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

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.


Effects of 4HR on the expressions of cMyc/MAX/MAD network proteins in RAW 264.7 cells. 4HR
increased the expressions of cMyc and MAD by 7.1 and 7.2% at 16 hours, respectively, but reduced the expression of MAX by 5.6% at 16 hours versus non-treated controls. MAD expression increased by a maximum of 7.6% after 8 hours of 4HR treatment and this was maintained until 24 hours, and cMyc expression increased by a maximum of 7.1% at 16 hours but decreased to the control level at 24 hours ( Fig. 1B1 and B2). These expressional changes of the cMyc/MAX/MAD network co-occurred inhibition of proliferation by 4HR ( Fig. 1A1 and A2).

Effects of 4HR on the expressions of epigenetic modification-related proteins in RAW 264.7 cells.
Histone H1 and histone deacetylase 10 (HDAC10) expressions increased to 108.9% and 106% of those in non-treated controls after 8 hours of 4HR treatment, respectively, but then gradually decreased to 93.8% and 103% at 24 hours, respectively. The expression of lysine-specific demethylase 4D (KDM4D) was reduced by 6.4% at 24 hours, while that of DNA methyltransferase 1-associated protein 1 (DMAP1) and was gradually increased by 11.1% at 16 hours and maintained at 109.6% at 24 hours, DNA (cytosine-5)-methyltransferase 1 (DNMT1) expression was increased by 7.2% at 24 hours, and also methyl-CpG binding domain 4 (MBD4) expression was increased by 9.4% at 16 hours and by 5.2% at 24 hours ( Fig. 1C1 and C2). These results suggest 4HR might inactivate DNA transcription in RAW 264.7 cells, and that this epigenetic effect of 4HR might be related to the downregulations of proliferation-related proteins.

Effects of 4HR on the expressions of translation-related proteins in RAW 264.7 cells. RAW
264.7 cells treated with 4HR showed gradual reductions in protein translation-related protein levels versus non-treated controls. Deoxyhypusine hydroxylase (DOHH) expression reduced by 3.9% and 7.1% at 16 and 24 hours, respectively, and deoxyhypusine synthase (DHS) expression was reduced by 6.9% and 5.5% at 16 and 24 hours, respectively. The protein expressions of objective factors of protein translation, that is, eukaryotic translation initiation factor 5A-1 (eIF5A-1) and eIF5A-2 proteins were also reduced by 7.9% and 3.9% at 16 hours, respectively, while that of eukaryotic translation initiation factor 2-α kinase 3 (eIF2AK3; an inactivator of eIF2) was increased by 11% at 16 hours ( Fig. 1D1 and D2). It was thought that the rapid reduction of the expressions of translation-related proteins by 4HR might induce global inactivation of cellular signaling, although these changes in protein levels tended to disappear after 24 hours of 4HR treatment.

Effects of 4HR on the expressions of p53/Rb/E2F signaling proteins in RAW 264.7 cells. 4HR
increased the expression of p53 in RAW 264.7 cells by 16% at 16 hours and by 12% at 24 hours versus non-treated controls. Rb-1 expression was also slightly increased by 6.9% and 6.5% at 8 and 16 hours, respectively, but then decreased to the control level (1.3% increase) at 24 hours. Notably, p21 expression increased by 7.7% at 16 hours, whereas CDK4 expression gradually decreased by 5.3% and 4.5% at 16 and 24 hours, respectively. The expression of the objective transcription factor E2F-1 decreased by 6.4% at 16 hours and by 4.5% at 24 hours ( Fig. 2A1 and A2). We supposed these protein expression changes in p53/Rb/E2F signaling, increases in the expressions of p53, Rb-1, and p21, but concurrent decreases in the expressions of CDK4 and E2F could contribute to 4HR-induced reductions in proliferation. www.nature.com/scientificreports www.nature.com/scientificreports/ (FGF-1), and estrogen receptor β (ERβ) by 2.9-8.7% during 24 hours of treatment versus non-treated controls, while the expressions of transforming growth factor-β1 (TGF-β1), TGF-β2, SMAD4 (mothers against decapentaplegic homolog 4), FGF-2, and Met were increased by 5.5%, 11.6%, 3.5%, 5.9%, and 7.7%, respectively. The expressions of other growth factor-related proteins, including HGFα, insulin-like growth factor-1 (IGF-1), IGFIIR, HER1, and HER2, like those of housekeeping proteins changed minimally (by ±5%), however, 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 GH, GHRH, PDGF-A, FGF-1, and ERβ, and positively for TGF-β1, TGF-β2, SMAD4, FGF-2, and Met. www.nature.com/scientificreports www.nature.com/scientificreports/ Effects of 4HR on the expressions of RAS signaling proteins in RAW 264.7 cells. 4HR gradually suppressed the expressions of RAS signaling proteins in RAW 264.7 cells. Most RAS signaling proteins were downregulated after 4HR treatment versus non-treated controls. The expressions of KRAS, NRAS, RAF-B, and STAT3 (signal transducer and activator of transcription-3) were moderately decreased by 10.3%, 12.2%, 13.4%, and 9.6%, respectively, at 16 to 24 hours. Similarly, the expressions of protein kinase C (PKC), AMP-activated protein kinase (AMPK), Jun N-terminal protein kinase-1 (JNK-1), extracellular signal-regulated kinase 1 (ERK1), and p-ERK were slightly decreased by 3.5%, 6.6%, 6.1%, 3.6%, and 3.2%, respectively. The expressions of SOS-1 (son of sevenless homolog-1), SOS-2, and specificity protein-1 (SP-1) decreased by <5% over 24 hours, while the expressions of p-PKC, and activating protein-1 (AP-1) increased slightly by 5.5% and 3.2% at 8 hours, but then gradually decreased to 99.6% and 100.3% at 24 hours, respectively ( Fig. 2D1 and D2). These results indicate major signaling for cellular growth and protection, that is, RAS signaling, was greatly inhibited by 4HR. On the other hand, the expression of SP3, a bifunctional transcription factor that either stimulates or represses the transcriptions of numerous genes gradually increased by 8.1% at 24 hours, and the expression of JAK2 (a non-receptor tyrosine kinase implicated in signaling by members of the type II cytokine receptor family) also increased by 10.8%.
Effects of 4HR on the expressions of NFkB signaling proteins in RAW 264.7 cells. 4HR gradually decreased the expressions of NFkB signaling proteins in RAW 264.7 cells. The expression of NFkB (nuclear factor kappa-light-chain-enhancer of activated B) was decreased by 5.6% at 16 hours versus non-treated controls, while the expressions of IKK (ikappaB kinase) and NRF2 (nuclear factor (erythroid-derived 2)-like 2) were slightly increased by 3% and 2.3% at 8 hours, respectively. Most proteins that activate NFkB signaling were downregulated by 4HR, that is, p38 by 5% at 8 hours, p-p38 by 8% at 8 hours, MDR (multi-drug resistance) by 9.5% at 24 hours, mTOR (mammalian target of rapamycin) by 8.8% at 24 hours, IL-1 by 6.3% at 24 hours, and GADD45 by 4.6% at 24 hours. Proteins that inhibit NFkB signaling were also downregulated by 4HR, that is, AMPK by 6.6% at 16 hours and 1.1% at 24 hours, ERK1 by 5.9% at 8 hours, and p-ERK by 5% at 16 hours ( Fig. 3A1 and A2). These results indicate major aspects of NFkB signaling were greatly inhibited by 4HR, but its effects were broad and not selective for NFkB signaling pathways.

Effects of 4HR on the expressions of cell protection-related proteins in RAW 264.7 cells.
The expressions of cell protection-related proteins in RAW 264.7 cells were reduced by 4HR; pAKT1/2/3 by 6.8% at 8 hours, PLC-β2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2) by 7% at 24 hours, PI3K (phosphatidylinositol-3-kinase) by 4.6% at 16 hours, and PKC (protein kinase C) by 4.2% at 16 hours versus non-treated controls. p-PKC expression was slightly increased by 5% at 8 hours but gradually decreased to 98.1% in 24 hours, and the expression of its downstream signaling component FAK was also increased by 6.9% at 16 hours and by 11.9% at 24 hours. The expression of the cellular chaperone protein HSP-70 (heat shock protein-70), increased by 3.3% at 8 hours but gradually decreased to 98.9% at 24 hours. HSP-27 expression slightly decreased to 97.2% at 24 hours, and HSP-90 expression decreased to 94.2% at 8 hours but then gradually increased to 102.6% at 24 hours. The expression of PGC-1α (master regulator of mitochondrial biogenesis) increased by 6% at 16 hours and by 11.8% at 24 hours, and that of its downstream target protein, HO-1 (heme oxygenase-1) was increased by 6.2% at 16 hours and by 11.4% at 24 hours. In addition, the expression of caveoin-1 (a competitive inhibitor of HO-1) was also increased by 10.9% at 16 hours and by 12.3% by 24 hours. On the other hand, TGase-2 expression was reduced by 5.3% at 16 hours, but the expressions of LC3 and p63 increased by 4.7% and 7.4%, respectively, at 24 hours ( Fig. 3B1 and B2).
Although 4HR appeared to induce only low levels of stress in RAW 264.7 cells, as indicated by decreases in the expressions of NFkB signaling proteins, it also induced slight increases in the expressions of cellular adaptation-related proteins. These observations suggest NFkB signaling adversely affected cells, and that the observed atypical expressions of NFkB signaling molecules were induced by 4HR.
Most of the proteins downregulated by 4HR were inflammatory cytokines, which suggested 4HR might inhibit inflammatory signaling. However, 4HR only mildly affected the expressions of COX1 (a constitutive enzyme for homeostasis), and COX2 (an inducer of inflammation). Therefore, we considered 4HR might have a potent www.nature.com/scientificreports www.nature.com/scientificreports/ anti-inflammatory effect on RAW 264.7 cells by suppressing TNFα signaling but not COX1 or COX2 signaling. On the other hand, the majority of proteins upregulated by 4HR were related to cellular immunity, innate immunity, and matrix regeneration. The observed anti-inflammatory effect of 4HR might appear to contradict with its immune stimulatory effect in RAW 264.7 cells. However, cells treated with 4HR showed reduced phagocytosis activity due to the downregulations of lysozyme, cathepsin-C, -G, and -K, LTA4H, and IL-8, but increased tissue degradation due to the upregulations of MMP-1, -2, -3, -9, -10, and -12 after 16 hours of treatment, and subsequently exhibited activations of cell-mediated immune proteins including CD3, CD28, CD31, CD34, CD40, CD56, CD68, CD80, and CD99.
4HR adherent assay using acrylamide beads in vitro. 4HR adherent assays were used to detect 4HR adherent or non-adherent proteins on 4HR coated acrylamide beads as compared with non-coated acrylamide beads. Of the proteins differentially expressed in 4HR-treated RAW 264.7 cells, many proteins (n = 86) were weakly adherent to 4HR coated beads by <±5%, though some proteins (n = 23) were more adherent to 4HR-coated acrylamide beads than non-coated beads by 5.1~18.2%, and the other proteins (n = 26) were less adherent to 4HR-coated acrylamide beads than to non-coated acrylamide beads by −5.2~−17.3% (Table 1).

Discussion
4HR is a phenolic compound with a hexane chain, and as a result is strongly hydrophobic, freely soluble in ether and acetone but only sparingly soluble in water (0.5 mg/mL at 18 °C). Recently, the use of 4HR was extended to local anesthetics, antiseptics, anthelmintics, throat lozenges, skincare products, and anti-aging creams 2 . 4HR has also been shown to exhibit estrogenic activity when used as a food additive 21 and has been suggested to have anticancer effects in animals by reducing the expression of NFkB 22 .
Our results show 4HR is biologically active and induces anti-proliferative and anti-inflammatory effects in RAW 264.7 cells, and suggest it may also induce a low level of cellular stress, because it upregulated the expressions of antioxidant and oncogenic-related proteins. The effect of 4HR on cellular stress was also observed to be compensated for by increases in the expressions of cellular adaptation and angiogenesis-related proteins. Furthermore, the protein expressional changes induced by 4HR were usually in the range <±10%, though some RAS signaling proteins (KRAS, NRAS, RAF-B, and JAK2), inflammatory proteins (IL-6, -10, -28, CD-34, M-CSF, MMP-10, and cathepsin G), apoptosis-related proteins (p53, BAX, and caspase 3), and oncogenic proteins (ATM, BRCA1, and BRCA2) exhibited expressional changes of around ±15%. These results indicate that 4HR has only mild effects on RAW 264.7 cells.
4HR slightly increased p53-mediated apoptosis and upregulated antioxidant-related proteins in RAW 264.7 cells, which indicates 4HR induced cellular scavenging under conditions that diminish proliferation and growth.  (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.
www.nature.com/scientificreports www.nature.com/scientificreports/ This is in-line with the reported induction of cellular dormancy by 4HR in micro-organisms 23,24 , and suggests 4HR might exhibit synergistic effects if administered in combination with anticancer drugs 22 .
Our 4HR adherence assay showed 4HR up-or down-regulated different proteins in RAW 264.7 cells, and that it positively or negatively interacted with proteins when coated on acrylamide beads. Because 4HR is strongly hydrophobic, it may interact with the hydrophobic domains of proteins 25 . We found 4HR-coated acrylamide beads markedly absorbed TNFα, PKC, osteopontin, and GADD45, and slightly absorbed lysozyme, OPG, osteocalcin, KDM4D, ET-1, PDGF-A, pAKT, Muc1, SMAD4, E2f-1, p-PKC, MAX, FLT-4, RANKL, Rb-1, p-ERK, caspase 8, ERβ, and NOS-1, and absorbed IL-6, c-caspase 3, CDK4, c-caspase 9 less than non-coated acrylamide beads. Although we could not explore molecular interactions affecting protein conformations, 4HR adherence and lack of adherence to proteins tended to be correlated with the negativity or positivity, respectively, of their expressional changes in 4HR-treated RAW 264.7 cells (Fig. 8). These results suggest 4HR influences protein expressions differentially in RAW 264.7 cells, and that these influences are related to adherence.
It has also been reported that 4HR negatively effects TNFα 26 , NFkB 22 , and TGase-2 27 levels but positively effects lysozyme levels 25,28 . The 4HR adherence assay performed in the present study showed 4HR influenced the levels of many proteins in RAW 264.7 cells, and our adherence results suggest its interaction with proteins is non-specific but depends on the strengths of hydrophobic interactions.
In the present study, 4HR adherence to different proteins was observed to be associated with global protein expressional changes in RAW 264.7 cells. These molecular interactions between 4HR and different proteins differ from those between molecular chaperones and target proteins 29 , and we formed the opinion that observed 4HR-induced protein expressional changes were derived from active molecular interference by 4HR. Molecular interactions between 4HR and proteins are likely to be dynamic and dependent on cell status and type, and therefore, we recommend further investigations to be conducted to elucidate the mechanism responsible for 4HR-induced changes in protein conformations.
In the present study, we explored the effects of 4HR on RAW 264.7 cells by IP-HPLC using 216 antisera. 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. In addition, 4HR suppressed the expressions of growth factors and proteins associated with RAS signaling, NFkB signaling, inflammation, and osteogenesis to different extents, but elevated the expressions of proteins associated with p53-mediated apoptosis, T-cell immunity, angiogenesis, antioxidant, and oncogenic signaling. The 4HR adherence assay showed 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. Furthermore, the expressions of most proteins that adhered to 4HR were down-regulated by 4HR, whereas non-adherent proteins were up-regulated. These observations  (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. 4HR adherence assay using acrylamide beads in vitro. In order to detect 4HR adherent proteins, acrylamide beads were coated with 4HR by adding 10 mL of ethanol saturated with 4HR to 1 L of normal saline solution containing acrylamide beads (30 mL, Sephacryl TM S-300, Amersham Pharmacia Biotech. Sweden) in chromatography columns (Bio-Rad, USA). Beads were coated with 4HR by stirring the mixture and became brown in color. 4HR coated acrylamide beads were washed with 1.5 M NaCl solution, incubated with total protein extract from RAW 264.7 cells in 50 mM Tris buffer (pH 7.5) for 1 hour, and washed with normal saline solution three times. They were then incubated with 1.5 M NaCl solution for 10 minutes to elute 4HR adherent proteins. Eluted proteins were analyzed by IP-HPLC as described above using 135 antisera. Results were compared with those obtained for non-coated acrylamide beads (Supplemental Data 1 and Fig. 1).
Briefly, protein samples were mixed with 5 mL of binding buffer (150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF and 0.5% NP-40) and incubated in protein A/G agarose (Amicogen, Korea) columns at 4 °C for 1 hour (columns were placed on a rotating stirrer during incubation). After washing each column with sufficient phosphate buffered saline solution, target proteins were eluted with 150 μL of IgG elution buffer (Pierce, USA). Immunoprecipitated proteins were analyzed using a HPLC unit (1100 series, Agilent, USA) equipped with a reverse phase column and a micro-analytical detector system (SG Highteco, Korea). Elution was performed using 0.15 M NaCl/20% acetonitrile solution at 0.4 mL/min for 30 min, and proteins were detected by UV spectroscopy at 280 nm. Control and experimental samples were run sequentially to allow comparisons 12,30,31 . For IP-HPLC, whole protein peak areas (mAU*s) were calculated after subtracting negative control antibody peak areas, and the square roots of protein peak areas were calculated to normalize concentrations ( Supplementary Fig. 2). Protein percentages in total proteins in experimental and control groups were plotted. Analyses were repeated two to six times to achieve mean standard deviations of ≤±5%. Results were analyzed using the Chi-squared test.
Statistical analysis. Proportional data (%) of experimental and control groups were plotted, and the analysis was repeated two to six times (until the mean and standard deviations were ≤±5%). The results were analyzed using the Chi-squared test. The expression of control housekeeping proteins, that is, β-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were relatively unchanged (≤5%) after 8, 16, or 24 hours of 4HR treatment.