Human Skin Keratinocytes Research Articles

Apoptotic and DNA-damaging effects of yttria-stabilized zirconia nanoparticles on human skin epithelial cells.

BackgroundYttria-stabilized zirconia (Y2O3/ZrO2) nanoparticles are one of the important nanoparticles extensively used in manufacturing of plastics, textiles, catalyst, etc. Still, the cytotoxic and apoptotic effects of yttria-stabilized zirconia nanoparticles have not been well identified on human skin keratinocyte (HaCaT) cells. Therefore, in this study, we have designed to examine the cytotoxic potential of yttria-stabilized zirconia nanoparticles in HaCaT cells.MethodsPrior to treatment, the yttria-stabilized zirconia nanoparticles were characterized by using different advanced instruments viz. dynamic light scattering (DLS), scanning electron microscope (SEM) and transmission electron microscope (TEM). Cell viability of HaCaT cells was measured by using MTS and NRU assays and viability of cells was reduced in a dose- and time-dependent manner.ResultsReduction in the viability of cells was correlated with the rise of reactive oxygen species generation, increased caspase-3, mitochondria membrane potential and evidence of DNA strand breakage. These were consistent with the possibility that mitochondria damage can play a significant role in the cytotoxic response. Moreover, the activity of oxidative enzymes such as lipid peroxide (LPO) was increased and glutathione was reduced in HaCaT cells exposed with yttria-stabilized zirconia nanoparticles. It is also important to indicate that HaCaT cells appear to be more susceptible to yttria-stabilized zirconia nanoparticles exposure after 24 hrs.ConclusionThis result provides a dose- and time-dependent apoptosis and genotoxicity of yttria-stabilized zirconia nanoparticles in HaCaT cells.

215 Identifying the regulatory elements at the keratin type I and II locus 12q13.13 using C-TALE method during human epidermal skin keratinocytes differentiation

215 Identifying the regulatory elements at the keratin type I and II locus 12q13.13 using C-TALE method during human epidermal skin keratinocytes differentiation

Decreased expression of GPC1 in human skin keratinocytes and epidermis during ageing.

Glypicans (GPCs) are heparan sulfate cell membrane proteoglycans containing glycosylphosphatidylinositol (GPI) anchor. They play important role in cell behavior by activating/presenting numerous growth factors and cytokines. The expression of GPCs was investigated in primary culture of skin keratinocytes sampled from healthy donors of different age. Primary keratinocytes from healthy female donors aged from 20 to 89 years old (n = 30) were either isolated from breast or abdominal skin samples (n = 27) or purchased (n = 3). GPCs expression was examined by qPCR, immunohistochemistry and western blot. Its role in proliferation induced by fibroblast growth factor 2 (FGF2) was also studied. Glypican 1 (GPC1) was the major expressed GPC in human keratinocytes. Its expression was up to two orders of magnitude higher than other GPCs and was significantly decreased with the age of the donors. It was localized at the cell surface and associated with intracellular granules. In skin sections, GPC1 was mainly localized in basal layer of epidermis. Shedding of GPCs decreased the proliferative effect of FGF2, confirming their role of modulator of growth factor effects on keratinocytes. These results established GPC1 as an important player in epidermis biology and skin ageing.

Mussel-Inspired Cell/Tissue-Adhesive, Hemostatic Hydrogels for Tissue Engineering Applications.

The combination of multiple physiological (swelling, porosity, mechanical, and biodegradation) and biological (cell/tissue-adhesive, cell proliferation, and hemostatic) properties on a single hydrogel has great potential for skin tissue engineering. Adhesive hydrogels based on polydopamine (PDA) have become the most popular in the biomedical field; however, integrating multiple properties on a single adhesive hydrogel remains a challenge. Here, inspired by the chemistry of mussels, we developed PDA–sodium alginate–polyacrylamide (PDA–SA–PAM)-based hydrogels with multiple physiological and biological properties for skin tissue engineering applications. The hydrogels were prepared by alkali-induced polymerization of DA followed by complexation with SA in PAM networks. The chemical composition of the hydrogels was characterized by X-ray photoelectron spectroscopy. PDA–SA complexed chains were homogeneously dispersed in the PAM network and exhibited good elasticity and excellent mechanical properties, such as a compressive stress of 0.24 MPa at a compression strain of 70% for 0.4PDA–SA–PAM. The adhesive hydrogel also maintained a highly interconnected porous structure (∼94% porosity) along with PDA microfibrils. The hydrogel possesses outstanding swelling and biodegradability properties. Owing to the presence of the PDA–SA complex in the PAM network, the hydrogels show good adhesion to various substrates (plastic, skin, glass, computer screens, and leaves); for example, the adhesive strength of the 0.4PDA–SA–PAM to porcine skin was 24.5 kPa. The adhesive component of the PDA–SA chains in the PAM network significantly improves the cell proliferation, cell attachment, cell spreading, and functional expression of human skin fibroblasts (CCD-986sk) and keratinocytes. Moreover, the PDA chains exhibited good hemostatic properties, resulting in rapid blood coagulation. Considering their excellent cell affinity, and rapid blood coagulation ability, these mussel-inspired hydrogels have substantial potential for skin tissue engineering applications.

Sunscreen-induced expression and identification of photosensitive marker proteins in human keratinocytes under UV radiation.

Solar ultraviolet (UV) radiation is the main factor of photocarcinogenesis, photoaging, and photosensitivity; thus protection from biological damaging UV radiation is a concern. Sunscreens containing UV filters are the most preferred means of photoprotection but the safety and efficacy of UV filters are in question. Benzophenone (BP) and its derivatives, namely, benzophenone 1 (BP1), is commonly used in sunscreens as a UV blocker. The aim of this study was to assess the effects of BP and BP1 on the differential expression of proteins in human keratinocytes (HaCaT cells) under exposure to ultraviolet A radiation. Photosensitive proteins were screened from HaCaT cells by two-dimensional (2-D) gel electrophoresis, and identification of these differentially expressed proteins was performed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/TOF mass spectrometry. Protein identification was performed using the search program MASCOT and a database made of SUMO and GhJMJ12 amino acid sequences. Our results showed that the proteins involved directly or indirectly in apoptosis are 70 kDa heat shock protein, long-chain specific acyl-CoA dehydrogenase, serine/threonine-protein kinase, and FAM78A protein, which were upregulated in comparison to control HaCaT cells. The expressions of binding immunoglobulin protein, podocalyxin-like protein, actin, cytoplasmic, and calreticulin precursors were downregulated. The altered protein expression indicated that cell growth arrest and apoptosis were potential mechanisms of cytotoxicity and genotoxicity of BPs. The results of 2-D gel electrophoresis followed by mass spectrometry showed expression of novel proteins involved in promoting or initiating apoptotic pathways. Hence, we conclude that BPs should be avoided as a UV blocker from sunscreens because of its potential to promote apoptotic proteins in human skin keratinocytes.

Efficacy of selenium in controlling Acinetobacter baumannii associated wound infections

Abstract Acinetobacter baumannii is a multi-drug resistant, nosocomial pathogen causing a variety of disease conditions, especially wound infections in humans. A. baumannii’s ability to form biofilms and colonize epithelial cells potentially makes it difficult to treat skin and soft-tissue infections of this pathogen. Thus, in light of the multidrug resistance and biofilm producing capacity, new strategies for controlling A. baumannii wound infections are necessary. This study investigated the efficacy of the essential mineral, selenium (Se) in inhibiting skin-colonizing and biofilm forming abilities of A. baumannii in vitro. The effect of Se on A. baumannii adhesion and invasion of human skin keratinocytes (HEK001) was studied. Additionally, the efficacy of Se in inhibiting A. baumannii biofilm formation was determined using an in vitro collagen matrix wound model, and scanning electron microscopy (SEM) was done to visualize its potential antibiofilm effect. The effect of Se on critical A. baumannii genes for biofilm synthesis was also determined using real-time qPCR (RT-qPCR). Selenium inhibited A. baumannii biofilm formation in the collagen-based wound model and reduced bacterial adhesion and invasion of HEK001 (P

Characterization of Chitosan-Gelatin Blend Scaffolds

Vacuum freeze-drying was used to prepare chitosan-gelatin (CG) scaffolds from hydrogels, with glutaraldehyde (GA) used as a crosslinker. The effects of the changes in volume ratios of the 2 wt% CG and GA solutions on scaffold performance were studied. The ratio of chitosan to gelatin solution volumes, vr(C/G), was adjusted to 1/2 or 1/1, with the 0.25 wt% GA volume at 3, 6, or 8% of the CG/GA volume. Six groups of CG scaffolds were fabricated and the scaffolds performance compared. After the cells were incubated for 4 days, hematoxylin eosin (HE) staining was used to observe the spreading of human skin keratinocyte (HaCaT) cells on these scaffolds, with the MTT method also used to detect the cells proliferation. The inhibition zone method was used on cells cultures to determine the antibacterial properties of the scaffolds against S. aureus and E. coli. Scaffolds were also examined for degradation in lysozyme and their compression properties were tested after degradation. The results showed that the HaCaT cells grew well on these scaffolds and proliferated significantly, indicating that these scaffolds possessed good cytocompatibility. With increased chitosan volume, the antibacterial properties of the scaffolds against S. aureus increased, however, there was no significant change in the antibacterial properties toward E. coli. Increased volumes of chitosan and GA decreased the scaffolds degradation rates and improved the elastic compressive moduli of the scaffolds after degradation. The scaffolds in the vr(C/G) = 1/1, 8% GA group have potential application prospects in the field of skin regeneration.

Electrochemical detection and photocatalytic performance of MoS2/TiO2 nanocomposite against pharmaceutical contaminant: Paracetamol

Rapidly increasing sternness of the water crisis and environmental degradation are stimulating rapid development of photocatalysts and sensors. In this article, we report a non-metal plasmonic MoS2/TiO2 nanocomposite for electrochemical sensing and sunlight-driven photocatalysis of pharmaceutical contaminant; paracetamol (PCM). Nanocomposite and its constituent materials were synthesized by wet chemical approach and characterized using a range of analytical techniques. Before performing electrochemical sensing of PCM, an insight into electrochemistry of PCM was developed for which cyclic voltammetry (CV) was used. The square wave voltammetric technique was used for electrochemical sensing of PCM, using oxidation peak current of PCM as a signal. Sensing platform revealed a linear dynamic range from 0.5 μM to 750 μM with regression coefficient (r2) of 0.980 and is able to perform real sample analysis. Moreover, this electrochemical sensor shows excellent reproducibility of 0.554% relative standard deviation and well-defined peak separation from the peaks of interfering biological species like dopamine and ascorbic acid for selective analytical determination of PCM. Photocatalysis performance of synthesized composite was evaluated under visible light conditions. The synthesized nanocomposite showed enhanced photocatalytic activity against PCM, attaining a degradation rate of 40% in 25 mins. Photostability of the composite was assessed for three consecutive runs, and a comparative study was performed against basal material (TiO2). Unlike numerous other reports on photocatalytic studies, the cytotoxic activity of synthesized nanocomposite was evaluated against human skin keratinocytes (HaCaT) cells to ensure its applicability as environment-friendly photocatalyst.

Effects of Sting Plant Extracts as Penetration Enhancers on Transdermal Delivery of Hypoglycemic Compounds.

Background and objectives: The percutaneous route is an interesting and inventive investigation field of drug delivery. However, it is challenging for drug molecules to pass through the skins surface, which is a characterized by its permeability barrier. The purpose of this study is to look at the effect of some penetration enhancers on in vivo permeation of insulin and insulin sensitizers (curcumin and rutin) through diabetes-induced mouse skin. Materials and Methods: Sting crude extracts of Dendrocnide meyeniana, Urtica thunbergiana Sieb. and Zucc, and Alocasia odora (Lodd.) Spach were used as the penetration enhancers. Mouse skin irritation was tested by smearing the enhancers for the measurements at different time points and the cell viability of the HaCaT human skin keratinocytes, which was determined by Trypan blue exclusion and MTT assays to evaluate human biosafety for these extracts after the mouse skin permeation experiments. Results: All enhancers induced a slight erythema without edema on the mouse skin that completely recovered after 6 h from the enhancer smears as compared with normal mouse skin. Furthermore, no damaged cells were found in the HaCaT keratinocytes under sting crude extract treatments. The blood sugar level in the diabetic mice treated with the insulin or insulin sensitizers, decreased significantly (p < 0.05) in the presence of enhancers. The area under the curve (AUC) values of transdermal drug delivery (TDD) ranged from 42,000 ± 5000 mg/dL x min without enhancers, to 30,000 ± 2000 mg/dL x min in the presence of enhancers. Conclusions: This study exhibited that natural plant extracts could be preferred over the chemically synthesized molecules and are safe and potent penetration enhancers for stimulating the transdermal absorption of drugs.

Enzymatic hydrolysates obtained from Trametes versicolor polysaccharopeptides protect human skin keratinocyte against AAPH-induced oxidative stress and inflammatory.

Polysaccharopeptides (PSPs) extracted from Trametes versicolor show antitumor, anti-inflammatory, and immunomodulation effects. According to our previous report, the enzymatic hydrolysates obtained from Tversicolor PSPs by 80U/mL β-1,3-D-glucanase (PSPs-EH80) did not change the functional groups of PSPs but enhanced their antioxidative activities. However, the mechanism elevating the antioxidant and anti-inflammatory effect of PSPs-EH80 is not clear. This research focused on the protective mechanism(s) of PSPs-EH80 against free radical and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative damage in human keratinocyte (HaCaT) cells. We evaluated the anti-inflammatory potential of PSPs-EH80 by assessing its free radical-induced oxidative damage. Using the HaCaT cell as the experimental system, we tested the protective effects of PSPs-EH80 on a model of AAPH-induced cellular oxidative damage through the assessment of cell survival rate. Heme oxygenase 1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), cyclooxygenase-2 (COX-2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase were determined using MTT assays and Western blotting. We demonstrated that PSPs-EH80 significantly enhanced keratinocyte viability, and augmented the antioxidant HO-1 expressions through upregulation of the Nrf2, compared with PSPs. Furthermore, PSPs-EH80 significantly reduced AAPH-induced COX-2 expressions through downregulation of the ERK, p38, and NF-κB signaling pathways. The PSPs-EH80 exhibits a stronger antioxidant and anti-inflammatory capacity than PSPs. Therefore, PSPs-EH80 could be effective for attenuating free radical-induced oxidative damage in human skin and can be applied widely in the fields of cosmetics and medicine.

Silver nanoparticle-mediated cellular responses in human keratinocyte cell line hacat in vitro

&#x0D; &#x0D; &#x0D; The interactions between cells and nanoparticles has been the focus of recent research in the area. The effects of AgNPs on skin cell lines for further potential biological applications are highlighted. This study aimed to investigate the mechanism of cytotoxic and genotoxic effects of AgNPs nanoparticles on human skin keratinocytes (HaCaT). Genocytotoxic effects of AgNPs was assessed using changes in various cellular parameters of HaCaT cells involving viability, superoxide anion radical production, lactate dehydrogenase release and the levels of the antioxidant enzymes, namely, Catalase, Glutathione peroxidase (GPX) and Superoxide Dismutase (SOD). Superoxide anion was detected using nitroblue tetrazolium NBT reduction assay. LDH levels was evaluated using the standard kit, and activity of antioxidant enzymes such as catalase (CAT), glutathione peroxidase 1 (GPX-1) and superoxide dismutase 1 (SOD-1) were quantified using qPCR. Our results indicated that AgNPs caused severe HaCaT oxidative damage, accompanied by increased the production of superoxide anion levels as well as significant decrease in endogenous antioxidant enzyme of SOD, CAT, GPX expression involved in HaCat cells in vitro. Our study suggests that AgNPs exposure increased oxidative stress levels. Moreover; the low cytotoxic effect observed on human HaCaT keratinocytes suggested that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure.&#x0D; &#x0D; &#x0D;

Side Effects of Frequently Used Antihypertensive Drugs on Wound Healing in vitro

Background: The number of patients who has a daily intake of antihypertensive drugs is rising, due to an also rising prevalence of lifestyle diseases. Interestingly, knowledge about effects of these drugs in terms of wound healing is low. Objective: Based on a few differing studies, the idea arose that antihypertensives may have side effects on wound healing. Methods: Five antihypertensive drugs from different substance classes (metoprolol, amlodipine, ramipril, hydrochlorothiazide, candesartan) were investigated, in terms of possible impacts on cell metabolism and migration of human skin fibroblasts and keratinocytes. Additionally, histological and immunohistochemical analyses were performed in a 3-dimensional (3D) wound model addressing the influence on regeneration processes, such as cell migration, metabolic activity, apoptosis and epidermal thickness. Results: Hydrochlorothiazide and ramipril exerted inhibiting effects in nearly all analyses, interestingly, in serum equivalent doses. In contrast, candesartan and amlodipine induced slight positive effects in 2D as well as in 3D models. The previously described positive effects of β-blockers could only partially be confirmed by metoprolol. Antihypertensive drugs affected fibroblasts more than keratinocytes – whether positively or negatively. Conclusion: Antihypertensive drugs have an influence on keratinocytes and fibroblasts; they are not neutral. Candesartan has the most positive effects on skin cells. For angiotensin-converting enzyme inhibitors and thiazide diuretics, wound healing in a 3D model is delayed. β-Receptor blockers seem to improve wound healing to a small extent just like calcium channel blockers. These results should be evaluated in a clinical trial to verify their clinical relevance.

317 Mechanism of SO2-induced oxidation and inflammatory injury of human skin keratinocytes

Objective: This study aimed to study the effect and mechanism of SO2-induced oxidation on human skin keratinocytes. Methods: Different concentrations of SO2 derivatives (0, 25, 50, 100, 200, 400, and 800μM) were used for treating HaCaTkeratinocytes for 24 h. MTT was used to evaluate the effect of each concentration on cell proliferation. Then, HaCaT cells were randomly divided into control and SO2 groups.The control group was without any treatment, and the SO2 group was treated with SO2 derivatives of selected concentrations for 24 h.The contents of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD), tumor necrosis factor TNF-α (TNF-α), and interleukin-1 (IL-1-β) in cell supernatants were detected by enzyme-linked immunosorbent assay.Real-time polymerase chain reaction was used to detect the expression of nuclear transcription factor (Nrf2) and heme oxygenase (HO)-1 mRNA.The Western blotanalysis was used to test the expression levels of Nrf2, HO-1, activated caspase-3, Bcl-2, Bax, IκB, NF-κB p65 (p65), ERK1/2, p38, phospho-NF-κB p65 (p-p65), p-ERK1/2, and p-p38. Results: SO2derivatives (100, 200, 400, and 800μM) could inhibit cell proliferation. SO2 derivatives increased the level of ROS, MDA, TNF-α, IL-1β, Nrf2, HO-1, and p-p65/p65 and decreased the levels of SOD, IκB, p-ERK1/2/ERK1/2, and p-p38/p38 compared with the control group, but they had no effect on the levels of caspase-3, Bcl-2, and Bax. Conclusions: SO2could inhibit the proliferation of human skin keratinocytes and induce oxidative stress and inflammation via the activation of NF-κB pathway to inhibit the ERK1/2 and p38 pathways.

Protective Effect of Phloroglucinol on Oxidative Stress-Induced DNA Damage and Apoptosis through Activation of the Nrf2/HO-1 Signaling Pathway in HaCaT Human Keratinocytes.

Phloroglucinol (PG) is a component of phlorotannins, which are abundant in marine brown alga species. Recent studies have shown that PG is beneficial in protecting cells from oxidative stress. In this study, we evaluated the protective efficacy of PG in HaCaT human skin keratinocytes stimulated with oxidative stress (hydrogen peroxide, H2O2). The results showed that PG significantly inhibited the H2O2-induced growth inhibition in HaCaT cells, which was associated with increased expression of heme oxygenase-1 (HO-1) by the activation of nuclear factor erythroid 2-related factor-2 (Nrf2). PG remarkably reversed H2O2-induced excessive ROS production, DNA damage, and apoptosis. Additionally, H2O2-induced mitochondrial dysfunction was related to a decrease in ATP levels, and in the presence of PG, these changes were significantly impaired. Furthermore, the increases of cytosolic release of cytochrome c and ratio of Bax to Bcl-2, and the activation of caspase-9 and caspase-3 by the H2O2 were markedly abolished under the condition of PG pretreatment. However, the inhibition of HO-1 function using zinc protoporphyrin, a HO-1 inhibitor, markedly attenuated these protective effects of PG against H2O2. Overall, our results suggest that PG is able to protect HaCaT keratinocytes against oxidative stress-induced DNA damage and apoptosis through activating the Nrf2/HO-1 signaling pathway.

The role of human immortal skin keratinocytes-acellular dermal matrix scaffold in skin repair and regeneration.

In this study, we aimed to investigate the phenotypic characteristics of human immortal skin keratinocytes (HaCaT) cells and the role of acellular dermal matrix (ADM) in coculture system of HaCaT cells and ADM. Flow cytometry was used to examine the cluster of differentiation (CD) makers of HaCaT cells. Apoptosis analysis was applied to detect the apoptosis rate of HaCaT cells. Morphological observation of ADM isolated from the reticular layer of Sprague-Dawley rat dermis was utilized to evaluate the morphological structure of ADM. Methylthiazolyl tetrazolium (MTT) assay and morphological experiments were further used to confirm the scaffold role of ADM in HaCaT cells. A wound-healing mice model accompanied by HaCaT-ADM scaffold transplantation was performed to further verify the function of HaCaT-ADM scaffold. Our results showed that CD71, CD49f, K19, and CD29 were highly expressed in HaCaT cells, and the percentage of apoptosis cells was significantly increased, which represented that HaCaT cells had much stronger capacities of adhesion and proliferation than normal human keratinocytes. Additionally, the morphological structure of ADM presented many natural microbores, which made cells rapidly grow on ADM. The results exhibited that the HaCaT cells indeed promptly proliferate on ADM and easily grow into the microbores of ADM. Finally, an in vivo experiment further confirmed that the transplantation of the HaCaT-ADM scaffold into the dorsal skin of a wound-healing mice model could gradually repair the injured wound. Thus, these findings indicated that HaCaT cells might be as seed cells to develop skin tissue engineering and the HaCaT-ADM scaffold might be a better candidate to promote skin repair and regeneration.

Tailored functionalization of poly(L-lactic acid) substrates at the nanoscale to enhance cell response

Poly(L-lactic) acid (PLLA) has been widely employed in tissue engineering due to its mechanical properties, biodegradability and biocompatibility. The layer-by-layer (LbL) technique was here proposed as a simple method to impart bioactivity to the surface of PLLA substrates. Aminolysis treatment was applied to introduce amino groups on the surface of PLLA solvent cast films. Then, PLLA films were coated with heparin (HE)/chitosan (CH) multilayer by the LbL technique. Each functionalization step was characterized through physico-chemical and morphological analyses. Aminolysis treatment increased film surface wettability (64.8° ± 2.4° against 74.6° ± 1.3° for untreated PLLA) due to the formation of surface amino groups, which were quantified by acid orange colorimetric assay (0.05 nmol/mm2). After the deposition of 9 layers, the static contact angle varied between values close to 40° C (HE-based layer) and 60 °C (CH-based layer), showing the typical alternate trend of LbL coating. The successful HE/CH deposition was confirmed by ATR-FTIR and X-ray photoelectron spectroscopy (XPS) analyses. Particularly, XPS spectra of coated samples showed the presence of nitrogen (indicative of HE and CH deposition), and sulfur (indicative of HE deposition). The amount of deposited HE was quantified by Taylor’s Blue colorimetric method: after the deposition of 19 and 20 layers the HE concentration was around 33 µg/cm2. Finally, in vitro studies performed using HaCaT immortalized human skin keratinocytes, C2C12 immortalized mouse myoblasts and human fibroblasts demonstrated that HE/CH multilayer-coated PLLA is a promising substrate for soft tissue engineering, as cell response may be modulated by changing the surface chemical properties.

Photoexcited triclosan induced DNA damage and oxidative stress via p38 MAP kinase signaling involving type I radicals under sunlight/UVB exposure

Triclosan (TCS) is an antimicrobial preservative used in personal care products. Here, we have studied the phototoxicity, photogenotoxicity of TCS and its molecular mechanism involving p38 mitogen activated protein kinase (MAPK) pathway under UVB/sunlight exposure. We found that TCS showed photodegradation and photoproducts formation under UVB/sunlight. In silico study suggests that photosensitized TCS loses its preservative property due to the formation of its photoproducts. Photosensitized TCS induces significant O2•−, •OH generation and lipid peroxidation via type-I photochemical reaction mechanism under UVB/sunlight exposure. We performed intracellular study of TCS on human skin keratinocytes (HaCaT cell-line) under the ambient intensity of UVB (0.6 mW/cm2) and sunlight exposure. Significant intracellular ROS generation was observed through DCFH2-DA/DHE assays along with a significant reduction in cell viability through MTT and NRU assays in photosensitized TCS. Photosensitized TCS also induces endoplasmic reticulum (ER) stress as shown through ER-tracker/DAPI staining and Ca2+ release. It further induced cell cycle arrest through the sub-G1 phase augmentation and caused lysosomal/mitochondrial destabilization. Photogenotoxicity was shown through significant tail DNA, micronuclei and cyclobutane pyrimidine dimers (CPDs) formations. Cell signaling mechanism implicated upregulated expression of cleaved Caspase-3, Bax, phospho-p38, phospho-JNK and cytochrome C, thereby downregulated Bcl-2 expressions. Results advocate that TCS induces phototoxic effects via type I mediated photodynamic mechanism and activation of MAPK pathway. We conclude that photoexcited TCS may be deleterious to human health at the ambient environmental intensities of sunlight reaching at the earth's surface. Therefore, it may be replaced by alternative safe preservative.

Rutin and ascorbic acid cooperation in antioxidant and antiapoptotic effect on human skin keratinocytes and fibroblasts exposed to UVA and UVB radiation

The combination of ascorbic acid and rutin is frequently used in oral preparations. However, despite numerous protective effects of each component individually, their combined effect on ultraviolet (UV)-irradiated skin cells has never been evaluated. The aim of this study was to evaluate the combined effect of ascorbic acid and rutin on human keratinocytes and fibroblasts exposed to UVA and UVB radiation. Skin keratinocytes and fibroblasts exposed to UVA and UVB radiation were treated with ascorbic acid or/and rutin. The total antioxidant properties of both components, as well as their effect on cellular pro- and antioxidant status, lipid and protein oxidation, transmembrane transport, and pro-inflammatory and pro/antiapoptotic protein expression were measured. The combination of ascorbic acid and rutin had higher antioxidant properties compared to the activity of the single compound alone, and showed a stronger effect against UV-induced reactive oxygen species generation. The ascorbic acid and rutin combination also showed increased antioxidant enzyme activity (catalase, superoxide dismutase, thioredoxin reductase), which was impaired following UV irradiation. Moreover, ascorbic acid additional stimulated UV-induced bilitranslocase activity responsible for rutin transport, and therefore favored rutin effect on Nrf2 pathway, simultaneously differentiating the reaction of keratinocytes and fibroblasts. In keratinocytes, Nrf2 is strongly activated, while in fibroblasts decreased Nrf2 activity was observed. Used mixture, also significantly silenced UV-induced expression of pro-inflammatory factor NFκB and pro-apoptotic proteins such as caspases 3, 8, and 9. These results showed that ascorbic acid and rutin are complementary in their antioxidant actions, transport and signaling functions. Their combined antioxidant, antiinflammatory and antiapoptotic actions suggest rutin and ascorbic acid are a potentially cytoprotective team against UV-induced skin damage.

Alleviation of Ultraviolet-B Radiation-Induced Photoaging by a TNFR Antagonistic Peptide, TNFR2-SKE.

Ultraviolet (UV) radiation of the sunlight, especially UVA and UVB, is the primary environmental cause of skin damage, including topical inflammation, premature skin aging, and skin cancer. Previous reports show that activation of nuclear factor-κB (NF-κB) in human skin fibroblasts and keratinocytes after UV exposure induces the expression and release of proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), and subsequently leads to the production of matrix metalloproteases (MMPs) and growth factor basic fibroblast growth factor (bFGF). Here, we demonstrated that TNFR2-SKEE and TNFR2-SKE, oligopeptides from TNF receptor-associated factor 2 (TRAF2)-binding site of TNF receptor 2 (TNFR2), strongly inhibited the interaction of TNFR1 as well as TNFR2 with TRAF2. In particular, TNFR2-SKE suppressed UVB- or TNF-α-induced nuclear translocalization of activated NF-κB in mouse fibroblasts. It decreased the expression of bFGF, MMPs, and COX2, which were upregulated by TNF-α, and increased procollagen production, which was reduced by TNF-α. Furthermore, TNFR2-SKE inhibited the UVB-induced proliferation of keratinocytes and melanocytes in the mouse skin and the infiltration of immune cells into inflamed tissues. These results suggest that TNFR2-SKE may possess the clinical potency to alleviate UV-induced photoaging in human skin.

Polydopamine-coated gold nanostar for combined antitumor and antiangiogenic therapy in multidrug-resistant breast cancer.

Cancer combination therapy can improve treatment efficacy and is widely utilized in the biomedical field. In this paper, we propose a facile strategy to develop a polydopamine (PDA)-coated Au nanostar (NS@PPFA) as a multifunctional nanoplatform for cancer targeting and combination therapy. The Au nanostar demonstrated high photothermal conversion efficiency because of the tip-enhanced plasmonic effect. Modification of PDA and folic acid on the NS surface improved its drug-loading efficiency and targeting capability. In vitro, compared with nontargeted cells, targeted breast cancer MCF-7 cells demonstrated efficient uptake of chemodrug-loaded NS-D@PPFA through the receptor-mediated endocytosis pathway. In combination with the photothermal effect induced by near-infrared laser irradiation, controlled payload release could be activated in response to both internal (acid) and external (photothermal) stimuli, leading to an efficient chemo-photothermal action against MCF-7 cells and drug-resistant MCF-7/ADR cells. By contrast, cellular damage was less obvious in normal HaCaT (human skin keratinocytes) and NIH-3T3 cells (murine fibroblasts). In addition, payload-free NS@PPFA exhibited a high binding affinity (Kd = 2.68 × 10-10 M) toward vascular endothelial growth factor (VEGF-A165), which was at least two orders of magnitude stronger than that of highly abundant plasma proteins, such as human serum albumin. Furthermore, in vitro study showed that NS@PPFA could effectively inhibit VEGF-A165-induced proliferation, migration, and tube formation of human umbilical vein endothelial cells, resulting in additional therapeutic benefits for eradicating tumors through a simultaneous antiangiogenic action in chemo-photothermal treatment. The combined treatment also exhibited the lowest microvessel density, leading to a potent antitumor effect in vivo. Overall, our “all-in-one” nanoplatform is highly promising for tumor therapy, enabling effective treatment against multidrug-resistant cancers.