Dehydroglyasperin D Suppresses Melanin Synthesis through MITF Degradation in Melanocytes.

Licorice (Glycyrrhiza) has been used as preventive and therapeutic material for hyperpigmentation disorders. Previously, we isolated noble compounds including dehydroglyasperin C (DGC), dehydroglyasperin D (DGD) and isoangustone A (IAA) from licorice hexane/ethanol extracts. However, their anti-melanogenic effects and underlying molecular mechanisms are unknown. The present study compared effects of DGC, DGD and IAA on pigmentation in melan-a melanocytes and human epidermal melanocytes (HEMn). DGD exerted the most excellent anti-melanogenic effect, followed by DGC and IAA at non-cytotoxic concentrations. In addition, DGD significantly inhibited tyrosinase activity in vitro cell-free system and cell system. Western blot result showed that DGD decreased expression of microphthalmia-associated transcription factor (MITF), tyrosinase and tyrosinase-related protein-1 (TRP-1) in melan-a cells and HEMn cells. DGD induced phosphorylation of MITF, ERK and Akt signal pathway promoting MITF degradation system. However, DGD did not influence p38 and cAMP-dependent protein kinase (PKA)/CREB signal pathway in melan-a cells. These result indicated that DGD inhibited melanogenesis not only direct regulation of tyrosinase but also modulating intracellular signaling related with MITF level. Collectively, these results suggested a protective role for DGD against melanogenesis.

A Genomics-Based Semirational Approach for Expanding the Postbiotic Potential of Collagen Peptides Using Lactobacillaceae.

Food-derived bioactive peptides (BPs) have received considerable attention as postbiotics for human gut health. Here we used a genomics-based semirational approach to expand the postbiotic potential of collagen peptides (CPs) produced from probiotic fermentation. In silico digestion revealed distinct BPs embedded in fish collagen in a protease-dependent manner. Anaerobic digestion of collagen by representative Lactobacillaceae species revealed differential substrate utilization and collagen degradation patterns. Nanoliquid chromatography-mass spectrometry analysis of CPs showed that each species exhibited different cleavage patterns and unique peptide profiles. Remarkably, the 1-10 kDa CPs produced by Lacticaseibacillus paracasei showed agonistic activities toward G protein-coupled receptor 35 (GPR35). These CPs could repair intestinal epithelium through the GPR35-mediated extracellular signal-regulated protein kinase (ERK) 1/2 signaling pathway, suggesting that probiotic-aided collagen hydrolysates can serve as postbiotics for host-microbe interactions. Therefore, this study provides an effective strategy for the rapid screening of CPs for gut health in the gastrointestinal tract.

In Vitro Prebiotic and Anti-Colon Cancer Activities of Agar-Derived Sugars from Red Seaweeds.

Numerous health benefits of diets containing red seaweeds or agar-derived sugar mixtures produced by enzymatic or acid hydrolysis of agar have been reported. However, among various agar-derived sugars, the key components that confer health-beneficial effects, such as prebiotic and anti-colon cancer activities, remain unclear. Here, we prepared various agar-derived sugars by multiple enzymatic reactions using an endo-type and an exo-type of ß-agarase and a neoagarobiose hydrolase and tested their in vitro prebiotic and anti-colon cancer activities. Among various agar-derived sugars, agarotriose exhibited prebiotic activity that was verified based on the fermentability of agarotriose by probiotic bifidobacteria. Furthermore, we demonstrated the anti-colon cancer activity of 3,6-anhydro-l-galactose, which significantly inhibited the proliferation of human colon cancer cells and induced their apoptosis. Our results provide crucial information regarding the key compounds derived from red seaweeds that confer beneficial health effects, including prebiotic and anti-colon cancer activities, to the host.

3,6-Anhydro-L-galactose increases hyaluronic acid production via the EGFR and AMPKα signaling pathway in HaCaT keratinocytes.

BACKGROUND: Hyaluronic acid (HA) is an important factor in skin hydration maintenance. In mammalian keratinocytes, hyaluronan synthase 2 (HAS2) is a critical enzyme in HA production. Therefore, the promotion of HAS2 expression in keratinocytes may be a strategy for maintaining skin moisture. OBJECTIVE: The aim was to determine the skin hydration effect and regulatory mechanisms of 3,6-anhydro-L-galactose (L-AHG), a main component of red macroalgal carbohydrates in human keratinocytes. METHODS: L-AHG was applied to an immortalized human epidermal keratinocyte cell line (HaCaT cells). HA production, HAS2 protein and mRNA levels, and the activation of the signaling pathways involved in HAS2 expression were measured. HA levels were also evaluated for three dimensional (3D) reconstructed human skin. RESULTS: Our results suggest that L-AHG upregulates HA production and may enhance HAS2 expression by activating EGFR-mediated ERK, PI3K/Akt, and STAT3 signaling pathways. We confirmed that L-AHG activated the AMPKα signaling pathway which in turn could regulate HAS2 expression in HaCaT cells. The effects of L-AHG on HA production were observed in the 3D reconstructed human skin model. CONCLUSION: Our results suggest that L-AHG may enhance skin moisture retention by increasing HA synthesis in human epidermal keratinocytes.

Penta-1,2,3,4,6-O-Galloyl-ß-D-Glucose Inhibits UVB-Induced Photoaging by Targeting PAK1 and JNK1.

Penta-O-galloyl-ß-D-glucose (PGG) is a gallotannin polyphenolic compound that occurs naturally in fermented Rhus verniciflua. The present study aimed to examine the effect of PGG on UVB-induced skin aging and its molecular mechanisms in HaCaT human keratinocytes and SKH-1 hairless mice models. PGG suppressed UVB-induced matrix metalloproteinase-1 (MMP-1) expression in HaCaT cells by inhibiting phosphorylation of RAF/MEK/ERK, MKK3/6/p38, and c-Jun. UVB-induced ERK and p38 signaling pathways that induce the MMP-1 expression were mediated by PAK1 in HaCaT cells. PGG suppressed PAK1 and JNK1 kinase activities, and directly bound both PAK1 in an ATP-competitive manner and JNK1 in an ATP-noncompetitive manner. Consistently, PGG decreased UVB-induced wrinkle formation, epidermal thickness, type 1 collagen and MMP-13 expression in mouse skin. Overall, these results indicate that PGG exhibits anti-photoaging effects in vitro and in vivo by the suppression of PAK1 and JNK1 kinase activities, and may be useful for the prevention of skin aging.

Identification of Matrix Metalloproteinase-1-Suppressive Peptides in Feather Keratin Hydrolysate.

Inhibition of matrix metalloproteinases (MMPs), which degrade collagen and elastin in the dermis of normal skin, is a key strategy for anti-skin aging. In this study, we identified five low-molecular-weight (LMW, <1 kDa) MMP-1-suppressive peptides in feather keratin hydrolysate (FKH) obtained by anaerobic digestion with an extremophilic bacterium. FKH was first subjected to ultrafiltration, followed by size-exclusion chromatography and liquid chromatography/electrospray ionization tandem mass spectrometry analysis. Chemically synthesized peptides identical to the sequences identified suppressed MMP expression in human dermal fibroblasts (HDFs). To investigate the impact of the MMP-1-suppressive peptides on the signaling pathway, we performed antibody array phosphorylation profiling of HDFs. The results suggested that the peptide GGFDL regulates ultraviolet-B-induced MMP-1 expression by inhibiting mitogen-activated protein kinases and nuclear factor κB signaling pathways as well as histone modification. Thus, LMW feather keratin peptides could serve as novel bioactive compounds to protect the skin against intrinsic and extrinsic factors.

Effect of 3,6-anhydro-l-galactose on α-melanocyte stimulating hormone-induced melanogenesis in human melanocytes and a skin-equivalent model.

3,6-Anhydro-l-galactose (l-AHG) is a bioactive sugar that is a major component of agarose. Recently, l-AHG was reported to have anti-melanogenic potential in human epidermal melanocytes (HEMs) and B16F10 melanoma cells; however, its underlying molecular mechanisms remain unknown. At noncytotoxic concentrations, l-AHG has been shown to inhibit alpha-melanocyte-stimulating hormone-induced melanin synthesis in various cell models, including HEMs, melan-a cells, and B16F10 cells. Although l-AHG did not inhibit tyrosinase activity in vitro, reverse transcription-polymerase chain reaction results demonstrated that the anti-melanogenic effect of l-AHG was mediated by transcriptional repression of melanogenesis-related genes, including tyrosinase, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and microphthalmia-associated transcription factor (MITF) in HEMs. Western blot analysis showed that l-AHG effectively attenuated α-melanocyte-stimulating hormone-induced melanogenic proteins by inhibiting cyclic adenosine monophosphate/cyclic adenosine monophosphate-dependent protein kinase, mitogen-activated protein kinase, and Akt signaling pathways in HEMs. Topical application of l-AHG significantly ameliorated melanin production in a 3D pigmented human skin model. Collectively, these results suggest that l-AHG could be utilized as novel cosmetic compounds with skin-whitening efficacy.

Different Levels of Skin Whitening Activity among 3,6-Anhydro-l-galactose, Agarooligosaccharides, and Neoagarooligosaccharides.

3,6-Anhydro-l-galactose (AHG), a major monomeric constituent of red macroalgae (Rhodophyta), was recently reported to possess skin whitening activity. Moreover, AHG-containing oligosaccharides, such as agarooligosaccharides (AOSs) and neoagarooligosaccharides (NAOSs), have various physiological activities, including anti-inflammatory, antioxidant, and skin moisturizing effects. In this study, AHG and NAOSs were produced from agarose by enzymatic reactions catalyzed by an endo-type ß-agarase, an exo-type ß-agarase, and a neoagarobiose hydrolase. In a cell proliferation assay, AHG, AOSs, and NAOSs at 12.5, 25, and 50 µg/mL concentrations did not exhibit cytotoxicity toward murine B16 melanoma cells or human epidermal melanocytes. In an in vitro skin whitening activity assay of AHG, AOSs, and NAOSs at 50 µg/mL, AHG showed the highest skin whitening activity in both murine B16 melanoma cells and human epidermal melanocytes; this activity was mediated by the inhibition of melanogenesis. Neoagarotetraose and neoagarohexaose also exhibited in vitro skin whitening activity, whereas neoagarobiose and AOSs with degrees of polymerization of 3 (agarotriose), 5 (agaropentaose), and 7 (agaroheptaose) did not. Therefore, AHG is responsible for the skin whitening activity of agar-derived sugars, and the structural differences among the AHG-containing oligosaccharides may be responsible for their different skin whitening activities.

A Combination of Soybean and Haematococcus Extract Alleviates Ultraviolet B-Induced Photoaging.

Soybean-derived isoflavones have been investigated for their preventative effects against UV-induced symptoms of skin damage including wrinkle formation and inflammation. Haematococcus pluvialis is a freshwater species of Chlorophyta that contains high concentrations of the natural carotenoid pigment astaxanthin. Astaxanthin is known to be involved in retinoic acid receptor (RAR) signaling and previously been associated with the inhibition of activator protein (AP)-1 dependent transcription. Based on previous studies, we hypothesized that a combination of soy extract (SE) and Haematococcus extract (HE) may prevent UVB-induced photoaging through specific signaling pathways, as measured by UVB-induced wrinkling on hairless mice skin and expression changes in human dermal fibroblasts (HDFs). The 1:2 ratio of SE and HE mixture (SHM) showed the optimal benefit in vivo. SHM was found to inhibit wrinkle formation via the downregulation of matrix metalloproteinase (MMP)-1 mRNA and protein expression. SHM also inhibited mitogen-activated protein kinase (MAPK) phosphorylation and the transactivation of AP-1 which plays an important role in regulating MMP expression. These results highlight the potential for SHM to be developed as a therapeutic agent to prevent UVB-induced skin wrinkling.

20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol, a metabolite of ginsenoside Rb1, enhances the production of hyaluronic acid through the activation of ERK and Akt mediated by Src tyrosin kinase in human keratinocytes.

The aim of the present study was to determine the mechanisms through which 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol (20GPPD) promotes the production of hyaluronic acid (HA) in human keratinocytes. 20GPPD is the primary bioactive metabolite of Rb1, a major ginsenoside found in ginseng (Panax ginseng). We sought to elucidate the underlying mechanisms behind the 20GPPD-induced production of HA. We found that 20GPPD induced an increase in HA production by elevating hyaluronan synthase 2 (HAS2) expression in human keratinocytes. The phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was also enhanced by 20GPPD in a dose-dependent manner. The pharmacological inhibition of ERK (using U0126) or Akt (using LY294002) suppressed the 20GPPD-induced expression of HAS2, whereas treatment with an epidermal growth factor receptor (EGFR) inhibitor (AG1478) or an intracellular Ca2+ chelator (BAPTA/AM) did not exert any observable effects. The increased Src phosphorylation was also confirmed following treatment with 20GPPD in the human keratinocytes. Following pre-treatment with the Src inhibitor, PP2, both HA production and HAS2 expression were attenuated. Furthermore, the 20GPPD-enhanced ERK and Akt signaling decreased following treatment with PP2. Taken together, our results suggest that Src kinase plays a critical role in the 20GPPD-induced production of HA by acting as an upstream modulator of ERK and Akt activity in human keratinocytes.

Ginsenoside F1 attenuates hyperpigmentation in B16F10 melanoma cells by inducing dendrite retraction and activating Rho signalling.

Ginsenoside F1 (GF1) is a metabolite produced by hydrolysis of the ginsenoside Re and Rg1 in Panax ginseng. According to various studies, high amounts of ginseng components are absorbed in the metabolized form, which are key constituents responsible for the biological effects of P. ginseng. Recently, GF1 was reported to have beneficial effects on skin. However, there has not been a sound understanding of its antimelanogenic effect and underlying molecular mechanisms. In this study, GF1 reduced α-melanocyte-stimulating hormone-induced melanin secretion in B16F10 cell culture media by 60%. However, it did not suppress intracellular melanin levels, tyrosinase activity and expression. Immunofluorescence assay showed that GF1 had no effect on melanosome transport, but significantly induced dendrite retraction. Pull-down assay demonstrated that GF1 primarily modulates the Rho family GTPases resulting in dendrite retraction. Collectively, these data suggest that GF1 could act as a potent skin-whitening agent.

20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol suppresses UV-Induced MMP-1 expression through AMPK-mediated mTOR inhibition as a downstream of the PKA-LKB1 pathway.

Various health effects have been attributed to the ginsenoside metabolite 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol (GPD); however, its effect on ultraviolet (UV)-induced matrix metalloproteinase (MMP)-1 expression and the mechanism underlying this effect are unknown. We examined the inhibitory effect of GPD on UV-induced MMP-1 expression and its mechanisms in human dermal fibroblasts (HDFs). GPD attenuated UV-induced MMP-1 expression in HDFs and suppressed the UV-induced phosphorylation of mammalian target of rapamycin (mTOR) and p70(S6K) without inhibiting the activity of phosphatidylinositol 3-kinase and Akt, which are well-known upstream kinases of mTOR. GPD augmented the phosphorylation of liver kinase B1 (LKB1) and adenosine monophosphate-activated protein kinase (AMPK), which are inhibitors of mTOR, to a greater extent than UV treatment alone. Similar to GPD, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranosyl 5'-monophosphate (AICAR), an activator of AMPK, augmented UV-induced AMPK phosphorylation to a greater extent than UV treatment alone, resulting in the inhibition of MMP-1 expression. AICAR also decreased the phosphorylation of mTOR and p70(S6K). However, compound C, an antagonist of AMPK, increased MMP-1 expression. In HDF cells with AMPK knock-down using shRNA, MMP-1 expression was increased. These results indicate that AMPK activation plays a key role in MMP-1 suppression. Additionally, the cAMP-dependent protein kinase (PKA) inhibitor, H-89, antagonized GPD-mediated MMP-1 suppression via the inhibition of LKB1. Our results suggest that the suppressive activity of GPD on UV-induced MMP-1 expression is due to the activation of AMPK as a downstream of the PKA-LKB1 pathway.

Quercetin suppresses invasion and migration of H-Ras-transformed MCF10A human epithelial cells by inhibiting phosphatidylinositol 3-kinase.

Quercetin is a major flavonoid compound found in red wine at a much higher concentration than the phytoalexin resveratrol. In this study, we examined potential anti-metastatic effects and found that compared to resveratrol, quercetin more potently inhibits H-Ras-induced invasion and migration in MCF10A human epithelial cells, an effect likely mediated by the mitigation of matrix metalloproteinase (MMP)-2 activation. We then measured Akt phosphorylation to investigate whether the decreased MMP-2 activation was attributable to the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signalling. Quercetin, but not resveratrol at equivalent concentrations, suppressed the phosphorylation of Akt and was a more potent inhibitor of PI3K activity than resveratrol. An ex vivo binding assay further revealed that quercetin directly binds to PI3K. Collectively, these results suggest that PI3K is a molecular target of quercetin for the inhibition of H-Ras-induced invasion and migration of MCF10A cells.

7,3',4'-Trihydroxyisoflavone Ameliorates the Development of Dermatophagoides farinae-Induced Atopic Dermatitis in NC/Nga Mice.

Atopic dermatitis is an inflammatory and chronically relapsing skin disorder that commonly occurs in children; the number of atopic dermatitis patients is increasing. The cause and mechanism of atopic dermatitis have not been defined clearly, although many studies are ongoing. Epidemiological studies suggest that soybean and its isoflavones have immunoregulatory activities. Here, we report that 7,3',4'-trihydroxyisoflavone (7,3',4'-THIF), a major metabolite of daidzin, effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO), tumor necrosis factor (TNF)- α , and interleukin (IL)-6 production in RAW 264.7 cells, and also reduced ß -hexosaminidase secretion in RBL-2H3 cells. Moreover, 7,3',4'-THIF significantly reduced scratching time, transepidermal water loss, and mast cell infiltration. It also decreased protease-activated receptor (PAR)-2 and IL-4 expression and increased filaggrin expression in skin lesions of NC/Nga mice. These results suggest that 7,3',4'-THIF improves Dermatophagoides farina body extract-induced atopic dermatitis in NC/Nga mice.

Rutin inhibits B[a]PDE-induced cyclooxygenase-2 expression by targeting EGFR kinase activity.

Rutin is a well-known flavonoid that exists in various natural sources. Accumulative studies have represented the biological effects of rutin, such as anti-oxidative and anti-inflammatory effects. However, the underlying mechanisms of rutin and its direct targets are not understood. We investigated whether rutin reduced B[a]PDE-induced-COX-2 expression. The transactivation of AP-1 and NF-κB were inhibited by rutin. Rutin also attenuated B[a]PDE-induced Raf/MEK/ERK and Akt activation, but had no effect on the phosphorylation of EGFR. An in vitro kinase assay revealed rutin suppressed EGFR kinase activity. We also confirmed direct binding between rutin and EGFR, and found that the binding was regressed by ATP. The EGFR inhibitor also inhibited the B[a]PDE-induced MEK/ERK and Akt signaling pathways and subsequently, suppressed COX-2 expression and promoter activity, in addition to suppressing the transactivation of AP-1 and NF-κB. In EGFR(-/-)mouse embryonic fibroblast cells, B[a]PDE-induced COX-2 expression was also diminished. Collectively, rutin inhibits B[a]PDE-induced COX-2 expression by suppressing the Raf/MEK/ERK and Akt signaling pathways. EGFR appeared to be the direct target of rutin.

Enzymatic production of 3,6-anhydro-L-galactose from agarose and its purification and in vitro skin whitening and anti-inflammatory activities.

3,6-Anhydro-L-galactose (L-AHG) constitutes 50% of agarose, which is the main component of red macroalgae. No information is currently available on the mass production, metabolic fate, or physiological effects of L-AHG. Here, agarose was converted to L-AHG in the following three steps: pre-hydrolysis of agarose into agaro-oligosaccharides by using acetic acid, hydrolysis of the agaro-oligosaccharides into neoagarobiose by an exo-agarase, and hydrolysis of neoagarobiose into L-AHG and galactose by a neoagarobiose hydrolase. After these three steps, L-AHG was purified by adsorption and gel permeation chromatographies. The final product obtained was 95.6% pure L-AHG at a final yield of 4.0% based on the initial agarose. In a cell proliferation assay, L-AHG at a concentration of 100 or 200 µg/ mL did not exhibit any significant cytotoxicity. In a skin whitening assay, 100 µg/ mL of L-AHG showed significantly lower melanin production compared to arbutin. L-AHG at 100 and 200 µg/ mL showed strong anti-inflammatory activity, indicating the significant suppression of nitrite production. This is the first report on the production of high-purity L-AHG and its physiological activities.

Structural and functional analysis of the natural JNK1 inhibitor quercetagetin.

c-Jun NH2-terminal kinases (JNKs) and phosphatidylinositol 3-kinase (PI3-K) play critical roles in chronic diseases such as cancer, type II diabetes, and obesity. We describe here the binding of quercetagetin (3,3',4',5,6,7-hydroxyflavone), related flavonoids, and SP600125 to JNK1 and PI3-K by ATP-competitive and immobilized metal ion affinity-based fluorescence polarization assays and measure the effect of quercetagetin on JNK1 and PI3-K activities. Quercetagetin attenuated the phosphorylation of c-Jun and AKT, suppressed AP-1 and NF-κB promoter activities, and also reduced cell transformation. It attenuated tumor incidence and reduced tumor volumes in a two-stage skin carcinogenesis mouse model. Our crystallographic structure determination data show that quercetagetin binds to the ATP-binding site of JNK1. Notably, the interaction between Lys55, Asp169, and Glu73 of JNK1 and the catechol moiety of quercetagetin reorients the N-terminal lobe of JNK1, thereby improving compatibility of the ligand with its binding site. The results of a theoretical docking study suggest a binding mode of PI3-K with the hydroxyl groups of the catechol moiety forming hydrogen bonds with the side chains of Asp964 and Asp841 in the p110γ catalytic subunit. These interactions could contribute to the high inhibitory activity of quercetagetin against PI3-K. Our study suggests the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases.

Myricetin is a potent chemopreventive phytochemical in skin carcinogenesis.

Myricetin is a widely distributed flavonol that is found in many plants, including tea, berries, fruits, vegetables, and medicinal herbs. Abundant sources provide interesting insights into the multiple mechanisms by which myricetin mediates chemopreventive effects on skin cancer. Myricetin strongly inhibited tumor promoter-induced neoplastic cell transformation by inhibiting MEK, JAK1, Akt, and MKK4 kinase activity directly. In a mouse skin model, myricetin attenuated the ultraviolet B (UVB)-induced COX-2 expression and skin tumor formation by regulating Fyn. Myricetin-mediated inactivation of Akt in the UVB response plays a role in regulating UVB-induced carcinogenesis. Recently, myricetin was found to inhibit UVB-induced angiogenesis by targeting PI3-K in an SKH-1 hairless mouse skin tumorigenesis model. Raf kinase is a critical target for myricetin in inhibiting the UVB-induced formation of wrinkles and suppression of type I procollagen and collagen levels in mouse skin. Accumulated data suggest that myricetin acts as a promising agent for the chemoprevention of skin cancer.

Luteolin, a novel natural inhibitor of tumor progression locus 2 serine/threonine kinase, inhibits tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in JB6 mouse epidermis cells.

Targeting tumor necrosis factor (TNF)-α-mediated signal pathways may be a promising strategy for developing chemopreventive agents, because TNF-α-mediated cyclooxygenase (COX)-2 expression plays a key role in inflammation and carcinogenesis. Luteolin [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone] exerts anticarcinogenic effects, although little is known about the underlying molecular mechanisms and specific targets of this compound. In the present study, we found that luteolin inhibited TNF-α-induced COX-2 expression by down-regulating the transactivation of nuclear factor-κB and activator protein-1. Furthermore, luteolin inhibited TNF-α-induced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/ERK/p90(RSK), mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase/c-Jun, and Akt/p70(S6K). However, it had no effect on the phosphorylation of p38. These effects of luteolin on TNF-α-mediated signaling pathways and COX-2 expression are similar to those achieved by blocking tumor progression locus 2 serine/threonine kinase (TPL2) using pharmacologic inhibitors and small interfering RNAs. Luteolin inhibited TPL2 activity in vitro and in TPL2 immunoprecipitation kinase assays by binding directly in an ATP-competitive manner. Overall, these results indicate that luteolin exerts potent chemopreventive activities, which primarily target TPL2.

7,3',4'-Trihydroxyisoflavone, a metabolite of the soy isoflavone daidzein, suppresses ultraviolet B-induced skin cancer by targeting Cot and MKK4.

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3',4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3',4'-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3',4'-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3',4'-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3',4'-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3',4'-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3',4'-THIF, a potential skin cancer chemopreventive agent.