Survival Of HaCaT Cells Research Articles

Protective effect of ginseng berry saponin conversion products on skin photodamage caused by UVB in vitro and in vivo

Ultraviolet (UV) B irradiation is closely related to skin aging and skin damage. Here, we report the photoprotective mechanism of action of ginseng berry rare saponins (GFRS) on UVB-induced damage to human keratinocytes and mouse skin. Several UVB irradiation–induced cytotoxicity and oxidative stress responses were assessed. GFRS preconditioning significantly improved HaCaT cell survival and reduced the levels of the DNA damage markers histone H2AX and cyclobutane pyrimidine dimer. Under oxidative stress, GFRS could reduce the transformation and loss of the mitochondrial membrane potential to the monomer form; effectively clear the expression of lipid reactive oxygen species, malondialdehyde, and other peroxides, and restore total superoxide dismutase, glutathione peroxidase, and catalase levels. The occurrence of ferroptosis after UVB induction was also studied. Erastin exacerbated the induced cellular iron overload, whereas GFRS and Fer-1 reversed this response to varying degrees. Mechanistically, GFRS activated the Nrf2/HO-1/GPX4 pathway and inhibited the phenomenon of ferroptosis in cells. Our findings were confirmed using a mouse model of UV induced skin injury. GFRS not only mitigated lipid peroxides and iron overload in tissues but also prevented skin barrier damage and collagen loss. Therefore, GFRS shows potential as a novel functional product as it protects the skin from UVB light–induced damage.

Ginseng glycoprotein and ginsenoside facilitate anti UV damage effects in diabetic rats.

Diabetes mellitus combined with ultraviolet (UV) radiation damage not only brings great mental stress to patients, but also seriously impairs their quality of life. A UV-irradiated diabetic rat trauma skin model was established by us to investigate the effects and possible mechanisms of ginsenoside and glycoprotein on skin trauma repair in UV-irradiated diabetic rats. In the study, ginsenosides and ginseng glycoproteins were extracted from different parts of ginseng roots. It found that it's easier to prepare saponins in ginseng bark and proteins in ginseng core in large quantities. Since glycoprotein-like metabolites are relatively novel ginseng extracts, specifically characterized its structures. It was verified that the ginseng glycoproteins are not toxic to HaCaT cells and can significantly increase the survival of HaCaT cells after UV damage at the in vitro cellular level. Experiments in vivo were conducted to evaluate the therapeutic effects of ginsenoside and ginseng glycoprotein in a rat model of diabetes mellitus combined with UV irradiation injury. Histopathological changes on rat skin after treatment with ginsenoside and ginseng glycoprotein were evaluated by hematoxylin and eosin (H&E) staining and aldehyde fuchsine staining. The expression levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), matrix metalloproteinases (MMPs), hydroxyproline (HYP), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were measured. The results indicate that both ginsenoside and ginseng glycoprotein could improve skin damage and ulcers caused by diabetes combined with UV irradiation and could alleviate a range of skin damage caused by the combination of diabetes and UV irradiation, including peroxidation and collagen fiber loss. Ginsenoside and ginseng glycoproteins can be considered as natural product candidates for the development of new drugs to treat diabetes combined with UV irradiation-induced skin damage.

UV-B Protective and Antioxidant Activities of Protein Hydrolysate From Sea Cucumber (Holothuria scabra) Using Enzymatic Hydrolysis

Sea cucumber is rich in protein that can be used to prepare a potential derived bioactive peptide for antioxidant and protective effect against UV-B induced skin cell damage. This study aimed to optimize preparation of sea cucumber hydrolysate with both UV-B protective and antioxidant activities using three commercial enzymes using response surface methodology (RSM) with a face-centered central composite design (face- centered CCD). Hydrolysis time and concentration of enzyme effects on the degree of hydrolysis (DH), yield, antioxidant and UV-B protective activities of sea cucumber hydrolysates were determined. The optimum conditions for sea cucumber hydrolysis using papain (SCP), alcalase (SCA), or flavourzyme (SCF) were 3.6, 5.0, and 4.1% (w/w protein), respectively, and a hydrolysis time of 360 min. The resulting hydrolysates had a DH of 81-91%, yield of 13-14%, IC50 for DPPH radical scavenging activity of 0.3-4.1 mg/mL, FRAP of 0.5-0.6 mmol FeSO4/mL, and IC50 for ABTS radical scavenging activity of 1.3-1.6 mg/mL. The UV-B protective activity was reported as the HaCaT cell viability percentage after UV-B treatment. The SCP, SCA, and SCF hydrolysates showed 72.4, 74.5, and 71.3% cell viability, respectively. The concentration of hydrolysates with 80% survival of HaCaT cells was 0.21, 0.15 and 0.20 mg/mL for SCP, SCA and SCF, respectively. Thus, the SCP was selected for bioactive peptide isolation and characterization. The SCP contained hydrophilic and hydrophobic amino acids of 42.4 and 57.6%, respectively. The ultrafiltration and Sephadex G-25 gel filtration chromatography were done for peptide isolation from the SCP. Six potential peptides were identified using LC-MS/MS as Leu-Val-Asn-Glu-Leu-Thr-Glu-Phe-Ala-Gln (1163 Da), Leu-Val-Asn-Glu-Val-Thr-Glu-Phe-Ala-Gln (1149 Da), Phe-Val-Asp-Ser-Ser-Ala-Thr-Thr (826 Da), Phe-Asn-Asp-Leu-Gly-Ala-Trp (821 Da), Phe-Pro-Asp-Thr-Thr-Thr-Leu (793 Da), and Lys-Phe-Gly-Glu-Gly-Lys (664).

Silibinin relieves UVB-induced apoptosis of human skin cells by inhibiting the YAP-p73 pathway.

Excessive exposure to UVB induces skin diseases. Silibinin, a flavonolignan used for treating liver diseases, is found to be effective against UVB-caused skin epidermal and dermal cell damage. In this study we investigated the molecular mechanisms underlying. Human nonmalignant immortalized keratinocyte HaCaT cells and neonatal human foreskin fibroblasts HFFs were exposed to UVB irradiation. We showed that pre-treatment with silibinin dose-dependently decreased UVB-induced apoptosis of HaCaT cells. Furthermore, we showed that silibinin treatment inhibited nuclear translocation of YAP after UVB irradiation. Molecular docking analysis and DARTS assay confirmed the direct interaction of silibinin with YAP. Silencing YAP by siRNA had no influence on the survival of HaCaT cells, whereas inhibiting classical YAP-TEAD signaling pathway by siRNA targeting TEAD1 or its pharmaceutical inhibitor verteporfin further augmented UVB-induced apoptosis, suggesting that YAP-TEAD pathway was prosurvival, which did not participate in the protective effect of silibinin. We then explored the pro-apoptotic YAP-p73 pathway. p73 was upregulated in UVB-irradiated cells, but reduced by silibinin cotreatment. The mRNA and protein levels of p73 target genes (PML, p21 and Bax) were all increased by UVB but decreased by silibinin co-treatment. Inhibiting p73 by using siRNA reduced UVB-induced apoptosis, suggesting that downregulation of p73 was responsible for the cytoprotective effect of silibinin. In HFFs, the upregulated YAP-p73 pathway by UVB irradiation was also suppressed by silibinin. Collectively, YAP-p73 pathway is a major cause of the death of UVB-exposed epidermal HaCaT cells and dermal HFFs. Silibinin directly inhibits YAP-p73 pathway, exerting the protective action on UVB-irradiated skin cells.

Malonic Acid Isolated from Pinus densiflora Inhibits UVB-Induced Oxidative Stress and Inflammation in HaCaT Keratinocytes.

Skin aging is caused by exposure to various external factors. Ultraviolet B (UVB) irradiation induces oxidative stress, photoaging, and inflammation in skin cells. Pinus densiflora Sieb. et Zucc. (red pine) has various antimicrobial and antioxidant activities. However, the anti-inflammatory effects of red pine on skin have rarely been reported. The protective effects of malonic acid (MA) isolated from Pinus densiflora were investigated against UVB-induced damage in an immortalized human keratinocyte cell line (HaCaT). MA increased levels of the antioxidant enzymes superoxide dismutase 1 (SOD-1) and heme oxygenase 1 (HO-1) via activation of nuclear factor-erythroid 2-related factor-2 (Nrf2), resulting in a reduction in UVB-induced reactive oxygen species (ROS) levels. Additionally, the inhibition of ROS increased HaCaT cell survival rate. Thus, MA downregulated the expression of ROS-induced nuclear factor-κB, as well as inflammation-related cytokines (interleukin-6, cyclooxygenase-2, and tumor necrosis factor-α). Furthermore, MA significantly suppressed the mitogen-activated protein kinase/activator protein 1 signaling pathway and reduced the expression of matrix metalloproteinases (MMPs; MMP-1, MMP-3, and MMP-9). In contrast, MA treatment increased the expression of collagen synthesis regulatory genes (COL1A1 and COL3A1) via regulation of Smad2/3 signal induction through transforming growth factor-β. In conclusion, MA protected against UVB-induced photoaging via suppression of skin inflammation and induction of collagen biosynthesis.

Influence of a combination of triptolide and ferulic acid on the activities of CYP450 enzymes and oxidative stress in HaCaT cells.

Topical administration of triptolide (TP) is effective in the treatment of rheumatoid arthritis (RA), but it can also induce skin irritation. Previous studies have used data mining strategies to analyze the application of Tripterygium wilfordii in the treatment of RA and have shown that TP and ferulic acid (FA) can be used in combination due to their component compatibility. The aims of the present study were to investigate the mechanisms underlying the effects of TP treatment and to identify its effects on metabolism and oxidative damage in the skin. MTT assay results suggested that the HaCaT cell survival rate was significantly increased when the compatibility ratio of TP to FA was 1:100. Moreover, the combination of TP with FA (TP + FA) did not significantly affect the activities of the cytochrome P40 (CYP) enzymes CYP family 1 subfamily A member 2 (CYP1A2), CYP2E1 and CYP3A4, when used as a ‘cocktail’. It was found that TP + FA significantly decreased the production levels of reactive oxygen species (ROS), superoxide dismutase and malondialdehyde in HaCaT cells, while significantly increasing levels of glutathione and catalase. In addition, TP + FA significantly increased nuclear factor erythroid 2-related factor 2 protein expression, compared with TP alone. Thus, the present results indicated that the underlying mechanism of TP + FA efficacy may be related to decreased ROS production level in HaCaT cells, increased production levels of key antioxidant factors and increased antioxidant activity of the epidermis, all of which were correlated with a protective effect against oxidative damage.

Oncostatin M upregulates Livin to promote keratinocyte proliferation and survival via ERK and STAT3 signalling pathways.

What is the central question of this study? What controls the proliferation and apoptosis in the pathogenesis of psoriasis? What is the main finding and its importance? The pathogenesis psoriasis involves abnormal homeostasis of keratinocytes, with hyperproliferation and decreasing apoptosis. An inhibitor of apoptosis protein family molecule, Livin, is highly expressed in psoriasis vulgaris lesional skin tissue. Expression of Livin was upregulated at transcription and protein levels after stimulation with oncostatinM (OSM). OSM promoted the survival of HaCaT cells in oxidative stress conditions. Expression of Livin and proliferation of HaCaT cells stimulated by OSM was regulated through ERK and STAT3 signalling pathways. This study might provide new insights into targeted therapy for psoriasis. Psoriasis is an immune-mediated chronic inflammatory disease. Abnormal homeostasis of keratinocytes, with hyperproliferation and decreasing apoptosis, is involved in the pathogenesis of psoriasis. Here, we report that an inhibitor of apoptosis protein family molecule, Livin, is highly expressed in psoriasis vulgaris lesional skin tissue at transcription and protein levels. Importantly, the expression level of Livin is related to the severity of psoriasis. The aim of the study was to investigate the regulation and functions of Livin in keratinocytes stimulated by the pro-inflammatory cytokine oncostatinM (OSM). The expression of Livin in HaCaT cells at mRNA and protein levels was measured by real-time PCR and Western blotting after OSM stimulation. The cell proliferation was measured by a 5-ethynyl-2'-deoxyuridine incorporation assay. Cell death was induced by the exogenous hydrogen peroxide (H2 O2 ) stress model, detected by 7-amino-actinomycinD staining and analysed by flow cytometry. Livin was overexpressed by a lentiviral transduction system to validate the roles of OSM and Livin in HaCaT cells. Specific inhibitors of ERK (U0126) and STAT3 (cryptotanshinone) were applied to investigate the signalling pathways involved in the regulation of Livin expression by OSM. The expression of Livin was upregulated after stimulation with OSM. OSM promoted the proliferation and survival of HaCaT cells. The expression of Livin and the proliferation of HaCaT cells induced by OSM were regulated through the ERK and STAT3 signalling pathways. We conclude that OSM promotes HaCaT cell proliferation and survival in conditions of oxidative stress.

Serotonin and 5-HT3 receptors sensitize human skin cells to direct irradiation cell death but not to soluble radiation-induced bystander signals

Ionizing radiation (IR) is an environmental carcinogen and the biological damages it elicits are mechanistically distinct between high and low doses. Non-targeted effects occurring in nonirradiated cells such as the radiation-induced bystander effect predominate at low doses of IR. However, the role of non-targeted effects in environmental radiation protection is often overlooked because the governing mechanisms are complex and multifactorial. An improved understanding of the signaling molecules and their capacity to sensitize specific cell types are essential in establishing environmental IR risks. In particular, serotonin (5-HT) has been identified to exacerbate both direct irradiation and bystander-induced cell death (CD) in certain cell types, although not all cell types are responsive to 5-HT in this respect. In this study, we further characterize the role of 5-HT and 5-HT receptors (5-HTR) in the amplification of CD following IR exposure in human keratinocytes. We examined the survival of HaCaT cells treated with 5-HT and the 5-HTR antagonists ketanserin (5-HT2A) and ondansetron (5-HT3) following exposure to direct IR and irradiated cell condition medium (ICCM). Nonirradiated cell survival was consistent with the vehicle control among 5-HT concentrations ranging from 0.001 to 100 μM. Significant 5-HT concentration-dependent increases in CD occurred following direct IR exposure. Nonirradiated ICCM-recipient CD was not altered by 5-HT (0.001–100 μM) when present during donor cell irradiation among all IR doses. Increases in direct irradiation CD evoked by 5-HT were significantly attenuated by ondansetron, blocking the effect of 5-HT, whereas ketanserin did not alter CD. Western blotting of these target 5-HTRs revealed protein expression of the 5-HT3 receptor, while the 5-HT2A receptor was not detected. We have demonstrated a definitive role for 5-HT in the exacerbation of CD following direct IR exposure and identified the 5-HT3 receptor as a potential target for ameliorating radiation damage in keratinocytes.

Inhibitory Effect of Galactooligosaccharide on Skin Pigmentation.

To investigate the effects of ingestion of galactooligosaccharide (GOS) on skin pigmentation, we conducted cell experiments and clinical trials. The effect of GOS on melanin accumulation was assessed in vitro using B16F10 cells. Moreover, melanin and erythema indexes following GOS consumption were explored during a double-blind, randomized, and placebo-controlled study, which included subjects divided by stratified block randomization to placebo or GOS. No cytotoxicity was observed at 70 mg/mL or lower GOS in B16F10 melanoma cells. Melanin accumulation was inhibited at 14 mg/mL or higher GOS. Upon ultraviolet B (UVB) irradiation, the survival of HaCaT cells (control) was reduced to 69.0% lower than baseline. A protective effect of GOS was observed upon treatment with 14~35 mg/mL GOS; however at 70 mg/mL, cells showed 64% viability compared to control cells irradiated with UVB. Delta values (Δ melanin index), which indicate the difference from the baseline melanin level, were significantly different to placebo (P<0.01) after 8 weeks. In the GOS group, delta values (Δ erythema index), which indicate the difference from baseline erythema level, also significantly differed from the placebo group (P<0.05) after 8 weeks. Our results suggest that intake of prebiotic GOS inhibits skin pigmentation and may represent a novel nutritional approach for skin care.

INF2 regulates oxidative stress-induced apoptosis in epidermal HaCaT cells by modulating the HIF1 signaling pathway

Promoting epidermal cell survival in an oxidative stress microenvironment is vital for skin regeneration after burns and/or wounds. However, few studies have explored the mediators related to epidermal cell apoptosis in an oxidative stress microenvironment. Cellular viability was determined using the MTT assay, TUNEL staining, western blot analysis and LDH release assay. Two independent siRNAs were transfected into HaCaT cell to repress INF2 and/or HIF1 in the presence of H2O2. Mitochondrial function was determined using JC-1 staining, mitochondrial ROS staining, immunofluorescence staining and western blotting. In the present study, our data demonstrated that the expression of inverted formin-2 (INF2) increased rapidly when the cells were exposed to H2O2. Interestingly, INF2 knockdown promoted HaCaT cell survival via reducing H2O2-mediated cell apoptosis. Molecular investigations demonstrated that INF2 deletion attenuated mitochondrial ROS overloading, restored the cellular redox balance, sustained the mitochondrial membrane potential, improved mitochondrial respiratory function and corrected the mitochondrial dynamics disorder in an H2O2-mimicking oxidative stress microenvironment. In addition, INF2 deletion upregulated the expression of HIF1. Interestingly, the inhibition of HIF1 increased cell death and caused mitochondrial stress despite the deletion of INF2, suggesting that the HIF1 signaling pathway is required for INF2 deletion-mediated HaCaT cell survival and mitochondrial protection. Altogether, our results identified INF2 as a novel apoptotic mediator for oxidative stress-mediated HaCaT cell death via modulating mitochondrial stress and repressing the HIF1 signaling pathway. This finding provides evidence to support the critical role played by the INF2-HIF1 axis in regulating mitochondrial stress and epidermal cell viability in an oxidative stress microenvironment.

MicroRNA-520a suppresses the proliferation and mitosis of HaCaT cells by inactivating protein kinase B.

Psoriasis is a chronic inflammatory disease of the skin for which an effective treatment strategy remains to be developed. Characteristics of psoriasis include an altered differentiation of keratinocytes and hyperplasia of the skin. The present study aimed to investigate the role served by miR-520a in psoriasis. The results demonstrated that miR-520a inhibited the proliferation of HaCaT cells. miR-520a directly regulated the mRNA and protein expression of its target gene, protein kinase B (AKT). The siRNA silencing of AKT expression in these cells was also evaluated. miRNA-520a repressed the proliferation and mitotic entry of HaCaT cells, and promoted cell apoptosis. AKT silencing suppressed the proliferation of HaCaT cells. These results suggest that miRNA-520a regulates the survival of HaCaT cells by inhibiting AKT expression. miRNA-520a and AKT may therefore be novel targets for the treatment of patients with psoriasis.

Enhancing the Antioxidant Ability of Trametes versicolor Polysaccharopeptides by an Enzymatic Hydrolysis Process.

Polysaccharopeptides (PSPs) are among the main bioactive constituents of Trametes versicolor (T. versicolor). The purpose of this research was to investigate the antioxidant activities of enzymatic hydrolysates obtained from T. versicolor polysaccharopeptides by 80 U/mL β-1,3-glucanase (PSPs-EH80). The half-inhibitory concentration (IC50) of PSPs-EH80 in metal chelating assay, ABTS and DPPH radical scavenging test results were 0.83 mg/mL, 0.14 mg/mL and 0.52 mg/mL, respectively, which were lower than that of PSPs-EH 20 U/mL. The molecular weights of the PSPs-EH80 hydrolysates were 300, 190, 140 and 50 kDa, respectively, and the hydrolysis of polysaccharides by β-1,3-glucanase did not change the original functional group. PSPs-EH80 reduced the reactive oxygen species (ROS) content at least twice that of treatment without PSPs-EH80. In addition, an oxidative damage test showed that PSPs-EH80 can improve HaCaT cell survival. According to our results, PSP demonstrates the potential of anti-oxidative damage; besides, enzyme hydrolysis can improve the ability of the PSP.

Mitochondria-Targeted Vitamin E Protects Skin from UVB-Irradiation.

Mitochondria-targeted vitamin E (MVE) is designed to accumulate within mitochondria and is applied to decrease mitochondrial oxidative damage. However, the protective effects of MVE in skin cells have not been identified. We investigated the protective effect of MVE against UVB in dermal fibroblasts and immortalized human keratinocyte cell line (HaCaT). In addition, we studied the wound-healing effect of MVE in animal models. We found that MVE increased the proliferation and survival of fibroblasts at low concentration (i.e., nM ranges). In addition, MVE increased collagen production and downregulated matrix metalloproteinase1. MVE also increased the proliferation and survival of HaCaT cells. UVB increased reactive oxygen species (ROS) production in fibroblasts and HaCaT cells, while MVE decreased ROS production at low concentration. In an animal experiment, MVE accelerated wound healing from laser-induced skin damage. These results collectively suggest that low dose MVE protects skin from UVB irradiation. Therefore, MVE can be developed as a cosmetic raw material.

Abstract 3809: Synthesis of the novel prostamide, 15-deoxy-Δ12,14 prostaglandin J2-ethanolamide, and characterization of its anti-tumor activity

Abstract Non-melanoma skin cancer (NMSC), is the most prevalent form of cancer in the United States. Many epithelial cancers including NMSC over-express the enzyme cyclooxygenase-2 (COX-2). COX-2 converts arachidonic acid (AA) to prostaglandins including prostaglandin E2 (PGE2). The interaction of PGE2 with the EP receptor has been shown to promote cancer by activating anti-apoptotic signals and angiogenesis. AA is also metabolized by COX-2 to 15deoxy, Δ12,14 prostaglandin J2 (15d-PGJ2) which promotes apoptosis via mechanisms including oxidative and endoplasmic reticulum (ER) stress. Arachidonoylethanolamide (AEA) is an endogenous cannabinoid that causes death in numerous cancer cell lines. Our lab previously demonstrated that AEA-induced apoptosis in NMSC cells occurs in a COX-2-dependent manner. Furthermore, mass spectral analysis of the products of AEA metabolism by COX-2 reveal the presence of a novel ethanolamide-conjugated J-series prostaglandin, (15d-PGJ2-EA). We hypothesize that the apoptotic effects of AEA are mediated by its primary cytotoxic metabolite, 15d-PGJ2-EA. To determine if 15d-PGJ2-EA mediates the effects of AEA we synthesized 15d-PGJ2-EA using 15d-PGJ2 as a substrate. The synthesis was accomplished using an uronium coupling reagent in the presence of ethanolamine with verification performed by H1-NMR. Tumorigenic JWF2 and non-tumorigenic HaCaT cell lines were treated with various concentrations of 15d-PGJ2-EA or 15d-PGJ2 and viability was measured using MTS assays. At 5 μM 15d-PGJ2-EA there was a 4.1-fold difference while at 10 μM there was a 217- fold difference in JWF-2 cell compared with HaCaT cell survival. In cells treated the AA metabolite, 15d-PGJ2, a 1.4-fold difference at 5 μM and a 1.7 fold difference at 10 μM in JWF2 compared to HaCaT cell survival was observed. To verify that cell death was due to apoptosis, the cleavage of caspase-3 and PARP was examined by conducting Western blot analysis. 15d-PGJ2-EA and 15d-PGJ2 increased caspase-3 and PARP cleavage in the tumorigenic but not in the non-tumorigenic cell line. To evaluate the mechanism of apoptosis, we examined oxidative- and ER-stress production in tumorigenic JWF2 cells. 15d-PGJ2-EA treated cells showed a 300% increase in DCF fluorescence (oxidative stress) and an 8.5-fold increase in CHOP-10 expression (ER stress) while 15d-PGJ2 treated cells showed 200% increase in DCF and a 3.5 fold increase in CHOP-10. Collectively, these findings suggest that AEA-induced apoptosis is mediated by 15d-PGJ2-EA since 15d-PGJ2-EA and AEA exhibit the same molecular effects. In addition, 15d-PGJ2-EA selectively induced cell death in tumorigenic but not non-tumorigenic keratinocytes. Furthermore, 15d-PGJ2-EA was more potent than 15d-PGJ2. Hence, the endocannabinoid metabolite 15d-PGJ2-EA may be an effective agent for eliminating cancer. Citation Format: Daniel Ladin, Colin Burns, Rukiyah T. Van Dross. Synthesis of the novel prostamide, 15-deoxy-Δ12,14 prostaglandin J2-ethanolamide, and characterization of its anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3809. doi:10.1158/1538-7445.AM2015-3809

Protective effects of citrus and rosemary extracts on UV-induced damage in skin cell model and human volunteers

Ultraviolet radiation absorbed by the epidermis is the major cause of various cutaneous disorders, including photoaging and skin cancers. Although topical sunscreens may offer proper skin protection, dietary plant compounds may significantly contribute to lifelong protection of skin health, especially when unconsciously sun UV exposed. A combination of rosemary and citrus bioflavonoids extracts was used to inhibit UV harmful effects on human HaCaT keratinocytes and in human volunteers after oral intake. Survival of HaCaT cells after UVB radiation was higher in treatments using the combination of extracts than in those performed with individual extracts, indicating potential synergic effects. The combination of extracts also decreased UVB-induced intracellular radical oxygen species (ROS) and prevented DNA damage in HaCaT cells by comet assay and decreased chromosomal aberrations in X-irradiated human lymphocytes. The oral daily consumption of 250mg of the combination by human volunteers revealed a significant minimal erythema dose (MED) increase after eight weeks (34%, p<0.05). Stronger protection was achieved after 12weeks (56%, p<0.01). The combination of citrus flavonoids and rosemary polyphenols and diterpenes may be considered as an ingredient for oral photoprotection. Their mechanism of action may deserve further attention.

Curcumin Protects Human Keratinocytes against Inorganic Arsenite-Induced Acute Cytotoxicity through an NRF2-Dependent Mechanism

Human exposure to inorganic arsenic leads to various dermal disorders, including hyperkeratosis and skin cancer. Curcumin is demonstrated to induce remarkable antioxidant activity in a variety of cells and tissues. The present study aimed at identifying curcumin as a potent activator of nuclear factor erythroid 2-related factor 2 (NRF2) and demonstrating its protective effect against inorganic arsenite- (iAs3+-) induced cytotoxicity in human keratinocytes. We found that curcumin led to nuclear accumulation of NRF2 protein and increased the expression of antioxidant response element- (ARE-) regulated genes in HaCaT keratinocytes in concentration- and time-dependent manners. High concentration of curcumin (20 μM) also increased protein expression of long isoforms of NRF1. Treatment with low concentrations of curcumin (2.5 or 5 μM) effectively increased the viability and survival of HaCaT cells against iAs3+-induced cytotoxicity as assessed by the MTT assay and flow cytometry and also attenuated iAs3+-induced expression of cleaved caspase-3 and cleaved PARP protein. Selective knockdown of NRF2 or KEAP1 by lentiviral shRNAs significantly diminished the cytoprotection conferred by curcumin, suggesting that the protection against iAs3+-induced cytotoxicity is dependent on the activation of NRF2. Our results provided a proof of the concept of using curcumin to activate the NRF2 pathway to alleviate arsenic-induced dermal damage.

Ultraviolet B‐induced LGI3 secretion protects human keratinocytes

Leucine-rich glioma inactivated 3 (LGI3) is known to be expressed mainly in the brain. However, the expression and physiological roles of LGI3 in skin cells remain unknown. In this study, it was found for the first time that LGI3 is expressed mostly by normal human keratinocytes. Furthermore, ELISA analysis showed that HaCaT human keratinocytes increased LGI3 secretion after exposure to ultraviolet B (UVB) in a time- and dose-dependent manner. We next investigated the possible role of LGI3 in keratinocytes. LGI3 (50ng/ml) increased survival of HaCaT cells by 20% after UVB irradiation (150mJ/cm(2) ). It was also found that LGI3 stimulates the phosphorylation of Akt, which is involved in the cell survival-signalling cascade. Furthermore, LGI3 led to the phosphorylation of MDM2 and subsequent p53 degradation. Taken together, the data suggest that LGI3 may regulate p53 levels and that keratinocyte-derived LGI3 may act as a novel cytokine for skin homoeostasis.

Models of the radiation-induced bystander effect

Purpose: To implement quantitative models of the Radiation-Induced Bystander Effects (RIBE) based on cellular excitation at a rate proportional to the concentration of signal molecules (called signals here) released by irradiated cells. Clonogenic cell survival and transformation frequency as a function of rescue time and dose were considered.Materials and methods: Our first stochastic model was based on the hypothesis that chemical signals are released into the extracellular medium by irradiated cells. These signals act on unirradiated cells switching them from the healthy to the dead state at rate R(t). We extended this model including a non-lethal transformed state in order to describe clonogenic cell survival and transformation frequency as a function of the number of alpha particles.Results: The first stochastic model was applied to an experiment on human keratinocyte (HaCat) cells yielding the half-life of at least one signal among the ensemble of possible candidates to trigger cell death in this cell culture. The second model yielded good fits to the data on clonogenic cell survival and transformation frequency in microbeam experiments with mouse embryo (C3H10T1/2) cells (Sawant et al. 2001a, 2001b).Conclusions: The fit of the first stochastic model to HaCat cell survival yielded a half-life of the order of minutes for possible signal candidates. This model also furnished the variance of the fraction of surviving cells.

Thrombomodulin exerts cytoprotective effect on low-dose UVB-irradiated HaCaT cells

Thrombomodulin (TM) is an endothelial cell surface anticoagulant glycoprotein that performs antimetastatic, angiogenic, adhesive, and anti-inflammatory functions in various tissues. It is also expressed in epidermal keratinocytes. We found that a physiological dose (10mJ/cm2) of mid-wavelength ultraviolet irradiation (UVB) significantly induced TM expression via the p38mitogen-activated protein kinase (MAPK)/cyclic AMP response element (CRE) signaling pathway in the epidermal keratinocyte cell line HaCaT; this shows that TM regulates the survival of HaCaT cells. SB203580, a p38MAPK inhibitor, significantly decreased TM expression and the viability of cells exposed to UVB. Furthermore, overexpression of TM markedly increased cell viability, and it was abrogated by TM small interfering RNA (siRNA), suggesting that TM may play an important role in exerting cytoprotective effect on epidermal keratinocytes against low-dose UVB.

EGFR-dependent downregulation of bim in epithelial cells requires MAPK and PKC-δ activities

Activation of the epidermal growth factor receptor (EGFR) provides a measure of protection to immortalized epidermal keratinocytes (HaCaT cells) against apoptosis induced by diverse cellular stressors. This effect is due, in part, to sustained MAPK-dependent Bcl-xL expression. Here, we report a second EGFR/MAPK-dependent signaling event that protects HaCaT cells against apoptosis incurred during forced suspension culture (anoikis). This pathway targets Bim, a pro-apoptotic BH3-only Bcl-2 family member. Bim expression was functionally relevant to HaCaT cell survival as demonstrated by partial protection against anoikis provided by siRNA-induced Bim downregulation. Growth factor starvation of attached and suspended cells was associated with enhanced Bim expression whereas EGFR activation reduced Bim expression by inducing Bim phosphorylation and proteasomal degradation. EGFR-dependent Bim phosphorylation required MAPK activation. Furthermore, PKC-δ activity contributed to both MEK/MAPK phosphorylation and Bim phosphorylation as demonstrated using both pharmacological inhibitors of PKC-δ and siRNA-mediated PKC-δ knockdown. In addition to HaCaT cells, EGFR activation supported survival and induced Bim phosphorylation in several squamous carcinoma cell lines in a strictly MAPK-dependent fashion. These results establish that EGFR activation attenuates susceptibility of immortalized and malignant keratinocytes to apoptosis by posttranslational control of Bim-EL expression through a pathway requiring PKC-δ and MEK/MAPK activation.