Alla I. Potapovich

Plant polyphenols differentially modulate inflammatory responses of human keratinocytes by interfering with activation of transcription factors NFκB and AhR and EGFR-ERK pathway.

Molecular mechanisms underlying modulation of inflammatory responses in primary human keratinocytes by plant polyphenols (PPs), namely the glycosylated phenylpropanoid verbascoside, the stilbenoid resveratrol and its glycoside polydatin, and the flavonoid quercetin and its glycoside rutin were evaluated. As non-lethal stimuli, the prototypic ligand for epidermal growth factor receptor (EGFR) transforming growth factor alpha (TGFalpha), the combination of tumor necrosis factor (TNFalpha) and interferon (IFNgamma) (T/I), UVA+UVB irradiation, and bacterial lipopolysaccharide (LPS) were used. We demonstrated differential modulation of inflammatory responses in keratinocytes at signal transduction, gene transcription, and protein synthesis levels as a function of PP chemical structure, the pro-inflammatory trigger used, and PP interaction with intracellular detoxifying systems. The PPs remarkably inhibited constitutive, LPS- and T/I-induced but not TGFalpha-induced ERK phosphorylation. They also suppressed NFkappaB activation by LPS and T/I. Verbascoside and quercetin invariably impaired EGFR phosphorylation and UV-associated aryl hydrocarbon receptor (AhR)-mediated signaling, while rutin, polydatin and resveratrol did not affect EGFR phosphorylation and further activated AhR machinery in UV-exposed keratinocytes. In general, PPs down-regulated gene expression of pro-inflammatory cytokines/enzymes, except significant up-regulation of IL-8 observed under stimulation with TGFalpha. Both spontaneous and T/I-induced release of IL-8 and IP-10 was suppressed, although 50μM resveratrol and polydatin up-regulated IL-8. At this concentration, resveratrol activated both gene expression and de novo synthesis of IL-8 and AhR-mediated mechanisms were involved. We conclude that PPs differentially modulate the inflammatory response of human keratinocytes through distinct signal transduction pathways, including AhR and EGFR.

Arachidonic acid‐induced carbon‐centered radicals and phospholipid peroxidation in cyclo‐oxygenase‐2‐transfected PC12 cells

Cyclo‐oxygenase‐2 (COX‐2) is believed to induce neuronal oxidative stress via production of radicals. While oxygen radicals are not directly involved in COX‐2‐catalytic cycle, superoxide anion radicals have been repeatedly reported to play a critical role in COX‐2‐associated oxidative stress. To resolve the controversy, we characterized production of free radicals in PC12 cells in which COX‐2 expression was manipulated either genetically or by direct protein transfection and compared them with those generated by a recombinant COX‐2 in a cell‐free system. Using spin‐traps α‐(4‐pyridyl‐1‐oxide)‐N‐t‐butylnitrone, 5,5‐dimethyl‐1‐pyrroline‐N‐oxide and 4‐((9‐acridinecarbonyl) amino)‐2,2,6,6‐ tetramethylpiperidine‐1‐oxyl (Ac‐Tempo), we observed arachidonic acid (AA)‐dependent production of carbon‐centered radicals by heme‐reconstituted recombinant COX‐2. No oxygen radicals or thiyl radicals have been detected. COX‐2 also catalyzed AA‐dependent one‐electron co‐oxidation of ascorbate to ascorbate radicals. Next, we used two different approaches of COX‐2 expression in cells, PCXII cells which express isopropyl‐1‐thio‐β‐d‐galactopyranoside inducible COX‐2, and PC12 cells transfected with COX‐2 using a protein delivery reagent, Chariot. In both models, COX‐2‐dependent AA‐induced generation of carbon‐centered radicals was documented using spin‐traps and Ac‐Tempo. No oxygen radical formation was detected in COX‐2‐transfected cells by either spin‐traps or fluorogenic probe, dihydroethidium. In the presence of ascorbate, AA‐induced COX‐2‐dependent ascorbate radicals were detected. AA caused a significant and selective oxidation of one of the major phospholipids, phosphatidylserine (PS). PS was not a direct substrate for COX‐2 but was co‐oxidized in the presence of AA. The radical generation and PS oxidation were inhibited by COX‐2 inhibitors, niflumic acid, nimesulide, or NS‐398. Thus, COX‐2 generated carbon‐centered radicals but not oxygen radicals or thiyl radicals are responsible for oxidative stress in AA‐challenged PC12 cells overexpressing COX‐2.

Antioxidant and signal modulation properties of plant polyphenols in controlling vascular inflammation.

Oxidized low-density lipoproteins (oxLDL) play a critical role in the initiation of atherosclerosis through activation of inflammatory signaling. In the present work we investigated the role of antioxidant and signal modulation properties of plant polyphenols in controlling vascular inflammation. Significant decrease in intracellular NO level and superoxide overproduction was found in human umbilical vein endothelial cells (HUVEC) treated with oxLDL, but not with LDL. The redox imbalance was prevented by the addition of quercetin or resveratrol. Expression analysis of 14 genes associated with oxidative stress and inflammation revealed oxLDL-mediated up-regulation of genes specifically involved in leukocyte recruitment and adhesion. This up-regulation could be partially avoided by the addition of verbascoside or resveratrol, while treatment with quercetin resulted in a further increase in the expression of these genes. Lipopolysaccharide (LPS)-treated HUVEC were also used for the evaluation of anti-inflammatory potency of plant polyphenols. Significant differences between HUVEC treaded with oxLDL and LPS were found in both the expression pattern of inflammation-related genes and the effects of plant polyphenols on cellular responses. The present data indicate that plant polyphenols may affect vascular inflammation not only as antioxidants but also as modulators of inflammatory redox signaling pathways.

Photo-Oxidation Products of Skin Surface Squalene Mediate Metabolic and Inflammatory Responses to Solar UV in Human Keratinocytes

The study aimed to identify endogenous lipid mediators of metabolic and inflammatory responses of human keratinocytes to solar UV irradiation. Physiologically relevant doses of solar simulated UVA+UVB were applied to human skin surface lipids (SSL) or to primary cultures of normal human epidermal keratinocytes (NHEK). The decay of photo-sensitive lipid-soluble components, alpha-tocopherol, squalene (Sq), and cholesterol in SSL was analysed and products of squalene photo-oxidation (SqPx) were quantitatively isolated from irradiated SSL. When administered directly to NHEK, low-dose solar UVA+UVB induced time-dependent inflammatory and metabolic responses. To mimic UVA+UVB action, NHEK were exposed to intact or photo-oxidised SSL, Sq or SqPx, 4-hydroxy-2-nonenal (4-HNE), and the product of tryptophan photo-oxidation 6-formylindolo[3,2-b]carbazole (FICZ). FICZ activated exclusively metabolic responses characteristic for UV, i.e. the aryl hydrocarbon receptor (AhR) machinery and downstream CYP1A1/CYP1B1 gene expression, while 4-HNE slightly stimulated inflammatory UV markers IL-6, COX-2, and iNOS genes. On contrast, SqPx induced the majority of metabolic and inflammatory responses characteristic for UVA+UVB, acting via AhR, EGFR, and G-protein-coupled arachidonic acid receptor (G2A). Conclusions/Significance Our findings indicate that Sq could be a primary sensor of solar UV irradiation in human SSL, and products of its photo-oxidation mediate/induce metabolic and inflammatory responses of keratinocytes to UVA+UVB, which could be relevant for skin inflammation in the sun-exposed oily skin.

Differential modulation of stress-inflammation responses by plant polyphenols in cultured normal human keratinocytes and immortalized HaCaT cells

BACKGROUND Environmental and endogenous stresses to skin are considered causative reasons for skin cancers, premature ageing, and chronic inflammation. Screening of substances with preventive and/or curative properties is currently based on mechanistic studies of their effects towards stress-induced responses in skin cell cultures. OBJECTIVE We compared effects of plant polyphenols (PPs) on the constitutive, UVA-, LPS-, or TNF-alpha-induced inflammatory responses in cultured normal human epidermal keratinocytes (NHEK) and immortalized HaCaT cells. METHODS Representatives of three classes of PPs, flavonoids, stilbenoids, and phenylpropanoids were studied. Their effects on mRNA were determined by qRT-PCR; protein expression was assayed by Western blot and bioplexed ELISA; phosphorylation of Akt1, ERK1/2, EGFR, and NFkappaB was quantified by intracellular ELISA or Western blot. RESULTS PPs or their combination with UVA or LPS induced strong up-regulation of stress responses in HaCaT but not in NHEK. In addition, compared to NHEK, HaCaT responded to TNF-alpha with higher synthesis of MCP-1, IP-10 and IL-8, concomitant with stronger NFkappaB activation. PPs down-regulated the chemokine release from both cell types, although with distinct effects on NFkappaB, Akt1, ERK, and EGFR activation. CONCLUSION Results of pharmacological screenings obtained by using HaCaT should be cautiously considered while extending them to primary keratinocytes from human epidermis.

Plant polyphenols effectively protect HaCaT cells from ultraviolet C-triggered necrosis and suppress inflammatory chemokine expression.

Oxidative stress is a common response of epidermal cells to a variety of noxious stimuli such as ultraviolet (UV) radiation from solar light and proinflammatory cytokines from skin‐infiltrating leukocytes. Here, we report that two types of plant‐derived antioxidants, the phenylpropanoid glycoside verbascoside as well as the flavonoids rutin and quercetin possess protective effects against UVC‐induced cell damage and proinflammatory activation. The molecules under investigation were effective against the loss of cell integrity associated with necrosis in doses consistent with their antioxidant activity, whereas they did not significantly oppose UVC‐induced proliferation arrest and apoptosis. By contrast, only verbascoside effectively inhibited cytokine‐induced release of proinflammatory mediators in a dose‐dependent fashion. Verbascoside and its homologue teupolioside dramatically impaired NF‐κB and AP‐1 DNA binding activity. These results suggest that plant polyphenols with antioxidant properties have distinct mechanisms in the suppression of oxidative stress induced in keratinocytes by different stimuli. Verbascoside and teupolioside are hence of potential interest in the protection of the skin from both environmental and inflammatory insults.

Plant polyphenols against UV-C-induced cellular death.

The glycosylated phenylpropanoid verbascoside isolated from cultured cells of the medicinal plant Syringa vulgaris (Oleaceae) has previously been characterized as an effective scavenger of biologically active free radicals such as hydroxyl, superoxide, and nitric oxide, as a chelator of redox active transition metal ions (Fe (2+), Fe (3+), Cu (2+), and Ni (2+)), and an inhibitor of lipid peroxidation. In the present work, we have compared the cytoprotective effects of the biotechnologically produced verbascoside with two commercially available polyphenols (the glycosylated flavonoid rutin and its aglycone quercetin) against free radical-mediated UVC-induced cellular death in cultures of human keratinocytes (HaCaT) and breast cancer cells (MCF 7). We have shown that all the polyphenols studied afforded effective protection against UVC-induced necrosis and did not prevent UVC-induced apoptosis in both normal and tumor cell lines. The cytoprotection did not correlate either with UVC absorbance by polyphenols or with their superoxide radical scavenging properties. However, UVC protection strongly depended on the lipid peroxidation inhibiting and Fe (2+) chelating properties of polyphenols. We suggest that these plant polyphenols could be feasible for a photoprotection of human skin.

Oxidation and cytotoxicity of 6-OHDA are mediated by reactive intermediates of COX-2 overexpressed in PC12 cells

Parkinsons disease is characterized by a progressive loss of dopaminergic neurons, likely associated with dysregulation of oxidation of catechols, such as dopamine (DA) and 6-hydroxydopamine (6-OHDA), and resulting in oxidative stress. The involvement of cyclooxygenase-2 (COX-2) in pathogenesis of Parkinsons disease has been suggested. However, specific COX-2 triggered mechanisms participating in catalysis of DA oxidation and enhanced catechol-induced cytotoxicity remain poorly characterized. Here, we demonstrate that in a model biochemical system, recombinant heme-reconstituted COX-2 induced oxidation of 6-OHDA in the course of its peroxidase (H(2)O(2)-dependent) and cyclooxygenase (arachidonic acid (AA)-dependent) catalytic half-cycles. Similarly, COX-2 was able to stimulate 6-OHDA oxidation during its peroxidase- and cyclooxygenase half-cycles and caused oxidative stress in homogenates of PC12 cells stably overexpressing the enzyme (but not in mock-transfected cells). In addition, the increased levels of COX-2 were associated with enhanced cytotoxicity of 6-OHDA in stably transfected PC12 cells. Finally, co-oxidation of 6-OHDA by COX-2 triggered production of superoxide radicals critical for both propagation of 6-OHDA oxidation and induction of oxidative stress in COX-2 overexpressing cells. Thus, we conclude that both peroxidase and cyclooxygenase half-cycles of COX-2-catalyzed reactions are essential for COX-2-dependent activation of 6-OHDA oxidation, oxygen radical production, oxidative stress, and cytotoxicity.

Protective effect of natural flavonoids on rat peritoneal macrophages injury caused by asbestos fibers.

Exposure of macrophages to asbestos fibers resulted in enhancement of the production of oxygen radicals, determined by a lucigenin enhanced chemiluminescence (LEC) assay, a formation of thiobarbituric acid reactive substances (TBARS), a LDH release into the incubation mixture, and a rapid lysis of the cells. Rutin (Rut) and quercetin (Qr) were effective in inhibiting LEC, TBARS formation, and reducing peritoneal macrophages injury caused by asbestos. The concentrations pre-treatment of antioxidants that were required to prevent the injury of peritoneal macrophages caused by asbestos by 50% (IC50) were 90 microM and 290 microM for Qr and Rut, respectively. Both flavonoids were found to be oxidized during exposure of peritoneal macrophages to asbestos and the oxidation was SOD sensitive. The efficacy of flavonoids as antioxidant agents as well as superoxide ion scavengers was also evaluated using appropriate model systems, and both quercetin and rutin were found to be effective in scavenging O2.-. These findings indicate that flavonoids are able to prevent the respiratory burst in rat peritoneal macrophages exposed to asbestos at the stage of activated oxygen species generation, mainly as superoxide scavengers. On the basis of this study it was concluded that natural flavonoids quercetin and rutin would be promising drug candidates for a prophylactic asbestos-induced disease.

Effects of pre- and post-treatment with plant polyphenols on human keratinocyte responses to solar UV.

AbstractsBackgroundThe understanding of the anti-inflammatory mechanisms of action of plant polyphenols (PPs) and clarification of the relationship between their anti-inflammatory and antioxidant properties may result in a new therapeutic approach to skin cancers.ObjectiveTo elucidate the underlying mechanism, we analyzed the ability of PPs to attenuate inflammatory, metabolic and oxidative cellular responses to UV irradiation.MethodsNormal human epidermal keratinocytes (NHEK) were exposed to physiologically relevant dose of solar-simulated UV irradiation. Effects of pre- and post-treatment with PPs on the overproduction of peroxides and inflammatory mediators (mRNA and protein) were analyzed using real-time RT-PCR, enzyme-linked immunosorbent and fluorometric techniques.ResultsDifferences between the effectiveness of pre- and post-treatment with polyphenols was found. In particular, PPs post-treatment, but not pretreatment, completely abolished overexpression of Cyp1a1 and Cyp1b1 genes and elevation of intracellular peroxides in NHEK irradiated by UV. Post-treatment with PPs also more efficiently than pretreatment prevented UV-induced overexpression of IL-1 beta, IL-6 and COX2 mRNAs.ConclusionOur data strongly suggest that PPs predominantly affect delayed molecular and cellular events initiated in NHEK by solar UV rather than primary photochemical reactions. PPs may be important component in cosmetic formulations for post-sun skin care.