Animals/declarations for the animal research
Six (per treatment group) seven (7)-weeks-old male C57BL/6 J mice (Charles River Laboratories Japan, Inc.) were housed individually in polycarbonate cage lined with paper bedding (Palsoft Oriental Yeast Co., Ltd., Tokyo, Japan) with stainless wire cover and maintained under standard conditions with free access to food and water, and housed in a 12-h light/dark cycle room. The animals were sacrificed using the cervical spine dislocation method. All the experiments complied with the guidelines of the University of Tsukuba’s Regulation of Animal Experiments and were approved by the University of Tsukuba’s Committee on Animal Care and Use (No. 16–046).
B16F10 murine melanoma cells (B16 cells) were obtained from RIKEN, Tsukuba (Catalog No. RCB2630:B16F10) and cultured in RPMI1640 (Gibco, Invitrogen GmbH, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidified atmosphere of 5% CO2. For mouse tail vein injection, 1 × 106 cells/ml B16 cells were resuspended in saline solution, which was then passed through a 79-μm-cell strainer (BD Falcon, BD Biosciences, San Jose, CA, USA) before injection to remove aggregated cells.
Daphnane diterpenes were treated as ethanolic extract of air-dried Thymelaea hirsuta leaves (10 g in 100 mL of 70% EtOH). T. hirsuta extract (TH) was passed through a 0.22 μm filter (Millipore, Germany) and stored at − 80 °C °C until use. Ethanol in the TH samples for oral administration was removed by evaporation (SCRUM Inc., Tokyo, Japan) and dissolved in distilled water. Gnidilatidin (MW: 648.749 g/mol) was extracted from T. hirsuta following a similar protocol for isolating hirsein A and hirsein B, as previously reported  TH (100 g/l 70% EtOH) contains about 0.5 mg gnidilatidin. Preliminary screening assays using different concentrations of gnidilatidin (data not shown) revealed that gnidilatidin was effective but not cytotoxic at concentrations of 0.1 to 1.0 μM so for this study, gnidilatidin was used at 0.1 μM concentration in all the experiments.
C57BL/6 J mice (7 mice/group) were allowed to acclimatize for a week before they were randomly divided into four groups: Controls of “(−) B16F10” group injected with water and given tap water and “(+)B16F10 group” injected with B16F10 cells and given tap water; “DTIC/(+)B16F10 group” injected with B16F10 cells and orally-administered with 70 mg/kg/day dacarbazine (DTIC) and is the positive control; the “TH/(+)B16F10 group” injected with B16F10 cells and orally-administered with 50 mg/kg/day T. hirsuta extract. After acclimatization, mice lateral tail veins were injected with B16F10 cells (2 × 105 tumor cells in 100 μl PBS, 0.2 ml/mouse). The day after B16F10 injection, every morning in the animal room, mice were orally administered, to ensure that each animal receive the exact dosage corresponding to each animal’s body weight, with DTIC or TH everyday for 20 days. Food and water were given ad libitum during the experimental period. The day after the last oral administration, the mice were sacrificed using the widely accepted method which is cervical spine dislocation and the lungs were collected, washed, the number of lung tumor colonies was counted, then frozen in liquid nitrogen and stored at − 80 °C for protein and RNA extractions. Mice body weight was recorded daily for 21 days.
Total protein was extracted from B16F10 cells or mice lungs using Radio-Immunoprecipitation Assay (RIPA) buffer following the manufacturer’s instructions. SDS-PAGE (10%) was then carried out to resolve 10 μg of extracted protein sample, which was then transferred to a PVDF membrane (Merck Millipore, USA). Membranes were incubated with primary antibodies against MMP2, MMP9, and CD44 (obtained from Abcam, Cambridge, UK), at 4 °C overnight, then washed and incubated with secondary antibody, IRDye 800CW Donkey anti-rabbit IgG (LI-COR, Inc., NE, USA), at room temperature for 30 min. The signal was detected using the OdysseyFc Imaging System (LI-COR, Inc., NE, USA). All protein quantifications were normalized to β-actin expression level.
Cell proliferation assay
Cell proliferation was assessed using the 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide or MTT (Dojindo, Japan) assay. B16F10 cells (3 × 104 cells/well) were seeded onto 96-well plates and cultured as described above. After 24 h of incubation, the medium was replaced with medium without or containing TH or gnidilatidin at various concentrations. MTT (5 mg/ml) was then added, the plates covered with aluminum foil, and incubated further for 48 h. Sodium dodecyl sulphate (SDS; 10%) was then added at 100 μl per well, and incubated overnight at 37 °C to dissolve the formazan completely. Absorbances were obtained at 570 nm using a microplate reader (Powerscan HT; Dainippon Pharmaceuticals USA Corp., NJ, USA). Blanks containing medium only was used to correct the absorbances.
Viable cell count
Viable cell count was performed using the ViaCount program of Guava PCA (Millipore, Billerica, MA, USA). B16F10 cells were seeded onto 10-mm dish (5 × 104 cells/ml) and incubated for 24 h at 37 °C, after which, medium was replaced with fresh medium without or containing TH or gnilatidin. After further incubation for 48 h, cells were collected and resuspended in 1 ml medium and stained with DNA-binding Guava ViaCount reagent (Millipore) and analyzed according to the manufacturer’s instructions.
Wound healing assay
B16F10 cells were seeded onto 6-well plates (5 × 105 cells/well) until they reach 80% confluence when a scratch was made on the cell monolayer using a sterile white pipette tip, and washed with PBS (−) to remove the detached cells. The growth medium was then replaced by fresh growth medium without or with 1000 v/v TH or gnidilatidin. The cells were photographed using Leica DFC290 HD camera (Beckman Coulter, CA, USA) before scratching and at 0 h, 12 h, and 24 h following sample treatment. The images of the cells before and after scratching in treated and untreated cells were then compared.
Cell adhesion assay
B16F10 cells were seeded onto 10-cm petri dishes (3 × 105 cells/ml) and incubated for 24 h at 37 °C. After overnight incubation, the medium was replaced with TH- or or gnidilatidin-containing medium. After further incubation (24 h) cells were trypsinized and resuspended in serum-free RPMI 1640 medium (2 × 105 cells/ml). The cell suspensions (100 μl) were then seeded onto fibronectin coated-plate (BD). After incubation for 1 h at 37 °C, medium was removed and washed three times with PBS (−) to remove the unattached cells, after which, MTT assay was perfomed as described above.
Invasion assay was performed using the 24-well Corning BioCoat Matrigel Invasion Chambers containing Falcon cell culture inserts (8 μh pore size PET membrane with a thin layer of Matrigel basement membrane matrix (Corning, Bedford, MA, USA), following the manufacturer’s instructions. B16F10 cells (5 × 105 cells/ml), in serum-free media, were seeded onto the Matrigel-coated chambers while 10% FBS was added to the lower chamber as chemoattractant. After 24 h incubation, the lower chamber was stained with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA), and the number of cells that had invaded moved to the lower chamber was counted using Leica DMI-4000B fluorescence microscope. Three fields in each sample were counted and the mean values from three independent experiments were used.
DNA microarray analysis
DNA microarray was performed using Affymetrix (Santa Clara, CA, USA) GeneChip Mouse Genome 430 2.0 Array following the manufacturer’s instructions. Total RNA was extracted from B16F10 cells (3 × 105 cells/ml) and the quality was assessed using Agilent 2100 bioanalyzer (Agilent Technologies). Biotin-labeled aRNA was synthesized by in vitro transcription and the purified aRNA (10 μg) was fragmented using the GeneAtlas 3’ IVT Express Kit, and was hybridized to the gene chip for 16 h at 45 °C. The chips were washed and stained in the GeneAtlas Fluidics Station 400 (Affymetrix) and the resulting image scanned using the GeneAtlas Imaging Station (Affymetrix). Identification of the differentially expressed genes was performed using Affymetrix® Expression Console™ Software and Affymetrix® Transcriptome Analysis Console (TAC) 2.0 Software (Affymetrix).
Quantitative real-time PCR
RNA (1 μg), extracted using Isogen reagent (Nippon Gene, Tokyo, Japan), was reverse transcribed using the SuperScript III reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA). Primers specific to Egr, Id2, Sytl2, Tyr, Trp1, Dct, and Mitf were used for real-time PCR performed using 7500 Fast Real-time PCR system using TaqMan Universal PCR mix and TaqMan probes (Applied Biosystems, Foster City, CA, USA). All reactions were run in triplicate, and data were analyzed using the 2 -Δ Δ CT values method.
Results were expressed as mean ± standard deviations (SD), and the statistical evaluation performed using Student’s t-test when two value sets were compared (Control vs treated cells). ANOVA was performed to assess the level of significance between groups’ number of nodules and weight. A P value of ≤0.05 was considered to be statistically significant.