Materials
Sorafenib, lycorine, Desmosterol and Compound C were purchased from MedChemExpress (Monmouth Junction, NJ, USA).
Cell culture
Human HCC cell lines PLC/PRF/5, HepG2 were obtained from the American Type Culture Collection (ATCC, VA, USA); MHCC-97H, SK-Hep1, and Huh7 from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Sorafenib-resistant clones were established by subjecting PLC/PRF/5 and MHCC-97H cells to continuous administration of gradually increasing Sorafenib concentrations and trained up to 8 μM Sorafenib. Cells were maintained in high glucose DMEM supplemented with 10% fetal bovine serum (FBS), 100 mg/mL of streptomycin, and 100 unit/mL of penicillin at 37 °C in 5% CO2. LDL was isolated from the plasma of healthy human volunteers by sequential ultracentrifugation. Informed consent (ethical approval: Sheffield REC 10/H1308/25 according to the principles outlined in the Declaration of Helsinki) was obtained from volunteers regarding the use of their plasma samples for research.
Patients
Datum of cases (78 cases) was obtained from randomly selected Sorafenib-treated HCC patients who under-treated at the Second Affiliated Hospital of Chongqing Medical University and Chongqing University Cancer Hospital between 2015 and 2021, with the approval of the Institutional Review Board of Chongqing Medical University and Chongqing University Cancer Hospital.
Patient samples
HCC tissues were obtained from 24 patients who under Sorafenib treatment at the Second Affiliated Hospital of Chongqing Medical University and Chongqing University Cancer Hospital between 2015 and 2021, with the approval of the Institutional Review Board of Chongqing Medical University. Patients provided informed consent. All specimens were frozen immediately after surgery and stored in liquid nitrogen until use.
Cell proliferation assay
Cells were seeded in 96-well plates at a density of 5000 cells/well. After 24 h, the cells were incubated in a serum-free medium for 12 h. Then, the cells were subjected to a concentration gradient of Sorafenib for 48 h. All experiments were carried out in a serum-free DMEM medium. The OD values were measured at 450 nm after incubation with CCK-8 reagent for 1 to 2 h at 37°C.
Oil Red O staining
Intracellular lipids were stained by means of Oil Red-O (Solarbio Life Science, Beijing, China). Cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 minutes. Fixed cells were incubated with Oil Red-O solution for 20 minutes at room temperature and then with 4',6-diamidino-2-phenylindole (DAPI) (Solarbio Life Science, Beijing, China) for 5 minutes. Frozen slices from euthanized mice were permeabilized with 4% paraformaldehyde for 20 min. After rinsing with PBS for 5 min approximately 3 times, the slices were stained with Oil Red O for 15 min at 37° C. Next, the slices were washed with ddH2O and counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 8 min. Finally, all the slides were examined under a light microscope.
Lipid analysis
Serum total cholesterol (TC), Serum triglycerides (TG) and total intracellular cholesterol (TC) were tested on enzymatic colorimetric methods using commercial kits purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Western blot analysis
Protein lysates of cells or tumor tissues were extracted by RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China). Equal volumes of protein samples were separated by 7.5% or 12.5% SDS/PAGE and electro-transferred to PVDF membranes (Millipore, Billerica, MA, USA). The immunoblots were probed with the indicated antibodies. Finally, the detection was performed using an ECL chemical luminescent detection kit (Bio-Rad), and the bands were further analyzed using ImageJ software. The expression of the target protein was normalized to β-actin expression.
Real-time quantitative PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Takara) and reverse transcribed into cDNA. Next, the cDNA products were subjected to 2-step PCR amplification. The relative expression of the genes was analyzed using the 2-∆∆ Ct method, and β-actin was used as the internal reference gene.
Gene silencing
Knockdown of SCAP in cells was achieved by using a reverse siRNA transfection procedure performed in six-well plates. Therefore, for each well to be transfected, 5 μl Lipofectamine RNAiMAX (Thermo Fisher Scientific, Eugene, OR) was mixed with 500 μl Opti-MEM (Thermo Fisher Scientific) and combined with 25 pmol siRNA (GenePharma, Shanghai, China). The transfection mixture was incubated at room temperature for 20 min. Cells were harvested in a complete growth medium without antibiotics and diluted so that 2 ml contained the appropriate number of cells to give 30% to 50% confluence 24 h after plating. Cell suspensions were mixed with the transfection mixture and incubated.
Colony formation assay
Cells were plated in 6-well plates at a density of 4000 cells/well with a medium containing 10% FBS. Then, the cells were treated with Sorafenib (6 µM) for 48 h and plated for 2 weeks. Colonies were fixed and stained with a 0.1% crystal violet solution and counted grossly.
Wound healing
Cells were seeded in 6-well plates and treated with Sorafenib (6 µM) for 48 h, and the monolayer was scratched with a pipette tip. After that, the cells were incubated in a serum-free medium for 0 to 72 h. Then, the wound areas were quantified using Image J software.
Transwell assays
For the transwell migration assays, cells in the upper chamber were treated with Sorafenib (6 µM) for 48 h in advance, while DEME containing 10% FBS was added to the lower chambers. For the transwell invasion assays, the upper membrane was coated with 50 µl Matrigel (BD Biosciences) in advance. After incubation, the cells were fixed and stained with trypan blue.
Flow cytometry analysis
Following treatment, cells were collected and resuspended in 1 ml of ice-cold PBS at a density of 1× 106 cells/mL or fixed with 75% alcohol, and the samples were immediately detected by flow cytometry (BD Biosciences, US) and the data were analyzed using FlowJo software version 10.
Transmission electron microscopy (TEM)
Fresh tissue and cells were placed in 4% glutaraldehyde overnight at 4°C. Ultrathin sections were cut and then stained. Images were acquired on a transmission electron microscope. For the quantification of autophagy, autophagic vacuoles (defined as autophagosomes, double-membraned structures surrounding cytoplasmic material, and autolysosomes, lysosomes containing cytoplasmic material) were counted.
Autophagic flux analysis
Cells were first transduced with siSCAP or siVector in a confocal dish. 24 h after the first transduction, the cells were then transduced with monomeric red fluorescent protein (mRFP)-GFP-LC3 adenoviral vectors (HanBio Technology, Shanghai, China). The principle of the assay is based on the different pH stability of red and green fluorescent proteins. The enhanced GFP signal could be quenched under the acidic condition (pH <5) inside the lysosome, whereas the mRFP signal did not change significantly in acidic conditions. In red- and green-merged images, autophagosomes are shown as yellow puncta, while autolysosomes are shown as red puncta. An enhancement of both yellow and red puncta in cells indicate that autophagic flux is increased, while autophagic flux is blocked when only yellow puncta are increased without alteration of red puncta, or when both yellow and red puncta are decreased in cells. Cells were incubated in 1 ml growth medium with the adenoviruses for 2 h at 37°C, and the growth medium was replaced with fresh medium. Cells were treated with Sorafenib (6μm) at the same time. Experiments were performed 48 h after the second transduction. LC3 puncta were examined with a Leica confocal microscope.
Animal model and treatment
Animal care and experimental procedures were performed with approval from the Animal Care Committee of Chongqing Medical University. All animal studies were conducted in accordance with institutional guidelines for the care and use of experimental animals. Animal experiments were conducted using 4–6-week-old BALB/c nude mice (male, 20–25 g). Mice were purchased from Gempharmatech Co., Ltd. (Nanjing, China). For the xenograft implantation model, a total of 20 nude mice were randomly divided into 4 groups, including control (con), lycorine (Ly), Sorafenib (Sora) and lycorine+Sorafenib (Ly+Sora). 2 × 106 cells were subcutaneously injected into the flanks of the mice. Tumor growth was measured every 2 days, and the volumes of the xenograft tumors were calculated using the following standard formula: length × width × width × 0.5. Treatment was initiated on the fifth day, tumor-bearing mice were intragastric administration with lycorine (10 mg/kg/day, 2.5 mg/mL in 5% dimethyl sulfoxide (DMSO)), Sorafenib (30 mg/kg/day, 1.2 mg/mL in 5% DMSO), both, or vehicle (5% DMSO dissolved in saline) (n=5 per group) for 30 days. After 30 days, the mice were sacrificed, and tumor tissues were harvested for histological analysis.
Immunofluorescent staining
Frozen slices or cells were fixed with 4% paraformaldehyde for 15 min and incubated with 0.3% Triton X100 for 15 min. After blocking with 3% bovine serum albumin, the slices were incubated with the following primary antibodies: anti-p-AMPK (1:100, CST), anti-P62(1:200 proteintech). After overnight incubation, the slices or cells were then incubated with fluorescence-conjugated secondary antibodies for 1 h. Finally, the slices or cells were incubated with Hoechst for 5 min, and then images were captured under a Zeiss fluorescence microscope and analyzed using Image Pro Plus software.
immunohistochemistry (IHC) staining
Tumor tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin according to standard procedures. Sections were incubated with the indicated primary antibodies overnight at 4°C. Subsequently, the slides were incubated with a secondary anti-rabbit or anti-mouse IgG (ZSGB-BIO, Beijing, China) and visualized using 3,3′-diaminobenzidine (ZSGB-BIO). Stained slides were scanned with a Pannoramic Scan 250 Flash or MIDI system and images were acquired using Pannoramic Viewer 1.15.2 (3DHistech, Budapest, Hungary). Images were analyzed using Image Pro Plus software.
Study approval
For patient samples, the study protocol was approved by the Medical Ethics Committee of Chongqing Medical University. Patients were provided informed consent. All experimental procedures performed on animals were approved by Institutional Animal Care and Use Committee at the Chongqing Medical University (license number: 2017011). All mice were maintained under specific pathogen-free conditions in the laboratory animal center of Chongqing Medical University. Animal care and use protocols adhere to National Regulations for the Administration of Laboratory Animal to ensure minimal suffering.
Statistical analysis
All data were presented as means ± SD. Sample sizes for relevant experiments were determined by power analyses conducted during experiment planning. Appropriate statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software Inc, La Jolla, CA, USA). Statistical significance was determined using one-way ANOVA for multiple comparisons. Student’s t-test was used to compare two groups. Pearson correlation was used to analyze the relationship between SCAP protein expression and sensibility of Sorafenib. The chi-square test and Student’s t-test was applied to determine the association between clinicopathological parameters and tumor development. Probability values (p) < 0.05 were considered statistically significant.