Chemicals and antibodies
Silymarin, Solutol® HS 15, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), dimethyl sulphoxide, epithelial growth factor (EGF), basic fibroblast growth factor (bFGF), β-actin, annexin V-PI kit, nestin and Ki-67 were purchased from Sigma-Aldrich, USA. RPMI-1640, DMEM F-12, phosphate-buffered saline, and the penicillin-streptomycin antibody cocktail solution used here were obtained from Welgene, Korea. Labrafac lipophile WL 1349 and Transcutol® HS 15 were procured from Gattefossé (France). H2DCFDA and DAFFM-DA were purchased from Molecular Probes, USA. Antibodies specific to c-Met, BRAF, YKL-40, N-cadherin, E-cadherin, and ABCB and PARP ELISA kits and anti-BRAF antibody were purchased from Abcam, Korea. FBS, Trypsin was procured from Hyclone (GE Healthcare Life Sciences). Anti-gamma H2AX antibody, anti-PD1 antibody and anti-CD133 antibody were obtained from BD Biosciences, USA. Anti-NAMPT antibody and anti-DNMT antibody were purchased by Novus Biological, USA. Caspase 3/7, 8 and 9 kits were obtained by Promega Corporation, USA. B27 supplement was purchased from Invitrogen and the antibody cocktail used in this study (penicillin & streptomycin) was procured from Gibco, Korea. Antibodies specific to PARP, Bcl-2, caspase-3, BAX, p53, p-c-Met (Tyr1234/1235), p-BRAF, p-AKT (T308), p-AKT(S473), AKT, and SNAI1 were procured from Cell Signaling Technology, USA. All primers were designed and purchased from DNA Macrogen, Korea.
G-361 cells and SK-MEL-5 were cultured in RPMI-1640 cell culture media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin and were maintained at 37οC in a 5% humidified CO2 environment. The cells were routinely tested for mycoplasma test (MycoAlert™ Mycoplasma Detection Kit, Lonza, Switzerland). The passage number for G-361 cells was 30 and SK-MEL-5 was 20 at the time of experiments. For co-culturing with SN, G-361 and SK-MEL-5 cells were placed separately for 4 h in the SN before the CAP. For the spherical cultures, the sphere permissive medium was achieved through serum-free DMEM-F12 supplemented with 20 ng/ml EGF, bFGF, B-27 supplement (50X), and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin containing an antimycotic solution).
μ-Dielectric barrier discharge (DBD) CAP device and irradiation
A CAP micro-dielectric barrier discharge (μ-DBD) surface CAP device consisting of electrodes with a silicon dioxide (SiO2) dielectric layer (30 mm) and hydration-prevention layers consisting of aluminum oxide (Al2O3) and a magnesium oxide (MgO) layer was used. The electrode gap was held at 200 µm. To generate the CAP, air was used as a feeder gas into the device at a flow rate of l.5 lpm and a 2-mm distance was maintained between the CAP source and the upper surface of the medium in the cell culture petri dish. No significant increase in the temperature of the treated media was observed after up to 5 min of CAP exposure. G-361 and SK-MEL-5 cells were seeded in culture dishes with a 35 mm diameter and treated for 300 s with μ-DBD CAP.
Formulation of the SN and its characterization
The SN was fabricated according to the process outlined in our previous publication . First, a pre-weighed amount of silymarin was dissolved in a calculated amount (15%) of oil (Labrafac Lipophile WL1349), a surfactant (50%) (Solutol HS 15) and a co-surfactant (35%) (Transcutol HP). The resulting emulsion was characterized in terms of the globule size, polydispersity index (PDI), and surface morphology and size by transmission electron microscopy (TEM). The globule size distribution and zeta potential were determined by photon correlation spectroscopy using a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK; dynamic light scattering). The droplet size distribution was determined at a refractive index 1.41, viscosity of 5.0 PaS, and a dielectric constant 79.4.
Metabolic cellular viability
The cytotoxic effects of CAP and the SN at different doses and time intervals on human melanoma G-361 and SK-MEL-5 cells were determined using a colorimetric MTT test which evaluates cellular metabolic activity based on the ability of mitochondrial succinate reductase to convert a yellow-colored dye (MTT) to purple formazan in living cells. Absorbance was recorded at 540 nm using a Synergy HT Biotek microplate reader. The metabolic activity is directly proportional to the tetrazolium reduction inside the cells and is calculated in terms of the percent of a control, which was arbitrarily assigned a value of 100% viability.
Intracellular RONS measurement
Free radicals of ROS and RNS were estimated using the fluorescent dye H2DCFDA and DAFFM-diacetate dye, respectively. G-361 and SK-MEL-5 cells were cultured in six-well plates and treated with CAP and the SN respectively. H2DCFDA was added 30 min prior, whereas DAF-FM-diacetate was added 15 min before the completion of the incubation period of the cells. Both cells were then trypsinized and all measurements were completed within 30 min. RONS generation was conducted by flow cytometry and the outcome was quantitatively measured using FACS Suite software (Becton Dickinson and Co., Franklin Lakes, NJ, USA).
Cellular damage studies
The induction and damage of DNA double-strand breaks (DSBs) in terms of γ-H2AX positive cells were analyzed in G-361 cells. The cells were harvested in the RPMI-1640 medium with 10% FBS overnight and acquired 24 h after treatment as per the manufacturer’s recommended procedures (BD Biosciences) using a FACSVerse flow cytometer. The number of γ-H2AX positive foci in the G-361 cells was visualized by immunocytochemistry using a fluorescent microscope along with DAPI dye (Fig. 3a).
To determine the melanoma-specific nuclear proteins, G-361 cells were treated with CAP and the SN at the described time intervals. They were then harvested using trypsinization, washed with phosphate-buffered saline, and correspondingly incubated with anti-PD-1 (Programmed cell death protein 1) antibody and anti-DNMT (DNA-methyl transferase) antibody as per the manufacturer’s protocol. Samples were immediately analyzed using BD FACSVerse software of the FACS suite.
To assess caspases, G-361 cells were seeded into 96-well plates and the caspase 3/7, 8 and 9 activities were estimated by measuring the luminescence (Promega, USA). The Caspase kits provided in lyophilized form and were converted to a working solution, and the luminescence levels were evaluated for all groups.
Sandwich ELISA assay for cleaved PARP
To check the level of cellular damage, G-361 cells were cultured in 96-well plates after the CAP and SN treatment by estimating the degree of cleaved PARP. The cells were washed, fixed, and treated with an antigen retrieval buffer before the addition of the PARP primary antibody. On the next day, after the washing of the primary antibody, the secondary antibody was diluted according to the standard procedure and finally read at 450 nm. Background subtraction was necessary and was done by adding 50 μl of Janus green, after which the absorbance recorded at 595 nm. A sandwich ELISA kit (Abcam) was used to evaluate the effect of the aforementioned group on the induction of apoptosis in the cells.
Immunoblotting and q-PCR analysis
Cell lysates were prepared by extracting proteins with lysis buffer (40 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.1% Nonidet-P40) supplemented with protease inhibitors for western blotting. Proteins were separated by SDS-PAGE and then transferred to a nitrocellulose membrane (Amersham, IL). The membranes were blocked with 5% skim milk in Tris-buffered saline and incubated overnight with primary antibodies at 4 °C. Blots were developed using a peroxidase-conjugated secondary antibody, and proteins were visualized using enhanced chemiluminescence (ECL) procedures (Amersham, IL) according to the manufacturer’s protocol. Total RNA was extracted using Trizol (Invitrogen, USA), after which qRT-PCR was performed using a Biorad 2X SYBR green mix. Reactions were carried out in a Biorad thermal cycler (Biorad, Korea), and the results were expressed as the fold change calculated using the ΔΔCt method relative to a control sample. β-actin was used as an internal normalization control. All primers were purchased from DNA Macrogen, Korea.
FITC Annexin V–propidium iodide (PI) staining for apoptosis assay
Apoptotic cell death was determined by staining the G-361 cells with Annexin V-PI. Cells were incubated in 35mm2 petri dishes after the CAP and SN treatment, washed with PBS, and then resuspended in annexin V binding buffer and incubated for 30 min at RT (room temperature). Subsequently, FITC-annexin V was added to each tube and the tubes were incubated for 20 min at RT. Propidium iodide (PI) was added to the cells with incubation for 20 min at RT, and cell apoptosis was analyzed by FACSVerse. Viable cells were negative for both PI and Annexin V; apoptotic cells were positive for Annexin V and negative for PI, whereas late apoptotic dead cells displayed both high Annexin V and PI labeling. Non-viable cells, which underwent necrosis, were considered as positive for PI and negative for Annexin V.
Melanoma -specific markers
To check for the specificity of melanoma, G-361 cells were washed, trypsinized, and pelletized for both melanoma-specific markers. The cells were fixed with 100 μl of 3.7% formaldehyde and vortexed to create a single cell suspension. After washing with 1XPBS, cells were resuspended in pre-chilled methanol and incubated for 30 min at 4 °C. They were then rewashed with 1XPBS two times, after which anti-BRAF antibody and anti-NAMPT antibodies were added independently. Subsequently, after antibody treatments a final wash with 1XPBS were done before acquiring the samples using FACSVerse (BD Biosciences, USA).
Migration and invasion assays
Polycarbonate filters (0.8 μm; Corning USA) were coated with a reconstituted growth-factor reduced matrigel (BD Biosciences). Afterward, 2 × 104 cells in 200 μl of serum-free growth medium were seeded into the upper chamber. The cells were then incubated at 37 °C and allowed to migrate towards the complete growth medium for 24 or 48 h. Non-invading cells were removed using cotton swabs. For migration assays, inserts were not coated with the matrigel.
To visualize the N-cadherin expression and γ-H2AX foci, cells were fixed in 4% paraformaldehyde for 10–15 min and blocked with goat serum (Sigma-Aldrich) for 30 min, after which they were incubated at 4 °C overnight with appropriate primary antibodies (1:200). Following two washes with 1X PBS, the cells were incubated with a conjugated secondary antibody (1:1000; Invitrogen at RT for 2 h and counter-stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma) for approximately 10 min for nuclear staining. The fluorescence staining intensity and intercellular location were examined using a fluorescence-inverted microscope (Olympus BX51, Japan).
Sphere culture and sphere-forming assay
For sphere-formation assays, the size of the spheres was determined using the Motic Images Plus 2.0 software in three randomly chosen visual fields until day 4. Clones were photographed using a phase-contrast microscope, and the sphere diameter was measured using the Motic Images Plus 2.0 software.
Expression of melanoma stem cell markers, were estimated by labeling G-361 cells with anti CD133 antibody (BD Biosciences, USA) or anti ABCB5 antibody (Abcam, Korea). All samples were incubated for 20 min at 4 °C, washed twice with PBS, and immediately analyzed using a BD FACSVerse cytometer and the FACS suite software.
Tumor xenografts in nude mice
G-361 cells (human melanoma, 1 X 106 cells/200 μl PBS) were injected intradermally into the upper right flank of nude male mice (CAnN.Cg-Foxn1/ 5 weeks of age; Orientbio, Korea). The mice were randomly divided into vehicle, PAM only, SN only and co-treatment groups after achieving a tumor size of 100mm3. Plasma activated media (PAM) were freshly prepared because of reduction in free radicals, with a 10-min CAP treatment of incomplete RPMI media using μ-DBD CAP. Mice were treated with 1 mg/kg of body weight of the SN 1 day before the PAM treatment to the co-treatment group. The vehicle group of mice received 200 μl of PAM once for each of the next 3 days by an intradermal injection into the tumor, and the control group received the same volume of RPMI medium into the right flank. Body weights were estimated on each subsequent day after the injections of the tumors, and tumor sizes were measured with Vernier calipers (calculated volume = shortest diameter X longest diameter/2) at two- day intervals. This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Center for Laboratory Animal Sciences, Medical Research Coordinating Center.
Tumor tissues were excised with proper precautions and fixed in formalin for the preparation of paraffin sections. Paraffin-embedded tumor sections were deparaffinized in xylene and then with 100, 90, 80 and 70% ethanol, followed by phosphate-buffered saline (PBS). Tumor sections were stained with hematoxylin and eosin (H & E) or immunostained overnight at 4οC with the BRAF antibody (1:300; Abcam), the cMET (1:300; Abcam), the CD133 (1:200; Abcam) antibody, and Ki67 (1:200; Abcam). After washing in PBS, a 1:200 dilution of biotinylated goat anti-rabbit IgG or anti-mouse IgG antibody in a blocking solution was applied to the sections and they were incubated for 30–40 min. Following the PBS treatment, ABC reagent was applied to the sections and they were incubated a further 30 min. Color reaction tests were performed with 3, 30-diaminobenzidine (Vector Laboratories) and the slides were washed twice with PBS. After hematoxylin counterstaining and clearing with a graded ethanol series and xylene, sections were mounted with Canada balsam. Images were photographed using an IX71 microscope (Olympus) equipped with the DP71 digital imaging system (Olympus).
Data were expressed as the means ± SD of triplicates. The statistical significance of the difference between the values of the control and treatment groups was determined using Student’s t-tests, and in each case the levels of significance are indicated as *, p < 0.05; δ, p < 0.01; and #, p < 0.001.