Cell culture

HTR-8/SVneo cell line (ATCC, Manassas, VA, USA) was maintained at 37 °C in a 5% CO2 humidified atmosphere with RPMI 1640 medium (Gibco/Invitrogen Corporation, Carlsbad, CA, USA). The cell culture medium was enriched with 10% fetal bovine serum (FBS) (PAN-Biotech, Aidenbach, Germany), 1% antibiotic–antimycotic solution (penicillin G, streptomycin, and amphotericin B, AB-AM) (PAN-Biotech, Aidenbach, Germany), 1.25% glucose (Gibco/Invitrogen Corporation, Carlsbad, CA, USA) and 1% sodium pyruvate (PAN-Biotech, Aidenbach, Germany). After adhesion, cells were exposed to different concentrations (1–10 µM) of CBDV and CBG (THC Pharm GmbH, Frankfurt, Germany) pre-diluted in cell culture medium with (1%) or without FBS for different time incubations (6, 24, or 48 h). The CBDV and CBG stock solutions, with a purity of 98.9% and 99.8%, respectively, were prepared in 100% dimethyl sulfoxide (DMSO, Sigma-Aldrich Co, Saint Louis, MO, USA) and stored at −20 °C. The final concentration of DMSO in the cell culture medium was below 0.05% for all experiments. DMSO at 0.05% was used in the control and per se had no impact on the viability of the HTR-8/SVneo cells.

Cell viability assays

HTR-8/SVneo cells were seeded in 96-well plates at a cellular density of 5 × 103 cells/well and exposed to CBDV and CBG (1–10 µM) for 24 and 48 h. After the incubation time, MTT (M5655, Sigma-Aldrich Co, Saint Louis, MO, USA) was added at a final concentration of 0.5 mg/mL and incubated for 3 h at 37 °C. The resulting purple formazan was subsequently dissolved in a solution of DMSO and isopropanol (3:1). Absorbance was measured at 540 nm, using a Biotek Synergy HTX Multi-Mode Microplate Reader (Biotek Instruments, Vermont, USA).

The activity of the cytoplasmic enzyme lactate dehydrogenase (LDH), released into the culture medium in case of cell necrosis, was evaluated using the CytoTox 96® non-radioactive cytotoxicity assay kit (G1781, Promega, Madison, WI, USA), following the manufacturer's provided guidelines.

Cell cycle analysis

HTR-8/SVneo cells were seeded in 6-well plates (4 × 105 cells/well) and exposed to CBDV and CBG (5 μM) for 48 h. As previously described (Alves et al. 2021), after this, cells were collected and fixed with cold ethanol 70%. Then, cells were treated with a DNA staining solution containing 5 μg/mL propidium iodide (PI) (P-4170, Sigma-Aldrich Co., Saint Louis, USA), 0.1% Triton X-100 (Sigma-Aldrich Co., Saint Louis, USA) and 200 μg/mL Dnase-free Rnase A (GE011.0100, GriSP Research Solutions, Porto, Portugal) diluted in PBS. DNA content was measured through flow cytometry in a BD Accuri™ C6 cytometer (San Jose, CA, USA). The cytometer, equipped with BD Accuri™ C6 software, featured detectors for three fluorescence channels (FL-1, FL-2, FL-3) and for forward (FSC) and side (SSC) light scatter channels set on a linear scale. Data was collected from 40 000 events/cells, and gates were applied to exclude debris, cell doublets, and aggregates. Singlet cells were analyzed using a two-parameter plot of FL-2-Area to FL-2-Width for PI fluorescence. The acquired data were processed using BD Accuri™ C6 software. Results are presented as percentage of total cells in the G0/G1, S and G2/M cell cycle phases.

RT‑PCR analysis

HTR-8/SVneo cells were seeded in 6-well plates (4 × 105 cells/well) and treated with CBDV and CBG at 5 µM, with or without the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (CPZ, 0.2 µM, 0464, Tocris Bioscience, Bristol, UK), the IRE1 inhibitor 4µ8c (1 µM, SML0949, Sigma-Aldrich Co, Saint Louis, MO, USA) or the selective inhibitor of PERK, GSK 2656157 (GSK, 0.5 µM, sc-490341, Santa Cruz Biotechnology, CA, USA), which were added 30 min before the co-incubation with cannabinoids. The ER stress inducer thapsigargin (TG, 0.1 µM, sc-24017, Santa Cruz Biotechnology, CA, USA) was used as positive control. After 48 h, cells were collected in TripleXtractor (GB23.0200, GRiSP, Porto, Portugal) and RNA was extracted, according to manufacturer’s instructions. RNA quantification was performed in the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc, Wilmington, DE, USA). cDNA was obtained through reverse transcription of RNA with the Xpert cDNA Synthesis Supermix (GK86.0100, GRiSP, Porto, Portugal). Amplification was achieved using Xpert Fast SYBR (GE20.2501, GRiSP, Porto, Portugal) and specific primers in the Applied Biosystems StepOnePlus™ Real-Time PCR system. Table 1 displays the primer sequences and qRT-PCR conditions. Gene expression was normalized using two housekeeping genes, ACTB (β-actin) and TUBA1A (α-tubulin) and analysis was carried out using 2−ΔΔCt method, with ACTB as reference gene. Results are presented as fold change in gene expression, in comparison with the control.

Table 1 Primer sequences and RT-PCR conditions used to assess the gene expression of HSPA5, DDIT3, ATF4, sXBP1, VEGFA, PGF, FLT1 and sFLT1. ACTB and TUBA1A were used as housekeeping controlsWestern blotting

Like previously described (Alves et al. 2021), HTR-8/SVneo cells (4 × 105/well) were seeded in 6-well plates and treated with CBDV and CBG (5 µM), with or without CPZ (0.2 µM), for 48 h. Protein samples (25 µg for most proteins and 50 µg for CHOP and tribbles-related protein 3, TRB3) were separated by 10 or 12% SDS–PAGE and transferred onto nitrocellulose membranes. Membranes were incubated with mouse monoclonal antibodies against CHOP (1:100, sc-7351; Santa Cruz Biotechnology, CA, USA) and TRB3 (1:100, sc-271572; Santa Cruz Biotechnology, CA, USA), or rabbit monoclonal or polyclonal antibodies against p-eIF2α (1:200, 3398S; Cell Signaling Technology, Leiden, The Netherlands), eIF2α (1:200, 5342S; Cell Signaling Technology, Leiden, The Netherlands), caspase-3 (1:200, sc-7148; Santa Cruz Biotechnology, CA, USA), poly (ADP-ribose) polymerase-1 (PARP-1) (1:200, 9542S; Cell Signaling Technology, Leiden, The Netherlands), p-AKT (Ser473) (1:200, 4060S; Cell Signaling Technology, Leiden, The Netherlands) and AKT (1:200, 4691S; Cell Signaling Technology, Leiden, The Netherlands) at 4 °C overnight. Then, membranes were washed and incubated with peroxidase-conjugated secondary antibody anti-rabbit or anti-mouse (1:1000 or 1:2000; Thermo Fisher, Waltham, MA, USA). Immunoreactive bands were visualized using a chemiluminescent substrate WesternBright™ ECL HRP substrate (K-12045-D20, Advansta, Menlo Park, USA) and a ChemiDoc™ Touch Imaging System (Bio-Rad, Laboratories Melville, NY, USA). Stripping was performed and the membranes were incubated with mouse monoclonal antibody against β-actin (1:500, sc-47778; Santa Cruz Biotechnology, CA, USA), used as loading control.

Evaluation of mitochondrial transmembrane potential (Δψm) and intracellular reactive oxygen species (ROS) production

HTR-8/SVneo cells were seeded in 96-well black plates (5 × 103 cells/well) and treated with CBDV and CBG (5 µM), with or without CPZ (0.2 µM), N-acetylcysteine (NAC, 1 mM, A0150000, Sigma-Aldrich Co, Saint Louis, MO, USA), 4µ8c (1 µM) or GSK (0.5 µM). Mitochondrial transmembrane potential (Δψm) was evaluated after 48 h of exposure, as previously described, using the fluorescent probe 3,3′-dihexyloxacarbocyanine iodide (DiOC6, 100 nM, D273, Thermo Fisher, Waltham, MA, USA) (Alves et al. 2021). The mitochondrial transmembrane depolarizing agent carbonyl cyanide m-chlorophenylhydrazone (CCCP, 30 µM, C2759, Sigma-Aldrich Co, Saint Louis, MO, USA) was used as a positive control. The generation of intracellular reactive oxygen species (ROS) was assessed by incubation with 2ʹ,7ʹ-dichlorodihydrofuoresceindiacetate (DCDHF-DA, 25 µM, D6883, Sigma-Aldrich Co, Saint Louis, MO, USA) for 1 h, as previously reported (Alves et al. 2021). The emitted fluorescence was measured at different time periods (0, 24 and 48 h). For positive control, cells were incubated with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, 79,346, Sigma-Aldrich Co, Saint Louis, MO, USA). Biotek Synergy HTX Multi-Mode Microplate Reader (Biotek Instruments, Vermont, USA) was used to measure fluorescence and the results were presented as relative fluorescence units (RFU).

Determination of caspases‑3/‑7, -8 and ‑9 activities

HTR-8/SVneo cells were seeded in 96-well white plates (5 × 103 cells/well) and incubated with CBDV and CBG at 5 μM for 48 h. In addition, CPZ (0.2 µM), NAC (1 mM), 4µ8c (1 µM) or GSK (0.5 µM) were also added to cells treated with or without cannabinoids. The activities of caspases-3/-7, -8 and -9 were assessed through a luminescence assay, using Caspase-Glo® 3/7 (G811C), Caspase-Glo® 8 (G815C) and Caspase-Glo® 9 (G816C) kits (Promega, Madison, WI, USA), according to manufacturer’s instructions. The resultant luminescence was measured in a Biotek Synergy HTX Multi-Mode Microplate Reader (Biotek Instruments, Vermont, USA). Staurosporine (STS, 10 μM, S4400, Sigma-Aldrich Co, Saint Louis, MO, USA), an inducer of apoptosis, was used as positive control. Results are expressed in relative luminescence units (RLU).

Tube formation assay

The impact of CBDV and CBG in the tube formation of HTR-8/SVneo cells was evaluated at 2 μM. For this, growth factor reduced Matrigel (50 μL, 3,56,230, Corning, NY, USA) was added to 96-well plates and incubated for 30 min at 37 °C to solidify, as described previously (Maia et al. 2022). After this step, cells were seeded (1.5 × 104 cells/well) and incubated with the compounds, in RPMI 1640 FBS-free medium, for 6 h. Images from three random different fields/well were acquired (× 100 magnification) under a phase contrast microscope (Eclipse 400, Nikon, Japan), using Nikon NIS Elements Software. Analysis was carried out using the Angiogenesis Analyzer plugin for Image J (Carpentier et al. 2020) and the results presented as total segment length (µm), in comparison with the control.

Statistical analysis

Statistical analysis was carried out by ANOVA, followed by Bonferroni post hoc-test to make pairwise comparisons of individual means (GraphPad PRISM v.8.0, GraphPad Software, Inc., San Diego, CA, USA). At least three independent experiments were performed in triplicate. Data are expressed as the mean ± SEM and differences were statistically significant at p < 0.05.