DNase and collagenase (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in Dulbecco's modified Eagle's medium (DMEM). NecroX-5 (LG Life Sciences, Ltd., Seoul, Korea), Mitoquinone (MitoQ; BioTrend, Switzerland), 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA), MitoSOX-red, Fluo4-AM (Thermo Fisher Scientific, Waltham, MA, USA), MK-2206 (Biovision, Palo Alto, CA, USA), and bepridil (bepridil hydrochloride; Sigma-Aldrich) were dissolved in dimethyl sulphoxide (DMSO) for each condition and dose. Mouse breast cancer 4T1 and human breast cancer HCC70, MDA-MB-231 and MDA-MB-453 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The TUBO-P2J cell line was established from a metastatic lung nodule that had spontaneously developed during a mechanistic study involving an anti-neu antibody in TUBO-bearing mice (13). Cells were grown in DMEM of RPMI with 10% fetal bovine serum and 1% penicillin, and cultured at 37°C with 5% CO₂.

Six-week-old female BALB/c mice were obtained from Orient Bio (Taejun, Korea). Tumours were established by the subcutaneous injection of 2×10⁵ TUBO-P2J cells into the backs of the mice. When the tumour volume reached 100–150 mm³ in the treatment group, NecroX-5 was dissolved in phosphate-buffered saline (PBS) and administered via intraperitoneal injection (2.5 mg/kg/day). The same volume of PBS was injected into the control group. Tumour volumes were measured along three orthogonal axes (x, y and z) and calculated as tumour volume = (xyz)/2. All animal procedures were performed in accordance with the animal experimental guidelines set by the Institutional Animal Care and Use Committee of the INJE University College of Medicine (protocol no. 2013-018). Lung metastasis was evaluated using a lung colony assay on day 23 following tumour implantation. Single cell suspensions were prepared from the lung tissue via enzyme digestion using a mixture of DNase (0.1 mg/ml) and collagenase (4 mg/ml) and seeded in 48-well culture plates in a 4-fold serial dilution. The plates were incubated in complete DMEM with 500 μg/ml of G418 (Sigma-Aldrich) and stained between days 10 and 14 with crystal violet (0.1% crystal violet acetate).

Breast cancer cell migration was evaluated with 24-well Transwell plates (Falcon, Bedford, MA, USA). The appropriate number of cells was added to the upper chamber and incubated for 6 or 24 h. The upper surface of the membrane was wiped with a cotton-tipped applicator to remove residual cells. Cells in the bottom compartment were fixed and stained with hematoxylin and eosin (H&E). Cells in four randomly selected fields at ×200 magnification were counted.

Cytoplasmic ROS (cytoROS) was measured with H₂DCFDA. Cells treated with Necrox-5 and MK-2206 for 1 h were harvested with trypsin (0.025%) and stained with 10 μM H₂DCFDA for 30 min at 37°C. Fluorescence intensity was measured using a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). MitoROS was measured with MitoSOX-red. Cells were treated with NecroX-5, MK-2206 and bepridil for 1 h in 96-well black microplates (Corning, Inc., Corning, NY, USA) and loaded with 5 μM MitoSOX-red. MitoROS levels were detected with a fluorescence reader (Molecular Devices, Sunnyvale, CA, USA) at 510/580 nm.

Whole-cell lysates were prepared in radio-immunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) on ice with an added phosphatase inhibitor cocktail (PhosSTOP, cat no. 04906845001; Roche, Basel, Switzerland). The protein concentrations for each sample were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) according to the manufacturer's directions. Equal amounts of protein (40 μg) were resolved by electrophoresis on 10% sodium dodecyl sulphate (SDS) polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in TBS and incubated at room temperature for 2 h with primary antibodies pAKT t308 (monoclonal, rabbit, dilution used 1:1,000, cat no. 2965), pAKT s473 (monoclonal, rabbit, dilution used 1:1,000, cat no. 4058), AKT (polyclonal, rabbit, dilution used 1:2,000, cat no. 9272), PI3K p85 (polyclonal, rabbit, dilution used 1:1,000, cat no. 4292), and pPI3K p110α (polyclonal, rabbit, dilution used 1:1,000, cat no. 4255) (Cell Signaling Technology). One hour with secondary antibodies were diluted 1:2,000 and incubated for 1 h (IgG HRP-linked, anti-rabbit and anti-mouse antibodies, cat no. 7074 and 7076, respectively; Cell Signaling Technology). The results were visualized using enhanced chemiluminescent (ECL) detection reagents (Millipore, Darmstadt, Germany).

The effects of NecroX-5 on intracellular calcium levels were analysed with Fluo-4 AM and a confocal microscope (LSM 700; Carl Zeiss, Oberkochen, Germany). Briefly, harvested cells were washed twice with PBS and incubated at 37°C with 5 μM Fluo-4 AM (excitation/emission, 494/506 nm). After washing twice with PBS, the stained cells were placed in a perfusion chamber at room temperature. Fluorescence intensity was measured every 30 sec using a confocal microscope with ZEN 2009 software. Regions of interest in the cells were selected to monitor changes in fluorescence intensity over time, and background was identified as an area without cells. Fluorescence intensity in cells treated for 10 min with normal tyrode solution [143.0 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 5.5 mM glucose, and 5.0 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid [HEPES, pH 7.4)] was used as the baseline value (F0). Fluorescence intensity during NecroX-5 perfusion (10 μM) was recorded every 30 sec for 20 min, and peak values (F1) were analysed relative to the baseline value (F1/F0) after subtracting the autofluorescence in the absence of any fluorescent dye. The temperature was set to 23±1°C while recording. To measure calcium influx, intracellular free Ca²⁺ was depleted with ethylene glycol tetraacetic acid (EGTA, 3 mM) and thapsigargin (TG, 2 μM). Ca²⁺ influx was induced by the subsequent addition of 2 mM Ca²⁺ with or without NecroX-5 and measured with a fluorescence reader (Molecular Devices) at 485/520 nm.

To test the anticancer effects of NecroX-5, TUBO-P2J tumour-bearing mice were treated three times with 500 μg of NecroX-5 every other day. NecroX-5 treatment did not inhibit tumour growth (Fig. 2A), however, it was able to significantly extend the mean survival from 40.5 to 55 days (p<0.05) (Fig. 2B). The number of cells that metastasized to the lung was also significantly reduced by 60% (Fig. 2C). These data suggested that NecroX-5 inhibited the metastasis of TUBO-P2J cells, without inhibiting the tumour growth. To determine whether the anti-metastatic effect of NecroX-5 was related to an inhibition of cell migration, we performed a Transwell migration assay. NecroX-5 significantly decreased the migration of the mouse breast cancer cell lines 4T1 and TUBO-P2J, in a dose-dependent manner (Fig. 3A and B). However, NecroX-5 had a different effect on the human breast cancer cells. While it reduced the number of migrated cells in HCC70, it had no effect in MDA-MB-231 (Fig. 3C).

To determine whether the observed decrease in cancer cell migration was related to the ROS scavenging activity of NecroX-5, the levels of cytoROS and mitoROS were measured with H₂DCFDA and MitoSOX-red staining, respectively. The intensity of H₂DCFDA decreased following NecroX-5 treatment in all the breast cancer cells tested (Fig. 4A). Interestingly, MDA-MB-231 cells exhibited the most dramatic decrease in H₂DCFDA intensity. Similarly, MitoSOX-red intensities significantly decreased in 4T1, TUBO-P2J and HCC70 cells, but appeared unchanged in the MDA-MB-231 cells, which were not susceptible to NecroX-5 (Fig. 4B). Next, we utilized MitoQ to test whether the observed decrease in migration was modulated by the reduction in mitoROS levels. MitoQ had a similar effect on mitoROS levels as NecroX-5 (Fig. 4C), however, it did not inhibit cell migration (Fig. 4D). These data suggested that the reduction in mitoROS caused by NecroX-5 was not a direct cause of cell migration inhibition.

NecroX-5 can reduce mitochondrial calcium (Ca²⁺) concentrations, which is important for mitoROS regulation and cell migration (10,14,15). Accordingly, we evaluated changes in intracellular free calcium concentrations using Fluo-4 AM staining. In the HCC70 cells, NecroX-5 decreased intracellular free Ca²⁺ by 50% within 20 min (Fig. 5A) but had no effect in MDA-MB-231 (Fig. 5B). To determine whether the decrease in Intracellular Ca²⁺ was mediated by a decrease in Ca²⁺ influx, 4T1 cells were treated with TG and ethylene glycol tetraacetic acid to chelate Ca²⁺ and its subsequent influx was followed. NecroX-5-treated cells significantly decreased the Ca²⁺ influx (Fig. 5C). To test if the decrease in Intracellular Ca²⁺ was related to the biological effect of NecroX-5, we utilized the calcium influx blocker bepridil. Bepridil treatment significantly reduced mitoROS levels (Fig. 5D) and decreased the migratory ability of 4T1, TUBO-P2J and HCC70 cells (Fig. 5E). These data suggest that the effect of NecroX-5 on cancer cell migration and mitoROS levels is mediated by a reduction in Intracellular Ca²⁺ concentrations.

One of the differences between the NecroX-5 responsive and non-responsive cell lines was the basal activity of AKT. 4T1, TUBO-P2J and HCC70 responded to NecroX-5 and demonstrated full AKT activation (phosphorylation at Thr308 and Ser473), whereas MDA-MB-231 did not respond to NecroX-5 or display AKT activation (Fig. 6A). Additionally, treatment with 10 μM NecroX-5 significantly reduced the AKT activation levels in all of the responsive cell lines (Fig. 6B). Unlike the downregulation of AKT, pPI3K levels were not changed by NecroX-5 (Fig. 6C). Furthermore, we found this PI3K-independent AKT inhibition was also inducible by bepridil (Fig. 6D). We used an AKT specific inhibitor (MK-2206) to test if AKT inhibition mediated the reduction in cell migration and mitoROS levels. The migratory ability of 4T1 cells was found to be significantly reduced upon treatment with 2.5 μM MK-2206 (Fig. 6E). In contrast, MK-2206 did not reduce mitoROS levels in any of the tested cells (Fig. 6F).