Animal experiments
Six-week-old male specific pathogen-free (SPF) hairless mice (SLC, Hamamatsu, Shizuoka, Japan) were individually bred in cages in an air-conditioned room at 23 ± 1°C under SPF and stress-free conditions with a 12-h light/12-h dark cycle. Mice were divided into seven groups, each consisting of five mice: control, 7,12-dimethylbenz[a]anthracene (DMBA)-treated, DMBA/blue light-treated, DMBA/blue light/tranexamic acid-treated, DMBA/blue light/carbazochrome-treated, DMBA/blue light/ DADA-treated, and DMBA/blue light/pantethine-treated groups. Tumor was initiated by topical application of 100 mg DMBA to the dorsal skin of mice. Although DMBA does not induce tumor development, it exerts a potent tumor-initiating effect [17]. Two weeks after tumor initiation, the dorsal skin of each mouse was locally exposed to blue LED light (wavelength, 380–500 nm; peak emission, 479 nm, 40 kJ/m2; ISLM-150X150-BB; CCS Inc., Kamikyo-ku, Kyoto, Japan) thrice a week for 15 weeks [18]. The control group was irradiated by visible light (wavelength: 400–700 nm). This study was approved by the Suzuka University of Medical Science Animal Experiment Ethics Committee (September 25, 2014) and was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Suzuka University of Medical Science (Approval number: 34). All surgeries on mice were performed under pentobarbital anesthesia, and efforts were made to minimize animal suffering.
Skin tumors
The induced skin tumors were examined on the last day of the experiment. Tumor data are expressed as mean tumor number and volume per mouse. Tumor volume was calculated using the following formula: (major axis 9 (minor axis) 2)/2.
Treatment of mice with tranexamic acid, DADA, carbazochrome, and pantethin
Mice were orally administered 12 mg·kg-1, 500 mg·kg-1, 167 mg·kg-1, or 500 mg·kg-1 of tranexamic acid, DADA, carbazochrome, or pantethine (Daiichi Sankyo Healthcare Co., Ltd., Tokyo, Japan), respectively, in distilled water thrice a week for 15 weeks. Control animals were administered distilled water [19]. Administration of these chemicals did not affect body weight or induce histological/biochemical alterations in the skin (data not shown). Accordingly, we reasoned that these chemicals were potentially nontoxic to the mice at the administered dose.
Preparation and staining of the dorsal skin
Skin samples were collected under anesthesia on the final day of the experiment and were fixed with 4% phosphate-buffered paraformaldehyde, embedded in Tissue Tek OCT compound (Sakura Finetek, Tokyo, Japan), and cryo-sectioned. These sections were stained with hematoxylin and eosin (HE) for histological analysis of the skin. The specimens were then stained using antibodies for immunohistological analysis as previously described [20]. Skin specimens were incubated with rabbit monoclonal anti-brain and muscle arnt-like 1 (Bmal1) (1:100; Cell Signaling Technology Inc., Danvers, MA, YSA), mouse monoclonal anti-lymphocyte antigen 6 complex locus G6D (Ly6G: marker of neutrophil) (1:100; BD Biosciences, Franklin Lakes, NJ, USA), rabbit monoclonal anti-beta 2 adrenergic receptor (β2-AR) (1:100; Abcam, Cambridge, MA, USA), rabbit monoclonal anti-intercellular adhesion molecule 1 (ICAM1) (1:100; Abcam), rabbit polyclonal anti-C-C motif chemokine ligand 2 (CCL2) (1:100; Abcam), rabbit polyclonal anti-citrullinated histone H3 (citH3) (1:100; Abcam), or rabbit polyclonal anti-protein arginine deiminase 4 (PAD4) (1:100; Abcam) primary antibodies. The sections were subsequently incubated with fluorescein isothiocyanate-conjugated antirabbit or anti-mouse secondary antibodies (1:30; Daco Cytomation, Glostrup, Denmark). Intensities of Bmal1, Ly6G, β2-AR, ICAM1, CCL2, PAD4, and citH3 were calculated based on five random visual fields with constant area using ImageJ software v.1.53 (National Institutes of Health, Bethesda, MD, USA). In brief, the original files were converted into monochrome 8-bit files. Next, the luminous intensity threshold was voluntarily established. Areas above the threshold were measured for each sample. These areas were defined as “intensity.”
Determining Ki-67, cyclin D1, and noradrenaline levels in the dorsal skin
Dorsal skin samples were collected on the final experimental day. First, 10 mg dorsal skin was rinsed with phosphate-buffered saline to remove excess blood before homogenization. The samples were centrifuged at 1,500 × g for 15 min at 4°C. The supernatant was collected, and the levels of Ki-67, cyclin D1, and noradrenaline were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits (Ki-67, Wuhan Fine Biotech, Hubei, China; cyclin D1, Novus, Centennial, CA, USA; noradrenaline, Abcam), according to the manufacturers’ instructions. Optical density was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Blue light-induced neutrophil extracellular trap (NET) formation in vitro
Cell culture
HL-60 human promyelocytic leukemia cells (RCB3683; RIKEN BioResource Center, Ibaraki, Japan) were cultured in RPMI1640 medium (Nacalai, Kyoto, Japan) containing 10% (v/v) inactivated fetal bovine serum and antibiotics [21]. Cells were maintained at 37°C in a humidified incubator with 5% CO2, and the medium was changed every 2 days. Cells were differentiated into nHL-60 cells by treating with 1.25% dimethyl sulfoxide for 3 days, as previously described [22].
Irradiation of LED blue light and addition of each sample
HL-60 cells (5 × 105/mL) were seeded in a 3-cm dish and differentiated into neutrophils, and each test substance (tranexamic acid, 0.012 mg/mL; carbazochrome, 4 mg/mL; pantethine, 12 mg/ml; and DADA, 0.2 mg/mL) was added. After 2 h, 1 kJ/m2 LED blue light was applied, and NETosis was measured after irradiation. Among 0.2, 0.4, 1.0, and 2.0 kJ/m2 irradiation doses, 1 kJ/m2 showed the maximum effect and did not affect cell viability.
Quantification of extracellular DNA
nHL-60 cells (1 × 106 cells/mL) treated with or without Aza for 72 h were seeded in 96-well plates and treated with 20 U/mL micrococcal nuclease (New England Biolabs Japan, Tokyo, Japan) for 20 min at 37°C. After centrifugation at 200 × g for 8 min at 4°C, the supernatant containing DNA was collected. Extracellular DNA was stained with SYTOX Green and measured using SpectraMax (excitation 485 nm and emission 525 nm; Molecular Devices Japan, Tokyo, Japan).
Quantification of NET-associated cell death (NETosis)
NETosis was analyzed using SYTOX Green fluorophotometry as previously described [23]. Briefly, nHL-60 cells (1 × 106 cells/mL), with or without Aza treatment, were treated with 5 µM SYTOX Green in 96-well plates. The PAD4 inhibitor, Cl-amidine, was treated with Aza for 30 min, and the mitochondrial reactive oxygen species scavenger, mito-TEMPO, or the NADPH oxidase inhibitor, diphenyleneiodonium chloride, was treated with A23187 for 30 min. After addition of 10 µM A23187 or control (no addition), changes in green fluorescence were measured for 4 h using SpectraMax (excitation 485 nm, emission 525 nm, Molecular Devices Japan). To determine total DNA concentration, nHL-60 cells were lysed with 1% (v/v) Triton X-100, and changes in fluorescence were measured. All values were normalized with respect to total DNA concentration in each experiment.
Statistical analyses
All data are presented as mean ± standard deviation (SD). Microsoft Excel 2010 (Microsoft Corp., Redmond, WA, USA) was used to analyze statistical significance of the data, along with one-way analysis of variance, followed by Tukey’s post-hoc test using SPSS v.20 (SPSS Inc., Chicago, IL, USA). Results with p-values < 0.05 (*) and 0.01 (**) were considered statistically significant.