Reagents and antibodies
The following reagents were used in the study: Penfluridol (PF), 2-Deoxy-D-glucose (2-DG), A-769662, dorsomorphin dihydrochloride/Compound C (CC) were purchased from MedChemExpress (MCE, Shanghai, China), Cell Counting Kit-8 (CCK-8) were obtained from Yeason (Shanghai, China), Annexin V/PI Apoptosis staining Kit, and cell cycle staining kit were purchased from MULTI SCIENCES (Hangzhou, China), si-AMPK (5’- GGTTGGCAAACATGAATTG 3’) were obtained from Ribio (Guangzhou, China), Glucose colorimetric assay kit, and L-Lactic Acid colorimetric assay kit were purchased from Elabscience (Wuhan, China), Lipofectamine 3000 reagent, and BCA protein assay kit were purchased from Invitrogen (CA, USA), 4% paraformaldehyde, 0.1% crystal violet, and RIPA were purchased from FUDE BIOLOGICAL (Hangzhou, China), ibidi Culture-insert was purchased from ibidi (Martinsried, Germany), transwell plate were purchased from Corning (Corning, NY, USA).
The anti-β-actin (FD0060), secondary antibodies goat-anti-rabbit (FD0128) and goat anti-mouse (FD0142) IgG-HRP antibody was purchased from FUDE BIOLOGICAL Technology (Hangzhou, China), Anti-Caspase-3 (ER30804), anti-activated caspase-3 (ET1602-47), anti-PARP (ET1608-56), anti-cleaved-PARP (ET1608-10), anti-activate + pro caspase-9 (ET-1610-95) were purchased from HUABIO (Hangzhou China). Anti-Bcl-2 (EPR17509), anti-AMPK (ab207442), and anti-PFKFB3 (ab181861) were purchased from Abcam (Cambridge, UK), anti-Noxa was purchased from Cell Signaling Technology (Beverly, MA, USA), anti-PFKFB3S461 (TA3581), and anti-AMPKT172 (TP56027) were purchased from Abmart (Shanghai, China), anti-HK2 (sc-374091) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Human GBC cell lines EH-GB1 and GBC-SD were purchased from a cell bank (Chinese Academy of Sciences, Shanghai, China). The SGC-996 cell line was provided by Dr. Ying-bin Liu’s lab at Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, China. These cells were maintained in DMEM (EH-GB1 and GBC-SD) and RPMI-1640 (SGC-996) medium containing 10% fetal calf serum (FBS) from cellMax (Beijing, China), penicillin (100 units/ml) and streptomycin (100 µg/ml) in a 5% CO2 humidified incubator at 37°C (Thermo Scientific).
Cell viability assay
Cell viability was assessed by CCK-8. Cells (3x103) were seeded in 96-well plates and allowed to attach overnight in a 5% CO2 incubator. The cells were then treated with agents at different concentrations for 24h or 48h. CCK-8 (100 µl/ml) was added to each well and incubated for 1h at 37°C. The absorbance at 450nm was measured with a Multiscan spectrophotometer (Thermo Scientific).
Colony formation assay
1x103 cells/well were seeded in triplicate in a six-well plate for 24h. Then, the original medium was replaced with medium-containing agents at each concentration tested. Cells were incubated for another 14 days, photographed after being fixed by 4% paraformaldehyde and stained with 0.1% crystal violet and counted.
GBC cells were pre-treated for 12h as indicated. Then, cells were digested and resuspended with a culture medium. 2x104 cells/well were seeded into ibidi Culture-insert on 12-well plates for 12h. Later, the insert was gently removed, and the medium was replaced with FBS-free medium. Photomicrographs were taken by a microscope (Zeiss, Germany) at the indicated time.
GBC cells were pre-treated for 12h as indicated. Then, cells were digested and resuspended with FBS-free medium. 2x104 cells/well were seeded into the upper chamber of transwell 24-well plates with 8 µm pore size. Then, the lower chamber was added with a medium containing 20% FBS. After incubation of 24h or 48h, the upper surface of the chamber was cleaned, and migrated cells of the lower surface were stained with 0.1% crystal violet for 4 minutes. The level of migration was observed by a microscope.
Detection of cell apoptosis
Cell apoptosis was detected by staining using Annexin V/PI Apoptosis staining Kit according to the manufacturer’s instructions. Briefly, GBC cells were digested with EDTA-free trypsin after agent treatment and resuspended in 500µL of binding buffer. After incubation with 5µL of Annexin V-FITC and PI staining for 15min at room temperature in the dark, data acquisition and analysis were carried out using flow cytometry.
Detection of cell cycle
Cell cycle was detected by staining using a cell cycle staining kit according to the manufacturer’s instructions. Briefly, GBC cells were digested with EDTA-free trypsin after agent treatment and resuspended in 1ml of DNA staining solution. After incubation with 10µL of Permeabilization solution for 30min at room temperature in the dark, data acquisition and analysis were carried out using flow cytometry.
Cells were seeded into a six-well plate (80,000 cells/well) overnight and treated with agents. After treatment, cells were collected by being scraped and washed with PBS. Then, the cells were lysed with RIPA buffer. Protein concentrations in the supernatants were determined with a BCA protein kit. Equal amounts of proteins were separated on SDS-polyacrylamide gels and then electroblotted onto polyvinylidene fluoride membranes (Sigma-Aldrich). The membranes were blocked with TBST plus 5% skimmed milk for 2h and incubated with primary antibodies overnight at 4°C. After being washed three times with TBST, the membranes were incubated with secondary antibodies (1:5000) for 1h at room temperature. Before development, the membranes were washed three times again, and the immunoblots were visualized with an ECL system.
Glucose consumption measurement
Cell glucose consumption was detected by Glucose colorimetric assay kit according to the manufacturer’s instructions. Briefly, cells were planted in the six-well plates (80,000 cells/well) and incubated for 24h. Then, the culture medium was replaced with a medium with different agents. After 24h, the supernatant was collected and measured for glucose concentration. The cells left were lysed by RIPA for the detection of protein concentration to standardize glucose consumption levels.
Lactic acid production measurement
Cell lactic acid production was detected by L-Lactic Acid colorimetric assay kit according to the manufacturer’s instructions. Briefly, cells were planted in the six-well plates (80,000 cells/well) and incubated for 24h. Then, the culture medium was replaced with a medium with different agents. After 24h, the supernatant was collected and measured for lactic acid concentration. The cells left were lysed by RIPA for the detection of protein concentration to standardize glucose consumption levels.
Patient-derived xenograft (PDX) model establishment
Tumor tissues from the patient were evaluated by pathologists and macro-dissected for implantation. 4-week-old female nude mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd (Hangzhou, China) and maintained in a pathogen-free environment. The mice were transplanted with tumor tissues subcutaneously. During the grafting, all mice were intact. Initial tumor growth was detected at 4 months post-implantation. The tumors were serially implanted into new female nude mice via the same procedure as the original implants. A line was considered established if there was active growth after at least five passages. Tumor samples were harvested from later passages (>3) for characterization.
In vivo study
Xenograft tumors were established by subcutaneous transplantation. After 10 days, to assess the anticancer effects of agents on tumor growth, tumor-bearing mice were randomly divided into four groups: the control group (10% DMSO, 40% polyethylene glycol, and 5% Tween-80 in saline), CC treatment group (10mg/kg CC, intratumor injection, twice every week), PF treatment group (10mg/kg, ig, qd), and CC plus PF treatment group. Tumor volumes were measured every 3 days and calculated by the followed equation: Volume = (Length x Width2)/2. On day 15, mice were sacrificed for the tumor tissue collection.
Tumor tissues were acquired and fixed overnight in 4% paraformaldehyde and then dehydrated and coated with wax. Paraffin-embedded specimens were cut into 3-µm thick sections. Slices were either dyed with hematoxylin and eosin or immunostained with the primary antibodies.
All experiments were performed at least three times in triplicate to ensure reproducibility. Statistical analyses used unpaired tailed student’s t-tests, Kaplan-Meier survival analysis, and log-rank tests with GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA). The results were shown as mean ± SD. P < 0.05 was considered statistically significant.