EpCAM plasmid resulted in stronger nuclear chromatin condensation than with 7.5 µg/mL 5-FU alone (Figure 1). We had the same results in both MCF-7 cells and MDA-MB-231 cells. These results indicated that the amount of EpCAM had an effect on cell apoptosis and N-glycosylation of EpCAM was an important factor associated with cell apoptosis in breast cancer cells.

Figure 1.

Effect of N-glycosylation of EpCAM on the morphology of breast cancer cells induced by 5-FU. Cells (MCF-7 and MDA-MB-231) were treated with EpCAM expression plasmid, si-EpCAM sequence, N-glycosylation mutation EpCAM plasmid, and 7.5 µg/mL 5-FU for 48 h. After staining with DAPI, the cells were observed under a fluorescent microscope. Magnification: 400×. The arrow indicates the apoptotic bodies of the apoptotic cells.

Deglycosylation of EpCAM decreased the viability of breast cancer cells

To evaluate the cytotoxicity of 5-FU and the effect of N-glycosylation of EpCAM, MCF-7 and MDA-MB-231 cells were incubated with 7.5 µg/mL 5-FU combined with or without EpCAM expression plasmid, si-EpCAM sequence, and N-glycosylation mutation EpCAM plasmid for 48 h. Cell viability was determined using the CCK-8 assay kit. The results (Figure 2) showed that 5-FU significantly decreased the viability. EpCAM overexpression increased the cell viability and knockdown of EpCAM, and deglycosylation of EpCAM decreased the cell viability. Treatment with si-EpCAM in combination with 5-FU further decreased cell viability compared with 5-FU or si-EpCAM treatment alone. Treatment with N-glycosylation mutation EpCAM plasmid in combination with 5-FU had the same result as treatment with si-EpCAM. Taken together, we concluded that N-glycosylation of EpCAM was the key functional point and played an important role in cell viability.

Figure 2.

Effect of N-glycosylation of EpCAM and/or 5-FU treatment on cell viability in vitro. MCF-7 and MDA-MB-231 cells were incubated with 7.5 µg/mL 5-FU combined with or without EpCAM expression plasmid, si-EpCAM sequence, and N-glycosylation mutation EpCAM plasmid for 48 h. Cell viability was determined using the CCK-8 assay kit. Results are presented as mean ± SEM.

Deglycosylation of EpCAM in combination with 5-FU promoted the chemosensitivity to 5-FU in breast cancer cells by regulating the apoptosis-related proteins

According to the results above, we suggested that N-glycosylation of EpCAM was associated with the cell apoptosis in breast cancer cells. We further investigated the effect of N-glycosylation of EpCAM on the apoptosis-related proteins. The results showed that the expression of apoptotic proteins Bax and Caspase 3 was decreased in EpCAM-overexpressed cells and increased in EpCAM-silenced and N-glycosylation mutation EpCAM cells, whereas the expression of anti-apoptotic protein Bcl-2 was increased in EpCAM-overexpressed cells and decreased in EpCAM-silenced and N-glycosylation mutation of EpCAM cells (Figure 3(a)). These results demonstrated that deglycosylation of EpCAM in combination with 5-FU may regulate cell apoptosis by modulating the expression of apoptosis-related proteins.

Figure 3.

Effect of N-glycosylation of EpCAM and/or 5-FU treatment on apoptosis-related proteins in breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with EpCAM expression plasmid, si-EpCAM sequence, N-glycosylation mutation EpCAM plasmid, and/or 5-FU (7.5 µg/mL) for 48 h; proteins were collected. (a) Expression of Bcl-2, Bax, and Caspase 3 was determined by immunoblotting. (b) pJNK and pERK were detected by immunoblotting.

In our previous article, we have drawn a conclusion that EpCAM knockdown enhanced the cytotoxic effect of 5-FU, promoting apoptosis by regulating the apoptosis-related proteins via the ERK1/2 and JNK mitogen-activated protein kinase (MAPK) signaling pathways in MCF-7 cells.²⁰ The expression of the pJNK and pERK1/2 was detected in cells treated with EpCAM expression plasmid, si-EpCAM sequence, N-glycosylation mutation EpCAM plasmid, and/or 5-FU. The results showed that 5-FU significantly increased the levels of pJNK, and this effect was dramatically attenuated by pretreatment with si-EpCAM and N-glycosylation mutation EpCAM plasmid. In addition, 5-FU decreased the levels of pERK1/2, and this effect was also attenuated by pretreatment with si-EpCAM and N-glycosylation mutation EpCAM plasmid (Figure 3(b)). These results indicated that through targeting apoptosis-related proteins, N-glycosylation of EpCAM contributed to the regulation of cell apoptosis via ERK1/2 and JNK signaling pathways in MCF-7 and MDA-MB-231cells.

Deglycosylation of EpCAM regulated the cell apoptosis via ERK1/2 and JNK signaling pathways

To test whether the ERK1/2 and JNK signaling pathways mediated the effect of deglycosylation of EpCAM on the apoptosis in breast cancer cells induced by 5-FU, cells were induced by 7.5 µg/mL 5-FU with or without the inhibitors of ERK1/2 (PD98059) and/or JNK (SP600125). The level of apoptosis-related proteins was analyzed by western blot. The apoptotic proteins Bax and Caspase 3 were decreased, and the anti-apoptotic protein Bcl-2 was increased in deglycosylation of EpCAM cells induced by 5-FU. When cells were treated with inhibitors of ERK1/2 (PD98059, 10 µM), pERK expression was inhibited and the expression change of apoptosis-related proteins was intensified (Figure 4). While treated with inhibitors of JNK (SP600125, 10 µM), the effect was attenuated (Figure 5). The results suggested that deglycosylation of EpCAM and 5-FU treatment induced JNK activation and inhibited ERK1/2 activation in breast cancer cells, which downregulated the expression of Bcl-2 and induced apoptosis. It thus concluded that deglycosylation of EpCAM may play an anti-apoptosis effect on cell apoptosis via ERK1/2 and JNK signaling pathways.

Figure 4.

Involvement of ERK signaling activation in the effect of N-glycosylation of EpCAM on apoptosis in breast cancer cells. Cell apoptosis was induced by 5-FU (7.5 µg/mL) followed by with or without the specific inhibitor of ERK1/2 (PD98059, 10 µM) and N-glycosylation mutation EpCAM plasmid for 24 h. Cell lysates were immunoblotted and detected with the antibodies for apoptosis analysis.

Figure 5.

Involvement of JNK signaling activation in the effect of N-glycosylation of EpCAM on apoptosis in breast cancer cells. Cell apoptosis was induced by 5-FU (7.5 µg/mL) followed by with or without the specific inhibitor of JNK (SP600125, 10 µM) and N-glycosylation mutation EpCAM plasmid for 24 h. Cell lysates were immunoblotted and detected with the antibodies for apoptosis analysis.

Discussion

EpCAM is an oncogene that is usually overexpressed in breast cancer and is involved in cancer growth.^21–23 There was accumulating evidence that EpCAM was associated with the prognosis and progression of breast cancer and may serve as future targets for gene therapy.^13,21,24,25 There were also a large number of reports that protein glycosylation modification would alter during the progression of cancer, which played roles in cancer cell signaling, tumor cell dissociation and invasion, cell–matrix interactions, angiogenesis, and metastasis.^26,27 Exploration of the role of EpCAM was also beginning, and few studies have unveiled the involvement of N-glycosylation of EpCAM in breast cancer. In this study, we aimed to understand the role of EpCAM N-glycosylation in the apoptosis process in breast cancer cells.

In our previous study, we have demonstrated that knockdown of EpCAM promoted apoptosis, inhibited cell proliferation, and caused cell-cycle arrest.²⁰ In this study, we showed that deglycosylation of EpCAM significantly inhibited the survival of MCF-7 and MDA-MB-231 cells. DAPI staining results showed that deglycosylation of EpCAM increased the degree of cell apoptosis (Figures 1 and 2). These results indicated that deglycosylation of EpCAM increased the chemosensitivity to 5-FU by increasing the rate of apoptosis in breast cancer cells. It suggested that N-glycosylation of EpCAM played an important role in the regulation of apoptosis in breast cancer cells. To further verify the function of N-glycosylation of EpCAM in 5-FU-induced apoptosis in breast cancer cells, we detected the relevant signaling pathways. MAPK signaling pathway is reported to be associated with the cell proliferation, differentiation, migration, senescence, and apoptosis, including ERK1/2 and JNK.²⁸ For example, it has been reported that curcumin suppresses proliferation and migration and induces apoptosis on human placental choriocarcinoma cells via ERK1/2 and stress-activated protein kinase (SAPK)/JNK MAPK signaling pathways.²⁹ Vitexin suppresses autophagy to induce apoptosis in hepatocellular carcinoma via activation of the JNK signaling pathway.³⁰ In our previous study, we draw a conclusion that knockdown of EpCAM and 5-FU treatment induced JNK activation and inhibited ERK1/2 activation in MCF-7 cells, which downregulated the expression of Bcl-2 and induced apoptosis.²⁰ Thus, we continued to investigate the effect of ERK1/2 and JNK signaling pathways on deglycosylation of EpCAM-mediated apoptosis induced by 5-FU. We used the inhibitors of ERK1/2 and JNK to test whether these two signaling pathways took part in the regulation process. Our results indicated that glycosylation of EpCAM may regulate the apoptosis-related proteins through the ERK1/2 and JNK signaling pathways. The apoptotic effects of deglycosylation of EpCAM were largely blocked by the JNK inhibitor (SP600125) and promoted by the ERK inhibitor (PD98059). These findings suggested that depletion of N-glycosylation of EpCAM could accelerate the process of apoptosis.

In summary, we have identified that deglycosylation of EpCAM significantly increased the chemosensitivity of breast cancer cells to 5-FU through a mechanism involving the glycosylation of EpCAM-mediated modulation of the expression of anti-apoptotic factors in tumor cells and the induction of apoptosis which was mediated by the ERK1/2 and JNK MAPK signaling pathways. These results indicated that N-glycosylation of EpCAM might be a protective factor against breast cancer cell apoptosis. Furthermore, our findings provided evidence that N-glycosylation of EpCAM contributed to the apoptotic signaling and EpCAM may be a potential target for the treatment of breast cancer.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported by grants from the Major State Basic Research Development Program of China (2012CB822103), the National Natural Science Foundation of China (No. 30800195, 31270866), and the National Natural Science Foundation of Liaoning Province (No. 201602242).

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