Cytotoxicity In HaCaT Cells Research Articles

Biomaterial-based dressings as vehicle for chitosan-encapsulated cabreuva essential oil: Cytotoxicity and regenerative activity

Chitosan-based emulsions functionalized with cabreuva essential oil (Myrocarpus fastigiatus) assembled with poly(vinyl alcohol) (PVA) were tailor-made to obtain a smooth, flexible and thin wound dressings capable for acting as a delivery vehicle. The cabreuva essential oil is composed mainly of trans-nerolidol, a natural compound with antimicrobial capacity that acts as a skin penetration enhancer. During the application of the dressing on skin explants, the penetration profile of cabreuva was analyzed through ex-vivo diffusion assays. Furthermore, the in-vivo essential oil delivery was followed by means of ATR-FTIR.The addition of chitosan-based emulsions produced modifications in the properties of the composite dressings associated with the water affinity and gas permeability as well as in the mechanical behavior. The dressing functionalized with cabreuva essential oil combined with chitosan proved its effectiveness against microorganisms such as S. aureus and S. epidermidis, capacity to produce cell regeneration after 24 h of contact time, and no cytotoxicity in HaCaT cells. The material developed could be used in superficial burns or minor wounds such as dressings due to its demonstrated functionality as controlled release devices of the cabreuva essential oil.

Evaluation of the Effectiveness of Crotoxin as an Antiseptic against Candida spp. Biofilms.

The growing number of oral infections caused by the Candida species are becoming harder to treat as the commonly used antibiotics become less effective. This drawback has led to the search for alternative strategies of treatment, which include the use of antifungal molecules derived from natural products. Herein, crotoxin (CTX), the main toxin of Crotalus durissus terrificus venom, was challenged against Candida tropicalis (CBS94) and Candida dubliniensis (CBS7987) strains by in vitro antimicrobial susceptibility tests. Minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and inhibition of biofilm formation were evaluated after CTX treatment. In addition, CTX-induced cytotoxicity in HaCaT cells was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay. Native CTX showed a higher antimicrobial activity (MIC = 47 μg/mL) when compared to CTX-containing mouthwash (MIC = 750 μg/mL) and nystatin (MIC = 375 μg/mL). Candida spp biofilm formation was more sensitive to both CTX and CTX-containing mouthwash (IC100 = 12 μg/mL) when compared to nystatin (IC100 > 47 μg/mL). Moreover, significant membrane permeabilization at concentrations of 1.5 and 47 µg/mL was observed. Native CTX was less cytotoxic to HaCaT cells than CTX-containing mouthwash or nystatin between 24 and 48 h. These preliminary findings highlight the potential use of CTX in the treatment of oral candidiasis caused by resistant strains.

Isolation, characterization and cytoprotective effects against UV radiation of exopolysaccharide produced from Paenibacillus polymyxa PYQ1

A novel exopolysaccharide (EPS) from Paenibacillus polymyxa PYQ1 was extracted, well purified and characterized. This EPS was homogeneous glucomannan-type polysaccharide with the average molecular weight of 4.38× 106Da. Structural characterization indicated that the monosaccharides of EPS were pyranoses connected by β-glycosidic linkages. Furthermore, our results showed the protective benefits of EPS against UVC induced cytotoxicity in HaCaT cells through scavenging excessive reactive oxygen species, mitigating the decrease of mitochondrial membrane potential, improving catalase activity and maintaining membrane integrity. Taken together, this study qualified EPS from P.polymyxa PYQ1 was a promising natural polymer which worth further investigation as a skin-care agent.

Phenolic-enriched maple syrup extract protects human keratinocytes against hydrogen peroxide and methylglyoxal induced cytotoxicity.

Reactive carbonyl species including methylglyoxal (MGO) are oxidation metabolites of glucose and precursors of advanced glycation end products (AGEs). They are important mediators of cellular oxidative stress and exacerbate skin complications. Published data supports that certain phenolic compounds can exert cellular protective effects by their antioxidant activity. A phenolic-enriched maple syrup extract (MSX) was previously reported to show protective effects against AGEs- and MGO-induced cytotoxicity in human colon cells but its skin protective effects remain unknown. The protective effects of MSX were evaluated against hydrogen peroxide (H2 O2 )- and MGO-induced cytotoxicity in human keratinocytes (HaCaT cells). Cellular viability and antioxidant activity were evaluated by the luminescent cell viability CellTiter-Glo assay and the reactive oxygen species (ROS) assay, respectively. A single-cell gel electrophoresis (Comet assay) was used to measure the strand breaks in the DNA of HaCaT cells. MSX (at 50 μg/mL) ameliorated H2 O2 - and MGO-induced cytotoxicity by increasing cell viability by 21.5% and 25.9%, respectively. MSX reduced H2 O2 - and MGO-induced ROS production by 69.4% and 56.6%, respectively. MSX also reduced MGO-induced DNA damage by 47.5%. MSX showed protective effects against H2 O2 - and MGO-induced cytotoxicity in HaCaT cells supporting its potential for dermatological and/or cosmeceutical applications.

Effects of Black Vinegar and Niacinamide on LPS-Induced Inflammation on Human Keratinocytes

In this study, the effects of black vinegar (BA) and niacinamide on lipopolysaccharide (LPS)-treated human keratinocytes, HaCaT cells, were investigated. First of all, BA and niacinamide have no cytotoxicity in HaCaT cells even at high concentrations. LPS treatment triggers the phosphorylation of p38 mitogen-activated protein kinases (MAPK) and the expression of inflammatory enzymes, iNOS and COX-2. In contrast, BA and niacinamide weakened the expression of LPS-induced COX-2 and iNOS. Based on the results, we concluded that BA and niacinamide have effective anti-inflammatory properties in HaCaT cells. Therefore, BA and niacinamide may be used as new alternative treatments for inflammatory skin diseases. Keywords: Black vinegar, HaCaT cell, Inflammation, Keratinocytes, Liposaccharides, Niacinamide

Biotinylated PAMAM G3 dendrimer conjugated with celecoxib and/or Fmoc-l-Leucine and its cytotoxicity for normal and cancer human cell lines

Tumors still remain one of the main causes of mortality due to the lack of effective anti-cancer therapy. Recently it has been shown, that overexpression of inducible cyclooxygenase-2 (COX-2) and decrease of peroxisome proliferator-activated receptor γ (PPARγ) expression accompany many malignances, therefore, it has been proposed, that COX-2 inhibitors and PPARγ agonists are potential candidates for anticancer therapy and their synergistic, antineoplastic action has been described. In the present study a COX-2 inhibitor (celecoxib) and/or PPARγ agonist (Fmoc-l-Leucine) were conjugated with the biotinylated G3 PAMAM dendrimer to form a three different constructs targeted to cells with increased biotin uptake. All conjugates were characterized by the NMR spectroscopy. Investigation of three types of human cells: normal skin fibroblasts (BJ), immortalized keratinocytes (HaCaT) and cancer lines: glioblastoma (U-118 MG) and squamous cell carcinoma (SCC-15) revealed similar biotin labeled ATTO590 accumulation (after 24 h), except for SCC-15 with significantly lower loading. Constitutive expression of COX-2 protein was confirmed in all tested cells with significantly higher levels (2–2.5 times) in both cancer lines.Comparison of cytotoxicity of the new synthetized dendrimers clearly documented the highest cytotoxicity of the G31B16C15L dendrimer conjugated with both drugs (1: 1) as compared with drugs alone and single conjugates. Additive effects of construct with both compounds were shown for fibroblasts and both cancer cell lines in the order BJ > U-118 MG > SCC-15 with IC50 in the range: 0.69, 1.44 and 2.22 μM, respectively and lowest cytotoxicity in HaCaT cells (IC50 = 2.88). Our results showed, that biotinylated G3 PAMAM dendrimers substituted with COX-2 inhibitor, celecoxib, and PPARγ agonist, Fmoc-l-Leucine (1:1) may be a good candidate for local therapy of glioblastoma but not a skin cancer. Since the effect of PPARγ agonists on COX-2 expression vary depending upon the cell type, specificity of used agonist and the presence of other environmental factors, it is necessary to carefully evaluate the response of chosen drugs on the target cells.

Effect of Prunus yedoensis Matsumura extract on the gene expression in HaCaT cells

The bark of Prunus yedoensis (PY) is used in herbal remedies for the treatment of inflammatory skin disorders. Previous studies have reported the antioxidative, anti-inflammatory, anticancer, and vasorelaxant effects of PY; however, the safety and toxicity of PY extracts have not yet been reported. In the present study, we explored whether PY bark extract induced toxicity in HaCaT, a human keratinocyte cell line. HaCaT cells were treated with PYE, the ethyl acetate (EtOAc) fraction of the crude extract (produced from 80% EtOH) of PY, for 24 h and cytotoxicity and ROS generation were determined. In addition, we evaluated the effect of a high concentration of PYE on the transcriptional profile to determine the action mechanism of the toxicity to HaCaT cells. PYE treatment significantly induced cytotoxicity in HaCaT cells in a dose-dependent manner. At 313 μg/mL, PYE generated intracellular ROS and up-regulated immediate early genes and oxidative stress-related genes, indicating the activation of stress signals by PYE. Moreover, PYE significantly changed the expression of genes that promote ECM degradation, inflammatory responses, and keratinocyte differentiation. The present study demonstrated that PYE at 313 μg/mL, a concentration which is not cytotoxic but generates intracellular ROS, affected gene regulation in HaCaT human keratinocytes through multiple toxicity mechanisms and could therefore disturb skin homeostasis or lead to skin aging. We propose that for the safe use of PYE in herbal cosmetic or medicines, further evaluations of the toxicity and safety in the skin must be performed.

Protective Effects of a Red Maple (Acer rubrum) Leaves Extract on Human Keratinocytes against H2O2‐induced Oxidative Stress

The red maple (Acer rubrum) species is endemic to eastern North America. It has been used as a folk medicine by the indigenous people of North America to treat dermatological ailments including skin irritation and inflammation. A series of glucitol‐core containing gallotannins (GCGs) has previously been identified from red maple aerial plant parts. Also, ginnalin A (GA), a major GCG in a proprietary phenolic‐enriched red maple leaves extract (Maplifa™), showed anti‐melanogenic effects in murine melanoma B16F10 cells by down‐regulation of tyrosinase and melanogenic gene expression as well as reduction of oxidative stress. However, the dermatological protective effects of Maplifa™ and GA on keratinocytes remain unknown. Herein, we report the protective effects of Maplifa™ and GA on hydrogen peroxide (H2O2)‐induced oxidative stress in human keratinocyte HaCaT cells. H2O2 (at 200 μM) induced cell damage of HaCaT cells and reduced cell viability by 14%. Maplifa™ (at concentrations ranging from 6.25–25 μg/mL) significantly reduced H2O2‐induced cytotoxicity in HaCaT cells and increased cell viability by 25–55%, respectively, as compared to the H2O2‐treatment group. Similarly, GA (at concentrations ranging from 6.25–50 μM) significantly increased cell viability by 34–47% respectively, as compared to the H2O2‐treatment group. In summary, Maplifa™ may have skin protective effects by reducing oxidative stress in keratinocyte HaCaT cells warranting further studies to evaluate its cosmetic applications.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Formulation and evaluation of gels containing coconut kernel extract for topical application

The biological activity of coconut (Cocos nucifera L.) extracts from its kernels and various parts was reported by many previous studies, it is therefore believable that the extracts of its kernels might show some activities in topical formulations. Among several kernel extracts, the TC06 extract prepared by soaking the steamed coconut kernels in hot water showed the highest total phenolic content (6.98 ± 0.30 mg GAE/g extract) and the strongest antioxidant activity as determined using FRAP and DPPH methods with a reducing power value of 4.12 ± 0.16 mg AAE/g of extract and an SC50 value of 2.38 ± 0.14 mg/ml, respectively. In addition, this extract did not display any cytotoxic effects in the concentration range of 50–3200 µg/ml. Meanwhile, it revealed cytoprotective effects against t-BHP-induced cytotoxicity in HaCaT cells at concentrations higher than 400 µg/ml. The results of phytochemical investigations including a chemical color test, TLC, 1H NMR and FTIR suggested that the TC06 extract was mainly composed of flavonoids and terpenoids. Furthermore, the concentrations of heavy metals including As, Cd, Hg, and Pb in the TC06 extract were below permissible limits. According to the solubility, the TC06 extract was incorporated into gels using Carbopol Ultrez 21 as a gelling agent. The formulated gel containing 3% (w/w) TC06 extract was stable at 4 °C and 25 °C with 75% RH throughout the storage period. It was found that the Carbopol Ultrez 21-based hydroalcoholic gel containing an aqueous extract of coconut kernels exhibited antioxidant activities in the two assays and showed a sufficient consistency, a pleasing color, and a non-oily perception during the period of observation.

Assessment of Pb, Cd and Hg soil contamination and its potential to cause cytotoxic and genotoxic effects in human cell lines (CaCo-2 and HaCaT).

Soil contamination by heavy metals is a serious global environmental problem, especially for developing countries. A large number of industrial plants, which continually pollute the environment, characterize Tuzla Canton, Bosnia and Herzegovina. The aim of this study was to assess the level of soil pollution by heavy metals and to estimate cytotoxicity and genotoxicity of soil leachates from this area. Lead (Pb), cadmium (Cd) and mercury (Hg) were analyzed by ICP-AES and AAS. Soil contamination was assessed using contamination factor, degree of contamination, geoaccumulation index and pollution load index. To determine the connection of variables and understanding their origin in soils, principal component analysis (PCA) and cluster analysis (CA) were used. The results indicate that Cd and Hg originated from natural and anthropogenic activities, while Pb is of anthropogenic origin. For toxicity evaluation, CaCo-2 and HaCaT cells were used. PrestoBlue assay was used for cytotoxicity testing, and γH2A.X for genotoxicity evaluation. Concerning cytotoxicity, Cd and Hg had a positive correlation with cytotoxicity in HaCaT cells, but only Hg induced cytotoxicity in CaCo-2 cells. We also demonstrate that soil leachates contaminated by heavy metals can induce genotoxicity in both used cell lines. According to these results, combining bioassays with standard physicochemical analysis can be useful for evaluating environmental and health risks more accurately. These results are important for developing proper management strategies to decrease pollution. This is one of the first studies from this area and an important indication of soil quality in Southeast Europe.

Evaluation of Mitochondrial Respiratory Chain on the Generation of Reactive Oxygen Species and Cytotoxicity in HaCaT Cells Induced by Nanosized Titanium Dioxide Under UVA Irradiation.

Nanosized titanium dioxide (nano-TiO2) is widely used in the chemical, electrical, and electronic industries. Nanosized TiO2 has been reported to be an efficient photocatalyst, which is able to produce reactive oxygen species (ROS) under UVA irradiation. In the present work, we evaluate the effect of mitochondrial respiratory chain on the generation of ROS and cytotoxicity in keratinocyte (HaCaT) cells induced by nano-TiO2 under UVA irradiation. HaCaT cells were pretreated with different inhibitors of mitochondrial respiratory chain and followed by treatment with 200 µg/mL nano-TiO2, then exposed to UVA (365 nm) for 1 hour and cultured for 24 hours. Our results demonstrated that the complexes I and III of the mitochondrial respiratory chain are the major site in the ROS generation induced by nano-TiO2 Our results also demonstrated that the uncouplers of mitochondrial oxidative phosphorylation resulted in obvious changes in the production of intracellular ROS induced by nano-TiO2 The ROS sources of lipoxygenase, cyclooxygenase, and nicotinamide adenine dinucleotide phosphate oxidase had no significant effect on the ROS production. To some extent, nitric oxide synthase had effect on the ROS production. These results indicated that mitochondrial respiratory chain may be the main source of intracellular ROS production induced by nano-TiO2.

Toxicity evaluation of the photoprotective compound LQFM048: Eye irritation, skin toxicity and genotoxic endpoints.

A new molecule, LQFM048, originally designed through molecular hybridization using green chemistry approach, is in development as a photoprotective agent. Eye irritation, skin toxicity and genotoxicity evaluations are mandatory for predicting health risks. In this context, the purpose of this study was to investigate the eye irritation potential of LQFM048 by combining Short Time Exposure (STE), Bovine Corneal Opacity and Permeability (BCOP) associated with corneal histomorphometry and Hen's Egg Test-Chorioallantoic Membrane (HET-CAM). Additionally, skin toxicity was evaluated by interleukin-18 production in the HaCaT keratinocyte, Local Lymph Node Assay (LLNA:BrdU-ELISA) method, 3T3 Neutral red uptake (NRU) assay and in vivo phototoxicity test. Genotoxic potential of LQFM048 was also analyzed by cytokinesis-block micronucleus assay (MNvit test-cytoB) in HepG2 cells. Our results showed that LQFM048 did not induce eye irritation and it was classified as UN GHS No Category for both STE and BCOP assays and non-irritating for HET-CAM test. LQFM048 showed non-potential skin sensitization with stimulation index (SI=0.7) in the LLNA:BrdU-ELISA method. Corroborating in vivo tests, it did not promote significant cytotoxicity in HaCaT cells and it showed similar levels of IL-18 when compared to control. Furthermore, LQFM048 induced non-phototoxic potential with photo-irritation factor (PIF) and mean photo effect (MPE) of 1 and -0.138, respectively, for 3T3 cells. Similarly, it was not phototoxic for in vivo testing with or without exposure to UVA, showing SI values of 1 and 1.2, respectively. The micronucleus test showed that LQFM048 was not genotoxic, under the conditions tested.In conclusion, LQFM048, a heterocyclic compound obtained through an environmentally acceptable simple synthetic route, seems to be safe for human use, especially for the development of a new sunscreen product, since it is neither an eye irritant, nor a contact allergen, nor mutagenic and nor phototoxic.

Physicochemical and biological characterizations of Pxt peptides from amphibian (Xenopus tropicalis) skin.

Pxt peptides (Pxt-1 through Pxt-12) have been isolated from amphibian, Xenopus tropicalis Pxt-related peptides (Pxt-2, Pxt-5, Pxt-12, reverse Pxt-2, reverse Pxt-5 and reverse Pxt-12) with significant foaming properties were further characterized. In the physicochemical experiments, all Pxt-related peptides formed significant amphiphilic α-helices in 50% 2,2,2-trifluoroethanol by circular dichroism measurements. Among Pxt-related peptides, both Pxt-5 and reverse Pxt-5 were the most effective in reducing their surface tensions. Moreover, Pxt-2, Pxt-5 and reverse Pxt-5 produced constant surface tensions above their critical association concentrations, suggesting the micelle-like assemblies. In the biological experiments, Pxt-5 possessed the most potent hemolytic activity, while reverse Pxt-5 exhibited the most remarkable gene expression of interleukin 8 and heme oxygenase 1 and the most potent cytotoxicity in HaCaT cells. In contrast, Pxt-12 and reverse Pxt-12 were much weaker in antimicrobial assays for Gram-negative bacteria, Gram-positive bacteria and yeasts, as well as in hemolytic, cell viability and cytotoxicity assays in HaCaT cells. All Pxt-related peptides exhibited about 20-50% of the total cellular histamine release at 10(-5) M, as well as mastoparan and melittin in mast cells. Real-time polymerase chain reaction analysis confirmed the gene expressions of Pxt-5 in testis and Pxt-12 in muscle, in addition to skin, while Pxt-2 was only in skin.

Anti-inflammatory Effects of Novel Polysaccharide Sacran Extracted from Cyanobacterium Aphanothece sacrum in Various Inflammatory Animal Models.

The goal of this study was to investigate the topical anti-inflammatory effects of the megamolecular polysaccharide sacran extracted from cyanobacterium Aphanothece sacrum using various inflammatory animal models. Sacran showed potent anti-inflammatory effects with optimum effective concentrations at 0.01 and 0.05% (w/v). Sacran markedly inhibited paw swelling and neutrophil infiltration in carrageenan-induced rat paw edema. Additionally, 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionyl-carboxylic acid (DMEQ)-labeled sacran had the ability to penetrate carrageenan-induced rat paw skin rather than normal skin. Also, sacran significantly suppressed kaolin-induced and dextran-induced rat paw edema throughout the duration of the study. Furthermore, sacran significantly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema and mRNA expression levels of cyclooxygenase (COX)-2 as well as pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. Safety of sacran solution was verified by negligible cytotoxicity in HaCaT cells. These results suggest that sacran may be useful as a therapeutic agent against inflammatory skin diseases with no life-threatening adverse effects.

A mechanism for nano-titanium dioxide-induced cytotoxicity in HaCaT cells under UVA irradiation.

Nano-TiO2 has been reported to be an efficient photocatalyst, which is able to produce reactive oxygen species (ROS) under UVA irradiation. In this study, we investigated the effects of nano-TiO2 on the cytotoxicity, induction of apoptosis, and the putative pathways of its actions in HaCaT cells. We show that nano-TiO2 is a potent inducer of apoptosis and that it transduces the apoptotic signal via ROS generation, thereby inducing mitochondrial permeability transition (MPT) and activating Caspase-3 from HaCaT cells. ROS production, mitochondrial alteration, and subsequent apoptotic cell death in nano-TiO2-treated cells were blocked by the MPT pore-blocker cyclosporin A. Taken together, our data indicate that nano-TiO2 induces the ROS-mediated MPT and resultant Caspase-3 activation.

HaCaT세포에서 TNF-α에 의해 유도되는 MMP-1에 대한 Gallic Aicd의 저해 효과

본 연구는 항주름 소재를 개발하기 위해서 목단피로부터 gallic acid (GA)를 분리하여 항산화능을 측정하였고, HaCaT 세포에서 세포독성을 측정하였다. 또한 HaCaT 세포에서 tumor necrosis factor alpha (TNF-<TEX>${\alpha}$</TEX>)에 의해 유도되는 matrix metalloproteinase-1 (MMP-1) mRNA 발현, protein 발현, 분비에 대한 GA의 영향을 관찰하였다. 결과로써 GA는 30 <TEX>${\mu}g/mL$</TEX>의 <TEX>$IC_{50}$</TEX>과 함께 항산화능을 나타내었고, 그것의 항산화능은 합성항산화제인 butylated hydroxyanisol (BHA)보다 높았다. GA는 HaCaT 세포에서 고농도인 200 <TEX>${\mu}g/mL$</TEX> 처리시 약한 세포독성을 나타내었다. 또한 HaCaT세포에서 TNF-<TEX>${\alpha}$</TEX> (10 ng/mL)의 처리에 의해 증가된 MMP-1의 mRNA 발현, protein 발현, 분비는 GA의 처리에 의해 농도 의존적으로 유의적인 감소를 나타내었다(p<0.05). 그러므로 GA는 항산화 효과와 TNF-<TEX>${\alpha}$</TEX>로부터 유도되는 MMP-1의 발현을 저해함으로써 피부 주름을 개선할 수 있는 주름개선제로서의 활용가능성을 확인하였다. In order to develop anti-wrinkle agent, we measured the anti-oxidative activity of gallic acid (GA) from Paeonia suffruticosa Andrews and investigated its cytotoxicity in HaCaT cells and then investigated its effect on tumor necrosis factor alpha (TNF-<TEX>${\alpha}$</TEX>)-induced matrix metalloproteinase-1 (MMP-1) mRNA, protein expressions and secretion in same cells. GA showed anti-oxidative activity with <TEX>$IC_{50}$</TEX> of 30 <TEX>${\mu}g/mL$</TEX> and its activity was higher than that of butylated hydroxyanisol (BHA). GA showed weak cytotoxicity with high concentration (200 <TEX>${\mu}g/mL$</TEX>) in HaCaT cells. MMP-1 mRNA, protein expression and secretion induced by tumor necrosis factor alpha (TNF-<TEX>${\alpha}$</TEX>) in HaCaT cells were significantly decreased by treatment of GA with dose-dependent manner(p<0.05). Therefore, our findings suggest that GA can be useful as an active ingredient for cosmeceuticals of anti-wrinkle effects.

Protective Effect of Vitamin C against Thermal Stimulus-induced Cytotoxicity in HaCaT Cells

Protective Effect of Vitamin C against Thermal Stimulus-induced Cytotoxicity in HaCaT Cells

Oxidative stress mediates chemical hypoxia-induced injury and inflammation by activating NF-κb-COX-2 pathway in HaCaT cells.

Hypoxia of skin is an important physiopathological process in many diseases, such as pressure ulcer, diabetic ulcer, and varicose ulcer. Although cellular injury and inflammation have been involved in hypoxia-induced dermatic injury, the underlying mechanisms remain largely unknown. This study was conducted to investigate the effects of cobalt chloride (CoCl(2)), a hypoxia-mimicking agent, on human skin keratinocytes (HaCaT cells) and to explore the possible molecular mechanisms. Exposure of HaCaT cells to CoCl(2) reduced cell viability and caused overproduction of reactive oxygen species (ROS) and oversecretion of interleukin-6 (IL-6) and interleukin-8 (IL-8). Importantly, CoCl(2) exposure elicited overexpression of cyclooxygenase-2 (COX-2) and phosphorylation of nuclear factor-kappa B (NF-κB) p65 subunit. Inhibition of COX-2 by NS-398, a selective inhibitor of COX-2, significantly repressed the cytotoxicity, as well as secretion of IL-6 and IL-8 induced by CoCl(2). Inhibition of NF-κB by PDTC (a selective inhibitor of NF-κB) or genetic silencing of p65 by RNAi (Si-p65), attenuated not only the cytotoxicity and secretion of IL-6 and IL-8, but also overexpression of COX-2 in CoCl(2)-treated HaCaT cells. Neutralizing anti-IL-6 or anti-IL-8 antibody statistically alleviated CoCl(2)-induced cytotoxicity in HaCaT cells. N-acetyl-L-cysteine (NAC), a well characterized ROS scavenger, obviously suppressed CoCl(2)-induced cytotoxicity in HaCaT cells, as well as secretion of IL-6 and IL-8. Additionally, NAC also repressed overexpression of COX-2 and phosphorylation of NF- B κ p65 subunit induced by CoCl(2) in HaCaT cells. In conclusion, our results demonstrated that oxidative stress mediates chemical hypoxia-induced injury and inflammatory response through activation of NF-κB-COX-2 pathway in HaCaT cells.

The novel synthetic ether lipid inositol-C2-PAF inhibits phosphorylation of the tyrosine kinases Src and FAK independent of integrin activation in transformed skin cells

New alkyl-phospholipids that are structurally derived from platelet-activating factor are promising candidates for anticancer treatment. The mechanism of action of derivatives of the platelet-activating factor is distinctly different from that of known DNA- or tubulin-targeting anticancer agents because they are incorporated into cell membranes, where they accumulate and interfere with a wide variety of key enzymes. We recently presented evidence of a novel group of alkyl-phospholipids, glycosidated phospholipids that efficiently inhibit cell proliferation. One member of this group, inositol-C2-PAF (Ino-C2-PAF), displays high efficacy and low cytotoxicity in HaCaT-cells, an immortalized non-tumorigenic skin keratinocyte cell line. Here, we show that Ino-C2-PAF also inhibits the motility of the skin-derived transformed cell lines HaCaT and squamous cell carcinoma (SCC)-25. This decrease in motility is accompanied by an altered F-actin cytoskeleton, increased clustering of integrins, and increased cell–matrix adhesion. Despite enhanced integrin clustering and matrix adhesion, we observed less phosphorylation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and Src, key regulators of cellular motility, at focal adhesion sites. Transient transfection of constitutively active variants of FAK and Src could at least in part bybass this inhibitory effect of Ino-C2-PAF. This fact indicates that Ino-C2-PAF interferes with the fine-tuned balance between adhesion and migration. Ino-C2-PAF at least partially uncouples integrin-mediated attachment from subsequent integrin-dependent signaling steps, which inhibits migration in transformed keratinocyte cell lines.

Hydrogen peroxide mediates EGCG-induced antioxidant protection in human keratinocytes

The beneficial health effects of (−)-epigallocatechin-3-gallate (EGCG), the main catechin of green tea, have been attributed to complex interactions with a focus on antioxidative properties. Susceptibility to autoxidation and production of cytotoxic reactive oxygen species (ROS), mostly H 2O 2, have been suggested to occur in vitro but also in vivo. In this study, we address whether autoxidation-derived H 2O 2 may be involved in the cytoprotective effects of EGCG. To that end we investigated keratinocyte-derived HaCat and HL-60 promyelocytic leukemia cells with significantly different sensitivities to H 2O 2 (IC 50 117.3 versus 58.3 μM, respectively) and EGCG (134.1 versus 84.1 μM). HaCat cells significantly resisted cytotoxicity and DNA damage based on enhanced H 2O 2 clearance, improved DNA repair, and reduced intracellular ROS generation. Cumulative versus bolus EGCG and H 2O 2 treatment and H 2O 2 pretreatment before subsequent high-dose EGCG and vice versa significantly reduced DNA damage and cytotoxicity in HaCat cells only. Addition of catalase abolished the protective activities of low-dose H 2O 2 and EGCG. In summary, our data suggest that autoxidative generation of low-dose H 2O 2 is a significant player in the cell-type-specific cytoprotection mediated by EGCG and support the hypothesis that regular green tea consumption can contribute as a pro-oxidant to increased resistance against high-dose oxidative stressors.