Chemical and reagents
CDDP was obtained from MedChemExpress (MCE, New Jersey, USA). ArBu with a molecular formula of C24H32O6 and a molecular weight of 416.51 was obtained from MedChemExpress (MCE, New Jersey, USA). The Cell-Counting Kit-8 (CCK-8) assay was purchased from MedChemExpress (MCE, New Jersey, USA). Dimethyl sulfoxide (DMSO) was procured from Good Laboratory Practice Bioscience (GLPBIO, Montclair, California, USA). BCA protein assay kits were obtained from Beyotime (Shanghai, China). Annexin V fluorescein-isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kits were purchased from Beyotime (Shanghai, China). Primary antibodies against CA9, IKBKB, NF-κB, MMP-2, MMP-9, E-cadherin and GAPDH were obtained from Affinity Biosciences (Cincinnati, OH, USA). Anti-rabbit IgG, and anti-mouse IgG, HRP-linked antibodies were purchased from Beyotime (Shanghai, China). Enhanced chemiluminescence (ECL) kits were purchased from Affinity Biosciences (Cincinnati, OH, USA).
Cell culture and treatments
Human gastric cancer cell line AGS and MKN-45 were obtained from Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences (Shanghai, China). AGS and MKN-45 cells were cultured in RPMI-1640 and DMEM medium (high glucose) supplemented with 10% heat-inactivated FBS and antibiotics (100 U/ml of penicillin and 100 μg/ml of streptomycin (Wako Pure Chemical Industries) in a humidified 5% CO2 atmosphere at 37°C, respectively. Cells were seeded in a 6-well plate at a density of 1×106 cells/2 ml media, which were treated with ArBu (10, 20, 40, 60, 80 nM), CDDP (10, 20, 40, 60, 80 μM), their combination or vehicle for 12, 24, 48 h.
Cell viability evaluation
Following treatment for 12, 24, 48 h, cell viability was measured using the CCK-8 kit. Cells were seeded in a 96-well plate with 1 × 105 cells per well. After treatments, CCK-8 solution (10 µl) was added to each well of the plate, which was then incubated for 1 h. Optical density of each well was determined by using a microplate reader at 450 nm. Relative cell viability was expressed as the ratio of the absorbance of each treatment group against those of the corresponding untreated control group. The ED50 values of the drugs were calculated using GraphPad Prism 8 software.
Wound healing assay
When AGS and MKN-45 cells reached approximately 80% confluence, the monolayer was scratched with a 200 μl pipette tip at the time recorded as 0 h. Then the cells were treated with DMSO or ArBu (40 nM), CDDP (40 uM), ArBu combination with CDDP (24.61 nM plus 28.37 uM) for 12 and 24 h. The culture medium was removed, and the plate was washed with phosphate buffered saline (PBS) three times. Cell debris produced during scratching was washed away, followed by the addition of serum-free culture medium and imaging at 12 and 24 h. ImageJ analysis software (Bethesda, MD, U.S.A) was used to calculate the migration distances.
Colony formation assay
A total of 1.5× 103 treated cells were coated into 6-well plates with three repetitions. After 14 days incubation, these plates were washed with PBS twice, fixed by methanol for 10 minutes and stained with 0.1% crystal violet solution within 10 minutes for further analysis. Briefly, AGS and MKN-45 cells were re-suspended in complete medium containing 10% FBS and were seeded into 12-well plates for 10 days. Using 0.1% crystal violet, cells fixed with methanol for 15 minutes were visualized under a dissection microscope (Olympus, Tokyo, Japan) and colonies consisting of 50 cells or more were counted.
Apoptosis assay
Apoptosis ratios of AGS and MKN-45 cells undergoing various treatments were measured using Annexin V-fluorescein (AV) and propidium iodide (PI) apoptosis detection kit Beyotime (Shanghai, China) by flow cytometry. Briefly, after stimulation, cells were washed with PBS and incubated with 10 µL of Annexin V-FITC and 5 µL of PI for 15 minutes at room temperature in the dark. Flow cytometry analysis was done by a FACScan flow cytometer (Beckman Coulter, Fullerton, CA, USA), and the data were analyzed by using FlowJo software (Tree Star, Ashland, OR, USA).
Cell cycle analysis
Treated cells were collected and fixed with chilled 75% ethanol at -20°C overnight. After ethanol discarded, cells were washed twice with PBS and resuspended with 250 μl DNA staining solution (Multiscience, China) at room temperature for 30 minutes. Cell cycle analysis was performed on the flow cytometry (FACS LSRII, BD Bioscience, USA).
Immunofluorescence
Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Following cell fixation, cells were incubated with the appropriate primary antibodies CA9, IKBKB, NF-κB (Proteintech, Chicago, USA) in a solution of PBS with 1% bovine serum albumin at 4°C overnight. Cells were further incubated with FITC (HY-66019, MCE, New Jersey, USA.) labeled secondary antibody for 1 h at room temperature. Nuclei were stained with DAPI for 5 minutes. Fluorescent signals were detected by confocal fluorescence microscopy.
qRT-PCR
Total RNA was isolated from AGS cells and MKN-45 cells as well as tumorigenic tissues of nude mice using TRIzol reagent (Invitrogen) according to manufacturer’s instructions. Reverse transcription was performed by the Multiscribe RT kit (Applied Biosystems, Foster, CA, USA). For analysis of CA9, NF-κB, IKBKB, MMP-2, MMP-9 and E-cadherin, the SYBR Green PCR kit (TaKaRa, Japan) was used to quantify the messenger RNA (mRNA) levels. GAPDH was used as a control and relative expression levels of CA9, NF-κB, IKBKB, MMP-2, MMP-9 and E-cadherin were calculated using the 2−ΔΔCT method. Primer sequences of these genes in human cells and nude mice were shown in Table 1 and in Table 2 separately. All experiments were performed independently and in triplicate.
Western blot assay
AGS and MKN-45 cells and tumorigenic tissues of nude mice were lysated using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime. Shanghai, China). Equal amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (GLPBIO, Montclair, California, USA). The membranes were incubated with the indicated primary and secondary antibodies. The primary antibodies used in this study included anti-CA9 (11071-1-AP, 1:1000, Proteintech, Chicago, USA), anti-NF-κB (10745-1-AP, 1:1000, Proteintech, Chicago, USA), anti-IKBKB (15649-1-AP, 1:1000, Proteintech, Chicago, USA), anti-MMP-2 (AF5330, 1:2000, Affinity Biosciences, Boston, USA), anti-MMP-9 (AF5228, 1:2000, Affinity Biosciences,, Boston, USA), anti-E-cadherin (AF0131, 1:2000, Affinity Biosciences, Boston, USA) and GAPDH (AF7021, 1:1000, Affinity Biosciences, Boston, USA) was used as the loading control. Immunoreactive bands were visualized using ECL detection reagent (Affinity Biosciences, OH, USA). The intensity of the bands was quantified by using Image LabTM Software (Bio-Rad, California, USA).
Mouse xenograft model and treatments
Mouse experiments were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Anhui Medical University (Approval Number: LLSC20180365) in accordance with the Guide for the Care and Use of Laboratory Animals. All male mice were fed in a pathogen-free environment with well-controlled temperature (20 - 24°C) and humidity (40% - 60%). A total of 1x107 AGS cells were injected subcutaneously under the right axilla of the nude mice. When the tumors reached sizes of approximately 200 mm3 after approximately 24 days, mice were randomly divided into four groups. Treatments in each group were shown as follows: control (0.1% dimethyl sulfoxide solvent), ArBu (6.4 mg/kg) [22], CDDP (3 mg/kg) [23] and ArBu combination with CDDP (6.4 mg/kg plus 3 mg/kg). All mice were intraperitoneal administration with drugs thrice a week for 3 weeks. Nude mice were monitored daily for tumor and body weight. At the end of the experiment, mice were euthanized. Tumors were removed for imaging and their size was measured.
Hematoxylin and eosin (H&E) staining
Heart, liver, and kidney were rinsed with cold saline and fixed in 4% paraformaldehyde. Paraffinized tissues were dehydrated in ethyl alcohol with gradient proportion, and embedded in paraffin. After that, the tissues sections of 4 µm thickness were prepared and stained with H&E routinely.
Blood biochemistry
Analysis of Serum creatinine and Blood Urea Nitrogen in blood of mice.
Statistical analysis
Data from this study were presented as mean ± standard deviation (SD) and statistically analyzed by one-way analysis of variance (ANOVA) using SPSS 23.0 statistical software (IBM Analytics, New York, USA). The difference between theoretical ED50 and experimental ED50 was examined by Student'st-test. A P value of less than 0.05 was considered to indicate a statistically significant result. All experiments were repeated at least 3 times.