Human gemcitabine-resistant CCA and non-cancerous fibroblast cell lines

The CCA cell line, KKU-213B, was obtained from Thai CCA patients. Informed consent from each patient was documented in writing, following the protocol established by Prof. Banchob Sripa at Khon Kaen University [27]. To create the KKU-213BGemR cell line, KKU-213B cells were grown using a stepwise dose-escalation process with gemcitabine, as previously described [28]. OUMS-24/P6X, transformed fibroblast cells originating from a human embryo, which are available from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan), were included for toxicity testing. The cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco) at 37 °C and 10% CO2 in a humidified incubator. Cells were maintained in the presence of gemcitabine and then in a drug-free medium for one passage immediately before use in an experiment.

Cell proliferation detection by MTT assay

Cells (3,000 cells/well) were seeded in flat-bottomed 96-well plates (Costar, Corning, NY, USA) and treated for 24, 48, or 72 h with either 0.5% dimethyl sulfoxide (DMSO) (Sigma Aldrich, Saint Louis, MO, USA), serving as a vehicle control, or various concentrations of CBD (2.5 to 30 µM) (DMSc reference standard was purchased from the Bureau of Drug and Narcotic, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand) dissolved in DMSO and diluted with culture media. Then, 24 µL of 2.5 mg/mL MTT (Invitrogen™, Thermo Fisher Scientific Inc., Waltham, MA, USA) was added to the cells and incubated for 2 h at 37 °C and 5% CO2 in a humidified incubator. DMSO was then added to dissolve the formazan, and absorbance was measured at 540 nm using a Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, MA, USA). Data derived from MTT assay was used to determine the half-maximal inhibitory concentration (IC50) of CBD.

Clonogenicity assay

Approximately 2,000 cells per well of KKU-213BGemR were grown in 6-well plates and treated with different concentrations of CBD (10, 20, and 30 µM). The culture medium was changed every 2 days, and the cells were cultured for approximately 2 weeks. Cells were then fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and dissolved with 33% acetic acid. Absorbance at 620 nm was measured using a Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific Inc.).

Apoptosis detection by flow cytometry

Apoptotic cells were detected by flow cytometry and a dual staining technique using Annexin V-FITC (BioLegend, San Diego, CA, USA) and propidium iodide (PI), following the manufacturer’s recommendations. In brief, 3 × 105 KKU-213BGemR cells were cultured for 24 h in six-well plates. Subsequently, these cells were treated with either 0.5% DMSO or different concentrations of CBD (10, 20, and 30 µM) for 24 h. After the treatment interval, cell pellets were resuspended in Annexin-V binding buffer and then subjected to dual staining with Annexin-V-FITC and PI in a light-protected environment, allowing 15 min incubation at room temperature. A BD FACSCanto II flow cytometer (BD biosciences, San Jose, CA, USA) was used to enumerate apoptotic cells, with subsequent data analysis performed using FlowJo™ software version 10.8.1 (BD biosciences, Ashland, OR, USA).

Cell cycle arrest

KKU-213BGemR cells (1 × 105 cells/well in 6-well plates) were treated for 24 h with either 0.5% DMSO (control) or CBD at concentrations of 10 to 30 µM. Cells were then fixed with 70% ethanol at 4 °C for 1 h. After fixation, cells were resuspended in FxCycle™ PI /RNase staining solution (Molecular Probes, Thermo Fisher Scientific Inc., Carlsbad, CA, USA) and incubated for 45 min in the dark at room temperature. Detection and analysis of stained cells were performed using a BD FACSCanto II flow cytometer, together with FlowJo™ software version 10.8.1 and FCS Express 7 (De Novo Software, Los Angeles, CA, USA).

ROS measurement

KKU-213BGemR cells were seeded in 12-well plates at a density of 2.5 × 104 cells per well. In brief, cells were exposed to either 0.5% DMSO or CBD 30 µM for 6 h. Subsequently, cells were incubated with dichloro-dihydro-fluorescein diacetate (DCFH-DA, #35845, Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 10 µM in DMEM serum-free medium at 37 °C and 10% CO2 in a humidified incubator for 30 min. Thereafter, the supernatant was aspirated, and washed once with DMEM serum-free media followed by two washes with 1X PBS. Each well was then treated with 500 µL of 1X PBS. Images of ten visual fields from each experimental group were acquired at a magnification of 10x using a fluorescence imaging microscope (ECLIPSE Ti-U, Nikon Instruments Inc., Japan). Fluorescence integrated intensity (fluorescence intensity normalized to the area) was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Western blot

KKU-213BGemR cells were treated with 0.5% DMSO and CBD at a concentration of 30 µM for different time periods (0, 2, 4, and 6 h). Protein extraction was performed using 1X RIPA reagent (#9806S, Cell Signaling Technology, Danvers, MA, USA) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail l (Thermo Fisher Scientific Inc., Rockford, IL, USA) and protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc). Protein (20–25 µg) was separated using SDS-PAGE. In brief, extracted proteins were separated by electrophoresis on an 8–12% gel based on their molecular weight at 90–110 volts for 1.30 h. Following electrophoresis, proteins were transferred from the gel onto a polyvinylidene fluoride (PVDF) membrane (#10600029, Cytiva, Dreieich, Germany) at 90 volts for 1.30 h. After separation, PVDF membrane was incubated with primary antibodies at a dilution of 1:1,000 for all. Primary antibodies used included PERK (#5683) purchased from Cell Signaling Technology, phospho-PERK (#AP0886), BiP (#A0241), ATF4 (#A0201), and CHOP (#A0221) from ABclonal in Wuhan, China. Additionally, BAX (#ab7977), Bcl-2 (#ab32124), and beta-actin (#ab3280) were purchased from Abcam, Cambridge Biomedical Campus in Cambridge, UK, while cytochrome c (#sc-7159) was sourced from Santa Cruz Biotechnology in Dallas, TX, USA. Next, the membrane was incubated with secondary antibodies, specifically HRP-conjugated goat anti-rabbit IgG (dilution 1:2,000, #111035003, Jackson Immuno Research Inc., West Grove, PA, USA) and HRP-conjugated horse anti-mouse IgG (dilution 1:3,000, #7076P2, Cell Signaling Technology).

CBD-induced CCA in xenograft mouse modelExperimental animals

Female nude mouse (BALB/CAJcl-Nu/Nu) aged 6–7 weeks weighing 22–25 g at the beginning of the experiment were obtained from Nomura Siam International, Bangkok, Thailand. The mouse was housed in individual ventilated cages under specific pathogen-free conditions at a room temperature of 23 ± 2 °C and a 12 h light and 12 h dark cycle with a light intensity of 350–400 lx. During the experimental period, the mouse had ad libitum access to water enriched with choline at a concentration of 3 to 4 ppm and their dietary requirements were met with commercially available pellets. This standardized housing and diet was designed to ensure the welfare of the mouse and provide consistent environmental conditions to minimize potential sources of variability in the experimental results. We made deliberate efforts to reduce animal utilization and alleviate any pain or discomfort.

Tumor xenograft and CBD treatment in a mouse model

After a 7-day acclimatization period, 16 mice were injected subcutaneously with KKU-213BGemR cells (1 × 106 cells in 50 µM serum-free medium mixed with Matrigel from Corning, Tewksbury, MA, USA) into the right axilla. The subsequent determination of the tumor volume was performed with a digital caliper and was calculated according to the following formula: Tumor volume = (length × width2)/2. When a tumor volume of 70 mm3 was reached, the animals were randomly assigned to one of three groups: Sesame oil (vehicle control) (#S3547, Sigma-Aldrich, Saint Louis, MO, USA) (n = 4), CBD 10 mg/kg·Bw (n = 6), or CBD 40 mg/kg·Bw (n = 6). Equal amounts of CBD or sesame oil were administered by oral gavage every other day for a period of 9 days. The tumor suppression rate (TSR %) was then calculated using a formula previously established in the study by Liu et al. (2012) [29].

Sample collection

At the end of the treatment period, the animals were sacrificed directly. Prior to sacrifice, the mouse was rendered unconscious in a plastic chamber containing 1% isoflurane (Attane, Minrad, NY, USA). Blood was then taken directly from the animals’ hearts. To ensure effective killing, the heart poles of the mouse was cut with scissors. Following this procedure, various tissues were removed from the mouse, including liver, kidney, intestine, tumor mass, feces and urine. The removed organs and tissues were weighed and immediately fixed in 10% formalin. The tumor masses were carefully arranged, photographed and safely stored for subsequent histological examination.

Immunohistochemistry study

Antigens were retrieved from tissue sections using citrate buffer followed by blocking with 5% FBS. Then tissues were incubated overnight with primary antibodies, proliferating cell nuclear antigen (PCNA) (diluted 1:750, #Ab2426, Abcam) and CHOP (diluted 1:30, #A0221, ABclonal). The resulting signal was visualized using diaminobenzidine substrate and counterstained with Mayer’s hematoxylin. Positive cells were identified by the presence of brown staining. Image analysis was performed on ten fields, each at 200× magnification, using ImageJ software for precise quantification and evaluation.

Statistical analysis

Data are expressed as mean ± SD. Student’s t-test was used to test for differences between experimental groups. Statistical comparisons involved one-way ANOVA followed by Tukey’s test for multiple group comparisons. A value of P < 0.05 was considered statistically significant. Nonlinear regression analysis was performed to calculate the IC50. All statistical analyses were performed using Graphpad Prism 9.0 for Mac (GraphPad Software, Inc., CA, USA).