The NIR dye IR-783 was obtained from Sigma-Aldrich (Merck KGaA) and dissolved in sterile distilled water as a stock solution. The antibodies used in the present study were as follows: Anti-Cyclin D1 (cat. no. sc-8396), anti-Cyclin E (cat. no. sc-481), anti-cyclin-dependent kinase 2 (CDK2; cat. no. sc-6248), anti-OPA1 (cat. no. sc-393296), anti-Mfn1 (cat. no. sc-1666447), anti-Mfn2 (cat. no. sc-515647), anti-MFF (cat. no. sc-398617), anti-Fis1 (cat. no. sc-376447) and anti-Drp1 (cat. no. sc-271583) were obtained from Santa Cruz Biotechnology, Inc. Anti-matrix metalloproteinase-2 (MMP-2; cat. no. 40994) and anti-MMP-9 (cat. no. 13667) were obtained from Cell Signaling Technology, Inc. Anti-GAPDH (cat. no. AG019-1) was obtained from Beyotime Institute of Biotechnology.

The human breast cancer cell lines MDA-MB-231 and MCF-7 were purchased from the American Type Culture Collection. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), all from Gibco (Thermo Fisher Scientific, Inc.). Cells were cultured at 37°C with 5% CO₂ and digested every 2 days by 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.).

Cells (2×10⁴ cells/well) were seeded onto 96-well plates and allowed to attach. After treatment with various concentrations of IR-783 (0, 20, 40, 60, 80, 100, 120, 140 and 160 μM) for 24 h or with 80 μM IR-783 for different time intervals (0, 3, 6, 12, 24, 36, 48 and 72 h) in a 5% CO₂ incubator at 37°C, cells were incubated with 20 μl MTT solution (5 mg/ml; Sigma-Aldrich; Merck KGaA), cells were treated at 37°C for 4 h. Then, the medium was removed slowly and 150 μl dimethyl sulfoxide (Eastman Kodak) was added to dissolve the formazan crystals. The proliferation of cells was determined using a microplate reader (Varioskan Flash; Thermo Fisher Scientific, Inc.) at 490 nm. All results were repeated in three independent experiments.

Cells were digested by 0.25% trypsin and seeded in a 5-cm sterile petri-dish at a density of 100 cells per dish overnight. Then, cells were treated with different concentrations of IR-783 (0, 80 and 160 μM) the next day and incubated at 37°C. After 24 h, the supernatant was discarded carefully and fresh DMEM with 10% FBS was added. Cells were cultured for 2-3 weeks until clones were visible. After removing culture medium, clones were washed for three times with PBS, fixed with 4% paraformaldehyde for 15 min, then stained with 0.1% crystal violet for 10 min at room temperature. Clones were counted using Photoshop CS6 software (version 13.0; Adobe Systems, Inc.). All results were performed for three independent times.

Cell proliferation was assessed with a BeyoClick™ EDU-488 imaging Kit (cat. no. C0071S; Beyotime Institute of Biotechnology). Cells were inoculated onto slides of 24-well plates at a density of 1×10⁴ cells/well and incubated overnight at 37°C. Then, IR-783 (0, 80 and 160 μM) was added to cells and cultured for 24 h. Subsequently, cells were exposed to 20 μM EdU for 2 h at 37°C. Cells were then fixed with 4% paraformaldehyde for 15 min, followed by the addition of 200 μl 0.5% Triton X-100 (AppliChem GmbH) for 10 min to increase cell permeability at room temperature. After washing with PBS for three times, 200 μl of click reaction solution was added according to the instructions and cells were incubated for another 30 min at room temperature in the dark. Next, cells were washed again and stained with 100 μl of Hoechst 33342 for 30 min at room temperature. The samples were analyzed and imaged with a fluorescence microscope (BX63; Olympus Corporation) at x20 magnification. EdU-positive cells were analyzed in three randomly selected field by using Photoshop CS6 software (version 13.0; Adobe Systems, Inc.).

Cell cycle analysis was performed by flow cytometry. Cells were seeded in a 6-well plate at a density of 1×106 cells/well and cultured overnight. IR-783 (0, 80 and 160 μM) was added and incubated for 24 h. Cells were collected and washed twice with cold PBS, then fixed in cold 75% ethanol at 4°C overnight. Subsequently, the cells were washed with cold PBS and incubated with 50 μg/ml RNase and 100 μg/ml propidium iodide (cat. no. 556547; BD Biosciences) for 30 min at 37°C in the dark. The samples were analyzed with a flow cytometer (FACScan) using CellQuest software (version 5.1) (both from BD Biosciences).

The migration ability of MDA-MB-231 cells was assessed using a wound healing assay. Cells (5×10⁵ cells/well) were cultured in 6-well plates and allowed to grow to 80-90% confluence. Scratches of a predetermined length were created with a 200 μl pipette tip. After washing the scratched cells with PBS, IR-783 (0, 80 and 160 μM) were added and incubated for 24 or 48 h at 37°C. The images of wounds were imaged using a Cell Imaging System (Carl Zeiss AG). The wound width was measured using ImageJ software (version 1.52a; National Institute of Health); wound healing rate (%) = 100% × (0 h width-24/48 h width)/0 h width.

Cells (2×10⁴ cells/well) were plated in the upper chamber of a Transwell chamber with 200 μl serum-free DMEM, while 600 μl media containing 30% FBS (Gibco; Thermo Fisher Scientific, Inc.) was added in the lower chamber. IR-783 (0, 80 and 160 μM) was applied to both chambers. After 24 h of treatment in a 37°C incubator, non-invasive cells on the upper surface of the membrane were gently wiped away with a cotton swab, while the invasive cells on the low surface of the membrane were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 10 min at room temperature. Three independent fields in each well were imaged under a light microscope (DP26; Olympus Corporation) at x20 magnification, and the number of cells in each field was counted.

The cellular ATP levels were measured with an ATP Assay Kit (cat. no. S0026; Beyotime Institute of Biotechnology). A standard curve of ATP concentration (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10 μM) was created from a known amount of ATP and then used to determine the ATP content of the samples. Briefly, after exposure to different concentrations of IR-783 (0, 40, 80, 120 and 160 μM) for 24 h, cells were harvested and washed twice with PBS. Then, cells were lysed and centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant was collected and mixed with 100 μl ATP detection working solution. The luminescence values were determined using a microplate reader at 420 nm (Varioskan Flash; Thermo Fisher Scientific, Inc.). The levels of ATP were expressed as a percentage of the control, which was set at 100%.

For electron microscopy, after 24 h incubation with 80 μM IR-783, MDA-MB-231 cells were collected and washed twice with PBS, and fixed in fresh 2.5% glutaraldehyde at 4°C overnight. Samples were fixed for 2 h in 2% osmium tetroxide at 4°C, dehydrated using a graded series of ethanol and embedded in Epon. Then, sections (70 nm) were made using an ultra-Microtome (Leica EM UC7; Leica Microsystems, Inc.), stained with 3% uranyl acetate and 3% lead citrate for 15 min at room temperature. The morphology of mitochondria was studied using a Tecnai 10 transmission electron microscope (Philips Healthcare).

Cells were inoculated into 24-well plates with coverslips overnight. After treatment with 80 μM IR-783, cells were stained with 100 nM MitoTracker Red CMXRos (Beyotime Institute of Biotechnology) for 30 min at 37°C in the dark, then washed three times with culture medium for 5 min each time. Subsequently, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS for three times, and observed using a laser-scanning confocal microscope (LSM780NLO; Carl Zeiss AG) at x63 magnification, the representative images were randomly selected from five fields of view. For staining of the actin cytoskeleton, cells were treated with 80 μM IR-783 for 24 h at 37°C, then cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. F-actin was incubated with Alexa Fluor® 488 Phalloidin (CST, 8878S) for 30 min at 37°C in the dark; cells were counterstained with DAPI for 5 min at room temperature (cat. no. C1002; Beyotime Institute of Biotechnology). Cell images were collected using a laser-scanning confocal microscope (LSM780NLO; Carl Zeiss AG) at x63 magnification, the representative images were randomly selected from five fields of view.

MDA-MB-231 cells (1×10⁶ cells/ml) were treated with IR-783 for 24 h, then harvested and lysed in cell lysis solution (cat. no. P0013; Beyotime Institute of Biotechnology) with PMSF. A BCA protein assay kit (cat. no. P0010, Beyotime Institute of Biotechnology) was used to quantify protein concentration. Proteins were quantified via a BCA assay. The proteins lysate samples (15, 30 or 60 μg) were separated via 10-12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked with 5% skim milk for 2 h at room temperature and incubated with specific primary antibodies overnight at 4°C. The following antibodies were used: Anti-Cyclin D1 (diluted 1:500), anti-Cyclin E (diluted 1:500), anti-CDK2 (diluted 1:500), anti-OPA1 (diluted 1:500), anti-Mfn1 (diluted 1:500), anti-Mfn2 (diluted 1:500), anti-MFF (diluted 1:500), anti-Fis1 (diluted 1:500), anti-Drp1 (diluted 1:500), anti-MMP-2 (diluted 1:1000), anti-MMP-9 (diluted 1:1,000) and anti-GAPDH (diluted 1:2,000). Membranes were washed three times with TBST and then incubated with anti-rabbit (diluted 1:50,000, cat. no. 150204) or anti-mouse (diluted 1:50,000, cat, no. 130639) horseradish peroxidase secondary antibodies (Kirkegaard & Perry Laboratories Inc.) for 2 h at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence kit (Amersham ECL or ECL Plus; GE Healthcare Life Sciences); protein expression was quantified using Quantity One software (version 4.6; Bio-Rad Laboratories, Inc.).

All data were presented as the mean ± standard deviation using GraphPad prism 6.0 software (GraphPad Software, Inc.). Statistical analysis between different groups were determined using one-way analysis of variance with a Dunnett's or Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

The chemical structure of IR-783 is presented in Fig. 1A. First, we evaluated the inhibitory effects of IR-783 on MDA-MB-231 and MCF-7 cells using an MTT assay. As shown in Fig. 1B and C, we reported that IR-783 treatment significantly decreased cell proliferation in a dose- and time-dependent manner compared with the control. To further confirm the effects of IR-783 on cell proliferation, a colony formation assay was performed. As presented in Fig. 1D, our results indicated that the number of colonies formed in IR-783-treated group was significantly reduced in MDA-MB-231 and MCF-7 breast cancer cells compared with the control. Furthermore, the effects of IR-783 on cell proliferation in MDA-MB-231 and MCF-7 cells was examined with an EdU assay. The results revealed significant fewer EdU-positive cells following treatment with IR-783 compared with the control (Fig. 1E). Taken together, these findings demonstrated that IR-783 suppresses MDA-MB-231 and MCF-7 cell proliferation.

To further determine the underlying mechanisms of IR-783-induced inhibition of the growth of MDA-MB-231 and MCF-7 cells, we examined the effects of IR-783 on MDA-MB-231 and MCF-7 cell cycle distribution. After treatment with IR-783 (0, 80 and 160 μM) for 24 h, the proportion of MCF-7 cells in G0/G1 phase significantly increased from 34.57% (without IR-783 treatment) to 57.45% (cells treated with 160 μM IR-783); the proportion of MDA-MB-231 cells in G0/G1 phase significantly increased from 37.04% (without IR-783 treatment) to 58.26% (cells treated with 160 μM IR -783) (Fig. 2A). No significant differences in the number of cells in S and G2/M phase were observed following treatment with IR-783. These results suggested that IR-783 induced MDA-MB-231 and MCF-7 cell cycle arrest at the G0/G1 phase. Furthermore, we detected alterations in the expression of G0/G1 phase-related proteins Cyclin D1, Cyclin E and CDK2 of MDA-MB-231 cells using western blotting. The results demonstrated that IR-783 significantly reduced the expression of Cyclin D1, Cyclin E and CDK2 compared with the control (Fig. 2B). These results suggested that IR-783 induces cell cycle arrest at G0/G1 phase.

In addition, a Transwell assay was conducted to examine the effects of IR-783 on MDA-MB-231 and MCF-7 cell migration; a wound healing assay was performed to determine the effects of IR-783 on MDA-MB-231 cell migration. The Transwell assay revealed that IR-783 significantly inhibited the migration of MDA-MB-231 and MCF-7 cells compared with the control (Fig. 3A). Furthermore, the wound healing assay indicated that IR-783 significantly decreased the migration of MDA-MB-231 cells in a dose- and time-dependent manner (Fig. 3B). MMPs, especially MMP-2 and MMP-9, have been reported to be critical regulators involved tumor migration (25). Thus, western blotting was conducted to investigate whether IR-783 affected the expression of MMP-2 and MMP-9 in MDA-MB-231 cells. The western blot assay demonstrated that IR-783 treatment significantly decreased the expression of MMP-2 and MMP-9 in a concentration-dependent manner compared with the control group (Fig. 3C). Collectively, these results indicated that IR-783 inhibits MDA-MB-231 and MCF-7 cell migration.

Filopodia are critical actin cytoskeletal protrusion structures, and are key factors involved in tumor metastasis (26). Metastatic tumor cells contain numerous filopodia, and the numbers of filopodia are correlated with the invasiveness and migration (27). F-actin is the fundamental component of the cytoskeleton that supports the structure of filopodia, stabilizing them in migrating cancer cells, and it is involved infilopodium formation via the polymerization of actin into bundles under the cell membrane (28). The present study reported that the control cell group exhibited a large number of F-actin filaments in the marginal zone and cytoplasm. Incubation with IR-783 induced MDA-MB-231 and MCF-7 cell shrinkage and a marked decrease in the abundance of F-actin (Fig. 4). Collectively, these data suggested that IR-783 inhibits filopodia formation by reducing F-actin formation.

ATP, which provides energy for cells, serves an important role in cell life and is central to the fate of cancer cells (29). It has been reported that cancer cells have higher ATP levels than normal cells, and decreases in ATP levels of cancer cells indicates alterations in cancer cell viability (30). As presented in Fig. 5A, the MDA-MB-231 and MCF-7 cellular ATP levels decreased in a dose-dependent manner in response to the IR-783. Mitochondria are the main organelle in cells involved in the synthesis of ATP. ATP depletion has been reported as an indicator of mitochondrial dysfunction (30). Our transmission electron microscopy results showed that mitochondria of MDA-MB-231 cells swelled; the cristae of mitochondria disintegrated partly as demonstrated by the transparent electronic density area in IR-783 treated cells. On the contrary, the mitochondria presented a slender flat structure in control cells, suggesting that IR-783 induced mitochondrial injury in MDA-MB-231 cells (Fig. 5B). The morphology of mitochondria is dynamically controlled by fission and fusion (20). When alterations in mitochondrial morphology occurred, the balance between fission and fusion is disrupted (20). Next, MDA-MB-231 and MCF-7 cells were stained with MitoTracker Red CMXRos to assess the influence of IR-783 on mitochondrial dynamics. Confocal laser scanning microscopy results revealed that IR-783 treatment promoted mitochondrial fission, as observed by a marked increase in number of small, punctate mitochondria in IR-783 treated cells compared with control cells, which exhibited stable and elongated structures (Fig. 5C). To further confirm these results, western blotting was used to evaluate the expression of mitochondrial fusion regulators OPA1, Mfn1 and Mfn2, and the expression of mitochondrial fission regulators MFF, Fis1 and Drp1 which affect mitochondrial dynamics at the outer mitochondrial membrane. We found that exposure of MDA-MB-231 cells to IR-783 resulted in a significant decrease in the expression of OPA1, Mfn1 and Mfn2, and increased the levels of MFF, Fis1 and Drp1 compared with the control (Fig. 5D and E). These results suggested that IR-783 induces mitochondrial fission and subsequent ATP depletion in MDA-MB-231 and MCF-7 cells.