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. 2021 Feb 2;22(3):1501.
doi: 10.3390/ijms22031501.

The Effect of Cannabidiol on UV-Induced Changes in Intracellular Signaling of 3D-Cultured Skin Keratinocytes

Agnieszka Gęgotek  1 , Sinemyiz Atalay  1 , Adelina Rogowska-Wrzesińska  2 , Elżbieta Skrzydlewska  1
Affiliations

The Effect of Cannabidiol on UV-Induced Changes in Intracellular Signaling of 3D-Cultured Skin Keratinocytes

Agnieszka Gęgotek et al. Int J Mol Sci. .

Abstract

Human epidermal keratinocytes are constantly exposed to UV radiation. As a result, there is a significant need for safe and effective compounds to protect skin cells against this environmental damage. This study aimed to analyze the effect of phytocannabinoid-cannabinoid (CBD)-on the proteome of UVA/B irradiated keratinocytes. The keratinocytes were cultured in a three-dimensional (3D) system, designed to mimic epidermal conditions closely. The obtained results indicate that CBD protected against the harmful effects of UVA/B radiation. CBD decreased the expression of proinflammatory proteins, including TNFα/NFκB and IκBKB complex and decreased the expression of proteins involved in de novo protein biosynthesis, which are increased in UVA/B-irradiated cells. Additionally, CBD enhanced the UV-induced expression of 20S proteasome subunits. CBD also protected protein structures from 4-hydroxynonenal (HNE)-binding induced by UV radiation, which primarily affects antioxidant enzymes. CBD-through its antioxidant/anti-inflammatory activity and regulation of protein biosynthesis and degradation-protects skin cells against UVA/B-induced changes. In the future, its long-term use in epidermal cells should be investigated.

Keywords: UV irradiated skin; cannabidiol; keratinocytes; lipid peroxidation products adducts formation; proteomics; three-dimensional in vitro culture.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structure of cannabidiol (CBD).
Figure 2
Figure 2
Venn diagram showing protein distribution in the keratinocytes cultured in a three-dimensional culture model and treated with cannabidiol (4 μM) following UVA (30 J/cm2) (A) or UVB (60 mJ/cm2) (B) radiation. Data analyzed using RStudio (R version 3.6.2). The names and abundance of proteins are shown in Supplementary Table S1. Abbreviations: Ctr, control; CBD, cannabidiol.
Figure 3
Figure 3
Principal component analysis (PCA) of keratinocytes cultured in a three-dimensional culture model and treated with cannabidiol (4 μM) following UVA (30 J/cm2) (A) or UVB (60 mJ/cm2) (B) radiation, as well as a hierarchical dendrogram (C) of these samples. Abbreviations: Ctr, control; CBD, cannabidiol; PC, principal component.
Figure 4
Figure 4
The point graph and heatmap showing the average intensity of proteins creating TNFα/NFκB (A) and IκBKB/HSP90 (B) complexes in keratinocytes following UVA (30 J/cm2) or UVB (60 mJ/cm2) radiation and treated with cannabidiol (CBD, 4 μM) in a three-dimensional culture model. Proteins IDs: O14920, inhibitor of nuclear factor κ-B kinase subunit B; P01375, tumor necrosis factor α; P07900, heat shock protein HSP 90A; P08238, heat shock protein HSP 90B; P19838, nuclear factor NFκB p105; P62888, 60S ribosomal protein L30; Q02878, 60S ribosomal protein L6; Q8N163, cell cycle and apoptosis regulator protein 2. Statistical significances showed only for the whole complexes. X: statistically significant differences vs. non-treated cells, p < 0.05; y: statistically significant differences vs. CBD-treated cells, p < 0.05; a: statistically significant differences vs. UVA-irradiated cells, p < 0.05; b: statistically significant differences vs. UVB-irradiated cells, p < 0.05.
Figure 5
Figure 5
The point graph and heatmap showing the average intensity of proteins creating multisynthetase (A) and ribosome (B) complexes in keratinocytes following UVA (30 J/cm2) or UVB (60 mJ/cm2) radiation and treated with cannabidiol (CBD, 4 μM) in a three-dimensional culture model. Proteins IDs: P07814, glutamate/proline-tRNA ligase; P14868, aspartate-tRNA ligase; P41252, isoleucine-tRNA ligase; P47897, glutamine-tRNA ligase; P54136, arginine-tRNA ligase; P56192, methionine-tRNA ligase; Q15046, lysine-tRNA ligase; Q9P2J5, leucine-tRNA ligase; P62081, P46781, P25398, P60866, 40S ribosomal proteins S7, S9, S12, S20; P39023, P27635, P30050, P35268, P46779, 60S ribosomal protein L3, L10, L12, L22, L28. Statistical significances showed only for the whole complexes. x: statistically significant differences vs. non-treated cells, p < 0.05; y: statistically significant differences vs. CBD-treated cells, p < 0.05; a: statistically significant differences vs. UVA-irradiated cells, p < 0.05; b: statistically significant differences vs. UVB-irradiated cells, p < 0.05.
Figure 6
Figure 6
The point graph and heatmap showing the average intensity of proteins creating 20S proteasome complex in keratinocytes following UVA (30 J/cm2) or UVB (60 mJ/cm2) radiation and treated with cannabidiol (CBD, 4 μM) in a three-dimensional culture model. Proteins IDs: P25786, P25788, P25789, P60900, proteasome subunit α type-1, -3, -4, -6; P20618, P49721, P49720, P28070, P28074, proteasome subunit β type-1, -2, -3, -4, -5. Statistical significances showed only for the whole complexes. x: statistically significant differences vs. non-treated cells, p < 0.05; y: statistically significant differences vs. CBD-treated cells, p < 0.05; a: statistically significant differences vs. UVA-irradiated cells, p < 0.05; b: statistically significant differences vs. UVB-irradiated cells, p < 0.05.
Figure 7
Figure 7
The level of 4-hydroxynonenal (4-HNE)-protein adducts in keratinocytes following UVA (30 J/cm2) or UVB (60 mJ/cm2) radiation and treated with cannabidiol (CBD, 4 μM) in a three-dimensional culture model. x: statistically significant differences vs. non-treated cells, p < 0.05; y: statistically significant differences vs. CBD-treated cells, p < 0.05; a: statistically significant differences vs. UVA-irradiated cells, p < 0.05; b: statistically significant differences vs. UVB-irradiated cells, p < 0.05.
Figure 8
Figure 8
The biological function of proteins forming adducts with 4-hydroxynonenal (4-HNE) in keratinocytes treated in a three-dimensional culture model with cannabidiol (CBD, 4 μM) (A) and following UVA (30 J/cm2) or UVB (60 mJ/cm2) (B) radiation. The size of the stamps indicates the statistically significant differences between the estimated amount of the 4-HNE-protein adducts in the samples non-treated or treated with CBD.
Figure 9
Figure 9
SDS–PAGE separation and staining with Coomassie brilliant blue R-250 of proteins from control keratinocytes and irradiated with UVA (30 J/cm2), UVB (60 mJ/cm2) or/and treated with cannabidiol (CBD, 4 μM) in a three-dimensional (3D) culture model. The grid indicates the borders of the protein migration zones. Original photo of the gel is added in Supplementary Figure S1.
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