Modulation of Lipopolysaccharide‐Induced NF‐κB Signaling Pathway by 635 nm Irradiation via Heat Shock Protein 27 in Human Gingival Fibroblast Cells

Heat shock protein‐27 (HSP27) is a member of the small HSP family which has been linked to the nuclear factor‐kappa B (NF‐κB) signaling pathway regulating inflammatory responses. Clinical reports have suggested that low‐level light therapy/laser irradiation (LLLT) could be an effective alternative treatment to relieve inflammation during bacterial infection associated with periodontal disease. However, it remains unclear how light irradiation can modulate the NF‐κB signaling pathway. We examined whether or not 635 nm irradiation could lead to a modulation of the NF‐kB signaling pathway in HSP27‐silenced cells and analyzed the functional cross‐talk between these factors in NF‐κB activation. The results showed that 635 nm irradiation led to a decrease in the HSP27 phosphorylation, reactive oxygen species (ROS) generation, I‐κB kinase (IKK)/inhibitor of κB (IκB)/NF‐κB phosphorylation, NF‐κB p65 translocation and a subsequent decrease in the COX‐1/2 expression and prostaglandin (PGE₂) release in lipopolysaccharide(LPS)‐induced human gingival fibroblast cells (hGFs). However, in HSP27‐silenced hGFs, no obvious changes were observed in ROS generation, IKK/IκB/NF‐κB phosphorylation, NF‐κB p65 translocation, nor in COX‐1/2 expression, or PGE₂ release. This could be a mechanism by which 635 nm irradiation modulates LPS‐induced NF‐κB signaling pathway via HSP27 in inflammation. Thus, HSP27 may play a role in regulating the anti‐inflammatory response of LLLT.     

Plantamajoside Inhibits UVB and Advanced Glycation End Products‐Induced MMP‐1 Expression by Suppressing the MAPK and NF‐κB Pathways in HaCaT Cells

Photoaging and glycation stress are major causes of skin deterioration. Oxidative stress caused by ultraviolet B (UVB) irradiation can upregulate matrix metalloprotease 1 (MMP‐1), a major enzyme responsible for collagen damage in the skin. Advanced glycation end products (AGEs) accumulate via gradual formation from skin proteins, especially from long‐lived proteins such as dermal elastin and collagen. Plantamajoside (PM), isolated from Plantago asiatica, has various biological effects including anti‐inflammatory and antioxidant effects. In this study, we assessed the protective effects of PM on a human keratinocyte cell line (HaCaT) and primary human dermal fibroblasts (HDF) against stress caused by glyceraldehyde‐induced AGEs (glycer‐AGEs) with UVB irradiation. We found that PM attenuated UVB‐ and‐glycer‐AGEs‐induced MMP‐1 expression in HaCaT and HDF cells and proinflammatory cytokines expression by inhibiting the phosphorylation of mitogen‐activated protein kinases (MAPKs) activated by reactive oxygen species. Specific inhibitors of NF‐κB and MAPKs attenuated the induced expression of MMP‐1. PM also inhibited the phosphorylation of IκBα, and reduced nuclear translocation of NF‐κB in these cells. Furthermore, PM attenuated the upregulation of receptor for AGEs (RAGE) by glycer‐AGEs with UVB irradiation. Therefore, our findings strongly suggest that PM is a promising inhibitor of skin photoaging.     

Mapping of phosphorylation sites in human MSK1 activated by a novel interaction with MRK‐β

The desire to map reliable phosphorylation signaling network has motivated the development of high‐performance techniques. Targeted biochemical studies and updated methods employing MS techniques are most used in mapping the phosphorylation sites and verifying novel interactions of kinases. Previously, we have established a novel method to efficiently facilitate more comprehensive, accurate phosphorylation site mapping of individual phosphoproteins by using combination of multiple stage MS analysis with target‐decoy database search against the much smaller targeted database. In this study, by applying this method, we have identified the phosphorylation sites in human MSK1 mitogen‐ and stress‐activated protein kinase 1), which has been proved to be a multi‐phosphorylated kinase that plays key roles in various cell functions, activated by a novel interaction with MRK‐β. The results show that this method can find out not only those previously identified active sites in MSK1, but also some novel phosphorylated sites, which correlates with biochemical evidence that, besides p38 and extracellular signal‐regulated kinase, MRK‐β could also activate MSK1 through direct interaction. Hence, we conclude this method is sensitive and reliable as expected and it can be further combined with automated screening and biochemical study in efficiently building up a more comprehensive phosphoprotein network.     

Salvianolic Acid B Protects Normal Human Dermal Fibroblasts Against Ultraviolet B Irradiation‐Induced Photoaging Through Mitogen‐Activated Protein Kinase and Activator Protein‐1 Pathways

Exposure to ultraviolet (UV) light causes increased matrix metalloproteinase (MMP) activity and decreased collagen synthesis, leading to skin photoaging. Salvianolic acid B (SAB), a polyphenol, was extracted and purified from salvia miltiorrhiza. We assessed effects of SAB on UVB‐induced photoaging and investigated its molecular mechanism of action in UVB‐irradiated normal human dermal fibroblasts. Our results show that SAB significantly inhibited the UVB‐induced expression of metalloproteinases‐1 (MMP‐1) and interleukin‐6 (IL‐6) while promoting the production of type I procollagen and transforming growth factor β1 (TGF‐β1). Moreover, treatment with SAB in the range of 1–100 μg/mL significantly inhibited UVB‐induced extracellular signal‐regulated kinase (ERK), Jun N‐terminal kinase (JNK) and p38 phosphorylation, which resulted in decreasing UVB‐induced phosphorylation of c‐Fos and c‐Jun. These results indicate that SAB downregulates UV‐induced MMP‐1 expression by inhibiting Mitogen‐activated protein kinase (MAPK) signaling pathways and activator protein‐1 (AP‐1) activation. Our results suggest a potential use for SAB in skin photoprotection.     

A Peptide YGDEY from Tilapia Gelatin Hydrolysates Inhibits UVB‐mediated Skin Photoaging by Regulating MMP‐1 and MMP‐9 Expression in HaCaT Cells

In this study, we investigated the protective effects of a peptide (YGDEY, Tyr‐Gly‐Asp‐Glu‐Tyr) isolated from tilapia skin gelatin hydrolysates (TGHs), against UVB‐induced photoaging in human keratinocytes (HaCaT) cells. Results showed that YGDEY significantly decreased levels of intracellular reactive oxygen species (ROS), increased antioxidant factors (Superoxide Dismutase, SOD and Glutathione, GSH) expression and maintained balance between GSH and GSSG in HaCaT cells. Comet assay shows that YGDEY can protect DNA from oxidative damage. Furthermore, it significantly inhibited MMP‐1 (collagenase) and MMP‐9 (gelatinase) expression and increased Type I procollagen production. In addition, the molecular docking study showed that YGDEY may form active sites with MMP‐1 and MMP‐9. Moreover, Western blot analysis was utilized to measure the protein levels of UVB‐induced mitogen‐activated protein kinase (MAPK) and nuclear factor‐kappa B (NF‐κB) signaling pathways. Therefore, these results suggested that YGDEY has a therapeutic effectiveness in prevention of UVB‐induced cellular damage, and it is a candidate worthy of being developed as a potential natural antioxidant and food additive.     

Fisetin Inhibits UVB‐induced Cutaneous Inflammation and Activation of PI3K/AKT/NFκB Signaling Pathways in SKH‐1 Hairless Mice

Solar ultraviolet B (UVB) radiation has been shown to induce inflammation, DNA damage, p53 mutations and alterations in signaling pathways eventually leading to skin cancer. In this study, we investigated whether fisetin reduces inflammatory responses and modulates PI3K/AKT/NFκB cell survival signaling pathways in UVB‐exposed SKH‐1 hairless mouse skin. Mice were exposed to 180 mJ cm^?2 of UVB radiation on alternate days for a total of seven exposures, and fisetin (250 and 500 nmol) was applied topically after 15 min of each UVB exposure. Fisetin treatment to UVB‐exposed mice resulted in decreased hyperplasia and reduced infiltration of inflammatory cells. Fisetin treatment also reduced inflammatory mediators such as COX‐2, PGE₂ as well as its receptors (EP1–EP4) and MPO activity. Furthermore, fisetin reduced the level of inflammatory cytokines TNFα, IL‐1β and IL‐6 in UVB‐exposed skin. Fisetin treatment also reduced cell proliferation markers as well as DNA damage as evidenced by increased expression of p53 and p21 proteins. Further studies revealed that fisetin inhibited UVB‐induced expression of PI3K, phosphorylation of AKT and activation of the NFκB signaling pathway in mouse skin. Overall, these data suggest that fisetin may be useful against UVB‐induced cutaneous inflammation and DNA damage.     

Requirement for Metalloproteinase‐dependent ERK and AKT Activation in UVB‐induced G1‐S Cell Cycle Progression of Human Keratinocytes

UVB (280–315 nm) in natural sunlight represents a major environmental challenge to the skin and is clearly associated with human skin cancer. Here we demonstrate that low doses of UVB induce keratinocyte proliferation and cell cycle progression of human HaCaT keratinocytes. Different from UVA, UVB irradiation induced extracellular signal‐regulated kinase (ERK) and AKT activation and their activation are both required for UVB‐induced cell cycle progression. Activation of epidermal growth factor receptor (EGFR) was observed after UVB exposure and is upstream of ERK/AKT/cyclin D1 pathway activation and cell cycle progression following UVB radiation. Furthermore, metalloproteinase (MP) inhibitor GM6001 blocked UVB‐induced ERK and AKT activation, cell cycle progression, and decreased the EGFR phosphorylation, demonstrating that MPs mediate the EGFR/ERK/AKT/cyclin D1 pathways and cell cycle progression induced by UVB radiation. In addition, ERK or AKT activation is essential for EGFR activation because ERK or AKT inhibitor blocks EGFR activation following UVB radiation, indicating that EGFR/AKT/ERK pathways form a regulatory loop and converge into cell cycle progression following UVB radiation. Identification of these signaling pathways in UVB‐induced cell cycle progression of quiescent keratinocytes as a process mimicking tumor promotion in vivo will facilitate the development of efficient and safe chemopreventive and therapeutic strategies for skin cancer.     

Gold‐Catalyzed Cycloisomerization and Diels–Alder Reaction of 1,4,9‐Dienyne Esters to 3 a,6‐Methanoisoindole Esters with Pro‐Inflammatory Cytokine Antagonist Activity

A synthetic method to prepare 3a,6‐methanoisoindole esters efficiently by gold(I)‐catalyzed tandem 1,2‐acyloxy migration/Nazarov cyclization followed by Diels–Alder reaction of 1,4,9‐dienyne esters is described. We also report the ability of one example to inhibit binding of tumor necrosis factor‐α (TNF‐α) to the tumor necrosis factor receptor 1 (TNFR1) site and TNF‐α‐induced nuclear factor κ‐light‐chain‐enhancer of activated B cells (NF‐κB) activation in cell at a half‐maximal inhibitory concentration (IC₅₀) value of 6.6 μM . Along with this is a study showing the isoindolyl derivative to exhibit low toxicity toward human hepatocellular liver carcinoma (HepG2) cells and its possible mode of activity based on molecular modeling analysis.     

Tualang honey protects keratinocytes from ultraviolet radiation-induced inflammation and DNA damage(†)

Malaysian tualang honey possesses strong antioxidant and anti‐inflammatory properties. Here, we evaluated the effect of tualang honey on early biomarkers of photocarcinogenesis employing PAM212 mouse keratinocyte cell line. Keratinocytes were treated with tualang honey (1.0%, v/v) before a single UVB (150 mJ cm^?2) irradiation. We found that the treatment of tualang honey inhibited UVB‐induced DNA damage, and enhanced repair of UVB‐mediated formation of cyclobutane pyrimidine dimers and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine. Treatment of tualang honey inhibited UVB‐induced nuclear translocation of NF‐κB and degradation of IκBα in murine keratinocyte cell line. The treatment of tualang honey also inhibited UVB‐induced inflammatory cytokines and inducible nitric oxide synthase protein expression. Furthermore, the treatment of tualang honey inhibited UVB‐induced COX‐2 expression and PGE2 production. Taken together, we provide evidence that the treatment of tualang honey to keratinocytes affords substantial protection from the adverse effects of UVB radiation via modulation in early biomarkers of photocarcinogenesis and provide suggestion for its photochemopreventive potential.     

Oral feeding of pomegranate fruit extract inhibits early biomarkers of UVB radiation-induced carcinogenesis in SKH-1 hairless mouse epidermis

Pomegranate from the plant Punica granatum L. possesses strong antioxidant and anti-inflammatory properties. Recently, we have demonstrated that treatment of normal human epidermal keratinocytes with pomegranate fruit extract (PFE) inhibited UVB-mediated activation of nuclear factor kappa B (NF-κB) and mitogen activated protein kinases pathways. Here, we evaluated the effect of PFE on early biomarkers of photocarcinogenesis employing SKH-1 hairless mice. PFE was provided in drinking water (0.2%, wt/vol) to SKH-1 hairless mice for 14 days before a single UVB (180 mJ cm⁻²) irradiation. We found that oral feeding of PFE inhibited UVB-induced: (1) skin edema; (2) hyperplasia; (3) infiltration of leukocytes; (4) lipid peroxidation; (5) hydrogen peroxide generation; (6) ornithine decarboxylase (ODC) activity; and (7) ODC, cyclooxygenase-2 and proliferating cell nuclear antigen protein expression. Oral feeding of PFE enhanced repair of UVB-mediated formation of cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). Importantly, PFE treatment further enhanced UVB-mediated increase in tumor suppressor p53 and cyclin kinase inhibitor p21. Furthermore, oral feeding of PFE inhibited UVB-mediated: (1) nuclear translocation of NF-κB; (2) activation of IKKα; and (3) phosphorylation and degradation of IκBα. Taken together, we provide evidence that oral feeding of PFE to mice affords substantial protection from the adverse effects of UVB radiation via modulation in early biomarkers of photocarcinogenesis and provide suggestion for its photochemopreventive potential.     

Angelica archangelia Prevented Collagen Degradation by Blocking Production of Matrix Metalloproteinases in UVB‐exposed Dermal Fibroblasts

Angelica archangelia (AA), a traditional herb, has attracted attention as an agent with potential for use in the prevention of chronic skin diseases. This study examined the photoprotective effects of AA on the inhibition of matrix metalloproteinases (MMPs) and collagen degradation in UVB‐irradiated normal human dermal fibroblasts. Our results showed that AA markedly blocked collagen degradation by restraining the production of MMPs in UVB‐exposed fibroblasts. We also investigated the underlying mechanism behind the effects of AA. AA attenuated UVB‐triggered interleukin‐6 (IL‐6) and promoted the expression of transforming growth factor β1. Application of AA extract (10, 100 μg mL^?1) significantly diminished UVB‐induced extracellular signal‐regulated kinase and Jun‐N‐terminal kinase phosphorylation, which consequently reduced phosphorylated c‐Fos and c‐Jun. Our results indicated that AA inhibited the UVB‐induced expression of MMPs by inhibiting mitogen‐activated protein kinase signaling pathways and activator protein‐1 activation. Our results suggest that AA is a promising botanical agent for use against skin photoaging.     

A Synthetic Loop Replacement Peptide That Blocks Canonical NF‐κB Signaling

Aberrant canonical NF‐κB signaling is implicated in diseases from autoimmune disorders to cancer. A major therapeutic challenge is the need for selective inhibition of the canonical pathway without impacting the many non‐canonical NF‐κB functions. Here we show that a selective peptide‐based inhibitor of canonical NF‐κB signaling, in which a hydrogen bond in the NBD peptide is synthetically replaced by a non‐labile bond, shows an about 10‐fold increased potency relative to the original inhibitor. Not only is this molecule, NBD2, a powerful tool for dissection of canonical NF‐κB signaling in disease models and healthy tissues, the success of the synthetic loop replacement suggests that the general strategy could be useful for discovering modulators of the many protein–protein interactions mediated by such structures.     

The Protective Effect of Violaxanthin from Nannochloropsis oceanica against Ultraviolet B‐Induced Damage in Normal Human Dermal Fibroblasts

Skin photoaging, which is mainly induced by ultraviolet B (UVB) radiation, is prevented by the application of UV‐protective agents. The microalga Nannochloropsis oceanica (N. oceanica) has been primarily reported as a potential biofuel; however, in this study, we investigated whether N. oceanica extracts exerted photoprotective effects against UVB‐irradiated human dermal fibroblasts (HDFs) and which single component was responsible for the protective effect of the extracts. Two extracts—pigment and nonpigment—were prepared from N. oceanica biomass. WST‐1 assay and expression analysis of interleukin genes showed that the pigment extracts were not significantly cytotoxic to HDFs. Further experiments revealed that treatment with the pigment extract upregulated the expression of collagen genes and significantly blocked UVB‐induced damage such as decreased cell viability and increased ROS production. Next, to investigate the pigment composition of the extracts, HPLC analysis was conducted and violaxanthin was identified as the major pigment. The UVB photoprotective effect of the pigment extracts was confirmed in violaxanthin‐treated HDFs. In addition, violaxanthin significantly attenuated UVB‐induced G1 phase arrest, senescence‐associated β‐galactosidase activation, p16 and p21 upregulation, ERK phosphorylation and the downregulation of ECM molecules in HDFs. Therefore, we concluded that violaxanthin was a potential antiphotoaging agent.     

UVA and UVB‐Induced 8‐Methoxypsoralen Photoadducts and a Novel Method for their Detection by Surface‐Enhanced Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry (SELDI‐TOF MS)

UVA‐activated psoralens are used to treat hyperproliferative skin conditions due to their ability to form DNA photoadducts, which impair cellular processes and may lead to cell death. Although UVA (320–400 nm) is more commonly used clinically, studies have shown that UVB (280–320 nm) activation of psoralen can also be effective. However, there has been no characterization of UVB‐induced adduct formation in DNA alone. As psoralen derivatives have a greater extinction coefficient in the UVB region (11 800 cm^?1 M^?1 at 300 nm) compared with the UVA region (2016 cm^?1 M^?1 at 365 nm), a greater extent of adduct formation is expected. SELDI‐TOF, a proteomic technique that combines chromatography with mass spectrometry, was used to detect photoadduct formation in an alternating A–T oligonucleotide. 8‐Methoxypsoralen (8‐MOP) and DNA solutions were irradiated with either UVA or UVB. An adduct peak was obtained with SELDI‐TOF. For UVB‐activated 8‐MOP, the extent of adducts was three times greater than for UVA. HPLC ESI‐MS analysis showed that UVB irradiation yielded high levels of 3,4‐monoadducts (78% of total adducts). UVA was more effective than UVB at conversion of 4′,5′‐monoadducts to crosslinks (17% vs 4%, respectively). This report presents a method for comparing DNA binding efficiencies of interstrand crosslink inducing agents.