4-hexylresorcinol-induced protein expression changes in human umbilical cord vein endothelial cells as determined by immunoprecipitation high-performance liquid chromatography

Abstract

4-Hexylresorcinol (4HR) is used as a food preservative and an ingredient of toothpaste and cosmetics. The present study was performed using 233 antisera to determine the changes in protein expression induced by 4HR in human umbilical cord vein endothelial cells (HUVECs), and evaluated the 4HR-induced effects in comparison with previous results (Kim et al., 2019). Similar to RAW 264.7 cells, 4HR-treated HUVECs showed decreases in the expression of the proliferation-related proteins, cMyc/MAX/MAD network proteins, p53/RB and Wnt/β-catenin signaling, and they showed inactivation of DNA transcription and protein translation compared to the untreated controls. 4HR upregulated growth factors (TGF-β1, β2, β3, SMAD2/3, SMAD4, HGF-α, Met, IGF-1) and RAS signaling proteins (RAF-B, p38, p-p38, p-ERK-1, and Rab-1), and induced stronger expression of the cellular protection-, survival-, and differentiation-related proteins in HUVECs than in RAW 264.7 cells. 4HR suppressed NFkB signaling in a manner that suggests potential anti-inflammatory and wound healing effects by reducing M1 macrophage polarization and increasing M2 macrophage polarization in both cells. 4HR-treated HUVECs tended to increase the ER stress mediators by upregulating eIF2AK3, ATF4, ATF6, lysozyme, and LC3 and downregulating eIF2α and GADD153 (CHOP), resulting in PARP-1/AIF-mediated apoptosis. These results indicate that 4HR has similar effects on the protein expression of HUVECs and RAW 264.7 cells, but their protein expression levels differ according to cell types. The 4HR-treated cells showed global protein expression characteristic of anticancer and wound healing effects, which could be alleviated simultaneously by other proteins exerting opposite functions. These results suggest that although 4HR has similar effects on the global protein expression of HUVECs and RAW 264.7 cells, the 4HR-induced molecular interferences in those cells are complex enough to produce variable protein expression, leading different cell functions. Moreover, HUVECs have stronger wound healing potential to overcome the impact induced by 4HR than RAW 264.7 cells.

4-Hexylresorcinol Inhibits Class I Histone Deacetylases in Human Umbilical Cord Endothelial Cells

“…In our previous study, 4HR increased the expression level of AIF in HUVECs [28,29]. In this study, TSA increased the expression level of AIF in HUVECs, too (Figure 3b).…”

“…HUVECs (Lonza, Walkersville, MD, USA) were cultured as previously described in our publications [28,29]. The medium was endothelial cell growth medium-2 (Clonetics, Lonza).…”

“…Antibodies for HDACs, TGF-β1, AIF, and Ac-lys were purchased from SantaCruz Biotechnology (Santa Cruz, CA, USA). The quantification of the proteins was done as described previously [28,29].…”

Increased Expression of TGF-β1 by 4-hexylresorcinol Is Mediated by Endoplasmic Reticulum and Mitochondrial Stress in Human Umbilical Endothelial Vein Cells

“…4HR exhibited a wide variety of functions, such as anti-cancer effects [21], pro-angiogenic effects [8,17], anti-inflammatory effects [30], and increased bone formation [31]. These divergent effects are likely to be involved in the broad-spectrum regulation of cellular events [24,32]. In this study, 4HR increased TGF-β1 expression via hyper-acetylated H3 and p-Sp1 (Figure 5).…”

“…HUVECs (Lonza, Walkersville, MD, USA) were cultured as described in our previous publications [15,24]. The medium was endothelial cell growth medium-2 (Clonetics ® , Lonza).…”