Monoclonal anti‑MUC1 antibody with novel octahydropyrazino[2,1‑a:5,4‑a']diisoquinoline derivative as a potential multi‑targeted strategy in MCF‑7 breast cancer cells
- Authors:
- Published online on: August 2, 2019 https://doi.org/10.3892/or.2019.7256
- Pages: 1391-1403
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Copyright: © Gornowicz et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Breast cancer represents the leading cause of death in women worldwide and has a poor prognosis. Estrogen and estrogen receptors (ERs) play key roles in breast cancer progression. Previous studies have demonstrated that ER is expressed in 75% of breast cancer overall (1–3). Conventional cytotoxic chemotherapy is still one of the key elements of therapeutic armamentarium, but the effectiveness of treatment is limited.
Mucin-1 (MUC1) is a transmembrane glycoprotein abnormally overexpressed in a wide range of human epithelial cancer types, including colon, breast, ovarian and pancreatic cancer (4,5). MUC1 is localized only on the apical membrane and functions via barrier formation and monitoring luminal events in healthy cells (4). Overexpression and aberrant glycosylation of MUC1 in cancer cells contribute to tumor progression and metastasis (6). MUC1 is also a heterodimer, which consists of two subunits: A long N-terminal fragment and short C-terminal fragment. Its cytoplasmic tail takes part in intracellular signaling by interfering with different proteins and affecting their function (4). The cytoplasmic tail of MUC1 interacts with different molecules, which are also overexpressed in cancer, such as ER, β-catenin and ErbB growth factor receptor tyrosine kinases (7).
Our recent study synthesized and evaluated the anticancer potential of novel diisoquinoline derivatives in breast and gastric cancer cells (8). The novel compounds were involved in the inhibition of AKT and ERK1/ERK2 (8). It was also demonstrated that the new diisoquinoline derivatives could modulate both apoptotic pathways. Another previous study demonstrated that all novel synthesized compounds activated the initiator and executioner caspases, such as caspase-8, caspase-9, caspase-10 and caspase-3, compared with untreated breast cancer cells (9).
The aim of the present study was to examine the multi-targeted potential of a monoclonal antibody against MUC1 and novel octahydropyrazin[2,1-a:5,4-a′]diisoquinoline derivative (OM-86II) in ER-positive MCF-7 human breast cancer cells. The results of the present study were compared with the findings concerning etoposide used together with anti-MUC1 antibody as well as with monotherapy. Etoposide is a widely used drug for chemotherapy and its molecular mechanism of action is associated with the inhibition of topoisomerase II (10).
Materials and methods
Materials
Stock cultures of human MCF-7 breast cancer cells were purchased from The American Type Culture Collection. DMEM and FBS used in cell culture were products of Gibco; Thermo Fisher Scientific, Inc. Glutamine, penicillin and streptomycin were obtained from Quality Biological, Inc. The JC-1 MitoScreen kit was supplied by BD Pharmigen; BD Biosciences. ELISA kits used to detect the concentrations of matrix metalloproteinase (MMP)-2, MMP-9, tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, mTOR and soluble intercellular adhesion molecule (sICAM)1 were obtained from Wuhan EIAab Science Co., Ltd. (cat. nos. E0100 h, E0553 h, E0133 h, E0699 h, E14969 h and EH0161). Etoposide was obtained from Sigma-Aldrich; Merck KGaA and the purity of the compound was >98%. Monoclonal anti-MUC1 antibody (cat. no. MA1-06503) was a product of Thermo Fisher Scientific, Inc.
Nuclear magnetic resonance (NMR) spectra were recorded using a Varian VNMR500 spectrometer (Varian, Inc.). Chemical shifts are quoted in parts per million relative to TMS for 1H and toluene-d8 for 13C NMR. Coupling constants J are reported in Hz. Mass spectra were recorded using an AMD-604 Intectra GmbH mass spectrometer (Waters Corporation).
Compounds
OM-86II was synthesized using previously standardized methods (9,11). The synthesis and physicochemical characterization of a compound 15a was presented in our previous study (11). Compound 15a (0.3 mmol; 169 mg) was dissolved in acetonitrile (15 ml) and cooled to 0°C. Into intensively stirred reaction mixture, a water solution (10 ml) of cerium ammonium nitrate (1 mmol; 326 mg) was added dropwise. Stirring was continued at room temperature until the reaction was over (~2 h), poured into cold sodium dithionate (60 ml; 1M), extracted with dichloromethane (3×40 ml), dried with magnesium sulphate, filtrated and concentrated. The crude product was purified on silica gel using gradient DCM/MeOH (1–10% MeOH) as an eluent (Fig. 1).
Yield: 79 mg, 45%. Semisolid. 1H NMR (toluene-d8, 80°C, 500 MHz): 7.84 - 7.80 (m, 2H), 7.66 - 7.1 (m, 2H), 7.17 - 7.11 (m, 3H), 7.11 - 7.00 (m, 3H), 6.80 (s, 1H), 6.78 (s, 1H), 6.54 (s, 1H), 6.37 (s, 1H), 3.85 (d, J=5.6 Hz, 1H), 3.70 (d, J=9.6 Hz, 1H), 3.56 (d, J=5.6 Hz, 1H), 3.46 (s, 3H), 3.44 (s, 3H), 3.37 (s, 3H), 3.26 - 3.21 (m, 4H), 3.10 - 2.91 (m, 2H), 2.90 - 2.71 (m, 3H), 2.58 - 2.50 (m, 2H). 13C NMR (toluene-d8, 80°C, 125 MHz): δ (ppm): 196.3, 151.5, 148.4, 148.3, 147.8, 147.0, 139.1, 134.1, 132.2, 132.0, 130.4, 130.1, 128.0, 128.0 127.7, 127.2, 126.3, 114.3, 113.7, 112.4, 111.9, 75.3, 69.0, 65.0, 57.0, 55.4, 55.1, 55.1, 55.1, 45.0, 32.9, 26.4. MS (ES, HR) m/z: (M+) calcd for C36H36N2O5: 576.6930; Found: 576.2626. Anal. Calcd for C36H36N2O5: C, 74.98; H, 6.29; N, 4.86; Found: C, 75.00; H, 6.20; N, 4.81.
Cell culture of MCF-7 cells
ER-positive breast cancer MCF-7 cells were maintained in DMEM supplemented with 10% FBS, 2 mM glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Sub-confluent cells were treated with 0.05% trypsin and 0.02% EDTA in calcium-free PBS, counted using a hemocytometer and seeded in 6-well plates (Nunc) in 2 ml growth medium (DMEM without phenol red with 10% CPSR1). The cells that reached ~80% confluency were used for the assays.
Treatment groups and conditions
MCF-7 breast cancer cells were incubated with anti-MUC1 (10 µg/ml), OM-86II (30 µM), OM-86II + anti-MUC1 (30 µM + 10 µg/ml), etoposide (30 µM) and etoposide + anti-MUC1 (30 µM + 10 µg/ml) for 24 and 48 h at 37°C in 5% CO2 in an incubator.
Cell viability assay
To examine the effect of the compounds on cell growth, MCF-7 cells were seeded in 6-well plates (2×106) and cultured as described. Cell cultures were incubated with varying concentrations of the compounds tested for 24 and 48 h. Then cells were washed three times with PBS and then incubated for 4 h in 1 ml MTT solution (0.5 mg/ml PBS) at 37°C in 5% CO2 in an incubator. The medium was removed and 1 ml 0.1 mol/l HCl in absolute isopropanol was added to the attached cells. The absorbance of the converted dye in living cells was measured at a wavelength of 570 nm (12).
[3H]thymidine incorporation assay
To examine the effect of the compounds on cell proliferation, MCF-7 cells were seeded (2×106) in 6-well plates and cultured as described. Cell cultures were incubated with varying concentrations of the tested compounds and 0.5 µCi [3H]thymidine for 24 h at 37°C. The cells were harvested by trypsinization and washed several times in cold PBS (10 min/1.500 g) until the dpm in the washes were similar to the reagent control. Radioactivity was determined by liquid scintillation counting. [3H]thymidine uptake is expressed as dpm/well (9).
Cell cycle analysis
The distribution of the cell cycle phases was analyzed by flow cytometry. Briefly, MCF-7 breast cancer cells were seeded into 6-well plates at a density of 2.5×105 cells/well and treated with the compounds for 24 and 48 h. After incubation, the cells were harvested and then fixed with 1 ml 70 % ethanol and kept overnight at −20°C. Before analysis, the cells were resuspended in PBS, treated with 50 µg/ml DNase-free RNase A solution (Promega Corporation), and stained for 30 min at 37°C with 100 µg/ml propidium iodide (PI; ImmunoChemistry Technologies, LLC; cat. no. 638). The FACSCanto II flow cytometer (BD Biosciences) was used to read the fluorescence and the results were analyzed using FACSDiva software (version 6.1.3; BD Biosciences Systems) (13).
Determination of mitochondrial membrane potential
Disruption of the mitochondrial membrane potential was assessed using the lipophilic cationic probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1 Mitoscreen kit; BD Biosciences) as previously described (14). Briefly, unfixed cells were washed and resuspended in PBS supplemented with JC-1. The cells were then incubated for 15 min. at room temperature in the dark, washed and resuspended in PBS for immediate flow cytometry analysis using a FACSCanto II flow cyometer. The percentage of cells with disrupted MMP was calculated using FACSDiva software (version 6.1.3; BD Biosciences).
Dual acridine orange/ethidium bromide fluorescent staining
To confirm that the compounds induce apoptosis, dual acridine orange/ethidium bromide fluorescent staining was assessed and visualized under a fluorescent microscope (Nikon Eclipse Ti; Nikon Corporation). MCF-7 breast cancer cells were treated with the compounds for 24 and 48 h. The cell suspension (250 µl) was stained for 10 min at room temperature in the dark with 10 µl dye mixture (10 µM acridine orange and 10 µM ethidium bromide), which was prepared in PBS. Cells cultured in a drug-free medium were used as controls. The morphology of two hundred cells per sample was examined by fluorescent microscopy (magnification, ×100) within 20 min. The results were analyzed with NIS-Elements software (version 3.10; Nikon Corporation).
Flow cytometry assessment of Annexin V binding
The effect of the compounds on the induction of apoptosis after 24 and 48 h of incubation was assessed using a Becton Dickinson FACSCanto II flow cytometer (BD Biosciences). The assessment allows checking the loss of asymmetry of phospholipids on the cell membrane. Cells were trypsinized, resuspended in DMEM and then in binding buffer. Next, they were stained with FITC Annexin V and PI for 15 min at room temperature in the dark, according to the manufacturer's protocol (FITC Annexin V Apoptosis Detection kit II; BD Biosciences). Cells cultured in a drug-free medium were used as controls. The optimal parameter settings were found using a positive control (cells incubated with 3% formaldehyde in buffer during 30 min on ice). Forward scatter and side scatter signals were detected on a logarithmic scale histogram. FITC was detected in the FL1 channel (FL1 539; Threshold-value 52). The results were analyzed with FACSDiva software (version 6.1.3; BD Biosciences).
Determination of matrix metalloproteinase (MMP)-2, MMP-9, tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, mTOR and soluble intercellular adhesion molecule (sICAM)1
High sensitivity assay ELISA kits (Wuhan EIAab Science Co., Ltd.) were used to determine the concentrations of proteins in supernatants from cell culture or in cell lysates (15) after 24 and 48 h of incubation with the compounds. There was no cross-reactivity or interference by other proteins present in biological samples. The microtiter plate provided in this kit was pre-coated with an antigen-specific antibody. Standards and samples were added to the appropriate microtiter plate wells. After 2 h of incubation at 37°C, the plate was incubated with biotin-conjugated antibody from the kits for 1 h at 37°C. Then, the microplate wells were aspirated and washed three times, and then incubated for 1 h at 37°C with avidin conjugated to horseradish peroxidase. Then, a 3,3′,5,5′-tetramethylbenzidine substrate solution was added to each well. The enzyme-substrate reaction was terminated by the addition of a sulfuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450±2 nm. The concentration of antigen in the samples was determined by comparing the optical density of the samples to the standard curve.
Statistical analysis
All numerical data are presented as the mean ± SD from three independent experiments. The statistical analysis was performed using GraphPad Prism Version 6.0 (GraphPad Software, Inc.). All datasets were analyzed using ANOVA and Tukey's test. P<0.05 was considered to indicate a statistically significant difference.
Results
Novel OM-86II combined with anti-MUC1 antibody decreases cell viability and proliferation of MCF-7 breast cancer cells
The effects of etoposide, a novel diisoquinoline derivative (OM-86II) and an anti-MUC1 antibody as well as etoposide or OM-86II in combination with anti-MUC1 antibody on cell viability and DNA biosynthesis in MCF-7 breast cancer cells were examined (Figs. 2 and 3). The cell viability was analyzed after 24 and 48 h of incubation with the agents tested. It was detected that the anti-MUC1 antibody used together with OM-86II represented the strongest cytotoxic and antiproliferative potential (Figs. 2 and 3). Such a combination decreased the number of viable cells to 47.1 and 38.8% after 24 and 48 h of incubation, respectively (Fig. 2). The combination of etoposide and anti-MUC1 antibody reduced the number of live cells to 87.4 and 73.2% after 24 and 48 h of incubation, respectively (Fig. 2). Monotherapy was not so efficient in decreasing the viability of MCF-7 cells. However, the most cytotoxic properties for monotherapy were observed after incubation with anti-MUC1 antibody, which reduced the viability of breast cancer cells to 53.4% after 48 h of incubation, respectively (Fig. 2).
The antiproliferative potential of the compounds tested was demonstrated by the incorporation of [3H]-thymidine into the DNA of MCF-7 cells (Fig. 3). The combination of novel OM-86II with anti-MUC1 represented the strongest antiproliferative activity in MCF-7 breast cancer cells. We detected that such a combination inhibited DNA biosynthesis to 47.15 and 26.5% after 24 and 48 h of incubation, while etoposide with anti-MUC1 reduced [3H]-thymidine incorporation to 53.64 and 32.12% after 24 and 48 h of incubation. Monotherapy was not so efficient, and in this case the most antiproliferative potential was demonstrated after incubation with OM-86II, which inhibited the [3H]-thymidine incorporation into DNA of breast cancer cells to 59.6 and 39.8% after 24 and 48 h of incubation (Fig. 3).
Novel OM-86II combined with anti-MUC1 antibody induces G2/M cell cycle arrest in MCF-7 cells
Anticancer agents can be designed to target specific cell cycle checkpoints in cancer cells and are able to induce cell death (16). Cell cycle analysis revealed that monotherapy as well as a combination of compounds (OM-86II, etoposide), together with anti-MUC1 antibody, induced G2/M arrest in MCF-7 breast cancer cells. As shown in Fig. 4A, the percentage of cells in the G2/M phase increased from 4.1% in the untreated control to 22.1% after treatment with OM-86II and anti-MUC1 (30 µM + 10 µg/ml), 13.6% after treatment with etoposide and anti-MUC1 (30 µM + 10 µg/ml), 8.3% after treatment with OM-86II (30 µM), 11% after treatment with etoposide (30 µM) and 9% after treatment with anti-MUC1 antibody (10 µg/ml). Upon prolongation of the exposure time to 48 h, the highest percentages of cells were arrested in the G2/M phase after treatment with OM-86II combined with anti-MUC1 antibody (33.0%; Fig. 4B).
Novel OM-86II combined with anti-MUC1 antibody induces apoptosis and decreases mitochondrial membrane potential in breast cancer cells
Dual acridine orange/ethidium bromide fluorescent staining was performed to visualize viable, apoptotic and necrotic cells after treatment with the compounds used alone and in combination with anti-MUC1 antibody (Fig. 5). Control (untreated) cells were displayed as green fluorescence. Bright green fluorescence was characteristic of early apoptotic cells, whereas an orange color was specific to late apoptotic cells. The present study demonstrated that the combination of OM-86II and anti-MUC1, resulted in the highest number of apoptotic cells. Bright green fluoresence as well as orange fluorescence was observed. The strongest pro-apoptotic effect was observed after 48 h of incubation (Fig. 5B).
An Annexin V binding assay was performed to confirm the results obtained by fluorescence microscopy. The results are presented in Fig. 6. The histograms showed viable, early and late apoptotic cells, and necrotic cells. In total, 11.3% apoptotic cells after treatment with etoposide and 17.9% with combination of etoposide and anti-MUC1 were observed after 24-h incubation. OM-86II demonstrated stronger pro-apoptotic potential and 38.9% of early and late apoptotic cells were detected. The most significant effect was observed after 24-h incubation with anti-MUC1 and OM-86II. In that case, 53.1% of apoptotic cells were detected (Fig. 6A). After the next 24 h of incubation, the pro-apoptotic effect was enhanced after all treatments, but the percentage of apoptotic cells (67.1%) was the highest after 48 h of incubation with anti-MUC1 and OM-86II (Fig. 6B).
The mitochondrial membrane potential of the cells was detected after 24 and 48 h of incubation (Fig. 7). It was identified that all the compounds significantly decreased the mitochondrial membrane potential compared with the control after 48 h of incubation (Fig. 7B). In the control MCF-7 cells, 7.4% of cells with reduced mitochondrial membrane potential were detected. After 48 h of incubation with etoposide and etoposide with anti-MUC1, 44.9 and 46.2% of cells, respectively, with reduced mitochondrial membrane potential were detected. The compound OM-86II led to a higher percentage of cells with decreased mitochondrial membrane potential (46.4%) as compared with etoposide and anti-MUC antibody. The combination of OM-86II with anti-MUC1 antibody reduced the mitochondrial membrane potential the most; it was observed that 55% of cells had reduced mitochondrial membrane potential (Fig. 7B).
Novel OM-86II combined with anti-MUC1 antibody decreases the concentration of MMP-2 and MMP-9 in supernatants from MCF-7 cell cultures
ELISA, a quantitative method, was chosen instead of western blotting to measure the concentration of analyzed proteins. The concentration of MMP-2 was detected after 24 and 48 h of incubation with etoposide, etoposide with anti-MUC1, OM-86II, OM-86II with anti-MUC1 and anti-MUC1 antibody (Fig. 8). It was identified that OM-86II with anti-MUC1 antibody decreased the concentration of MMP-2 in supernatants from the cell cultures (1.5 ng/ml) the most in comparison with control, where the concentration of MMP-2 was 1.8 ng/ml (Fig. 8B).
The concentration of MMP-9 was additionally detected (Fig. 9). The most significant change in the concentration of MMP-9 was observed after 48 h of incubation with the compounds tested (Fig. 9B). The concentration of MMP-9 in the control sample after 48 h was 24 ng/ml. It was demonstrated that novel OM-86II reduced the concentration of MMP-9 to 16 ng/ml and the combination of OM-86II with anti-MUC1 antibody reduced the concentration to 13 ng/ml. Etoposide alone and in combination with anti-MUC1 antibody significantly increased the concentration of MMP-9 to 48 ng/ml.
Novel OM-86II combined with anti-MUC1 antibody decreases the concentration of TNF-α and COX-2
The prolonged exposition of epithelial cells to different factors and activation of inflammatory pathways are associated with tumorigenesis (17). Several previous studies have demonstrated an anticancer effect after the inhibition of TNF-α and its receptors; such an effect was observed in animal models of breast cancer (18–25). In the present study, the concentration of TNF-α was detected after 24 and 48 h of incubation with the different treatments (Fig. 10). The most significant changes in TNF-α concentration were detected after 48 h of incubation with the compounds tested (Fig. 10B). The strongest inhibition of TNF-α release was observed after combined treatment with OM-86II and anti-MUC1 antibody. The concentration of TNF-α was 15 pg/ml, as compared with 19.5 pg/ml in the control sample.
Cancer cells with overexpressed COX-2 are resistant to apoptosis, and COX-2 acts as a key driver in increased growth and invasion of cancer cells via different molecular signaling pathways (17). The present study demonstrated that the combination therapy based on OM-86II and anti-MUC1 antibody led to decreased concentration of COX-2 in cell lysates in comparison with the control (Fig. 11). Etoposide and anti-MUC1 as well as OM-86II and anti-MUC1 decreased the concentration of COX-2 to 9.4 ng/ml after 24-h incubation compared with the control, where the concentration of COX-2 was 11.4 ng/ml (Fig. 11A). After 48 h of incubation, both analyzed combinations of the compounds (etoposide and anti-MUC1, and OM-86II and anti-MUC1) significantly decreased the COX-2 level. However, the strongest effect was observed after treatment with OM-86II and the anti-MUC1 antibody; 5 ng/ml of COX-2 was detected in the cell lysates. The concentration of COX-2 after treatment with etoposide and anti-MUC1 was 8 ng/ml and the difference was also statistically significant in comparison with the control (P<0.05; Fig. 11B).
Novel OM-86II combined with anti-MUC1 antibody decreases the concentration of mTOR
The activated PI3K/Akt/mTOR signaling pathway is responsible for tumor growth and cancer progression (26,27). Several agents targeted to one or more components of the PI3K/AKT/mTOR pathway were examined for the treatment of ER-positive breast cancer in clinical trials (28). The concentration of mTOR in cell lysates was determined after 24 and 48 h of incubation with the compounds tested (Fig. 12). Both monotherapy and combined therapy significantly decreased the concentration of mTOR in comparison with the untreated control after 24 h of incubation (P<0.05; Fig. 12A). The concentration of mTOR in the cell lysates was 949 pg/ml after treatment with OM-86II and anti-MUC1, while in the control sample the concentration was 1,131 pg/ml. The concentration of mTOR was 861 pg/ml after treatment with etoposide and anti-MUC1 antibody (Fig. 12A). After 48 h, it was observed that all the compounds tested except for etoposide significantly reduced the concentration of kinase. The lowest concentration of mTOR was detected after incubation with OM-86II and anti-MUC1 (826 pg/ml) compared with the control (3,729 pg/ml; Fig. 12B).
Novel OM-86II combined with anti-MUC1 antibody decreases the concentration of sICAM1
MUC1 interacts with ICAM-1 to facilitate the migration of tumor cells (29). The Src-CrkL-Rac1/Cdc42 signaling pathway plays the most significant role in promoting the migratory behavior of breast cancer cells, and upon ligation with sICAM-1 it connects with the MUC1 cytoplasmic domain and initiates cytoskeletal rearrangements (30). The present study demonstrated that all the tested compounds decreased sICAM1 concentration in supernatants from cell cultures (Fig. 13). The most significant change was observed after combined treatment. The concentration of sICAM1 after 24 and 48 h of incubation with etoposide and anti-MUC1 was 16.8 and 5.5 ng/ml, respectively. After 24 and 48 h of incubation OM-86II used together with anti-MUC1 significantly decreased the sICAM1 concentration to 11.6 and 11 ng/ml, respectively.
Discussion
Current evidence suggested that MUC1 is involved in growth, invasion, promotion of angiogenesis and chemoresistance to programmed cell death, induced by DNA damage, oxidative stress and hypoxia (31–35). Therapies targeting MUC1 include monoclonal antibodies, vaccines or small molecules (aptamers). However, none of them are currently used in clinical application and there is still a need to evaluate the most promising strategy in anticancer treatment based on MUC1 as a target (36). Combination of a monoclonal antibody with novel chemotherapeutic agents represents a more efficient approach in cancer treatment.
Disorders of cell cycle control and resistance to apoptosis represent the most characteristic features of cancer cells (37). The present study demonstrated that combined treatment based on an anti-MUC1 antibody with a novel diisoquinoline derivative (OM-86II) is an effective strategy in decreasing the number of viable cells and inhibiting the proliferation of MCF-7 breast cancer cells. The tested compounds led to the induction of apoptosis, decreased the mitochondrial membrane potential and induced a G2/M cell cycle arrest in MCF-7 cells.
The extracellular matrix (ECM) plays an essential role in the regulation of different signaling pathways, such as PI3K/AKT, ERK and Src-FAK, and its function in tumor progression has also been demonstrated in many previous studies (38,39). MMPs are the enzymes responsible for degradation of ECM proteins and promotion of breast cancer progression (40). Metalloproteinases take part in the remodeling of the ECM in tumor invasion (41). A high serum MMP-2 level is associated with an adverse prognosis in node-positive breast carcinoma (42). MMP-9 plays a crucial role in cancer growth and invasion. Its overexpression was correlated with poor prognosis and worse patient survival (43). MMP-9 is also responsible for destruction of collagen type IV and other ECM components (44). It is known that both MMP-2 and MMP-9 are key players of breast cancer invasion and metastasis (45). The present study demonstrated that OM-86II with an anti-MUC1 antibody significantly decreased the level of the MMPs analyzed. The association between the process of chronic inflammation and tumor progression is still of interest; anti-inflammatory agents can be beneficial in cancer therapy. The expression of pro-inflammatory cytokine TNF-α was increased in 85% of breast tumors in patients, whereas it was only minimally expressed in normal breast epithelial cells (46). A previous study conducted by Hosseini et al (47) demonstrated that β-D mannuronic acid decreased the relative mRNA expression level of inflammatory chemokines and other factors responsible for tumor growth, such as vascular endothelial growth factor, MMP-2, MMP-9 and hypoxia-inducible factor-1α. In the present study, it was demonstrated that the combination of anti-MUC1 with OM-86II decreased the concentration of pro-inflammatory cytokine TNF-α in the cell culture media.
In a previous study where COX-2 expression was analyzed, it was identified that COX-2 was only expressed in tumors, and its expression was correlated with unfavorable prognosis (17). The effect of treatments on the concentration of COX-2 was studied in the present study and it was identified that anti-MUC1 used together with OM-86II significantly decreased the concentration after 24 and 48 h of incubation. Such a strategy was more efficient than monotherapy and the combination of anti-MUC1 with etoposide.
In MCF-7 breast cancer cells, the activated PI3K/AKT/mTOR signaling pathway (48) leads to increased cellular growth and survival (26,27). Therefore, the effect of the compounds tested on the concentration of mTOR was determined in MCF-7 cell lysates. After 48 h of incubation with the combination of anti-MUC1 and OM-86II, the highest decrease in mTOR concentration was observed compared with the other compounds. The inhibitory effect was also much stronger than the combination of anti-MUC1 and etoposide.
Some researchers have shown that breast cancer types, which exhibit increased expression of MUC1, are more likely to metastasize. MUC1 is able to induce the Src-CrkL-Rac1/Cdc42 signaling pathway upon ligation to the ICAM-1 (29,30). The activated pathway leads to increased migration of breast cancer cells (30). Rahn et al (49) showed that breast cancer cells with overexpressed MUC1 were able to migrate through a layer of sICAM-1 expressing cells in an in vitro transendothelial migration assay. Thielemann et al (50) assessed the concentrations of the sICAM-1 in the serum of female patients with breast cancer. They identified increased concentrations of sICAM-1 in the serum of women with breast cancer compared with the serum of healthy controls (50). In the present study, it was observed that combination of anti-MUC1 antibody with etoposide or OM-86II significantly decreased the level of sICAM1 after 24 and 48 h of incubation in breast cancer cells.
Existing literature has suggested that the addition of monoclonal antibody to chemotherapeutic agents represents a promising strategy in anticancer treatment. The addition of anti-MUC1 antibody to cisplatin or a novel platinum(II) complex resulted in better pro-apoptotic activity and was more efficient than monotherapy in breast cancer cells (15,51). Another previous study showed that anti-MUC1 monoclonal antibody (C595) with docetaxel reduced the tumor burden and ascites in an in vivo ovarian cancer model (52). Slamon et al (53) demonstrated that the addition of trastuzumab to chemotherapy had more benefits than monotherapy. The final effect of such a treatment was longer survival of patients as well as decreased risk of death (53). The role of MUC1 in resistance to trastuzumab (Herceptin) is well documented (35,54). Fessler et al (35) noticed that cancer cells, which exhibit Herceptin resistance, were also resistant to doxorubicin and cyclophosphamide. The resistance to these chemotherapeutic agents was decreased by the combination of the original drug and MUC1 inhibitor (35).
The present study demonstrated that combination therapy based on anti-MUC1 antibody and a novel diisoquinoline derivative (OM-86II) inhibited the proliferation of breast cancer cells. Its inhibitory effects were associated with induction of cell cycle arrest and apoptosis. Moreover, such a combination was able to block the multiple intracellular signaling pathways responsible for tumor growth promotion and breast cancer progression. It was demonstrated that anti-MUC1 antibody with OM-86II decreased the concentration of MMP-2, MMP-9, sICAM1 and mTOR. In addition, combined therapy exhibited anti-inflammatory activity; decreased concentrations of pro-inflammatory cytokine TNF-α and COX-2 were observed. The present study suggested that the combination of anti-MUC1 with novel OM-86II represents a potential multi-targeted strategy in breast cancer treatment.
Acknowledgements
Not applicable.
Funding
The present study was funded by The National Science Centre (grant no. DEC-2017/01/X/NZ7/01315).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions
AG, AB and KB designed the study. AG wrote the manuscript, and performed the experiments and statistical analysis of the data. WS, RC and AS performed the in vitro experiments. AB analyzed the results and coordinated the study. ZK analyzed data and described the synthesis process. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
COX-2 |
cyclooxygenase-2 |
|
ECM |
extracellular matrix |
|
ER |
estrogen receptor |
|
MMP-2 |
matrix metalloproteinase-2 |
|
MMP-9 |
matrix metalloproteinase-9 |
|
MUC1 |
mucin-1 |
|
sICAM1 |
soluble intercellular adhesion molecule 1 |
|
TNF-α |
tumor necrosis factor-α |
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