Human Keratinocyte Cell Line Research Articles

The Ca2+ channel TRPV4 is dispensable for Ca2+ influx and cell volume regulation during hypotonic stress response in human keratinocyte cell lines

Cell swelling as a result of hypotonic stress is counteracted in mammalian cells by a process called regulatory volume decrease (RVD). We have recently discovered that RVD of human keratinocytes requires the LRRC8 volume-regulated anion channel (VRAC) and that Ca2+ exerts a modulatory function on RVD. However, the ion channel that is responsible for Ca2+ influx remains unknown. We investigated in this study whether the Ca2+-permeable TRPV4 ion channel, which functions as cell volume sensor in many cell types, may be involved in cell volume regulation during hypotonic stress response of human keratinocytes. We interfered with TRPV4 function in two human keratinocyte cell lines (HaCaT and NHEK-E6/E7) by using two TRPV4-specific inhibitors (RN1734 and GSK2193874), and by creating a CRISPR/Cas9-mediated genetic TRPV4−/− knockout in HaCaT cells. We employed electrophysiological patch clamp analysis, fluorescence-based Ca2+ imaging and cell volume measurements to determine the functional importance of TRPV4. We could show that both hypotonic stress and direct activation of TRPV4 by the specific agonist GSK1016790A triggered intracellular Ca2+ response. Strikingly, the Ca2+ increase upon hypotonic stress was neither affected by genetic knockout of TRPV4 in HaCaT cells nor by pharmacological inhibition of TRPV4 in both keratinocyte cell lines. Accordingly, hypotonicity-induced cell swelling, downstream activation of VRAC currents as well as subsequent RVD were unaffected both in TRPV4 inhibitor-treated keratinocytes and in HaCaT-TRPV4−/− cells. In summary, our study shows that keratinocytes do not require TRPV4 for coping with hypotonic stress, which implies the involvement of other, yet unidentified Ca2+ channels.

Bioactive exopolysaccharide-composites based on gold and silver nanoparticles tailored for wound healing

The marine isolate Alteromonas macleodii Mo 169 secretes a exopolysaccharide (EPS) mainly composed of glucuronic acid and containing sulphate, pyruvate, and lactate as acyl substituents. The high density of negatively charged functional groups of the EPS renders it the ability to function as a stabilizing and reducing agent for the synthesis of metallic nanoparticles (NP). In this study, silver and gold NP (AgNP and AuNP, respectively) were successfully synthesized and stabilized by A. macleodii Mo 169 EPS. The obtained AuNP and AgNP presenting a spherical shape with an average particle size of 16 and 15 nm, respectively, displaying a polysaccharide layer that apparently contributed to reduce their aggregation. The cytotoxicity of the EPS and the bio-nanocomposites was evaluated on human keratinocyte (HaCaT) and fibroblast (CCD-1079Sk) cell lines. None of the samples demonstrated cytotoxicity for concentrations up to 1000 mg L-1. In vitro wound healing experiments demonstrated that both bio-nanocomposites promoted cell migration for concentrations above 100 mg L-1, thus contributing for a faster wound recovery. These findings demonstrate for the first time the potential of A. macleodii Mo169 EPS to synthesize and stabilize nanoparticles that possess promising physical-chemical and biological characteristics that render them valuable for the development of novel biomaterials for several applications, including wound care.

Expression of Interleukin-1β and Histological Changes of the Three-Dimensional Oral Mucosal Model in Response to Yttria-Stabilized Nanozirconia.

Over the years, advancement in ceramic-based dental restorative materials has led to the development of monolithic zirconia with increased translucency. The monolithic zirconia fabricated from nano-sized zirconia powders is shown to be superior in physical properties and more translucent for anterior dental restorations. Most in vitro studies on monolithic zirconia have focused mainly on the effect of surface treatment or the wear of the material, while the nanotoxicity of this material is yet to be explored. Hence, this research aimed to assess the biocompatibility of yttria-stabilized nanozirconia (3-YZP) on the three-dimensional oral mucosal models (3D-OMM). The 3D-OMMs were constructed using human gingival fibroblast (HGF) and immortalized human oral keratinocyte cell line (OKF6/TERT-2), co-cultured on an acellular dermal matrix. On day 12, the tissue models were exposed to 3-YZP (test) and inCoris TZI (IC) (reference material). The growth media were collected at 24 and 48 h of exposure to materials and assessed for IL-1β released. The 3D-OMMs were fixed with 10% formalin for the histopathological assessments. The concentration of the IL-1β was not statistically different between the two materials for 24 and 48 h of exposure (p = 0.892). Histologically, stratification of epithelial cells was formed without evidence of cytotoxic damage and the epithelial thickness measured was the same for all model tissues. The excellent biocompatibility of nanozirconia, as evidenced by the multiple endpoint analyses of the 3D-OMM, may indicate the potential of its clinical application as a restorative material.

Studies on anti-inflammatory activity of herbal extract mixture using inflammation induced human keratinocytes (HaCaT) cells

Inflammation is a general name for reactions occurring in several types of tissue injuries, infections, or immunologic stimulation as a defense against foreign or altered Endogenous substances. This comprises of a series of changes of the terminal tissues, which tend to eliminate the injurious agents and to repair the damaged tissue. Chemokines are a family of small proteins secreted by various cell types that play a role in the immune cell infiltration into inflammatory or infectious sites [1, 2]. Skin inflammation is closely associated with the production of Th2 chemokines [3]. These chemokines released from keratinocytes bind to and attract CCR4-positive Th2 cells into inflammatory tissues. It was reported that GJBRH inhibited the production of inflammatory cytokines by dermal endothelial cells. This study aimed to investigate whether GJBRH has inhibitory effects on TNF-α and IFN-γ-induced Th2 chemokine production and to identify the underlying molecular mechanisms in HaCaT human keratinocyte cell line. The present study was planned to evaluate the Herbal Extract sample for anti inflammatory activity using human skin keratinocytes

Wound Healing Activity of the Flavonoid-Enriched Fraction of Selaginella bryopteris Linn. against Streptozocin-Induced Diabetes in Rats

Diabetes and its complications, such as delayed wound healing, are increasing at an alarming rate in India, putting an enormous strain on the country’s limited healthcare resources. Hence, the present study proposes to screen/identify the possible mechanisms and to study the effect of the flavonoid-enriched fraction of Selaginella bryopteris extract against human keratinocyte cell lines (HaCaT) and streptozocin (STZ)-induced diabetic wounds in a male Wistar rat model. Chemical profiling was performed by an MTT assay. The obtained GC–MS analysis results showed the presence of amentoflavone, gallic acid, imidazole, palmitic acid, catechine, L-fucitol, lupeol, and myo-inositol as the major bioactive phytoconstituents. S. bryopteris induces the generation of ROS, the condensation of chromatin in the nucleus, and changes in the membrane potential of mitochondria in HaCaT cell lines. An S. bryopteris-dependent induction of apoptosis-mediated cell death in HaCaT cell lines was confirmed by an AO/PI analysis. Mitochondrial depolarization was reflected in JC-1 staining of cells. The wound size was reduced and epithelialization was enhanced. Keratinocyte migration decreased interleukins, TNF-α, IL-2, and IL-6 and the expression of pro-apoptotic (p53, caspase-3, caspase-9, and Bax) and anti-apoptotic (Bcl-2) genes in a dose-dependent manner. Keratinocyte migration increased antioxidant enzyme levels (CAT, SOD, MDA, and GSH). Wound healing is facilitated through the mitochondria-mediated apoptosis pathway, revealing a new area of diabetic wound therapy.

Auranofin ameliorates psoriasis-like dermatitis in an imiquimod-induced mouse by inhibiting of inflammation and upregulating FA2H expression

Psoriasis vulgaris (PV) is a common inflammatory skin disorder with immune imbalance contributing to its pathology. It has been reported that auranofin, a known anti-rheumatic drug, has the therapeutic effects on inflammatory diseases. However, the effects and underlying mechanisms of auranofin on PV have not been reported. Here, we showed that auranofin significantly attenuated the mixture of five proinflammatory cytokines (termed as M5) induced hyperproliferation and inflammation in HaCaT cells, a kind of human immortalized keratinocyte cell line. Topical administration of auranofin markedly alleviated the imiquimod (IMQ)-induced mouse psoriatic dermatitis both in appearance and in pathology. The psoriasis area and severity index (PASI) score, acanthosis, dermal infiltrated inflammatory cells and Ki-67 expression were improved obviously. The results of RNA-sequencing from both M5-induced HaCaT cells and IMQ-treated mouse dermatitis showed that auranofin could regulate inflammation-related signaling pathway. Meanwhile, we observed that fatty acid 2-hydroxylase (FA2H), a key molecule in lipid metabolism, was significantly increased after auraonfin treatment in both M5-induced HaCaT cells and IMQ-treated mouse skin. Knockdown of FA2H in HaCaT cells remarkably inhibited cell apoptosis, increased cell proliferation and the expression profiles of inflammatory-related genes, whereas FA2H overexpression had opposite effect. In conclusion, auranofin notably alleviated IMQ-induced psoriasis-like skin inflammation, which might be mediated by increasing FA2H expression, suggesting its potential to be a novel strategy for psoriasis treatment.

One-pot microwave synthesis of chitosan-stabilized silver nanoparticles entrapped polyethylene oxide nanofibers, with their intrinsic antibacterial and antioxidant potency for wound healing

Different physical and chemical techniques could be used to prepare chitosan/Silver nanoparticle (CHS/AgNPs) nanocomposite. The microwave heating reactor was rationally adopted as a benign tool for preparing CHS/AgNPs owing to less energy consumption and shorter time required for completing the nucleation and growth particles. UV–Vis, FTIR, and XRD, provided conclusive evidence of the AgNPs creation, while TEM micrographs elucidated that the size was spherical (20 nm). CHS/AgNPs were embedded in polyethylene oxide (PEO) nanofiber via electrospinning, and their biological properties, cytotoxicity evaluation, antioxidant, and antibacterial activity assays were investigated. The generated nanofibers have mean diameters of 130.9 ± 9.5, 168.7 ± 18.8, and 186.8 ± 8.19 nm for PEO, PEO/ CHS, and PEO/ CHS (AgNPs), respectively. Because of the tiny AgNPs particle size loaded in PEO/CHS (AgNPs) fabricated nanofiber, good antibacterial activity with ZOI against E. coli was 51.2 ± 3.2, and S. aureus was 47.2 ± 2.1 for PEO/ CHS (AgNPs) nanofibers. Non-toxicity was observed against Human Skin Fibroblast and Keratinocytes cell lines (>93.5 %), which justifies its great antibacterial potential to remove or prevent infection in wounds with fewer adverse effects.

Peptide-Protected Gold Nanoclusters Efficiently Ameliorate Acute Contact Dermatitis and Psoriasis via Repressing the TNF-α/NF-κB/IL-17A Axis in Keratinocytes

Immune-mediated skin diseases have a high prevalence and seriously affect patients' quality of life. Gold compounds have been considered promising therapeutic agents in dermatology, but the high incidence of adverse reactions have limited their clinical application. There is a great need to develop more effective and less toxic gold-based drugs. Gold nanoclusters fabricated by using peptides (pep-AuNCs) have appeared as potential biomedical nanomaterials because of their excellent biocompatibility, ease of fabrication and unique physicochemical properties. Glutathione (GSH) is an endogenous tripeptide and has been used for lightening the skin color. Therefore, we fabricated a well-defined gold nanocluster with GSH as an example to explore the immunomodulatory effect of AuNCs on a TNF-α-treated human keratinocyte cell line (HaCaT) in vitro, the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritant contact dermatitis (ICD) model and the oxazolone (OXA)-induced psoriatic model in vivo. The results indicated that topically applied AuNCs successfully attenuated the severity of ICD and psoriasis-like lesions. In vitro and in vivo, AuNCs effectively inhibited the abnormal activation of the NF-κB pathway and the consequent overexpression of proinflammatory cytokines in keratinocytes. In particular, the transactivation of IL-17A, the most important cytokine in psoriasis pathology, was effectively inhibited by AuNCs treatment. In addition, AuNCs did not show any obvious cytotoxicity in HaCaT cells at doses even up to 100 µM and did not induce any irritation in the healthy skin and major organs, which indicated their favorable biosafety. These results indicate that biocompatible pep-AuNCs might be a promising gold-based nanomedicine for the treatment of inflammatory skin diseases.

Solvent System-Guided Extraction of Centaurium spicatum (L.) Fritch Provides Optimized Conditions for the Biological and Chemical Characteristics of the Herbal Extracts.

Spiked centaury (Centaurium spicatum) is a well-known medicinal plant from the Mediterranean region with various bioactivities, but there are no studies addressing the use of different solvent systems to improve its pharmacological potential. Nine extraction procedures were adapted to study the effects of solvent composition on the content of bioactive compounds in C. spicatum extracts and on corresponding bioactivities. Targeted metabolomics was performed to obtain information on the chemical composition of extracts. Ethanol-water-based extraction procedures were the most efficient in isolating polyphenols, while less polar butanol extract contained the highest amount of iridoids. Antioxidant potential analysis revealed stronger activity in extracts with higher polyphenol content. Bacillus cereus and Staphylococus aureus were designated as the most sensitive bacterial strains to the activity of extracts, while among the micromycetes tested, Penicillium funiculosum was the most susceptible strain. Butanol extract showed antivirulence potential on Candida albicans morphological transition from yeast to hyphal form, and selected extracts were effective against biofilm formation in two Candida species. All the extracts tested in this study showed no cytotoxic activity to immortalize human skin keratinocyte cell line (HaCaT), whereas extracts obtained by ethanol-water extraction stand out for their potent wound healing effects. Moreover, the influence of the extraction solvent system on various bioactivities of C. spicatum is reported herein for the first time. Overall, the results presented in this study promote the use of C. spicatum as a source of natural products with potential antioxidant, wound healing, and antimicrobial applications that are potentially safe for human use.

Colloidal silver against macrophage infections and biofilms of atypical mycobacteria.

Skin and soft tissue infection (SSTI) caused by atypical mycobacteria such as Mycobacterium abscessus and Mycobacterium avium intracellulare complex (MAIC) have increased in recent years. Current therapeutic options are limited, and hence new and better therapies are urgently required. Colloidal Silver (CS) has been identified for its widespread antibacterial properties and silver-impregnated dressings have been used for SSTIs caused by various pathogens. The efficacy of Green Synthesized Colloidal Silver (GSCS) was investigated for bacterial growth inhibition (BGI) using a microdilution method and minimum biofilm eradication concentration (MBEC) using resazurin assay and confocal scanning laser microscopy (CSLM) of M. abscessus (n = 5) and MAIC (n = 5). The antibacterial effect of GSCS against M. abscessus infected macrophages was also evaluated. The in vitro cytotoxicity of GSCS on a human keratinocyte cell line (HaCaT) and neonatal foreskin fibroblasts was analyzed by the crystal violet proliferation assay. Average BGI and MBEC of GSCS varied between 0.7 and 22 ppm for M. abscessus and MAIC. The concentration of 3 ppm reduced M. abscessus-infection in macrophages significantly. GSCS was not cytotoxic to HaCaT and neonatal foreskin fibroblast cells at concentrations < 3 ppm up to 2h exposure time. GSCS therefore, has the potential for topical application against atypical mycobacterial SSTI.

Selenium Bio-Nanocomposite Based on Alteromonas macleodii Mo169 Exopolysaccharide: Synthesis, Characterization, and In Vitro Antioxidant Activity.

In this study, the novel exopolysaccharide (EPS) produced by the marine bacterium Alteromonas macleodii Mo 169 was used as a stabilizer and capping agent in the preparation of selenium nanoparticles (SeNPs). The synthesized nanoparticles were well dispersed and spherical with an average particle size of 32 nm. The cytotoxicity of the EPS and the EPS/SeNPs bio-nanocomposite was investigated on human keratinocyte (HaCaT) and fibroblast (CCD-1079Sk) cell lines. No cytotoxicity was found for the EPS alone for concentrations up to 1 g L-1. A cytotoxic effect was only noticed for the bio-nanocomposite at the highest concentrations tested (0.5 and 1 g L-1). In vitro experiments demonstrated that non-cytotoxic concentrations of the EPS/SeNPs bio-nanocomposite had a significant cellular antioxidant effect on the HaCaT cell line by reducing ROS levels up to 33.8%. These findings demonstrated that the A. macleodii Mo 169 EPS can be efficiently used as a stabilizer and surface coating to produce a SeNP-based bio-nanocomposite with improved antioxidant activity.

Characterization of a Chimeric MmuPV1 Genome with HPV-16E6E7

Background/Objectives: Papilloma Viruses (PVs) are double-stranded DNA viruses that infect cutaneous and mucosal epithelium. In humans, there are over 100 types of PVs. HPV-16 is a high-risk type that causes ~50% of cervical cancers and ~70% of oropharyngeal cancers by expressing viral oncogenes E6 and E7 in replicating keratinocytes. The mouse PV, MmuPV1, displays species tropism and causes neoplastic lesions. Its discovery in 2011 introduced the opportunity to study PVs from early stages of infection to cancer development. Such an in vivo model of HPV-16 could uncover novel mechanisms for treatment intervention and disease prevention. Our study aims to characterize a small-animal infection model using a chimeric MmuPV1 genome with HPV-16 E6 and E7 in place of mouse E6 and E7 (MmuPV1-16E6E7). The goal of our study is to investigate the genome’s ability to express the HPV-16 oncogenes in vitro and to cause tumors in vivo. Methods: To increase selective pressure in vitro, we replaced the MmuPV1 L1 and L2 genes with a neomycin cassette (MmuPV1-16E6E7neo). We packaged these genomes into infectious quasiviruses using a HEK 293TTF viral packing cell line. After isolating quasiviruses, we infected two donor human foreskin keratinocytes (HFK) cell lines. Forty-eight hours post infection, we isolated mRNA for qPCR to quantify HPV-16 E6 expression. To investigate whether MmuPV1-16E6E7 can cause tumors in in vivo, we infected athymic nude (N/J) mice orally with quasiviruses to evaluate infection and viable genome persistence. Results: HPV-16 E6 cDNA was detected in three HFK isolates. Conclusions/Future Directions: The presence of HPV-16 E6 cDNA indicates that the gene can be successfully transcribed from MmuPV1-16E6E7neo in HFKs. Future studies will assess this ability in primary mouse keratinocytes. If tumors are observed, we can use this model to study the efficacy of antiviral compounds to inhibit HPV16 E6 in vivo.

Manuka Honey/2-Hydroxyethyl Methacrylate/Gelatin Hybrid Hydrogel Scaffolds for Potential Tissue Regeneration.

Scaffolding biomaterials are gaining great importance due to their beneficial properties for medical purposes. Targeted biomaterial engineering strategies through the synergy of different material types can be applied to design hybrid scaffolding biomaterials with advantageous properties for biomedical applications. In our research, a novel combination of the bioactive agent Manuka honey (MHo) with 2-hydroxyethyl methacrylate/gelatin (HG) hydrogel scaffolds was created as an efficient bioactive platform for biomedical applications. The effects of Manuka honey content on structural characteristics, porosity, swelling performance, in vitro degradation, and in vitro biocompatibility (fibroblast and keratinocyte cell lines) of hybrid hydrogel scaffolds were studied using Fourier transform infrared spectroscopy, the gravimetric method, and in vitro MTT biocompatibility assays. The engineered hybrid hydrogel scaffolds show advantageous properties, including porosity in the range of 71.25% to 90.09%, specific pH- and temperature-dependent swelling performance, and convenient absorption capacity. In vitro degradation studies showed scaffold degradability ranging from 6.27% to 27.18% for four weeks. In vitro biocompatibility assays on healthy human fibroblast (MRC5 cells) and keratinocyte (HaCaT cells) cell lines by MTT tests showed that cell viability depends on the Manuka honey content loaded in the HG hydrogel scaffolds. A sample containing the highest Manuka honey content (30%) exhibited the best biocompatible properties. The obtained results reveal that the synergy of the bioactive agent, Manuka honey, with 2-hydroxyethyl methacrylate/gelatin as hybrid hydrogel scaffolds has potential for biomedical purposes. By tuning the Manuka honey content in HG hydrogel scaffolds advantageous properties of hybrid scaffolds can be achieved for biomedical applications.

Cytotoxic, cytostatic, and keratolytic activity of anti-dandruff shampoo formulations

Background: Dandruff is characterized by increased sebum production, yeast colonization, and hyper-keratinization leading to an itchy-flaky scalp. Ancillary non-microbial reasons for flaky scalp include ultraviolet rays and excessive heat from the sun. Thus, apart from traditional anti-fungal activity, anti-dandruff formulations should also possess the keratolytic activity and should be safe to use. Methods: Selsun, a shampoo formulation with 2.5% selenium sulfide (SS2.5), was tested in-vitro for its cytotoxic and cytostatic activities via cell viability of primary human dermal fibroblasts (HDFs). Its keratolytic activity was determined by assessing anti-proliferative activity on immortalized human keratinocyte (HaCaT) cell line. The shampoo was compared with 3 other shampoo formulations, one containing 2% ketoconazole (K2), second containing 2% ketoconazole with 1% zinc pyrithione (K2ZP1), and third containing 1% zinc pyrithione (ZP1). Results: Of the tested samples, SS2.5 and K2ZP1 showed cytostatic and keratolytic activity where the IC50 values on normal HDFs i.e., 0.521 mg/ml and 0.220 mg/ml, respectively, were higher than those on HaCaT cells (0.162 mg/ml and 0.198 mg/ml, respectively). SS2.5 displayed the highest keratolytic activity at IC50 of 0.162 mg/ml, followed by K2ZP1 (0.198 mg/ml) and K2 (0.267 mg/ml). On normal HDFs, SS2.5 showed the highest IC50 value (0.521 mg/ml), followed by K2 at 0.264 mg/mL, K2ZP1 at 0.220 mg/ml, and ZP1 at 0.139 mg/ml. Conclusions: SS2.5 was found to possess the highest cytostatic and keratolytic activities among all the shampoo formulations tested, thereby confirming its safety profile.

Immunomodulatory effect of dopamine in human keratinocytes and macrophages under chronical bisphenol-A exposure conditions

Dopamine is a key neurotransmitter that links the nervous and the immune system. Bisphenol A (BPA) is an endocrine disruptor with a wide distribution in the environment that is used in the manufacturing of plastic products. Evidence shows that BPA can interfere with the central dopaminergic transmission; however, there are no previous reports of this effect outside the central nervous system. Thus, the aim of this work was to investigate the in vitro mechanisms of action involved in the response to dopamine in both human keratinocyte and macrophage cell lines chronically exposed to BPA. Dopamine modulates cytokine secretion and NF-κB expression in BPA-treated HaCaT keratinocytes, without modifying these parameters in BPA-treated THP-1 macrophages. In addition, dopamine increases MMP activity in both BPA-treated cell lines, although it decreases keratinocytes migration. We suggest the immunomodulatory effect of dopamine might be different in keratinocytes and macrophages under chronical BPA exposure conditions. These findings revealed for the first time the in vitro immunomodulatory effect of dopamine in the presence of BPA at peripheral level.

Chemical Profile and Biological Effects of an Herbal Mixture for the Development of an Oil-in-Water Cream.

Three individual hydroalcoholic extracts derived from Hamamelis virginiana leaves, Krameria lappacea root, Salix alba bark, and the resulting herbal mixture (HM) were assessed for the phytochemical profile as well as for antibacterial and cytotoxic potential. The chemical composition of the individual extracts and of their mixture was analyzed by chromatographical (LC-MS) and spectrophotometrical methods. The antimicrobial properties were evaluated by using the agar-well diffusion and the broth microdilution assays, whereas the potential cytotoxicity was investigated on human keratinocyte cell line by MTT method and apoptosis test. The HM composition revealed important amounts of valuable polyphenolic compounds provided from the individual extracts, having synergistic biological effects. All tested extracts displayed in vitro antimicrobial properties, with a significantly higher efficacy noticed for the HM when tested against Staphylococcus aureus. Moreover, none of the tested extracts was responsible for in vitro cytotoxicity against the human keratinocytes in the selected concentration range. Furthermore, the HM was included in an oil-in-water cream for the nonpharmacological treatment of seborrheic dermatitis, developed and optimized by using a QbD approach. A D-optimal experimental plan with four factors that varied on two levels was used to investigate the effect of the quantitative variation of the formulation factors (emulsifier, co-emulsifier, thickening agent, oily phase ratio) on the characteristics of the cream in terms of firmness, consistency, adhesiveness, stringiness, spreadability, and viscosity. Based on the experimental results, an optimal formulation containing 2.5% emulsifier and 20% oily phase was prepared and analyzed. The obtained results showed appropriate quality characteristics of this novel cream, which may be used in the future to manage the associated symptoms of seborrheic dermatitis.

Glucocorticoid reduces the efficacy of afatinib on the head and neck squamous cell carcinoma

Glucocorticoids (GC) are widely used to counter the adverse events during cancer therapy; nonetheless, previous studies pointed out that GC may reduce the efficacy of chemotherapy on cancer cells, especially in epidermal growth factor receptor (EGFR)-targeted therapy of head and neck squamous cell carcinoma (HNSCC) remaining to be elucidated. The primary aim of the present study was to probe into the GC-induced resistance of EGFR-targeted drug afatinib and the underlying mechanism. HNSCC cell lines (HSC-3, SCC-25, SCC-9, and H-400) and the human oral keratinocyte (HOK) cell lines were assessed for GC receptor (GR) expression. The promoting tumor growth effect of GC was evaluated by the CCK-8 assay and flow cytometry. Levels of signaling pathways participants GR, mTOR, and EGFR were determined by quantitative polymerase chain reaction and western blotting. GC increased the proliferation of HNSCC cells in a GR-dependent manner and promoted AKT/mTOR signaling. But GC failed in counteracting the inhibition of rapamycin in the mTOR signaling pathway. Besides, GC also induced resistance to EGFR-targeted drug afatinib through AKT/mTOR instead of the EGFR/ERK signaling pathway. Thus, GCs reduce the efficacy of afatinib on HNSCC, implicating a cautious use of glucocorticoids in clinical practice.

Kukoamine B from Lycii Radicis Cortex Protects Human Keratinocyte HaCaT Cells through Covalent Modification by Trans-2-Nonenal

The unsaturated aldehyde trans-2-nonenal is known to be generated by lipid peroxidation at the surface of the skin in an aging-related manner and has harmful effects on keratinocytes in the skin. In this study, the protective effect of a Lycii Radicis Cortex (LRC) extract against trans-2-nonenal-induced cell damage on human keratinocyte cell lines (HaCaT) was investigated. Notably, treatment with the LRC extract resulted in an increase in cell survival, while trans-2-nonenal decreased the viability of HaCaT cells. For identification of interaction between the LRC extract and trans-2-nonenal, this mixture was incubated in simulated physiological conditions, showing a strong decrease in the amount of trans-2-nonenal by the LRC extract. Subsequent LC-ESI-MS analysis revealed that kukoamine B (KB) formed Schiff base-derived pyridinium adducts with trans-2-nonenal. Thus, these results suggest that KB could be a potential agent that may protect HaCaT cells by forming new products with trans-2-nonenal.

Mulberroside F from In Vitro Culture of Mulberry and the Potential Use of the Root Extracts in Cosmeceutical Applications

Mulberry (Morus spp.) is primarily used in sericulture, and its uses also extend to the food, pharmaceutical, and cosmetic industries. Mulberry extracts are rich in many bioactive compounds that exhibit a wide range of biological properties. Mulberroside F (Moracin M-6, 3'-di-O-β-D-glucopyranoside), one of the bioactive compounds found in mulberry, has previously been reported as a whitening agent by inhibiting melanin synthesis and exhibiting antioxidant effects. However, there is still limited information on the presence of this compound in plants cultured in vitro. In this study, the mulberroside F content, biochemical, and cytotoxic properties of the extracts from mulberry cultured in vitro were determined. The results revealed that both root and callus were found to be a potential source of mulberroside F. Furthermore, the mulberroside F content was positively correlated with the inhibitory effects on tyrosinase activity. Cell viability assay also revealed that crude extract of the mulberry root has no cytotoxicity in both human keratinocyte cell line (HaCaT) and Vero cells. Taken together, mulberry tissue culture represents a possible alternative and continuous production of mulberroside F, which could be further utilized in cosmeceutical applications.

Particulate matter 2.5 induces the skin barrier dysfunction and cutaneous inflammation via AhR- and T helper 17 cell-related genes in human skin tissue as identified via transcriptome analysis.

Particulate matter (PM2.5) is an environmental pollutant causing skin inflammatory diseases via epidermal barrier damage. However, the mechanism and related gene expression induced by PM2.5 remains unclear. Our aim was to determine the effect of PM2.5 on human skin tissue ex vivo, and elucidate the mechanism of T helper 17 cell-related inflammatory cytokine and skin barrier function. We verified the expression levels of gene in PM2.5-treated human skin tissue using Quantseq (3' mRNA-Seq), and Gene Ontology (GO) terms and protein-protein interaction (PPI) networks were performed. The PM2.5 treatment significantly enhanced the expression of Th 1, 2, 17 and 22 cell-related genes (cut-off value: │1.2 │ > fold change and p < 0.05). Most of all, Th17 cell-related genes are upregulated and those genes are associated with skin epidermal barrier function and Aryl hydrocarbon receptor (AhR), a xenobiotic receptor, pathway. In human keratinocyte cell lines, AhR-regulated genes (e.g. AhRR, CYP1A1, IL6 and IL36G), Th17 cell-related genes (e.g. IL17C) and epidermal barrier-related genes (e.g. SPRR2A and KRT71) are significantly increased after PM2.5. In the protein level, the secretion of IL-6 and IL-36G was increased in human skin tissue following PM2.5 treatment, and the expression of SPRR2A and KRT71 was significantly increased. PM2.5 exposure could ruin the skin epidermal barrier function via AhR- and Th17 cell-related inflammatory pathway.