Catechins inhibit angiotensin II-induced vascular smooth muscle cell proliferation via mitogen-activated protein kinase pathway

Catechins, components of green tea, reduce the incidence of cardiovascular diseases such as atherosclerosis. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMC), resulting in atherosclerosis. The acting mechanisms of the catechins remain to be defined in the proliferation of VSMC induced by Ang II. Here we report that catechin, epicatechin (EC), epicatechingallate (ECG) or epigallocatechingallate (EGCG) significantly inhibits the Ang II-induced [3H]thymidine incorporation into the primary cultured rat aortic VSMC. Ang II increases the phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK 1/2), c-jun-N-terminal kinase 1/2 (JNK 1/2), or p38 mitogen-activated protein kinases (MAPKs) and mRNA expression of c-jun and c-fos. The EGCG pretreatment inhibits the Ang II-induced phosphorylation of ERK 1/2, JNK 1/2, or p38 MAPK, and the expression of c-jun or c-fos mRNA. U0126, a MEK inhibitor, SP600125, a JNK inhibitor, or SB203580, a p38 inhibitor, attenuates the Ang II-induced [3H]thymidine incorporation into the VSMC. In conclusion, catechins inhibit the Ang II-stimulated VSMC proliferation via the inhibition of the Ang II-stimulated activation of MAPK and activator protein-1 signaling pathways. The antiproliferative effect of catechins may be associated with the reduced risk of cardiovascular diseases by the intake of green tea. Catechins may be useful in the development of prevention and therapeutics of vascular diseases.     

PKCalpha induces differentiation through ERK1/2 phosphorylation in mouse keratinocytes

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKCa isoform. When PKCalpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKCalpha kinase active mutant (PKCalpha- CAT) in the undifferentiated keratinocyte, but not PKCbeta-CAT, also increased differentiation marker expressions. On the other hand, PKCalpha dominant negative mutant (PKCbeta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKCbeta-KR did not, suggesting that PKCalpha is responsible for keratinocyte differentiation. When downstream pathway of PKCalpha in Ca2+ -mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+ -mediated differentiation, and that only ERK1/2 pathway is specific for PKCalpha-mediated differentiation in mouse keratinocytes.     

Bucillamine Inhibits UVB-Induced MAPK Activation and Apoptosis in Human HaCaT Keratinocytes and SKH-1 Hairless Mouse Skin

Ultraviolet B (UVB) radiation is known as a culprit in skin carcinogenesis. We have previously reported that bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine), a cysteine derivative with antioxidant and anti-inflammatory capacity, protects against UVB-induced p53 activation and inflammatory responses in mouse skin. Since MAPK signaling pathways regulate p53 expression and activation, here we determined bucillamine effect on UVB-mediated MAPK activation in vitro using human skin keratinocyte cell line HaCaT and in vivo using SKH-1 hairless mouse skin. A single low dose of UVB (30 mJ cm⁻²) resulted in increased JNK/MAPK phosphorylation and caspase-3 cleavage in HaCaT cells. However, JNK activation and casaspe-3 cleavage were inhibited by pretreatment of HaCaT cells with physiological doses of bucillamine (25 and 100 µm ). Consistent with these results, bucillamine pretreatment in mice (20 mg kg⁻¹) inhibited JNK/MAPK and ERK/MAPK activation in skin epidermal cells at 6–12 and 24 h, respectively, after UVB exposure. Moreover, bucillamine attenuated UVB-induced Ki-67-positive cells and cleaved caspase-3-positive cells in mouse skin. These findings demonstrate that bucillamine inhibits UVB-induced MAPK signaling, cell proliferation and apoptosis. Together with our previous report, we provide evidence that bucillamine has a photoprotective effect against UV exposure.     

UVB-induced epidermal growth factor receptor phosphorylation is critical for downstream signaling and keratinocyte survival

We have recently shown that UVB radiation activates epidermal growth factor receptor (EGFR)/extracellular regulated kinase 1 and 2 (ERK1/2) and p38 signaling pathways in keratinocytes. However, the functional relevance of these processes for downstream signaling and cell survival remains to be determined. The specific EGFR inhibitor PD153035 markedly decreased UVB-induced phosphorylation of EGFR, ERK1/2 and shc, whereas p38 activation was unaffected. PD153035 pretreatment followed by UVB reduced clonogenic potential and enhanced peroxide production, apoptosis and cell death. Our data suggest that ligand-independent phosphorylation of EGFR and likely dependent downstream signaling pathways regulate cellular defense mechanisms important for cell survival following oxidative stress.     

Protective Effect of 3,4-Dihydroxybenzoic Acid Isolated from Cladophora wrightiana Harvey Against Ultraviolet B Radiation-Induced Cell Damage in Human HaCaT Keratinocytes

The aim of the present study was to elucidate the protective properties of 3,4-dihydroxybenzoic acid (DBA) isolated from Cladophora wrightiana Harvey (a green alga) against ultraviolet B (UVB)-induced damage to human HaCaT keratinocytes. DBA exhibited scavenging actions against the 1,1-diphenyl-2-picrylhydrazyl radical, the superoxide anion, and the hydroxyl radical. Furthermore, DBA decreased the levels of intracellular reactive oxygen species generated by hydrogen peroxide or UVB treatment of the cells. DBA also decreased the UVB-augmented levels of phospho-histone H2A.X and the extent of comet tail formation, which are both indications of DNA damage. In addition, the compound safeguarded keratinocytes from UVB-induced injury by reversing the production of apoptotic bodies, overturning the disruption of mitochondrial membrane potential, increasing the expression of the anti-apoptotic protein, B-cell lymphoma 2, and decreasing the expression of the pro-apoptotic proteins, Bcl-2-associated X and cleaved caspase-3. Taken together, these results demonstrate that DBA isolated from a green alga protects human keratinocytes against UVB-induced oxidative stress and apoptosis.     

Green tea polyphenols: DNA photodamage and photoimmunology.

Green tea is a popular beverage consumed worldwide. The epicatechin derivatives, which are commonly called 'polyphenols', are the active ingredients in green tea and possess antioxidant, anti-inflammatory and anti-carcinogenic properties. Studies conducted by our group on human skin have demonstrated that green tea polyphenols (GTP) prevent ultraviolet (UV)-B-induced cyclobutane pyrimidine dimers (CPD), which are considered to be mediators of UVB-induced immune suppression and skin cancer induction. GTP treated human skin prevented penetration of UV radiation, which was demonstrated by the absence of immunostaining for CPD in the reticular dermis. The topical application of GTP or its most potent chemopreventive constituent (-)-epigallocatechin-3-gallate (EGCG) prior to exposure to UVB protects against UVB-induced local as well as systemic immune suppression in laboratory animals. Additionally, studies have shown that EGCG treatment of mouse skin inhibits UVB-induced infiltration of CD11b+ cells. CD11b is a cell surface marker for activated macrophages and neutrophils, which are associated with induction of UVB-induced suppression of contact hypersensitivity responses. EGCG treatment also results in reduction of the UVB-induced immunoregulatory cytokine interleukin (IL)-10 in skin as well as in draining lymph nodes, and an elevated amount of IL-12 in draining lymph nodes. These in vivo observations suggest that GTPs are photoprotective, and can be used as pharmacological agents for the prevention of solar UVB light-induced skin disorders associated with immune suppression and DNA damage.     

Inhibition of UVB-mediated oxidative stress and markers of photoaging in immortalized HaCaT keratinocytes by pomegranate polyphenol extract POMx

In recent years there has been an increase in use of botanicals with antioxidant properties as skin photoprotective agents. Pomegranate (Punica granatum L.) fruit possesses strong antioxidant and antiinflammatory properties. Recently, we have shown that pomegranate-derived products rich in anthocyanidins and ellagitannins inhibit UVB-mediated activation of nuclear factor kappa B and modulate UVA-mediated cell proliferation pathways in normal human epidermal keratinocytes. In this study, we evaluated the effect of polyphenol-rich pomegranate fruit extract (POMx) on UVB-induced oxidative stress and photoaging in human immortalized HaCaT keratinocytes. Our data show that pretreatment of HaCaT cells with POMx (10-40 microg mL(-1)) inhibited UVB (15-30 mJ cm(-2))-mediated (1) decrease in cell viability, (2) decrease in intracellular glutathione content and (3) increase in lipid peroxidation. Employing immunoblot analysis we found that pretreatment of HaCaT cells with POMx inhibited UVB-induced (1) upregulation of MMP-1, -2, -7 and -9, (2) decrease in TIMP-1, (3) phosphorylation of MAPKs and (iv) phosphorylation of c-jun, whereas no effect was observed on UVB-induced c-fos protein levels. These results suggest that POMx protects HaCaT cells against UVB-induced oxidative stress and markers of photoaging and could be a useful supplement in skin care products.     

Oligonol inhibits UVB-induced COX-2 expression in HR-1 hairless mouse skin--AP-1 and C/EBP as potential upstream targets

Oxidative stress and inflammatory tissue damage are two major events frequently implicated in carcinogenesis. Numerous polyphenolic compounds derived from plants possess antioxidant and anti-inflammatory activities and are hence effective in preventing cancer. Oligonol is a polyphenol formulation enriched with catechin-type oligomers. As an initial approach to assess the chemopreventive potential of oligonol, we have determined its effects on inflammatory as well as oxidative damage in mouse skin irradiated with UVB. Topical application of oligonol onto the dorsal skin of male HR-1 hairless mice 30 min prior to UVB exposure diminished epidermal hyperplasia and formation of 4-hydroxynonenal, a biochemical hallmark of lipid peroxidation. Topical application of oligonol also significantly inhibited UVB-induced cyclooxygenase (COX-2) expression in mouse skin. Oligonol diminished the DNA binding of activator protein-1 (AP-1) and CCAAT/enhancer binding protein (C/EBP), and the expression of C/EBPdelta in mouse skin exposed to UVB. Our study also revealed that oligonol attenuated UVB-induced catalytic activity as well as expression of p38 mitogen-activated protein (MAP) kinase. Moreover, UVB-induced phosphorylation of another upstream kinase Akt was attenuated by oligonol. Taken together, oligonol showed antioxidative and anti-inflammatory effects in UVB-irradiated mouse skin by inhibiting COX-2 expression via blockade of the activation of AP-1 and C/EBP, and upstream kinases including p38 MAP kinase and Akt.     

Ionizing radiation induces blockade of c-Jun N-terminal kinase-dependent cell death pathway in a manner correlated with p21(Cip/WAF1) induction in primary cultured normal human fibroblasts

During radiotherapy of cancer, neighboring normal cells may receive sub-lethal doses of radiation. To investigate whether such low levels of radiation modulate normal cell responses to death stimuli, primary cultured human fibroblasts were exposed to various doses of gamma-rays. Analysis of cell viability using an exclusion dye propidium iodide revealed that the irradiation up to 10 Gy killed the fibroblasts only to a minimal extent. In contrast, the cells efficiently lost their viability when exposed to 0.5-0.65 mM H(2)O(2). This type of cell death was accompanied by JNK activation, and was reversed by the use of a JNK-specific inhibitor SP600125. Interestingly, H(2)O(2) failed to kill the fibroblasts when these cells were pre-irradiated, 24 h before H(2)O(2) treatment, with 0.25-0.5 Gy of gamma-rays. These cytoprotective doses of gamma-rays did not enhance cellular capacity to degrade H(2)O(2), but elevated cellular levels of p21(Cip/WAF1), a p53 target that can suppress H(2)O(2)-induced cell death by blocking JNK activation. Consistently, H(2)O(2)-induced JNK activation was dramatically suppressed in the pre-irradiated cells. The overall data suggests that ionizing radiation can impart normal fibroblasts with a survival advantage against oxidative stress by blocking the process leading to JNK activation.     

Electrophoretic studies on the phosphorylation of stathmin and mitogen-activated protein kinases in neuronal cell death induced by oxidized very-low-density lipoprotein with apolipoprotein E

In the central nervous system, stressful conditions can easily cause the oxidation of lipoprotein particles, followed by the oxidative modification of apolipoproteins such as apolipoprotein E (apoE) and the production of free radicals and aldehydes. We have confirmed that oxidized very-low-density lipoprotein (VLDL) inhibits the proliferation, viability and differentiation of neuronal PC12 cells leading to cell death. The cells internalized intact apoE, but did not internalize oxidized apoE. The phosphorylation of stathmin and various mitogen-activated protein (MAP) kinases including extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) was examined in PC12 cells exposed to native and oxidized VLDL, H(2)O(2) (which generates free radicals), and 4-hydroxy-2-nonenal (HNE) (an aldehyde). Oxidized VLDL and H(2)O(2) reduced stathmin phosphorylation while HNE increased it, suggesting that oxidized VLDL and H(2)O(2) stimulated similar signal transduction pathways. Based on the results, free radicals, but not aldehydes may play a major role in the neuronal cell death induced by lipoprotein oxidation. Furthermore, the phosphorylation status of MAP kinases indicated that the activation of the JNK cascade might be required for neuronal cell death.     

绿茶多酚对自由基诱导的红细胞膜过氧化的抑制作用

采用水溶性偶氮引发剂2,2'-偶氮二(2-脒基丙烷)二盐酸盐(AAPH)在37引发入血红细胞膜的过氧化,通过测定氧气吸收及维生素E的消耗研究了过氧化过程的动力学,并对从绿茶中提取的主要多酚类化合物的抗氧化活性做了定量研究。使用的绿茶多酚有:(-)-表儿茶素(EC),(-)-表儿茶素(EGC),(-)-表儿茶素酸酯(ECG)和(-)-表儿茶素培酸酯(EGCG)。结果表明,这些绿茶多酚能够显著缩短过氧化反应的动力学链长,有效地抑制红细胞膜的过氧化。抗氧化活性顺序为:EC〉GCG〉EGCG〉EGC。     

EGCG在Tween80胶束体系中的性质(英文)

通过离子萃取技术,从黄山绿茶中提取高纯度儿茶素没食子酸酯(EGCG).在Tween 80/EGCG/H2O体系中,随着EGCG浓度增加,Tween 80临界胶束浓度和胶束动力学半径增大;随着Tween 80浓度增加,EGCG和Tween 80扩散系数减小,而EGCG的紫外-可见吸收光强度和荧光强度增加.对EGCG在Tween 80胶束中的定位也进行了讨论.     

Activation of c-Jun N-terminal kinase is required for ultraviolet B-induced apoptosis of murine peritoneal macrophages in vitro

The mechanisms of ultraviolet B (UVB)-induced apoptosis and the role of c-Jun N-terminal kinase (JNK) mitogen activated protein kinase (MAPK) in murine peritoneal macrophages, the terminally differentiated non-dividing cells were investigated. Exposure of macrophages to UVB 100 mJ/cm2 induced rapid apoptosis concurrent with activation of JNK and mitochondrial cytochrome c release leading to procaspase-3 activation. Late into the UVB-induced apoptosis, a caspase-mediated cleavage of Bid was observed. Caspase inhibitors N-Benzylocarbonyl-Val-Asp-fluoromethyl ketone and N-Acetyl-Asp-Glu-Val-Asp-aldehyde inhibited the UVB-induced apoptosis without preventing the release of cytochrome c and JNK activation. The inhibition of JNK MAPK prevented UVB-induced apoptosis, concomitant with inhibition in cytochrome c release and procaspase-3 activation. However, it had no effect on procaspase-8 activation. These results indicate that activation of JNK MAPK upstream of caspases might play an important role in the apoptotic process of macrophages exposed to UVB irradiation.     

Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea reduces UVB-induced inflammatory responses and infiltration of leukocytes in human skin

Identification of natural products capable of affording protection against UVB radiation-induced inflammatory responses and generation of oxidative stress may have important human health implications. The UVB exposure-induced skin injury and oxidative stress has been associated with a variety of skin disease conditions including photoaging, inflammation and cancer. Tea is a popular beverage consumed worldwide. In several mouse skin models, topical application as well as oral consumption of green tea has been shown to afford protection against chemical and UVB-induced carcinogenesis and inflammatory responses. In the present study, we investigated in human skin, whether topical application of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent in green tea, inhibits UVB-induced infiltration of leukocytes (macrophage/neutrophils), a potential source of generation of reactive oxygen species (ROS), and generation of prostaglandin (PG) metabolites. Human subjects were UVB irradiated on sun-protected skin to four times their minimal erythema dosage (MED) and skin biopsies or keratomes were obtained either 24 h or 48 h later. We found that topical application of EGCG (3 mg/2.5 cm2) before UVB (4 MED) exposure to human skin significantly blocked UVB-induced infiltration of leukocytes and reduced myeloperoxidase activity. These infiltrating leukocytes are considered to be the major source of generation of ROS. In the same set of experiments we found that topical application of EGCG before UVB exposure decreased UVB-induced erythema. In additional experiments, we found that microsomes from EGCG pretreated human skin and exposed to UVB, compared to UVB exposure alone, produced significantly reduced PG metabolites, particularly PGE2. The PG metabolites play a critical role in free radical generation and skin tumor promotion in multistage skin carcinogenesis. Careful microscopic examination of skin sections, stained with hematoxylin and eosin, under higher magnification (x400) also revealed that EGCG pretreated and UVB-exposed human skin contained fewer dead cells in the epidermis with comparison to nonpretreated UVB-exposed skin. Taken together, our data demonstrate that EGCG has the potential to block the UVB-induced infiltration of leukocytes and the subsequent generation of ROS in human skin. This may explain the possible mechanism involved in anti-inflammatory effects of green tea. We suggest that EGCG may be useful as a topical agent for protection against UVB-induced ROS-associated inflammatory dermatoses, photoaging and photocarcinogenesis. Further studies are warranted in this direction.     

Wnt5a stimulates chemotactic migration and chemokine production in human neutrophils

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (β-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.     

Nondenatured soy extracts reduce UVB-induced skin damage via multiple mechanisms

UV irradiation results in DNA damage, inflammation and immunosuppression, leading to the development of basal and squamous cell carcinomas. Earlier data show that topical treatment with nondenatured soy extracts reduced the incidence and delayed the development/progression of already-initiated skin tumors in high-risk hairless mice. Here we show that pretreatment with nondenatured soy extracts reduced UVB-induced Thymine-Thymine (TT) dimer formation. In vitro, nondenatured soy extracts enhanced UVB-induced checkpoint kinase-1 (Chk1) activation, suggesting a delay in cell cycle progression that enables longer time for DNA repair. Soy also reduced UVB-induced cyclo-oxygenase-2 (COX-2) expression and prostaglandin E2 secretion, and inhibited p38 MAP kinase activation, suggesting its anti-inflammatory activity. Mice pretreated topically with nondenatured soy extracts had reduced levels of UVB-induced TT dimers and COX-2 expression in their skins compared to UVB alone. The nondenatured soy extracts also inhibited vascular endothelial growth factor-induced endothelial tube formation in Matrigel, suggesting a possible inhibitory effect on angiogenesis and tumor progression. Taken together, nondenatured soy extracts could prevent or reduce UVB-induced skin damage via multiple mechanisms, affecting both the initiation and the progression of skin cancer. These data suggest that topical application of nondenatured soy extracts could potentially reduce the incidence of skin cancer.