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.
Highlights
Multiple trials have shown that immunoprecipitation high-performance liquid chromatography (IP-HPLC) can be used to rapidly determine multiple protein levels accurately (±5% standard deviation) and reproducibly
It was found that 4HR differentially influenced the expressions of many essential proteins, and 4HR adherence assays were performed to investigate interactions between 4HR and proteins. 4HR adhered to the surfaces of acrylamide beads in 50 mM Tris buffer and treated with protein extract of RAW 264.7 cells
The expressions of other growth factor-related proteins, including HGFα, insulin-like growth factor-1 (IGF1), IGFIIR, HER1, and HER2, like those of housekeeping proteins changed minimally, the expressions of many growth factors and related proteins tended to increase slightly (Fig. 2C1 and C2). These results indicate 4HR alters the expressions of growth factors required for the growth and regeneration of RAW 264.7 cells, but does so negatively for growth hormone (GH), growth hormone-releasing hormone (GHRH), platelet-derived growth factor-A (PDGF-A), FGF-1, and estrogen receptor β (ERβ), and positively for transforming growth factor-β1 (TGF-β1), TGF-β2, SMAD4, FGF-2, and Met
Summary
Multiple trials have shown that IP-HPLC can be used to rapidly determine multiple protein levels accurately (±5% standard deviation) and reproducibly. The human body is believed to be relatively tolerant to 4HR, which is being increasingly used as a food additive and antiseptic agent, and 4HR has been suggested to have anti-inflammatory[18], anticancer[19], and angiogenesis[20] effects. Its molecular interactions and signaling in cells are not well understood and its biochemical properties remain ambiguous. The present study was undertaken to investigate and compare the cellular effects of 4HR, and to elucidate the molecular mechanism responsible for the effect of 4HR in RAW 264.7 cells using IP-HPLC
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