Cell lines and culture

The human ESCC cell lines KYSE450 and TE-1 were purchased from the Cell Bank of the Shanghai Institute in People’s Republic of China. The cells were cultured in Roswell Park Memorial Institute 1640 enriched with 1% penicillin/streptomycin (Sigma-Aldrich Co., St Louis, MO, USA), 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 1% L-glutamine. Cell culture plates were maintained in humidified incubators at 37°C in a 5% CO₂ incubator.

Immunohistochemistry and immunofluorescence

The immunohistochemistry studies of MUC1-C and MYC were performed by the streptavidin peroxidase method. The formalin-fixed, paraffin-embedded tissues were cut into sections, which then were deparaffinized and incubated with hydrogen peroxide. Rabbit anti-MUC1-C/anti-MYC antibodies (Abcam, Cambridge, MA, USA) were diluted to 1:100 and incubated at 4°C overnight. The subsequent steps were followed according to the instructions of the secondary biotinylated antibody kit (Zhongshan Biotech, Guangzhou, People’s Republic of China). Stained slides were scored by two blinded, independent observers. Integrated optical density was measured in three random fields from each patient using Image-Pro Plus 6.0. Values were represented as mean ± SD. Immunofluorescence process was also performed to verify the expression of MUC1-C and MYC protein in frozen sections. Following fixation with 4% paraformaldehyde, the frozen sections were treated with 0.25% Triton and blocked in 10% goat serum. Rabbit anti-MUC1-C/anti-MYC antibodies (Abcam) were diluted to 1:150, and the goat anti-rabbit DyLight 594/488 antibody (Abcam) was diluted to 1:300 for 1 h followed by counter staining of DAPI for 5 min. The images were examined using a fluorescence microscope (Olympus BX51; Olympus, Tokyo, Japan).

Protein extraction and western blotting

Protein was extracted both from tissues and cells by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA). The extracted proteins were electrophoresed on SDS polyacrylamide gels and transferred onto PVDF membrane filters (Millipore Corporation, Billerica, MA, USA). Membranes were blocked with non-fat dried milk and incubated overnight with antibodies (anti-MUC1-C, anti-MYC and anti-PCNA were obtained from Abcam; anti-β-actin was obtained from Boster Biological Technology Co. Ltd., CA, USA) in blocking buffer. After being washed, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase anti-mouse/rabbit IgG (Boster). The bands were visualized by an enhanced chemiluminescence detection system (Amersham imager 600; General Electric, Fairfield, CT, USA).

Colony formation assays

Cells were seeded in 6-well plates for 24 h and then treated with GO-201 (NJpeptide, Nanjing, People’s Republic of China), CP-1 (NJpeptide) or pure culture media every 3 days. The plates were incubated at 37°C in a 5% CO₂ incubator for 10 days. Then, colonies were stained with crystal violet, and those with >50 cells were scored as surviving colonies. The cloning efficiency was calculated by dividing the average number of colonies/dish by the number of plated cells. This experiment was performed in triplicates.

CCK-8 assay

Cell proliferation was detected by using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). The logarithmically growing cells were seeded in 96-well plates and incubated with GO-201, CP-1 or pure culture media, respectively, for 24, 48 and 72 h. At different time intervals, the cells were incubated with CCK-8 reagent for 1 h at 37°C. The absorbance of each well was measured using Thermo Scientific Varioskan Flash (Thermo Fisher Scientific, Vantaa, Finland). This experiment was performed in triplicates.

Wound-healing assay

Cells were treated with GO-201, CP-1 or pure culture media in plates separately until 95% confluence. A straight “wound” was made by scratching with a plastic pipette tip. Having cultured in FBS-free medium for 12 and 24 h, cell migration was analyzed by using light microscopy. The residual area of the scratch at the same position was measured by using ImageJ software. The migration rate was calculated by the following formula

The area of initial wound.

Cell apoptosis detection

Flow cytometric assays were conducted using the Annexin V-FITC apoptosis detection kit I (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instructions. Cells from the three groups (treated with GO-201, or treated with CP-1, or left untreated) were harvested by trypsinization and resuspended in 1X binding buffer. After double staining with P-phycoerythrin (PE) and 7-Amino-actinomycin D (7-AAD), the samples were analyzed by flow cytometry and the data were analyzed by using FlowJo software.

Real-time PCR

After being treated with GO-201, CP-1 or left untreated, the whole-cell RNA was extracted using the RNAiso Plus (TaKaRa, Shiga Japan). Then quantitative polymerase chain reaction (PCR) was carried out with SYBR Green PCR kit according to the manufacturer’s instructions (TaKaRa). Amplifications were performed in ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Relative transcript quantities were calculated by using the DDCt method with β-actin as the endogenous reference gene. PCR primers were as follows: MYC, 5′-GGAGGCTATTCTGCCCATTTG-3′ (forward primer) and 5′-CGAGGTCATAGTTCCTGTTGGTG-3′ (reverse primer); β-actin, 5′-AGAGCCTCGCCTTTGCCGATCC-3′ (forward primer) and 5′-ATACACCCGCTGCTCCGGGTC-3′ (reverse primer). This experiment was performed in triplicates.

Cell migration and invasion assay

For both migration and invasion assays, the cells treated with GO-201, CP-1 or left untreated were precultured in FBS-free medium for 24 h. The uncoated transwells were used in the migration assay, while Matrigel (1:4; BD Biosciences) was pre-coated to the upper surface of transwells in the invasion assay. Then, starved cells were seeded into the upper chamber of 24-well transwells (8 mm pore size polycarbonate membrane; Merck KGaA, Darmstadt, Germany) with FBS-free medium, and medium with FBS was added to the low chamber. The migration assay was allowed to incubate for 24 h, and the invasion assay was allowed to proceed for 48 h. Then, cells on the lower surface of membrane were fixed and stained. The cell numbers were calculated from three independent visual fields under light microscopy.

ESCC tumor xenograft treatment studies

Six-week-old female BALB/c nude mice were injected with 2×10⁶ ESCC cells subcutaneously in the flank. When the tumors reached a size of ~150 mm³, the mice were pair matched into 3 groups of 5 mice each, and treated by 1) control vehicle, 2) 15 mg/kg GO-201 administered intraperitoneally (IP) each day or 3) 15 mg/kg CP-1 administered IP daily. We measured the size of tumors every 4 days and weighed the tumors after 20 days. Tumor volumes were calculated by using the formula V=L×W²/2, where L and W are the larger and smaller diameters, respectively. All procedures relating to animal handling, care and treatment were performed in strict accordance with the recommendations of regulations on the management of experimental animals, which were approved by State Council of the People’s Republic of China on October 31, 1988, and promulgated by Decree No. 2 of the State Science and Technology Commission on November 14, 1988. The protocol was approved by the ethics committee of Provincial Hospital affiliated to Shandong University.

Targeting MUC1-C inhibits MYC transcription and thereby downregulates the level of MYC in ESCC cells

The co-expression of MUC1-C in the nucleus and MYC in ESCC suggests a potential interaction of these two proteins, which has been studied by targeting MUC1-C. The MUC1-C cytoplasmic domain contains a CQC motif that is necessary for MUC1-C homodimerization and function9–11 (Figure 2A). Accordingly, the cell penetrating peptide, designated as GO-201, was developed to block the MUC1-C CQC motif in the ESCC and other carcinoma cells7,11 (Figure 2A). Notably, treatment of KYSE450 and TE-1 cells with GO-201, but not the control peptide CP-1, was obviously inhibiting the entry of MUC1-C into the nucleus, which is confirmed by western blot analysis (Figure 2B and C). However, there was no significant difference in the level of MUC1-C in the cytoplasm (Figure 2D and E). Meanwhile, the downregulation of MYC (Figure 2F and G) was also detected by western blot analysis. The same result is further approved by the quantitative real-time PCR. Targeting MUC1-C by using GO-201 in the cells resulted in downregulation of MYC mRNA levels (Figure 2H); thereby, it has obviously suppressed the MYC transcription.

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Figure 2

Targeting MUC1-C downregulates the expression of MYC in ESCC cell lines.

Notes: (A) Schematic representation of the MUC1-C subunit and the 72 amino acid sequence of MUC1-CD is shown. The N-terminal 15 amino acid GO-201 and mutated CP-1 peptides were synthesized with the poly-dArg transduction domain.11 (B–G) KYSE450 and TE-1 cells were treated each day with 10 μM GO-201 or CP-1 or left untreated for 3 days, and lysates were immunoblotted with indicated antibodies. (B) The bands of MUC1-C and PCNA in the nucleus. (C) Quantitative analysis of MUC1-C protein in the nucleus normalized to PCNA. (D) The bands of MUC1-C and β-actin in the cytoplasm. (E) Quantitative analysis of MUC1-C protein in the cytoplasm normalized to β-actin. (F) The bands of MYC and β-actin in the whole cell. (G) Quantitative analysis of MYC protein of the whole cell normalized to β-actin. (H) MYC mRNA level, which is from the cells treated each day with 10 μM GO-201 or CP-2 or left untreated for 3 days, was determined by qRT-PCR. The results are expressed as relative MYC mRNA levels (mean ± SD of three determinations) compared with that obtained for β-actin as a control. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Abbreviations: MUC1-C, mucin 1 C-terminal transmembrane subunit; ESCC, esophageal squamous cell carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction.

Targeting MUC1-C decreases ESCC cell lines’ proliferation ability

To determine if inhibiting the MUC1-C into the nucleus could suppress the proliferation of ESCC cells, clonogenic assay and CCK-8 were conducted. The colony formation assays showed that treatment of KYSE450 and TE-1 cells with GO-201 was apparently suppressed in clonogenic survival compared to that obtained with CP-1 or left untreated (Figure 3A and B). The similar result was obtained from the CCK-8 assay. The OD values (450 nm) of the KYSE450 and TE-1 cells with GO-201 showed significant decrease at 24, 48 and 72 h, compared with those cells with CP-1 or without penetrating peptide (Figure 3C).

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Figure 3

Targeting MUC1-C inhibits cell proliferation in ESCC cell.

Notes: (A) The KYSE450 and TE-1 cells were seeded at 600 cells/well (6-well plate), treated with 10 μM GO-201 or CP-1 or left untreated for every 3 days. After 10 days, the cells were stained with crystal violet. (B) Colony formation (>50 cells) rates are expressed as mean ± SD of three replicates. (C) The KYSE450 and TE-1 cells were treated each day with 10 μM GO-201 or CP-1 or left untreated for three days, and then were seeded at 5×10³ cells/well (96-well plate). OD values were detected after 24, 48 and 72 h. The results are expressed as the mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Abbreviations: MUC1-C, mucin 1 C-terminal transmembrane subunit; ESCC, esophageal squamous cell carcinoma.

Targeting MUC1-C inhibits the migration and invasion of ESCC cell lines

To evaluate the migration and invasion abilities of ESCC cells, which are the signs of a malignant degree, the wound-healing and transwell assays were performed. With respect to wound-healing assay, the closure of the wounds was apparently slower in ESCC cells treated by GO-201, in which most wounds remained open, whereas most space along the migrating fronts fused in the control group (Figure 4A and B). In the transwell assay, compared to the control group, the number of ESCC cells treated with GO-201 that penetrated the transwell membrane was significantly lower (Figure 4C–F). These results indicated that inhibiting MUC1-C into the nucleus by GO-201 could remarkably suppress the migration and invasion of ESCC cells.

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Figure 4

Targeting MUC1-C inhibits the migration and invasion of ESCC cell lines.

Notes: (A) The KYSE450 and TE-1 cells treated with 10 μM GO-201 or CP-1 or left untreated were grown until confluence. “Wound” with a uniform width was made on the monolayer cells by scratching with a plastic pipette tip. The cells were cultured in FBS-free medium for 12 and 24 h. Pictures were taken using light microscopy (×100). (B) Migration rate of the cells was calculated from three independent experiments using the following formula: % Migration rate= (the area of initial wound – the area of wound after 12 h/24 h) ×100/the area of initial wound. (C) The indicated cells were seeded into the uncoated upper chamber of 24-well transwells at 1.5×10⁵ cells/well with 200 μL FBS-free medium, and 600 μL of medium with 15% FBS was added to the lower chamber. After 24 h, cells on the lower surface of membrane were fixed and stained. (D) The cell numbers on the lower surface of the membrane were counted in three randomly selected fields. (E) The indicated cells were seeded into the upper chamber pre-coated with 40 μL of Matrigel at 1.5×10⁵ cells/well with 200 μL FBS-free medium, and 600 μL of medium with 15% FBS was added to the low chamber. After 48 h, cells on the lower surface of membrane were fixed and stained. Pictures were taken using light microscopy (×200). (F) The cell numbers on the lower surface of the membrane were counted in three randomly selected fields. Each data point represents mean ± SD of 3 repeated experiments. ^**p < 0.01, ^***p < 0.001.

Abbreviations: MUC1-C, mucin 1 C-terminal transmembrane subunit; ESCC, esophageal squamous cell carcinoma; FBS, fetal bovine serum.

Targeting MUC1-C promotes apoptosis in ESCC cells

In order to determine whether apoptosis resulted in proliferation inhibition, a flow cytometric apoptosis assay was performed. The data showed that in contrast to control groups the number of apoptotic KYSE450 and TE-1 cells treated with GO-201 for 4 days increased significantly (Figure 5A and B). Based on these results, we concluded that inhibiting MUC1-C into the nucleus promotes apoptosis of KYSE450 and TE-1 cells.

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Figure 5

Targeting MUC1-C promotes apoptosis in ESCC cells.

Notes: (A) The cells treated with 10 μM GO-201 or CP-1 or left untreated for 4 days were incubated with PI/Annexin V and analyzed by flow cytometry. The percentage of PI and/or Annexin V-positive cells is included in the panels. (B) The results are expressed as the percentage (mean ± SD of 3 repeated experiments) of apoptotic/necrotic cells. ^***p < 0.001.

Abbreviations: MUC1-C, mucin 1 C-terminal transmembrane subunit; ESCC, esophageal squamous cell carcinoma; PI, propidium iodide.

This work was supported by grants from Natural Science Foundation of Shandong Province (2014ZRB14125). The authors appreciate the help from the Department of Thoracic Surgery, Pathology Department and Central Laboratory of Shandong Provincial Hospital affiliated to Shandong University.