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. 2019 Mar 4;9(1):3379.
doi: 10.1038/s41598-019-38946-4.

Effects of 4-Hexylresorcinol on Protein Expressions in RAW 264.7 Cells as Determined by Immunoprecipitation High Performance Liquid Chromatography

Min Keun Kim  1 , Cheol Soo Yoon  2 , Seong Gon Kim  1 , Young Wook Park  1 , Sang Shin Lee  2 , Suk Keun Lee  3
Affiliations

Effects of 4-Hexylresorcinol on Protein Expressions in RAW 264.7 Cells as Determined by Immunoprecipitation High Performance Liquid Chromatography

Min Keun Kim et al. Sci Rep. .

Abstract

4-Hexylresorcinol (4HR) is a small organic compound that is used as an additive antiseptic and antioxidant, but its molecular properties have not been clearly elucidated. The present study explored the cellular effects of 4HR on RAW 264.7 cells by immunoprecipitation high-performance liquid chromatography (IP-HPLC) using 216 antisera. 4HR-treated cells showed significant decreases in the expressions of proliferation-related proteins, cMyc/MAX/MAD network, p53/Rb/E2F and Wnt/β-catenin signalings, epigenetic modifications, and protein translation. Furthermore, 4HR suppressed the expressions of growth factors and proteins associated with RAS signaling, NFkB signaling, inflammation, and osteogenesis, but elevated the expressions of proteins associated with p53-mediated and FAS-mediated apoptosis, T-cell immunity, angiogenesis, antioxidant, and oncogenic signaling. In a 4HR adherence assay, TNFα, PKC, osteopontin, and GADD45 were strongly adherent to 4HR-coated beads, whereas IL-6, c-caspase 3, CDK4, and c-caspase 9 were not. Many 4HR adherent proteins were expressed at lower levels in 4HR treated RAW 264.7 cells than in non-treated controls, whereas 4HR non-adherent proteins were expressed at higher levels. These observations suggest 4HR affects the expressions of proteins in an adhesion-dependent manner and that its effects on proteins are characteristic and global in RAW 264.7 cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Expressions of proliferation-related proteins (A1 and A2), cMyc/MAX/MAD network proteins (B1 or B2), epigenetic modification proteins (C1 and C2), and protein translation-related proteins (D1 or D2) in 4HR-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs, (A1, B1, C1, and D1) show protein expressional changes in the same scale (%) with respect to culture time (8, 16, or 24 hours), whereas circular graphs (A2, B2, C2, and D2) show protein expressions after 16 hours of culture.
Figure 2
Figure 2
Expressions of p53/Rb/E2F signaling proteins (A1 and A2), Wnt/β-catenin signaling proteins (B1 or B2), growth factors (C1 and C2), and RAS signaling proteins (D1 or D2) in RAW 264.7 cells treated with 4HR, as determined by IP-HPLC. Line graphs, (A1, B1, C1, and D1) show protein expressional changes in the same scale (%) with respect to culture time (8, 16, or 24 hours), whereas circular graphs (A2, B2, C2, and D2) showed protein expression levels after 16 hours of culture.
Figure 3
Figure 3
Expressional changes in NFkB signaling proteins (A1 and A2), cell protection-related proteins (B1 or B2), and antioxidant-related proteins (C1 and C2) induced by 4HR in RAW 264.7 cells, as determined by IP-HPLC. Line graphs, (A1, B1, and C1) show protein expressional changes in the same scale (%) with respect to culture time (8, 16, or 24 hours), while circular graphs (A2, B2, and C2) showed protein expression levels after 16 hours of culture.
Figure 4
Figure 4
Expressions of downregulated (A1 and A2) and upregulated (B1 or B2) inflammatory proteins, p53-mediated (C1 and C2) and FAS-mediated (D1 or D2) apoptosis-related proteins by 4HR in RAW 264.7 cells, as determined by IP-HPLC. Line graphs, (A1, B1, C1, and D1) showed protein expressional changes in the same scale (%) with respect to culture time (8, 16, or 24 hours), whereas circular graphs (A2, B2, C2, and D2) showed protein expression levels after 16 hours of culture.
Figure 5
Figure 5
Changes in the expressions of angiogenesis-related proteins (A1 and A2), osteogenesis-related proteins (B1 or B2), and oncogenic proteins (C1 or C2) induced by 4HR in RAW 264.7 cells, as determined by IP-HPLC. Line graphs (A1, B1, and C1) show protein expressional changes in the same scale (%) with respect to culture time (8, 16, or 24 hours), whereas circular graphs (A2, B2, and C2) showed protein expression levels after 16 hours of culture.
Figure 6
Figure 6
4HR adherence assay results obtained by IP-HPLC. (A1 and B1), line graphs. (A2 and B2), rod graphs. Protein adherences to 4HR-coated acrylamide beads and to non-coated acrylamide beads (positive control) were compared. Line graphs were normalized versus negative control using non-coated beads with no protein application (100%). Rod graphs showing adherence and lack of adherence to 4HR as compared with positive control (100%) as determined by IP-HPLC. A: 4HR adherence to cellular proliferation, epigenetic modification, and protein translation-related proteins. B: 4HR adherence to growth factors, RAS signaling, and NFkB signaling proteins.
Figure 7
Figure 7
4HR adherence assay results obtained by IP-HPLC. (A1 and B1), line graphs. (A2 and B2), rod graphs. Protein adherences to 4HR-coated acrylamide beads and non-coated acrylamide beads were compared. Line graphs were normalized versus negative control using non-coated beads with no protein application (100%). Rod graphs showing adherence and lack of adherence to 4HR as compared with positive control (100%) as determined by IP-HPLC. A: 4HR adherence to inflammation and apoptosis-related proteins. B: 4HR adherence to angiogenesis, osteogenesis, oncogenesis, and cell protection-related proteins.
Figure 8
Figure 8
STAR plot representation of 4HR adherence assay results. Many proteins that bound to 4HR were downregulated when RAW 264.7 cells were treated with 4HR, and conversely most proteins that did not bind to 4HR were upregulated by 4HR. Blue line: protein expressional changes (%) in RAW 264.7 cells induced by treatment with 4HR for 16 hours. Red line: 4HR adherence (%).
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