# 1 CBD Products

2.2. Cannabinoid and Terpene Profiles of CBD Oils

The chemical profiles of CBD oils (V1-V6) were analyzed at three independent laboratories at the Aromatic Plant Research Center (APRC, Lehi, UT, USA), Botanacor (BTNCR, Denver, CO, USA), and dōTERRA (DTRR, Pleasant Groves, UT, USA). At all three laboratories, the cannabinoid and terpene profiles were analyzed with high-performance lipid chromatography (HPLC) and gas chromatography coupled with mass spectrometry (GC-MS) methods, respectively. Laboratory reports of cannabinoid and terpene profiles of all six CBD oils are provided in the Supplementary Materials.

2.3. GC-MS Analysis of Terpene Profiles

The terpene profiles of CBD oils were analyzed using a GC-MS method described previously [14]. In addition, GC-MS experimental parameters are listed on the laboratory reports of terpene profiles from APRC in the Supplementary Materials.

2.4. HPLC Analysis of Cannabinoid Profiles

The cannabinoid profiles of CBD oils were analyzed using an Agilent 1290 Infinity II HPLC with a diode array detector (Agilent, Santa Clara, CA, USA). Sample preparation consisted of dissolving 1.0 ml of CBD oil into 10.0 mL methanol. Approximately 5 µL of sample was injected into a Kinetex C-18 4.6 mm × 150 mm × 2.6 µm column. Each injection was repeated three times. The mobile phases comprised 0.1% trifluoroacetic acid for mobile phase A and acetonitrile for mobile phase B. The samples were run using a gradient; initial A: 100%, 0.5 mL/min to 4 min; and 4.10 min A: 100%, 1.0 mL/min to 10 min, A: 97% to 15 min, and A: 75% hold until 17 min; and 17.5 min A: 100%, 0.5 mL/min. Data was processed using OpenLab software. Identification and quantification of compounds in samples were compared to the standard using retention time and area of absorbance peak.

2.5. Cell Line and Culturing Condition

The SH-SY5Y human neuroblastoma cell line was obtained from the American Type Tissue Collection (cat. no. CRL2266, ATCC, Manassas, VA, USA). The SH-SY5Y cells were cultured in a 1:1 mixture of ATCC-formulated Eagle’s minimum essential medium (cat. no. 302003) and F12 medium (cat. no. 30-2004, ATCC) supplemented with 10% fetal bovine serum (cat. no. SH30088.03, GE Healthcare Life Sciences, Pittsburgh, PA, USA).

2.6. Treatment Condition

The SH-SY5Y cells were cultured to approximately 70% confluence prior to the replacement of the culturing medium. The new culturing medium contained isolated CBD or CBD oils at the desired final CBD concentrations. The claimed CBD concentrations were used for the dilution calculation. The incubation time in the new culturing medium was the same as the time after treatment. For the experiments using isolated CBD, the control groups were treated with culturing medium mixed with methanol at dilutions that matched those of the isolated CBD. For experiments using CBD oils, the control groups were treated with culturing medium alone.

2.7. Cell Proliferation and Cytotoxicity Assays

MTS and crystal violet assay kits were obtained from Promega (cat. no. G3581, Madison, WI, USA) and Fisher Scientific (cat. no. C581, Waltham, MA, USA) and performed according to manufacturers’ protocols. MTS signals were detected with a multimode microplate reader (Synergy 2, BioTek, Winooski, VT, USA). Cell density following crystal violet staining was examined with a standard cell culture microscope equipped with a digital camera. All MTS and crystal violet assays were performed in triplicate, and duplicate experiments were performed per treatment condition, producing six repeated measurements per treatment condition.

2.8. Preparation of Cell Lysates

Approximately 1 million SH-SY5Y cells were incubated with 60 µL of lysis buffer (Bicine/CHAPS, cat. no. 040-764, Protein Simple, Santa Clara, CA, USA) on ice for 10 min, sonicated four times for a duration of 5 s, and rotated for 2 h, at 4 °C. Following centrifugation at 12,000 rpm in an Eppendorf 5430R microfuge for 20 min at 4 °C, the supernatants were collected as cell lysates. The total protein concentration in cell lysates was determined with Bradford assays and adjusted to a final concentration of 0.3 µg/µL with separation gradients for charge-based immunoassays or 0.4 µg/µL with denaturing buffers for size-based immunoassays.

2.9. Antibodies and Biomarker Proteins

The primary and secondary antibodies are listed in Supplemental Table S1. The name and function of biomarker proteins are listed in Supplemental Table S2.

2.10. cIEF Immunoassays

Cell lysates were added to separation gradients that contained pI standards (Premix G2, pH 5 to 8 or pH 3 to 10, Protein Simple) and transferred into 384-well plates for cIEF immunoassays using the NanoPro 1000 system (Protein Simple). Approximately 400 nanoliters of cell lysate were loaded automatically into each capillary of the 96-capillary system. Following isoelectric focusing at 15 mW for 50 min, proteins were crosslinked to inner capillary walls using ultraviolet irradiation for 80 s. Primary and horseradish peroxidase-conjugated secondary antibodies were sequentially introduced into each capillary, with incubation times of 120 min and 60 min, respectively. After incubation with chemiluminescence detection reagents, the protein signal was detected with an average exposure time of 240 s. All cIEF immunoassays were performed in triplicate for each protein, and duplicate experiments were performed per treatment condition, producing six repeated measurements per protein. Hsp70 was used as a loading control for all cIEF immunoassays. In the NanoPro 1000 system, the loading control (Hsp70) and proteins of interest were detected with chemiluminescence in separate capillaries.

2.11. Capillary Western Immunoassays

Cell lysates were mixed with denaturing buffers (cat. no. PS-ST01EZ or PS-ST03EZ, Protein Simple) and denatured at 95 °C for 5 min and, then, transferred to assay plates containing either 12 to 230 kDa or 66 to 440 kDa Separation Modules (cat. no. SM-W004 or SM-W008, Protein Simple). Blocking reagents, wash buffers, primary antibodies, secondary antibodies, and chemiluminescence substrates were prepared and dispensed into the same assay plates. Assay plates were loaded into the Jess system (Protein Simple). Size-based protein separation and detection were performed automatically in the individual capillaries using the default protocols. Typically, the protein separation time was 30 min at 475 volts. Incubation with primary and secondary antibodies was sequential with an incubation time of 30 min per antibody. The Jess system is capable of simultaneous near infrared fluorescence and chemiluminescence detection of loading controls and proteins of interest, respectively, within the same capillaries. HSP60 and β-actin were used as loading controls. All capillary Western immunoassays were performed in triplicate for each protein, and duplicate experiments were performed per treatment condition, producing six repeated measurements per protein.

2.12. Data Analysis

Assignment of pI values to protein phosphoisoforms was based on the literature and our own data [15,16,17,18,19,20,21,22,23,24,25,26,27]. Quantitation of protein expression and phosphorylation levels were performed using Compass software from Protein Simple. The protein expression or phosphorylation levels were adjusted with loading controls. The expression level of phosphorylation at a specific residue in capillary Western immunoassays was further adjusted with the expression level of total protein.

2.13. Statistical Analysis

Quantitative data were presented as mean values ± standard deviations across six repeated measurements. Statistical significance was calculated with a Student’s t-test and thresholding at p ≤ 0.05 versus control.

3.2. Cytotoxicity of Isolated CBD to Cultured Neuronal Cells

Next, we evaluated the effects of isolated CBD and all six CBD oils on the viability of SH-SY5Y cells in culture. The SH-SY5Y cells were cultured to approximately 70% confluence and, then, treated with titrating concentrations of isolated CBD or CBD oils for 24 h. Cell viability was assessed with a colorimetric MTS assay that measured cell metabolic activity. We found that isolated CBD was highly cytotoxic to SH-SY5Y cells, with an effective EC₅₀ concentration of approximately 12.5 µg/mL or 40 µM (Figure 2A). In contrast, treatment with the CBD oils at CBD concentrations up to 400 µg/mL or 1.27 mM had no effect on the viability of SH-SY5Y cells. The MTS data were further corroborated by a crystal violet assay, which stained remaining viable and adherent cells with a histological dye. Observation with brightfield microscopy revealed that isolated CBD dramatically reduced the density of SH-SY5Y cells compared with that of control cells (Figure 2B). At the same CBD concentration of 100 µg/mL or 318 µM, CBD oils had no effect on the density of SH-SY5Y cells as compared with that of the control cells. Therefore, CBD in the CBD oils did not have the same level of cytotoxicity as isolated CBD.

 

Figure 2

Toxicity of isolated CBD on neuronal cells. (A) Viability of SH-SY5Y cells as a function of treatment with isolated CBD and CBD oils. Error bars were standard deviations across 6 repeated measurements per experimental condition. Cell viability was measured with MTS assays; (B) density of SH-SY5Y cells as a function of treatment with a CBD isolate and CBD oils. SH-SY5Y cells were stained with crystal violet dye and examined with a standard brightfield cell culture microscope. The image field of view has a xy dimension of 1500 µm x 1125 µm.

3.3. Downregulation of the PI3K/Akt/mTOR Signaling Pathway

We further evaluated the effects of isolated CBD and CBD oils on the signaling activity of the pI3K/Akt/mTOR pathway, which is critical for the regulation of neuronal cell growth, longevity, and energy metabolism [28,29]. The expression levels of the phosphoisoforms of Akt, mTOR, and p70S6K were used to monitor the activity of the PI3K/Akt/mTOR signaling pathway. To minimize cytotoxicity, SH-SY5Y cells were treated with 6.25 µg/mL isolated CBD or 100 µg/mL CBD oils for 24 h prior to the collection of cell lysates for analysis. Akt phosphoisoforms were detected with cIEF immunoassays using primary antibodies against Akt1 isoforms, Akt2 isoforms, Akt3 isoforms, pan-Akt, or all Akt isoforms. Peak assignment of Akt isoforms on the electropherograms was based on the experimental data herein (Figure 3A), as well as on previously published data from multiple independent research groups [15,17]. In SH-SY5Y cells, the Akt3 isoforms were the dominant isoforms, accounting for 50%, followed by Akt1 isoforms at 40% and Akt2 isoforms at 10% (Figure 3A). Treatment of SH-SY5Y cells with isolated CBD and CBD oils generally reduced the expression of Akt phosphoisoforms, with the strongest suppressive effects induced by CBD oil V1 (Figure 3B and Supplemental Figures S1 and S2). Treatment of SH-SY5Y cells with CBD oil V1 strongly reduced the phosphorylation of mTOR at residue Ser2448 and of p70S6K at residue Thr389, which occur downstream of Akt, as detected with capillary Western immunoassays (Figure 3C). In contrast, isolated CBD and other CBD oils had either undetectable or weak suppressive effects on mTOR and p70S6K phosphorylation. Fold changes of protein phosphoisoforms after treatment, as compared with the control, were calculated to the yield the relative concentrations of individual signaling proteins. Quantitative analysis data further revealed strong, consistent, and statistically significant effects of CBD oil V1 on the reduction in the protein phosphoisoforms in the PI3K/Akt/mTOR signaling cascade (Figure 3D).

 

Figure 3

Downregulation of the PI3K/Akt/mTOR signaling pathway by isolated CBD and CBD oils. (A) AKT isoform identification in SH-SY5Y cell lysates using capillary isoelectric focusing (cIEF) immunoassays. Antibodies against total Akt (pan-Akt, blue), Akt1 (green), Akt2 (gray), or Akt3 (pink) were used to identify Akt isoforms; (B) the total Akt profiles of SH-SY5Y cells before (C, blue) and after 24 h of treatment with isolated CBD (green) or with various CBD oil samples, V1 (pink), V2 (gray), V3 (deep red), V4 (dark blue), V5 (red), and V6 (dark yellow); (C) capillary Western immunoassays using antibodies against mTOR, p-mTOR (Ser2448), p70S6K, p-p70S6K (Thr389), and β-actin (loading control). Values on the left side of the panels are molecular weights in kilodaltons (kDa); (D) the relative expression levels of the phosphoisoforms of Akt1 (p-Akt1, light blue), Akt2 (p-Akt2, orange), Akt3 (p-Akt3, gray), mTOR (p-mTOR (Ser2448), yellow), and p70S6K (p-p70S6K (Thr389), blue) before (control) or after 24 h of treatment with isolated CBD or CBD oil samples V1-V6. Relative concentration describes fold change of protein phosphoisoforms after treatment as compared with the control. Error bars are the standard deviations of six repeated measurements per experimental condition. Asterisks denote statistical significance for p < 0.05 versus controls. SH-SY5Y cells were treated with 6.25 µg/mL isolated CBD or CBD oils at 100 µg/mL final CBD concentration.

3.4. Dose- and Time-dependent Suppression of the PI3K/Akt/mTOR Signaling Pathway

CBD oil V1 exhibited both dose- and time-dependent suppression of the PI3K/Akt/mTOR signaling pathway. Following treatment of SH-SY5Y cells with serial dilutions of CBD oil V1 for 24 h, we observed the suppression of phosphoisoforms of Akt1, Akt2, Akt3, mTOR, and p70S6K with an effective CBD concentration (EC₅₀) of approximately 45 µg/mL or 143 µM (Figure 4A–C and Supplemental Figure S3). However, time-dependent measurements following the treatment of SH-SY5Y cells with 100 µg/mL CBD oil V1 revealed a slow-acting mechanism, in which 4 h were required to reach 50% suppression and 24 h to reach maximal suppression (Figure 5A–C and Supplemental Figure S4). Therefore, CBD oil V1 exerted negative regulatory control of the PI3K/Akt/mTOR signaling pathway.

 

Figure 4

Dose-dependent negative regulation of the pI3K/Akt/mTOR signaling pathway by a CBD oil. (A) The pan-Akt profiles of SH-SY5Y cells as a function of CBD oil V1 dosages; (B) the expression levels of mTOR and p70S6K phosphoisoforms as a function of CBD oil V1 dosages; (C) the relative concentrations of Akt, mTOR, and p70S6K phosphoisoforms as a function of CBD oil V1 dosages. Relative concentration describes fold change of protein phosphoisoforms after treatment as compared with the control. Error bars are the standard deviations of six repeated measurements per experimental condition. SH-SY5Y cells were treated with various dosages of CBD oil V1 for 24 h.

 

Figure 5

Time-dependent negative regulation of the pI3K/Akt/mTOR signaling pathway by a CBD oil. (A) The pan-Akt profiles of SH-SY5Y cells as a function of time after treatment with CBD oil V1; (B) the expression levels of mTOR and p70S6K phosphoisoforms as a function of time after treatment with CBD oil V1; (C) the relative concentrations of Akt, mTOR, and p70S6K phosphoisoforms as a function of time after treatment with CBD oil V1. Relative concentration describes fold change of protein phosphoisoforms after treatment as compared with the control. Error bars are the standard deviations of six repeated measurements per experimental condition. SH-SY5Y cells were treated with CBD oil V1 at 100 µg/mL final CBD concentration.

The authors thank Russel J. Osguthorpe and Nicole Stevens (dōTERRA International) and Chikao Morimoto (University of Tokyo) for stimulating discussions and Alec Anderson (Aromatic Plant Research Center) for help on HPLC and GC-MS data analysis of CBD oils.