Plasmids, Cell lines and Reagents
The encoding sequence of mouse FAK was amplified from cDNA of B16F10 cells and cloned into pRK5-Flag vector. The promoter sequence of human and mouse FAK was amplified and cloned into pGL3 reporter vector just as the previous article (13). Actinomycin D (Act D) was purchased from Sigma. B16F10 and B16F1 (murine melanoma cells), A375 (human melanoma cells) and MEF (murine embryonic fibroblast cells) were purchased from American Type Culture Collection (ATCC, USA) or maintained in our laboratory. All cells were cultured at 37 °C in 5% CO2 in a humidified atmosphere in Dulbecco’s modified Eagle’s media (DMEM, Gibco, Shanghai, China) with 10% fetal bovine serum (FBS, Gibco, Australia), penicillin (100 IU/ml) and streptomycin (100 μg/ml).
Real-time quantitative PCR assay
Total RNA was extracted with TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions. Quantitative real-time PCR was performed using reverse transcription kit (Takara, Japan) and SYBR Green PCR Master Mix (Roche, Germany). The primers were listed in Supplementary Table S1. Data were analyzed by StepOne 2.1 software (Applied Biosystems, USA) according to the manufacturer’s specifications, and were normalized using β-actin.
Protein extraction and immunoblotting
Cells were collected and homogenized with whole cell lysate prepared with RIPA lysis buffer (Santa Cruz Biotechnology) containing protease inhibitors, PMSF and orthovanadate. After 30 minutes of incubation on ice, the whole-cell extract was pelleted in an Eppendorf microcentrifuge and centrifuged at 12,000 rpm for 10 minutes at 4 °C. Supernatants were collected and protein concentration was determined by the Bio-Rad protein assay method (Bio-Rad, Hercules, CA). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes that were blocked with 5% non-fat milk in TBS containing 0.1% Tween-20, and incubated with primary antibodies: p-FAK, FAK, Akt, p-Akt (Ser 473), Paxillin, E-cadherin, N-cadherin, Vimentin (Cell Signaling Technology, Beverly, MA, USA), β-actin (ABGENT, San Diego, USA). Secondary antibodies were coupled to horseradish peroxidase, and were goat anti-rabbit or goat anti-mouse. All experiments were done in triplicate and performed at least three times independently.
LC-MS/MS analysis and bioinformatics analysis
The LC-MS/MS analysis was performed as previously described (13). Briefly, after collecting the cells and obtaining the lysate, the peptide was purified using a Strata-XC18 column, and then the obtained peptide was labeled with the iTRAQ Reagent-8 plex Multiplex Kit (AB Sciex U.K. Limited) according to the manufacturer’s instructions. The samples and labeled markers in this article were as follows: the samples of MEF negative control cells were labeled with iTRAQ tag 115, the samples of MEF ADT-OH (2 μM, 15 min)-treated cells were labeled with iTRAQ tag 117, the samples of MEF ADT-OH (2 μM, 2 h)-treated cells were labeled with iTRAQ tag 118, the samples of MEF ADT-OH (50 μM, 15 min)-treated cells were labeled with iTRAQ tag 119 the samples of MEF ADT-OH (50 μM, 2 h)-treated cells were labeled with iTRAQ tag 121. All the labeled samples were mixed in equal amounts and then fractionated using a Durashell C18 (5 μm, 100 Å, 4.6 × 250 mm) though high-performance liquid chromatography (HPLC) system (Thermo DINOEX Ultimate 3000 BioRS). Then the Triple TOF 5600 System (AB SCIEX, Concord, ON) was used for data acquisition, and ProteinPilot Software 5.0 (AB SCIEX) was used for protein identification and iTRAQ relative quantification. High abundance (> 1.5-fold, p < 0.05) and low abundance (< 0.67-fold, p < 0.05) proteins were used to identify differentially expressed proteins (DEPs). Finally, MetaCoreTM version 5.4 (GeneGo, St. Joseph, MI) was used to further analyze the identified differentially expressed proteins through pathway analysis.
Cell cycle, cell apoptosis and cellular reactive oxygen species (ROS) levels analysis
B16F10, B16F1 and A375 cells were treated with different concentrations of ADT-OH (0, 6.3, 25, 100 μM) for 24 h. For cell cycle analysis, cells were fixed overnight at 4 °C using 70% pre-chilled ethanol (1 mL). After PI staining, cells were analyzed on a FACSCalibur flow cytometer with CellQuest software (BD Biosciences, CA, USA). For the detection of apoptosis, the treated cells were incubated with enhanced green fluorescent protein-coupled annexin V (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions, and then analyzed by flow cytometry (BD Biosciences, California, USA). For the detection of ROS, the cells treated with the above drugs were resuspended in serum-free DMEM medium containing 10 μM MDCFH-DA and incubated at 37°C in the dark for 30 minutes. After collecting and washing the cells, 400 μL of single cell suspension was analyzed using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences, CA, USA). The level of intracellular reactive oxygen species was determined based on the fluorescence intensity of the FL1 channel.
Luciferase reporter assay
B16F10, B16F1 and A375 cells were cotransfected with pGL3-basic, FAK-promoter luciferase (pGL3-FAK) and control pRL-SV40 reporter for 24 h. Luciferase activities were measured consecutively by using Dual-Luciferase assays (Promega, USA). All measurements were normalized for Renilla luciferase activity to correct the variations in transfection efficiencies.
Cell proliferation assay
CCK8 assay was used to measure cell proliferation. Cells in the exponential growth phase were seeded into a 96-well plate at a density of 5000 cells per well. The cells were incubated with ADT-OH (0.8-100 μM) for 24 h before adding 10 μl of CCK8 (Sigma, Milan, Italy), and cell viability was measured by using a microplate spectrophotometer (Titertek Mul-tiskan MCC / 340) equipped with a 450 nm filter. Each experiment was performed in quadruplicate and repeated at least three times.
Cell migration assay
The cell migration assay was performed using transwell inserts (8.0 mm pore size, Millipore, Billerica, MA, USA). The cells were starved for 12 h prior to the experiment. Cells were then harvested and resuspended in a cell suspension diluted to 5 × 105 cells / mL with serum-free DMEM containing 1% BSA. 200 μL of cell suspension were pipetted into the upper chamber, and the lower chamber was filled with 600 μL of 10% FBS supplemented medium. After incubation at 37°C for 10 h or 16 h, cells on the upper surface of the membrane were removed. The migrant cells attached to the lower surface were fixed in 4% paraformaldehyde at room temperature for 30 min, and stained for 30 min with a solution containing 1% crystal violet and 2% ethanol in 100 mM borate buffer (pH 9.0). The number of cells migrating to the lower surface of the membrane was photographed in five fields under a microscope with a magnification of × 100. The chamber was then purged with 33% HAC (100 μL). After the crystal violet was completely dissolved and the cells were evenly distributed in the HAC solution, the assay was performed at 570 nm using a microplate reader (TECNA, Switzerland) and quantitative analysis was performed using GraphPad Prism 8.0 software.
Cells were plated in 6-well culture plates to form cell monolayer (near 90% confluence). A sterile P-200 micropipette was used to scrape off the cells to make wounds. Then the wells were washed three times with PBS to remove non-adherent cells. The progress of wound closure was monitored with a ×10 microphotographs taken by the light microscope (Carl Zeiss Axioplan 2) at the beginning and the end of the experiments after washing with PBS. Image J software was used to calculate the scratch area of the melanoma cells treated with or without ADT-OH at 0 h and 24 h, and then the migration rates were calculated using the following formula：Migration rate = (S1-S2)/S1, where S1 represents the scratch area at 0 h; S2 represents the scratch area at 24 h.
Colony-forming assay was performed as previously described (14, 15). Briefly, about 300 cells in log phase were plated into 60 mm tissue culture Petri-dish (Greiner) in triplicate with 3 mL of culture medium and grown at 37°C with 5% CO2. 48 h later, cells were rinsed with fresh medium, and ADT-OH was added at different concentrations (0, 3.2, 6.3, 12.5, 25, 50 μM). After incubation for 48 h, the cells were washed twice with PBS and then incubated in drug-free medium. The medium was changed every 5 days. After culturing for additional 10-14 days, the medium was discarded and each dish was washed twice with PBS carefully. The cells were fixed with methanol for 15 min and stained with a 1:10 dilution of Giemsa regent (Merck, Germany) for 10 min. Any grouping of cells containing 30 or more cells was counted as a colony. Colony numbers were determined from triplicate plates. Colony growth was related to the control value without any treatment.
Immunofluorescence assay (IFA)
The immunofluorescence assay was performed as previously described (16). B16F10, B16F1 and A375 cells were treated with ADT-OH at different concentrations. After 48 h, cells were harvested and immunostained with anti-E-cadherin and vimentin antibody, respectively. The cells were then incubated with Alexa Fluor 488-labeled (#A21202, Thermo Fisher Scientific Inc.) and Alexa Fluor 594-labeled (#A21207, Thermo Fisher Scientific Inc.) secondary antibody for 1 h at room temperature. After the cell nucleus was counterstained with DAPI, images were obtained using the ZEN 2009 Light Edition software (Carl Zeiss) through an inverted Zeiss LSM710 confocal microscope (40x lens) (Carl Zeiss).
Development of mouse tail vein injection model
B16F10 and A375 cells (2×106) suspended in 200 μL of PBS were injected into the tail vein of the C57BL/6 mice or nude mice. Beginning on the second day, the mice were administered with vehicle, 17.5 mg/kg ADT-OH, 37.5 mg/kg ADT-OH, 75 mg/kg ADT-OH, 1.4 mg/kg NaHS, 2.8 mg/kg NaHS and 5.6 mg/kg NaHS five times a week for a total of 3 weeks with at least 8 mice per group. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. At the end of study, mice were euthanized and the lungs and livers were harvested, fixed in 10% formalin, and paraffin embedded for pathological examination of H&E slides.
In vivo footpad injection model
B16F10 cells (2×105 cells in 50 μl PBS) were implanted into the right hind footpads of C57BL/6 mice using a Hamilton syringe and 25‐gauge needle. The detailed treatment was described in Fig. 1b. All mice were euthanized 21 days after tumor inoculation in order to assess the number of spontaneous metastases in the lungs. The following formula was used to measure and calculate the volume of the tumor in the sole of the foot: 0.5236×L1×(L2)2, where L1 is the long axis of the tumor and L2 is the short axis of the tumor.
In a parallel animal assay (totally three groups, and three mice per group), the tumor establishment and drug treatment are the same as described above. On the 21th day, mice were euthanized. Tumors were collected, fixed with 4% formaldehyde and then embedded in paraffin. Tissue sections (5 μm in thickness) were prepared according to standard protocols for hematoxylin/eosin (H&E) staining. Apoptotic cells in tumor sections (two sections per mouse, three mice in total) were visualized by the TUNEL technique according to the manufacturer's instruction (Merck).
Data were presented as means ± SD. Comparisons within groups were done with a t-test with repeated measures; p-values indicated in figures are <0.05 (*), <0.01(**), and <0.001 (***).