• Cell Culture and Chemicals

The human breast cancer cell lines MCF-7, MDA-MB-361 and MDA-MB-231 were purchased from the American Type Culture Collection (ATCC). These cell lines were maintained in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% (v/v) fetal bovine serum, 2% (v/v) l-glutamine 200 mM and 1% (v/v) penicillin-streptomycin (5000 U/mL). Cells were maintained in a humidified incubator with 5% CO₂ at 37 °C. The monoacylglycerol lipase (MAGL) inhibitor, MJN110, was purchased from SIGMA and used at the concentration of 1 µM. N-acetyl cysteine (NAC) was purchased from SIGMA and used at 1 mM and 1–0.1 mM, respectively. Nat CBD and syn CBD were kindly provided by CBDepot (Teplice, Czech Republic). Reagents and solvents used in the synthesis were of reagent grade and used without further purification.

• Synthesis and Characterization of CBD Homologs: CBDB and CBDP

CBDB and CBDP were synthesized as reported in previous works [4,7]. Briefly, the synthetic procedure to obtain CBD involved a dropwise addition of a solution of (1S,4R)-1-methyl-4-(prop-1-en-2-yl) cycloex-2-enol and 5-butylbenzene-1,3-diol (76 mg, 0.50 mmol, 1 eq.) in 5 mL of dry drydichloromethane (DCM) to a solution of 5-butylbenzene-1,3-diol (83 mg, 0.50 mmol, 1eq.) and p-toluenesulfonic acid (9 mg, 0.05 mmol, 0.1 eq.) in DCM (5 mL) at −10 °C, under argon atmosphere. The mixture was stirred for 1 h and then quenched with saturated NaHCO₃ (10 mL). Extraction of the mixture with diethylether (2 × 10 mL) was followed by purification over silica gel (crude:silica gel ratio 1/200, eluent: cyclohexane:DCM 8/2). The chromatographic fractions were analyzed by HPLC-UV and HPLC-HRMS and those containing exclusively CBDB without impurities were collected to give 48 mg of a reddish oil (32% yield, purity > 99%). The same procedure was carried out for the synthesis of CBDP, but 5-heptylbenzene-1,3-diol was used in place of 5-butylbenzene-1,3-diol to obtain a linear heptyl side chain. CBDP was obtained with a 23% yield (76 mg) as a colorless oil (purity > 99%).

The purity of CBDB and CBDP was checked by HPLC-HRMS analysis using an Ultimate 3000 liquid chromatograph (Thermo Fisher Scientific, Grand Island, NY, USA), equipped with a vacuum degasser, a binary pump, a thermostated autosampler, and a thermostated column compartment. The chromatographic system was interfaced to a heated electrospray ionization source and a Q-Exactive Orbitrap mass spectrometer (HPLC-HRMS). The chromatographic separation was carried out on a Poroshell 120 SB-C18 (3.0 × 100 mm, 2.7 μm, Agilent, Milan, Italy). The same instrumental parameters used in previous works were applied to confirm the identity of the synthesized compounds [4,7]. In detail, an isocratic elution of 30% water with 0.1% formic acid (A) and 70% acetonitrile (ACN) with 0.1% of formic acid (B) was set for 10 min, then 95% B was pumped for 5 min and lastly, the column was re-equilibrated for 2 min with the initial conditions for a total run time of 17 min. The flow rate was maintained constant at 0.5 mL/min. 5 µL of 0.1 µg/mL solutions of CBDB and CBDP were separately injected into the analytical system. The parameters of the heated electrospray ionization source were set as follows: capillary temperature, 320 °C; vaporizer temperature, 280 °C; electrospray voltage, 4.2 kV for positive mode and 3.8 kV for negative mode; sheath gas, 55 arbitrary units; auxiliary gas, 30 arbitrary units; S lens RF level, 45. Analyses were acquired using the Xcalibur 3.0 software (Thermo Fisher Scientific, San Jose, CA, USA) in full scan data-dependent acquisition (FS-dd-MS²) in positive and negative mode at a resolving power of 70,000 FWHM at m/z 200. The parameters of the Orbitrap mass analyzer were as follows: scan range of m/z 250–400, AGC of 3e6, injection time 100 ms, and isolation window for the filtration of the precursor ions of m/z 0.7. Normalized collision energy (NCE) of 20 was used to fragment the precursor ions. Extracted ion chromatograms (EIC) of the [M + H]+ and [M − H]− molecular ions were derived from the total ion chromatogram with a 5-ppm mass tolerance.

• Western Immunoblot Analysis

Cells were lysed in a RIPA buffer (Cell Signaling) supplemented with protease inhibitors Cocktail (1×) and sodium fluoride (NaF, 16 µL/mL). Protein concentration was determined by the Bradford protein assay (BIO-RAD, Hercules, CA, USA). Samples were mixed 1:1 with Laemmli buffer (SIGMA, St. Louis, MO, USA), boiled for 5 min, 90 °C and 25 μg of proteins were separated onto Mini-PROTEAN^® TGX™ Precast Gels (BIO-RAD, Hercules, CA, USA). Electrophoresis was run at 200 V for 60 min (IEF Cell Protean System, BIO-RAD, Hercules, CA, USA) and consequently total protein bands were visualized by 2.5 min of UV exposure, for gel activation. The bands were then transferred to the Midi Nitrocellulose membrane Trans-Blot Turbo (BIO-RAD, Hercules, CA, USA). The membranes were blocked for 1 h in Blotto A (Santa Cruz, CA, USA) at room temperature and subsequently probed for 1 h by the appropriately diluted primary antibodies. After three washes with a solution containing 10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20 (TBST solution), blots were incubated with secondary antibody HRP-conjugated for 1 h at room temperature (1:2000 dilution). Blots were then developed using the Clarity Enhanced chemiluminescence (ECL) (BIO-RAD, Hercules, CA, USA). Primary antibodies (1:1000 dilution) were: from Santa Cruz CB1 (2F9) sc-293419, CB2 (3C7) sc-293188 and from cell signaling p38 MAPK (#8690). Secondary antibodies (HRP-conjugated) were from Bethyl Laboratories (1:5000 dilution) (mouse IgG-heavy and light chain antibody, A90-116P). Images shown in the paper are representative of three independent replicates. Densitometric quantification of the band intensity was normalized to p38 levels using ImageJ.

• Cell Viability Assay

Changes in viability after the various treatments were measured using the Thiazolyl blue tetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at a density of 1 × 10⁴/well and incubated at 37 °C in 5% CO₂. After overnight incubation, the medium was replaced with vehicle control or drug at different concentrations in DMEM and supplemented with 10% (v/v) fetal bovine serum, 2% (v/v) L-glutamine 200 mM and 1% (v/v) penicillin-streptomycin (5,000 U/mL). The cell lines were maintained in a humidified incubator with 5% CO₂ at 37 °C. After 24 h, upon completion of the drug treatments, the medium was removed and replaced with a serum-free medium containing 2 mg/mL MTT and incubated for 2 h at 37 °C. The MTT reagent was then removed and the formazan crystals were solubilized using dimethyl sulfoxide. The absorbance was read using the CLARIO star Plus microplate reader (570 nm). The absorbance of the vehicle control was subtracted and the percentage control was calculated as the absorbance of the treated cells/control cells × 100.

• Measurement of Reactive Oxygen Species (ROS)

To detect the changes in intracellular ROS levels, 2′,7′- dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich) staining was used (Hyeoncheol Kim et al.). DCFH-DA is a stable, fluorogenic and non-polar compound that can readily diffuse into the cells and get deacetylated by intracellular esterases to a non-fluorescent 2′,7′- dichlorodihydrofluorescein (DCFH) which is later oxidized by intracellular ROS into highly fluorescent 2′,7′-dichlorofluorescein (DCF). The intensity of fluorescence is proportional to intracellular ROS levels. MCF-7 cells were seeded at a density of 2 × 10⁵ cells per well in 24 well plates and were allowed to attach overnight. On the first day of treatment, the medium was replaced with the fresh ones containing the vehicle control, CBD homologs (10 µM), NAC (1 mM), PMA (1 µM) and MJN110 (1 µM) respectively. After 24 h, upon completion of the drug treatment, the spent medium was removed. The cells were washed once with fresh DMEM, and twice with 1X PBS and incubated with DCFH-DA in a final concentration of 10 µM for 30 min. Cells were rinsed with PBS and representative fluorescent images for each well using the green fluorescent protein (GFP) channel on an Evos m7000 fluorescence microscope were taken. After taking images, PBS was removed and a radioimmunoprecipitation assay (RIPA) buffer was added to each well. The collected cells were incubated at −80 °C for 20 min and then centrifuged at 21,130× g for 10 min at 4 °C. The collected supernatant was transferred to a black 96 well plate and the fluorescence intensity measured using the CLARIO star Plus microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. After fluorescence recording, 5 µL of supernatant were transferred to a clear 96 well plate containing 195 μL of 1× protein assay solution to measure the protein concentration using the BCA assay. The fluorescence intensity was normalized to the protein concentration.

• MitoTracker Staining

The determination of the mitochondrial membrane potential was performed with the Mitotracker assay. MCF-7 cells were seeded at a density of 2 × 10⁵ cells per well in a 24-well plate and were allowed to attach overnight, 37 °C. On the first day of treatment, the medium was replaced with vehicle control or CBD homologs (10 µM) in DMEM. After 24 h, upon completion of drug treatments, the spent medium was removed. The cells were then washed with fresh DMEM prior to incubating them with 75 nM of either MitoTracker™ Green FM or MitoTracker™ Red CMXRos in pre-warmed medium, without FBS, for 30 min, at 37 °C. Then the medium was removed, the cells were rinsed with PBS and representative fluorescent images for each well using respectively the GFP and the RFP channel on an Evos m7000 fluorescence microscope were taken.

• CellTiter-Glo 2.0 Cell Viability Assay

The quantification of the cellular ATP was performed using the CellTiter-Glo 2.0 cell viability assay. Cells were seeded in 96-well plates at a density of 1 × 10⁴ /well and incubated at 37 °C in 5% CO₂. After overnight incubation, the medium was replaced with either vehicle control or drug at different concentrations in DMEM and supplemented with 10% (v/v) fetal bovine serum, 2% (v/v) L-glutamine 200 mM and 1% (v/v) penicillin-streptomycin (5000 U/mL). The cell lines were maintained in a humidified incubator with 5% CO₂, at 37 °C. After 24 h, upon completion of the drug treatments, the CellTiter-Glo 2.0 reagent was added into each well at the equivalent volume of cell culture medium in the well. Then, the contents were mixed vigorously for 5 min to induce cell lysis, and the plate was incubated at room temperature for an additional 25 min to stabilize the luminescent signal. Afterward, the supernatants were transferred in technical replicates into the 96-well opaque white-walled plate and the luminescence was measured using the CLARIO star Plus microplate reader.

• Ultrastructural Analysis of the Cellular Samples

MCF-7 cells were seeded at a density of 5 × 10⁵ cells in Primo TC flasks 25 cm² and were allowed to attach overnight. Control and CBD homologs treated cells were grown to 80% confluency and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 2 h at 4 °C. The cells were washed three times, 5–10 min each, in ice-cold PBS buffer and then post-fixed in ice-cold 1% osmium tetroxide. After 1 h, the samples were washed three times in PBS and dehydrated in an acetone series for 15 min each with 25, 50, 75, 90 and 100% acetone. Three steps of infiltration in a mixture of resin/acetone (1/2, 1/1 and 2/1 ratios) were performed and finally, the specimens were embedded in 100% resin at 60 °C for 48 h. Ultrathin sections (70 nm thick) were cut with an Ultramicrotome. TEM images were recorded on a JEOL Jem1011 microscope operating at an accelerating voltage of 100 kV (Tokyo, Japan).

• Lysotracker Assay

The mitochondrial-lysosomal axis theory of aging postulates that oxidized material accumulates in lysosomes as cells age, which results in a decreased degradative capacity of lysosomes. This behavior was studied using Lysotracker staining. MCF-7 cells are seeded at a density of 2 × 10⁵ cells per well in a 24-well plate and are allowed to attach overnight, at 37 °C. On the first day of treatment, the medium was replaced with vehicle control, CBD homologs (10 µM), NAC (1 mM), PMA (1 µM) and MJN110 (1 µM) in DMEM. After 24 h, upon completion of the drug treatments, spent media was removed. The cells were washed once with fresh DMEM. Cells were incubated with HOECHST (0.5 mg/mL in PBS), for 5 min, at 37 °C. The Hoechst was removed and replaced with a pre-warmed medium, without FBS, containing 100 nM LysoTracker Red DND-99 (Cat. No. L-7528). Cells are incubated at 33 °C for 30 min with LysoTracker. Then the medium was removed, the cells were rinsed with PBS and representative fluorescent images for each well using the RFP channel on an Evos m7000 fluorescence microscope were taken.

• Endoplasmic Reticulum Staining

MCF-7 cells are seeded at a density of 2 × 10⁵ cells per well in a 24-well plate and are allowed to attach overnight, at 37 °C. On the first day of treatment, the medium was replaced with vehicle control, CBD homologs (10 µM), NAC (1 mM), PMA (1 µM) and MJN110 (1 µM) in DMEM. After 24 h, upon completion of the drug treatments, spent media was removed. The cells were washed once with fresh DMEM. Cells were stained with ER-Tracker™ Green (glibenclamide BODIPY^® FL), 1 µM in pre-warmed medium, without FBS for 15 min at 37 °C. Then the medium was removed, the cells were rinsed with PBS and representative fluorescent images for each well using the GFP channel on an Evos m7000 fluorescence microscope were taken.