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Filaggrin (FLG) is an essential structural protein expressed in differentiated keratinocytes. Insufficient FLG expression contributes to the pathogenesis of chronic inflammatory skin diseases. Saikosaponin A (SSA), a bioactive oleanane-type triterpenoid, exerts anti-inflammatory activity. However, the effects of topically applied SSA on FLG expression in inflamed skin remain unclear. This study aimed to evaluate the biological activity of SSA in restoring reduced FLG expression. The effect of SSA on FLG expression in HaCaT cells was assessed through various biological methods, including reverse transcription PCR, quantitative real-time PCR, immunoblotting, and immunofluorescence staining. TNFα and IFNγ decreased FLG mRNA, cytoplasmic FLG protein levels, and FLG gene promoter–reporter activity compared to the control groups. However, the presence of SSA restored these effects. A series of FLG promoter–reporter constructs were generated to investigate the underlying mechanism of the effect of SSA on FLG expression. Mutation of the AP1-binding site (mtAP1) in the −343/+25 FLG promoter–reporter abrogated the decrease in reporter activities caused by TNFα + IFNγ, suggesting the importance of the AP1-binding site in reducing FLG expression. The SSA treatment restored FLG expression by inhibiting the expression and nuclear localization of FRA1 and c-Jun, components of AP1, triggered by TNFα + IFNγ stimulation. The ERK1/2 mitogen-activated protein kinase signaling pathway upregulates FRA1 and c-Jun expression, thereby reducing FLG levels. The SSA treatment inhibited ERK1/2 activation caused by TNFα + IFNγ stimulation and reduced the levels of FRA1 and c-Jun proteins in the nucleus, leading to a decrease in the binding of FRA1, c-Jun, p-STAT1, and HDAC1 to the AP1-binding site in the FLG promoter. The effect of SSA was evaluated in an animal study using a BALB/c mouse model, which induces human atopic-dermatitis-like skin lesions via the topical application of dinitrochlorobenzene. Topically applied SSA significantly reduced skin thickening, immune cell infiltration, and the expression of FRA1, c-Jun, and p-ERK1/2 compared to the vehicle-treated group. These results suggest that SSA can effectively recover impaired FLG levels in inflamed skin by preventing the formation of the repressor complex consisting of FRA1, c-Jun, HDAC1, and STAT1.

Thymic stromal lymphopoietin (TSLP) is a cytokine derived from epithelial cells and plays an essential role in the onset and activation of Th2-derived allergic inflammatory conditions, including atopic dermatitis. Despite their potential as drug targets, well-defined small molecules that effectively block TSLP expression are still lacking. A plant-derived secondary metabolite, aurone, was derivatized based on bioisosteric replacement to identify compounds that inhibit the promoter activity of TSLP. Thirteen (E)-2-benzylidene-1-indanones were designed and synthesized, and their structures were identified using NMR spectroscopy and mass spectrometry. Inhibition of the expression of TSLP triggered by interleukin-4 (IL-4) caused by (E)-2-benzylidene-1-indanones was measured using a TSLP gene promoter-reporter activity assay. Because compound 12, (E)-5-methoxy-2-(3-methoxybenzylidene)-2,3-dihydro-1H-inden-1-one, showed the best activity, further biological experiments, including RT-PCR analysis, quantitative real-time PCR, and inhibitory effects on IL-4-induced early growth response-1 (EGR-1) expression, EGR-1 DNA-binding activity, and IL-4-induced phosphorylation of the mitogen-activated protein kinase (MAPK) signaling cascade were performed. This study demonstrated that compound 12 acts on MAPK to block IL-4-triggered mRNA expression of TSLP via the MAPK-EGR-1 signaling pathway in HaCaT keratinocytes.

The circadian clock system is closely associated with inflammatory responses. Dysregulation of the circadian clock genes in the skin impairs the skin barrier function and affects the pathophysiology of atopic dermatitis. Interleukin 4 (IL-4) is a proinflammatory cytokine derived from T-helper type 2 cells; it plays a critical role in the pathogenesis of atopic dermatitis. Agerarin (6,7-dimethoxy-2,2-dimethyl-2H-chromene) is a natural JAK1/2/3 inhibitor isolated from Ageratum houstonianum that has a protective effect on the epidermal skin barrier. However, it remains unclear whether agerarin affects the circadian clock system. The aim of this study is to investigate the effect of agerarin on IL-4-induced PER2 gene expression in human keratinocytes through reverse transcription (RT)-PCR, quantitative real-time PCR (qPCR), immunoblotting, immunofluorescence microscopic analysis, and real-time bioluminescence analysis. We found that agerarin reduced IL-4-induced PER2 mRNA expression by suppressing the JAK-STAT3 pathway. In addition, real-time bioluminescence analysis in PER2:luc2p promoter-reporter cells revealed that agerarin restored the oscillatory rhythmicity of PER2 promoter activity altered by IL-4. These findings suggest that agerarin may be useful as a cosmeceutical agent against inflammatory skin conditions associated with disrupted circadian rhythms, such as atopic dermatitis.

The spiro compound, 5,5′-dimethoxy-1,3-bis(3-(trifluoromethyl)phenyl)-3,3a-dihydro-1H-spiro[cyclopenta[a]indene-2,2′-indene]-1′,8(3′H,8aH)-dione (4), was synthesized and identified by NMR spectroscopy, mass spectrometry, and X-ray crystallography. Compound 4, C36H26F6O4, was crystallized in the triclinic space group P-1with the cell parameters a = 8.8669(5) Å, b = 10.5298(8) Å, c = 17.0135(11) Å, α = 91.396(2)°, β = 90.490(2)°, γ = 109.235°, V = 1499.14(17) Å3, Z = 2. In an asymmetric unit, two molecules are packed by short contacts to form an inversion dimer. The molecules are linked into chains along the a- and b-axis directions by additional short contacts in the crystal. Compound 4 was synthesized by the dimerization of (E)-5-methoxy-2-(3-(trifluoromethyl)benzylidene)-2,3-dihydro-1H-inden-1-one (3). (E)-5-Methoxy-2-(3-methoxybenzylidene)-2,3-dihydro-1H-inden-1-one (5), one of the analogs of compound 3, was compared with compound 4 based on in vitro experiments, DFT calculations, and an in silico docking study. The HOMO/LUMO energy difference and binding energy difference between the two compounds are consistent with the results obtained from an in vitro assay where 4 showed a better effect than 5. To evaluate the biological activity of 4, we examined its inhibitory effects on Early Growth Respone-1 (EGR-1)-regulated gene expression in HaCaT keratinocytes. Treatment of cells with 4 reduced interleukin-4 (IL-4)-induced thymic stromal lymphopoietin (TSLP) mRNA levels, as revealed by reverse transcription-polymerase chain reaction and quantitative real-time PCR. Furthermore, the electrophoretic mobility shift assay demonstrated that 4 inhibited IL-4-induced DNA binding of EGR-1 to the promoter region of the TSLP gene.

Chalcones have a skeleton of diphenyls connected via an α,β-unsaturated carbonyl group. Many chalcone derivatives have been shown to interact with tubulin. Based on previous reports, chalcones derived by substitution of a carbonyl group with 2-pyrazoline can be expected to inhibit tubulin polymerization. Therefore, 3,5-diphenyl-2-pyrazolines were prepared to investigate their ability to inhibit tubulin polymerization. The clonogenic long-term survival assay showed that derivative 4, 5-(3,5-dimethoxyphenyl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide, was the most effective at inhibiting the clonogenicity of HCT116 human colon cancer cells. Derivative 4 induced G2/M cell cycle arrest. In addition, derivative 4 caused dispersed microtubules, a disorganized spherical arrangement of chromosomes, and inhibition of mitotic spindle formation. The binding mode between tubulin and derivative 4 was elucidated by in silico molecular docking. Derivative 4 was superimposed with colchicine and entered the colchicine-binding site well. These results suggest that derivative 4 inhibits tubulin polymerization by binding to the colchicine binding site of tubulin, thus preventing mitotic spindle formation during mitosis in HCT116 colon cancer cells. We propose that derivative 4 could be used as a promising antimitotic chemotherapeutic agent.

The flavone–chalcone hybrid compound, (E)-6-bromo-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl)-4H-chromen-4-one (3), was synthesized and its three dimensional structure was identified by X-ray crystallography. The compound 3, C19H13BrO4, was crystallized in the triclinic space group P-1 with the following cell parameters: a = 8.2447(6) Å; b = 8.6032(6) Å; c = 11.7826(7) Å; α = 92.456(2)°; β = 91.541(2)°; γ = 106.138(2)°; V = 801.42(9) Å3 and Z = 2. In an asymmetric unit, two molecules are packed by a pi–pi stacking interaction between two flavone rings that are 3.790 Å apart from each other. In the crystal, two hydrogen bonds form inversion dimers and these dimers are extended along the a axis by another hydrogen bond. Hirshfeld analysis revealed that the H–H (34.3%), O–H (19.2%) and C–H (16.7%) intermolecular contacts are the major dominants, while the C–O (6.7%) and C–C (6.5%) are minor dominants. When HCT116 cells were treated with various concentrations of hybrid compound 3, reduced cell viability and induced apoptosis in HCT116 cells were observed in a dose-dependent manner. The treatment of HCT116 colon cancer cells with compound 3, decreased the intracellular glutathione (GSH) levels and generated a reactive oxygen species (ROS). In silico docking experiments between the compound 3 and glutathione S-transferase (GST) containing glutathione were performed to confirm whether the compound 3 binds to glutathione. Their binding energy ranged from −6.6 kcal/mol to −5.0 kcal/mol, and the sulfur of glutathione is very close to the Michael acceptor regions of the compound 3, so it is expected that they would easily react with each other. Compound 3 may be a promising novel anticancer agent by ROS generation through glutathione depletion.

Mesenchymal stem cells (MSCs) can differentiate into endothelial cells; however, the mechanisms underlying this process in the tumor microenvironment (TME) remain elusive. This study shows that tumor necrosis factor alpha (TNF-α), a key cytokine present in the TME, promotes the endothelial differentiation of MSCs by inducing vascular endothelial growth factor receptor 2 (VEGFR2) gene expression. EGR1 is a member of the zinc-finger transcription factor family induced by TNF-α. Our findings indicate that EGR1 directly binds to the VEGFR2 promoter and transactivates VEGFR2 expression. We also demonstrate that EGR1 forms a complex with c-JUN activated by c-JUN N-terminal kinase (JNK) to promote VEGFR2 transcription and endothelial differentiation in MSCs in response to TNF-α stimulation. The shRNA-mediated silencing of EGR1 or c-JUN abrogates TNF-α-induced VEGFR2 transcription and the endothelial differentiation of MSCs. To further evaluated the role of EGR1 in the endothelial differentiation of BM-MSCs, we used a syngenic tumor implantation model. 4T1 mouse mammary tumor cells were injected subcutaneously into BALB/c mice with primary mBM-MSCs isolated from wild-type (Egr1+/+) or Egr1-null (Egr1−/−) mice. CD31-positive cells were predominantly observed at the border of the tumor in the 4T1 plus wild-type MSC group, while staining less in the 4T1 alone or 4T1 plus Egr1-null MSC group. Collectively, these findings demonstrate that the JNK-EGR1 signaling axis plays a crucial role in the TNF-α-induced endothelial differentiation of MSCs in the TME, which could be a potential therapeutic target for solid tumors vasculatures.

Filaggrin (FLG), an essential structural protein for skin barrier function, is downregulated under chronic inflammatory conditions, leading to disruption of the skin barrier. However, the detailed molecular mechanisms of how FLG changes in the context of chronic inflammation are poorly understood. Here, we identified the molecular mechanisms by which inflammatory cytokines inhibit FLG expression in the skin. We found that the AP1 response element within the −343/+25 of the FLG promoter was necessary for TNFα + IFNγ–induced down-regulation of FLG promoter activity. Using DNA affinity precipitation assay, we observed that AP1 subunit composition binding to the FLG promoter was altered from c-FOS:c-JUN (at the early time) to FRA1:c-JUN (at the late time) in response to TNFα + IFNγ stimulation. Knockdown of FRA1 or c-JUN abrogated TNFα + IFNγ–induced FLG suppression. Histone deacetylase (HDAC) 1 interacted with FRA1:c-JUN under TNFα + IFNγ stimulation. Knockdown of HDAC1 abrogated the inhibitory effect of TNFα + IFNγ on FLG expression. The altered expression of FLG, FRA1, c-JUN, and HDAC1 was confirmed in mouse models of 2,4-dinitrochlorobenzene–induced atopic dermatitis and imiquimod-induced psoriasis. Thus, the current study demonstrates that TNFα + IFNγ stimulation suppresses FLG expression by promoting the FRA1:c-JUN:HDAC1 complex. This study provides insight into future therapeutic strategies targeting the FRA1:c-JUN:HDAC1 complex to restore impaired FLG expression in chronic skin inflammation.

Prospero homeobox 1 (PROX1) is a member of the homeobox transcription factor family that plays a critical role in the development of multiple tissues and specification of cell fate. PROX1 expression is differentially regulated based on the cellular context and plays an antagonistic role as a tumour promoter or suppressor in different tumour types. In human breast cancer, PROX1 expression is suppress­ed; however, the molecular mechanism by which it is down-regulated remains poorly understood. Here, we show that ectopic expression of PROX1 reduces the motility and invasiveness of MDA-MB-231 human breast cancer cells, suggesting that PROX1 functions as a negative regulator of tumour invasion in MDA-MB-231 cells. Treatment with histone deacetylase (HDAC) inhibitors up-regulates PROX1 mRNA and protein expression levels. Knockdown of HDAC1 using short hairpin RNA also up-regulates PROX1 mRNA and protein expression levels. We found that HDAC1 interacted with c-JUN at the activator protein (AP)-1-binding site located at –734 to –710 in the PROX1 promoter region to suppress PROX1 expression. In addition, c-JUN N-terminal kinase-mediated c-JUN phosphorylation was found to be crucial for silencing PROX1 expression. In conclusion, PROX1 expression can be silenced by the epigenetic mechanism involved in the complex formation of HDAC1 and c-JUN at the AP-1 site in the PROX1 promoter region in MDA-MB-231 human breast cancer cells. Therefore, this study revealed the epigenetic regulatory mechanism involved in the suppression of PROX1 expression in breast cancer cells.