Chemicals, kits, and cell culture media
Bovine lactoferrin (LF) with a purity > 98% was supplied by Phermpep Co., Ltd. (Taichung, Taiwan). A stock LF solution (250 mg/ml) was prepared in phosphate-buffered saline (PBS) and stored at -20 °C until use. Modified Eagle’s medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 100× MEM-nonessential amino acid (MEM-NEAA) solution, 100 mM sodium pyruvate solution, 100× penicillin/streptomycin solution, and the BCA protein assay kit were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Propidium iodide (PI)/RNase staining buffer was purchased from BD Pharmingen (San Diego, CA, USA). A mitochondrial membrane potential assay kit was purchased from Cell Signaling Technology (Danvers, MA, USA). An Annexin V-FITC/PI apoptosis detection kit was purchased from Elabscience Biotechnology (Wuhan, China). The Presto™ DNA/RNA/protein extraction kit was purchased from Geneaid Biotech Ltd. (Taipei, Taiwan). The MMLV Reverse Transcription Kit and 2× qPCRBIO SyGreen Mix were purchased from Protech Technology Enterprise Co., Ltd. (Taipei, Taiwan). The Novolink™ Polymer Detection System was purchased from Leica Biosystems (Deer Park, IL, USA). Inhibitors for p38 MAPK (SB203580) and JNK (SP600125) were purchased from Merck (Darmstadt, Germany). ERK inhibitor (LY3214996) was purchased from Cayman Chemical (Ann Arbor, MI, USA).
Cell culture
The cell lines used in this study, including FL83B (normal liver cells), HepG2 (hepatocellular carcinoma), Hep3B (hepatocellular carcinoma), and SK-Hep1 (liver adenocarcinoma), were purchased from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. FL83B cells were maintained in DMEM supplemented with 10% FBS, 1% MEM-NEAA, 1 mM sodium pyruvate, and 1% penicillin/streptomycin. HepG2, Hep3B, and SK-Hep1 cells were maintained in MEM supplemented with 10% FBS, 1% MEM-NEAA, 1 mM sodium pyruvate, and 1% penicillin/streptomycin. All of the cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2.
Cell viability assay
Cell viability was determined using a CCK-8 kit. Briefly, cells were seeded in 96-well plates (3000 cells/well) and treated with different concentrations of LF (1, 2.5, 5, and 10 mg/ml) or an equal volume of DPBS as a control. At the indicated time points (0, 24, and 48 h), the old media was replaced with new media, and 10 µl of CCK-8 solution was subsequently added. After 2 h of incubation at 37 °C, the absorbance at 450 nm was measured using a microplate spectrophotometer.
Colony formation assay
Cells were seeded in 6-well plates (500 cells/well) and allowed to grow in the presence of LF (5 and 10 mg/ml) or not (using PBS as a control). The incubation persisted for 9 days, and the media were refreshed every 3 days until the end of the incubation. Afterward, the cells were washed with PBS and then stained with 0.1% crystal violet for 15 min. Images were acquired using a Lionheart FX automated microscope (BioTek Instruments, Winooski, VT, USA), and the surface areas of the cell colonies were measured using ImageJ software.
Apoptosis analysis
For apoptosis analysis, cells were grown in 6-well plates at an initial cell density of 106 cells per well for 24 h and then treated with different concentrations of LF for another 48 h. The same procedures were also applied to the other cell experiments presented in this study. Apoptosis analysis was performed using an Annexin V-FITC/PI apoptosis detection kit. Briefly, the collected HepG2 cells were washed twice with cold PBS and resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) at a cell density of nearly 106 cells per ml. Aliquots of the cell suspension (100 µl) were then mixed with Annexin V-FITC antibody (20-fold dilution) and PI (50 µg/ml) and incubated for 30 min at room temperature under light protection. Afterward, the sample volume was brought up to 500 µl with binding buffer, followed by analysis using a BD Accuri C6 Plus flow cytometer.
Mitochondrial membrane potential assay
A mitochondrial membrane potential assay kit was used to assess changes in mitochondrial membrane potential. This assay was performed by incubating an adequate number of LF-treated cells in a tetramethylrhodamine (TMRE, 200 mM) labeling solution at 37 °C for 20 min. Cells were harvested, washed three times with ice-cold PBS, and then analyzed by flow cytometry. For microscopic observation, cells were grown on 18-mm sterile coverslips (5000 cells/slip) and treated with the same procedures as described above. The fluorescence intensities of TMRE (650–690 nm) were evaluated by cellSens imaging software (Olympus).
Cell cycle analysis
For the cell cycle analysis, the collected HepG2 cells were washed with ice-cold PBS, fixed with 70% ethanol for 2 h, and finally resuspended in PI/RNase staining buffer at a cell density of nearly 106 cells per ml. After incubation at 37 °C for 30 min, samples were subjected to cell cycle analysis using a BD Accuri C6 Plus flow cytometer (BD Biosciences, Mountain View, CA, USA).
Western blot analysis
The preparation of protein lysates and the related procedures for Western blot analysis were performed as previously described56. Briefly, protein lysates were prepared by lysing cells in 1× RIPA buffer containing protease and phosphatase inhibitors. The total protein concentration was determined using a BCA protein assay kit. Samples of 35 µg total protein were loaded, separated by SDS‒PAGE, and then transferred to PVDF membranes. The PVDF membranes were then sequentially treated by blocking (5 min), primary antibody incubation (4 °C, overnight), TBS-T wash (10 min, 3 times), secondary antibody incubation (10000-fold dilution, 2 h), TBS-T wash (10 min, 3 times), and detection of chemical luminescence immediately. Appropriate dilutions of the primary antibodies used in this study are listed in Supplementary Table S1.
RNA isolation and quantitative real-time PCR (qRT–PCR)
Total RNA was extracted using a Presto™ DNA/RNA/Protein Extraction Kit according to the manufacturer’s instructions. Two micrograms of total RNA from each sample was transcribed into cDNA using an MMLV Reverse Transcription Kit. qRT‒PCR was performed by mixing an aliquot of cDNA sample with target gene primers and 2× qPCRBIO SyGreen Mix in a total volume of 20 µl, followed by PCR running using the QuantStudio™ 6 Pro Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The primers used in this study are listed as follows: p21, 5’-CGATGGAACTTCGACTTTGTCA-3’ and 5’-CACAAGGGTACAAGACAGTG-3’; p27, 5’-CCGGTGGACCACGAAGAGT-3’ and 5’-GCTCGCCTCTTCCATGTCTC-3’; Cyclin A, 5’-GCCATTAGTTTACCTGGACCCAGA-3’ and 5’-ACTGACATGGAAGACAGGAACCT-3’.
Coimmunoprecipitation (Co-IP)
The harvested HepG2 cells were resuspended in lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA, pH 8.0) at 4 °C. The cell suspensions were then centrifuged (12,000 × g, 15 min) to collect the supernatants. Antibodies targeting the protein of interest were added and allowed to incubate at 4 °C overnight. Protein G magnetic beads (Cytiva 28-9440-08, Merck) were then introduced to the protein mixtures and incubated for 1 h at 4 °C with gentle mixing. Afterward, the beads were washed with lysis buffer to remove any nonspecifically bound proteins. Finally, proteins bound to beads were eluted with 50 µl of elution buffer (100 mM Tris-HCl, 4% SDS, 0.2% bromophenol blue, 20% glycerol). The samples were subjected to Western blot analysis after heating at 50 °C for 10 min.
RNA interference (RNAi)
The shRNA lentivirus particles targeting LRP1 (sh-LRP1) and lentivirus particles used for control (NC), as well as shRNA plasmid clones targeting INTL1 (sh-INTL1), were obtained from the RNA Technology Platform and Gene Manipulation Core, Academia Sinica, Taipei, Taiwan (http://rnai.genmed.sinica.edu.tw). shRNA-related information is presented in Supplementary Table S2.
For sh-LRP1 lentivirus transduction, cells were cultured in 6-well plates to a confluency of 70 ‒ 80%, followed by the addition of sh-LRP1 lentivirus particles at an MOI (multiplicity of infection) of 5 and incubation for 24 h at 37 °C in a humidified incubator. The media were refreshed with puromycin (2 µg/ml)-containing media every 3 days until puromycin-resistant colonies formed. The resistant cell colonies were selected and expanded, and the efficiency of LRP1 knockdown was determined by Western blot analysis.
For the transfection of sh-INTL1 plasmids, cells were cultured in 6-well plates to a confluency of 70‒80%, followed by transfection with a TransIT-X2 dynamic delivery system (Mirus Bio LLC., Madison, WI, USA) based on the recommended conditions. The efficiency of INTL1 knockdown was determined by Western blotting after 48 h of transfection.
Orthotopic mouse experiment
In vivo studies were performed in nude mice (BALB/c, male, 4‒5 weeks old, 18–22 g) that were obtained from the National Laboratory Animal Center, Taipei, Taiwan. Procedures such as housing and feeding were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of National Chung Hsing University (IACUC No. 104 − 091). The study is reported in accordance with ARRIVE guidelines.
To generate tumors, 40 µl of HepG2 cells (approximately 1 × 106 cells) mixed with 60 µl of Matrigel matrix (Corning) were implanted into the right lobe of the liver through an abdominal incision. One week after injection, mice were randomly divided into three groups (each n = 3). Our previous study demonstrated that a dose of 900 mg/kg LF produced no significant side effects in mice57; however, to minimize potential side effects, we selected doses within the EFSA-specified safety range for bovine lactoferrin18. Based on this information, nude mice were treated with PBS (mock group), 100 mg/kg/day LF (low-dose group), and 200 mg/kg/day LF (high-dose group) for thirty days. During oral administration, mouse body weight was recorded. At the end of the treatment, the mice were anesthetized with inhaled isoflurane and sacrificed by cervical dislocation to collect tissue samples for further analysis. Liver samples were collected, which were either immediately frozen in liquid nitrogen and stored at -80 °C until further use or fixed in 10% neutral formalin for subsequent paraffin embedding and tissue section staining.
Immunohistochemistry (IHC) staining
The paraffin-embedded liver sections were cut at 5-µm thickness in glass slides and subjected to IHC staining using a Novolink™ Polymer Detection System according to the manufacturer’s instructions. After dewaxing, liver sections on the slides were incubated in peroxidase-blocking buffer for 5 minutes to eliminate endogenous peroxidase activity. The slides were then incubated overnight at 4°C with primary antibody solutions (100-fold dilution) and subsequently incubated with HRP-conjugated secondary antibody solutions (500-fold dilution) for 2 h. After three PBS washes, 3,3’-diaminobenzidine (DAB) was added for color development. After being thoroughly washed with water, the slides were counterstained with hematoxylin, dehydrated, mounted, and observed under a microscope. The positively stained areas of IHC were quantitatively measured using ImageJ software.
Statistical analysis
All experiments were repeated independently at least three times. The data are presented as the mean ± SD and were plotted using GraphPad Prism 8.0 software (Boston, MA, USA). Statistical analyses were performed by one-way or two-way ANOVA with Tukey’s multiple comparison. Statistical significance is marked by * (*p < 0.05, **p < 0.01, and ***p < 0.001) and # (#p < 0.05, ##p < 0.01, and ###p < 0.001).
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- Source: https://www.nature.com/articles/s41598-024-82514-4