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Sample records for human hacat cells

  1. Effects of Ambient Fine Particles PM2.5 on Human HaCaT Cells

    PubMed Central

    Li, Qiao; Kang, Zhihua; Jiang, Shuo; Zhao, Jinzhuo; Yan, Shuxian; Xu, Feng; Xu, Jinhua

    2017-01-01

    The current study was conducted to observe the effects of fine particulate matter (PM2.5) on human keratinocyte cell line (HaCaT) cells. The potential mechanism linking PM2.5 and skin was explored. HaCaT cells were cultured and then accessed in plate with PM2.5. Cell viability was tested by Cell Counting Kit-8. The mRNA and protein expression of Filaggrin, Loricrin, Involucrin, and Repetin were analyzed. The levels of Granulocyte-macrophage Colony Stimulating Factor, Thymic Stromal Lymphopoietin, Tumor Necrosis Factor-α, Interleukin-1α, and Interleukin-8 were detected in the supernatant of the HaCaT cell with enzyme-linked immunosorbent assay kits. Cell viability decreased with the increase in PM2.5. Compared with the control group, the protein expression of Filaggrin, Repetin, Involucrin, and Loricrin showed different expression patterns in PM2.5 treatment groups. The level of Tumor Necrosis Factor-α, Thymic Stromal Lymphopoietin, Interleukin-1α, and Interleukin-8 significantly increased in the cells treated with PM2.5. Ambient PM2.5 may increase the risk of eczema and other skin diseases. The relative mechanism may be associated with the impairment of the skin barrier and the elevation of inflammatory responses. PMID:28085100

  2. Synergistic phototoxic effects of glycolic acid in a human keratinocyte cell line (HaCaT).

    PubMed

    Lai, Wan-Wen; Hsiao, Yu-Ping; Chung, Jing-Gung; Wei, Yau-Huei; Cheng, Ya-Wen; Yang, Jen-Hung

    2011-12-01

    Glycolic acid (GA) has been widely used in cosmetic agents and superficial chemical peeling in recent years. It has long been concerned that UV irradiation would enhance the photosensitivity of GA on human skin. Therefore, it is mandatory to explore the biologic effects of concomitant exposure of GA and UV irradiation in human keratinocytes. The aim of the study is to explore the effects of concomitant exposure of GA and UVB in a human keratinocyte cell line (HaCaT). We used HaCaT to investigate the effects of GA (5mM), UVB (50mJ/cm(2)), and co-treatment with GA and UVB (GA+UVB) in human keratinocytes. We used a phase contrast microscope to observe morphological changes of the cells, and employed flow cytometry to detect cell viability, cell cycle, and mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) levels. Cell damage was detected by DAPI stain, and Western blot was used to detect the activities of apoptosis- related and cycle checkpoint-related proteins such as Bax, Bcl-2, caspases-3, -4, -9, Endo G, AIF, and p21, p27, p53, cdk2, cyclin E, cyclin A. We found that either GA or UVB alone had inhibitory effect on cell proliferation, and co-treatment with GA and UVB had synergistic anti-proliferative effect. GA alone did not affect the cell cycle, and UVB induced HaCaT cells accumulated at S phase, and co-treatment with GA and UVB arrested cells at S phase more prominently. Moreover, all the treatment with GA, UVB, and GA+UVB in HaCaT cells induced apoptosis. We further demonstrated that GA had synergistic apoptotic effect in human keratinocytes. GA and UVB both had effects on the decline of MMP and increase of ROS release, and GA had synergistic increase in the level of ROS in UVB-treated HaCaT cells. Besides, co-treatment with GA and UVB had synergistic effect on apoptosis through the over-expressions of Bax, p21, p53, caspases-3, -4, -9, Endo G and AIF, and confocal microscopy disclosed translocation of AIF and Endo G from

  3. Propolis Inhibits UVA-Induced Apoptosis of Human Keratinocyte HaCaT Cells by Scavenging ROS

    PubMed Central

    Kim, Han Bit; Yoo, Byung Sun

    2016-01-01

    Propolis is a resinous material collected by honeybees from several plant sources. This research aimed at showing its protective effect against UVA-induced apoptosis of human keratinocyte HaCaT cells. Using Hoechst staining, it was demonstrated that propolis (5 and 10 μg/mL) significantly inhibited the apoptosis of HaCaT cells induced by UVA-irradiation. Propolis also showed the protective effect against loss of mitochondrial membrane potential induced by UVA-irradiaiton in HaCaT cells. Propolis also inhibited the expression of activated caspase-3 induced by UVA-irradiation. To investigate the role of ROS in UVA-induced apoptosis and protection by propolis, the generation of ROS was determined in cells. The results showed that the generation of ROS was markedly reduced in cells pretreated with propolis. Consequently, propolis protected human keratinocyte HaCaT cells against UVA-induced apoptosis, which might be related to the reduction of ROS generation by UVA-irradiation. PMID:27818737

  4. Propolis Inhibits UVA-Induced Apoptosis of Human Keratinocyte HaCaT Cells by Scavenging ROS.

    PubMed

    Kim, Han Bit; Yoo, Byung Sun

    2016-10-01

    Propolis is a resinous material collected by honeybees from several plant sources. This research aimed at showing its protective effect against UVA-induced apoptosis of human keratinocyte HaCaT cells. Using Hoechst staining, it was demonstrated that propolis (5 and 10 μg/mL) significantly inhibited the apoptosis of HaCaT cells induced by UVA-irradiation. Propolis also showed the protective effect against loss of mitochondrial membrane potential induced by UVA-irradiaiton in HaCaT cells. Propolis also inhibited the expression of activated caspase-3 induced by UVA-irradiation. To investigate the role of ROS in UVA-induced apoptosis and protection by propolis, the generation of ROS was determined in cells. The results showed that the generation of ROS was markedly reduced in cells pretreated with propolis. Consequently, propolis protected human keratinocyte HaCaT cells against UVA-induced apoptosis, which might be related to the reduction of ROS generation by UVA-irradiation.

  5. Cytogenotoxicity of selected organophosphate insecticides on HaCaT keratinocytes and NL-20 human bronchial cells.

    PubMed

    Arteaga-Gómez, Eduardo; Rodríguez-Levis, Alejandra; Cortés-Eslava, Josefina; Arenas-Huertero, Francisco; Valencia-Quintana, Rafael; Gómez-Arroyo, Sandra

    2016-02-01

    Organophosphate insecticides (OI) are widely used. To humans the main routes of exposure are skin and inhalation. For this, keratinocytes (HaCaT) and bronchial cells (NL-20) were used as cell culture models to evaluate the effects of OI. The aim of this study was to evaluate the effect of four OI on HaCaT and NL-20 cells: azinphos-methyl, (AM); parathion-methyl (PM); omethoate (OM); and methamidophos (MET). Cells were exposed to 0.1, 1 and 10 μg/μL of each. Results showed a decrease in cell viability in both cell lines. Viability of the NL-20 cell line decreased with the three concentrations of OM. All differences were significant (p < 0.05). Genotoxic damage, evaluated through the comet assay, was observed in both cell lines with AM. NL-20 cell line was more sensitive than HaCaT. Higher concentrations of the insecticides except MET, induced cell death. MET caused DNA damage in HaCaT cells at all concentrations. Differences were significant (p < 0.05). Both cell lines revealed the presence of single membrane vacuoles of different sizes when exposed to 1 μg/μL of each insecticide. Quantitative real time-polymerase chain reaction (RT-qPCR) showed an increase of BN1 gene in HaCaT by effect of AM and MET at 1 μg/μL. In conclusion, all the insecticides induced different levels of cyto and genotoxic effects in both cell lines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. MiADMSA protects arsenic-induced oxidative stress in human keratinocyte 'HaCaT' cells.

    PubMed

    Pachauri, Vidhu; Srivastava, Priyanka; Yadav, Abhishek; Kushwaha, Pramod; Flora, Swaran J S

    2013-06-01

    Arsenic toxicity may lead to skin manifestations and arsenic accumulation in keratinised tissue. Thus human keratinocytes has been extensively used to study dermal effects of arsenic exposure. The present study was aimed to investigate time and dose-dependent effects of arsenic using HaCaT cell line. Another major focus of the study was to evaluate if treatment with monoisoamyl dimercaptosuccinic acid (MiADMSA) offers protection against arsenic-induced oxidative stress and apoptotic cell death using HaCaT cells. HaCaT cell lines were incubated to three different concentrations of arsenic (10, 30 and 50 μM) for 24 h to identify the toxic dose by measuring oxidative stress variables. Later, MiADMSA pre-incubation for an hour preceded arsenic exposure (30 μM). We evaluated cell morphology, lactate dehydrogenase, glutathione linked enzyme and antioxidant enzyme activities to measure oxidative stress status, while MTT assay and caspase 9 and 3 levels were determined for cell viability and apoptotic status. The present study suggests arsenic-induced toxicity in a concentration-dependant manner. Arsenic also caused a significant increase in lactate dehydrogenase accompanied by an elevated antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase and caspase activity). Interestingly, pre-treatment of cell with MiADMSA elicited significant protection against arsenic-induced oxidative stress and apoptotic cell death. The present findings are of clinical relevance and suggest MiADMSA to be a promising candidate in protecting skin against arsenic-induced toxic effects, which need further exploration using in vivo experimental models.

  7. Mycobacterium ulcerans mycolactone interferes with adhesion, migration and proliferation of primary human keratinocytes and HaCaT cell line.

    PubMed

    Graziola, Francesca; Colombo, Elena; Tiberio, Rossana; Leigheb, Giorgio; Bozzo, Chiarella

    2017-04-01

    The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) is closely associated with the secretion of exotoxin mycolactone. The cytotoxicity of mycolactone has been linked to its apoptogenic activity. We explored if low mycolactone concentrations, which are not able to induce apoptosis, can influence other essential activities on two primary human keratinocyte populations, keratinocyte stem cells (KSC) and transit amplifying cells (TAC), and on a human keratinocyte line, HaCaT. We demonstrated that 0.01 and 0.1 ng/ml mycolactone A/B are not able to induce apoptosis in primary human keratinocytes, but interfere with KSC wound repair. Moreover, the same toxin concentrations reduce cell proliferation of KSC and TAC and their ability to adhere to type IV collagen. HaCaT cells are more resistant to the toxin; nevertheless, they show a delayed woud repair when treated with 1 and 10 ng/ml mycolactone A/B. Moreover, these sub-apoptotic concentrations affect their ability to proliferate and adhere to collagen IV. Wound healing is a complex mechanism, which occurs "in vivo" as the outcome of many co-ordinated events. Sub-apoptotic mycolactone concentrations can affect essential mechanisms, which are required to achieve wound repair, such as adhesion, migration and proliferation of human keratinocytes.

  8. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

    PubMed

    Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

    2013-10-01

    Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

  9. Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes.

    PubMed

    Micallef, Ludovic; Belaubre, Françoise; Pinon, Aline; Jayat-Vignoles, Chantal; Delage, Christiane; Charveron, Marie; Simon, Alain

    2009-02-01

    The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro, the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time-period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 mm. In NHK, addition of calcium-induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2-M phases. Furthermore, RT-PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium-induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.

  10. Impact of Different Spa Waters on Inflammation Parameters in Human Keratinocyte HaCaT Cells.

    PubMed

    Zöller, Nadja; Valesky, Eva; Hofmann, Matthias; Bereiter-Hahn, Jürgen; Bernd, August; Kaufmann, Roland; Meissner, Markus; Kippenberger, Stefan

    2015-12-01

    The treatment of different skin conditions with spa waters is a long tradition dating back to at least late Hellenism. Interestingly, independent scientific examinations studying the effect of spa waters are scarce. In the present in vitro study, we compared the effect of culture media supplemented with (a) thermal spa waters (La Roche-Posay, Avène) and (b) two natural mineral drinking waters (Heppinger, Adelholzener) on physiological parameters in HaCaT keratinocytes. The different medium preparations were investigated with regard to cell proliferation and cell damage. Moreover, the impact on inflammation parameters with and without ultraviolet B (UVB) irradiation was examined. Two popular thermal spring waters were found to suppress cell proliferation and cell damage. Moreover, these waters reversed the induction of interleukin-6, as measured using enzyme-linked immunosorbent assay and promoter transactivation, and the formation of reactive oxygen species after UVB stimulation. Of note, the two natural mineral waters, which are distributed as drinking waters, had some effect on the above-mentioned parameters but to a lesser extent. In summary, our results show that spa waters, and particularly those derived from thermal springs, reduce parameters associated with inflammation. It seems likely that trace elements such as selenium and zinc are critical for the observed effects.

  11. Possible Role of PAPR-1 in Protecting Human HaCaT cells Against Cytotoxicity of SiO2 Nano.

    PubMed

    Gong, Chunmei; Yang, Linqing; Zhou, Jichang; Xiang, Guo; Zhuang, Zhixiong

    2017-07-27

    Nano-SiO2 materials play a significant role in the engineered nanomaterials (ENMs) field. The ease of their production as well as their relatively low cost has promoted the wide use of these products in many fields. Nano-SiO2 exposure is known to cause severe DNA damage; however, the underlying mechanisms remain poorly understood. In a previous study, we found that nano-SiO2 exposure regulate the expression of the poly(ADP-ribose) polymerases-1 (PARP-1), a pivotal DNA repair gene, in human HaCaT cells. Here, we employed lentivirus-mediated RNA interference (RNAi) to knock down PAPR-1 expression in HaCaT cells and explored the potential role of PARP-1 in nano-SiO2 induced cytotoxicity. We found that nano-SiO2 treatment of HaCaT cells causes decreased cell viability, increased apoptosis and DNA damage. Nano-SiO2-treated HaCaT cells were also found to have slightly changed cell cycle distribution. Lentivirus-mediated PAPR-1 knockdown partially aggravated cytotoxicity and increased apoptosis induced by nano-SiO2 treatment. Nano-SiO2 had significant toxicity to human HaCaT cells and causes DNA damage. PAPR-1 knock-down cell line appears more sensitive to nano-SiO2 than the control cells in DNA damage. The results suggest that PAPR-1 is involved in protecting cells from damage caused by nano-SiO2. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Eicosapentaenoic acid inhibits TNF-{alpha}-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

    SciTech Connect

    Kim, Hyeon Ho; Lee, Youngae; Eun, Hee Chul Chung, Jin Ho

    2008-04-04

    Eicosapentaenoic acid (EPA) is an omega-3 ({omega}-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-{kappa}B activation induced by tumor necrosis factor (TNF)-{alpha} or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-{alpha}-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-{alpha} induced MMP-9 expression by NF-{kappa}B-dependent pathway. Pretreatment of EPA inhibited TNF-{alpha}-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect I{kappa}B-{alpha} phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-{kappa}B. EPA inhibited TNF-{alpha}-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKK{alpha}-dependent event. Taken together, we demonstrate that EPA inhibits TNF-{alpha}-induced MMP-9 expression through inhibition of p38 and Akt activation.

  13. Down-regulation of HLA class I antigen in human papillomavirus type 16 E7 expressing HaCaT cells: correlate with TAP-1 expression.

    PubMed

    Li, Wei; Deng, Xiao-Mei; Wang, Chuan-Xin; Zhang, Xiao; Zheng, Gui-Xi; Zhang, Jian; Feng, Jin-Bo

    2010-02-01

    High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer, and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein of high-risk HPV types disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting a role for E7 in tumor immune evasion. We previously reported that HPV-16 E7 expression and down-regulation of HLA class I was highly correlated in cervical lesions. This study was aimed to determine whether HPV-16 E7 oncoprotein could down-regulate surface HLA class I antigen in HPV-16 E7-transfected cells, and whether it had correlation with the expression of the transporter associated with antigen processing (TAP). The HPV-16 E7 open reading frame was transfected into HaCaT cells. After G418 selection, resistant colonies were individually picked and expanded into clonal cell lines. Using the fluoresence-activated cell sorting analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 and TAP-2 expressions were detected. Compared with the empty vector control, a statistical significant decrease of approximately 50% in cell surface HLA class I expression was observed in HPV-16 E7 expressing HaCaT cells (P < 0.001). Moreover, the expression of HPV-16 E7 in HaCaT cells resulted in decreased expression of TAP-1 that was essential for HLA class I expression at the cell surface, a statistical significant decrease of approximately 40% compared with that with the empty vector control (P < 0.001). Our finding demonstrates that HPV-16 E7 down-regulates surface HLA class I antigen, which in part correlates with the decrease of TAP-1.

  14. Differential expression and regulation of prohibitin during curcumin-induced apoptosis of immortalized human epidermal HaCaT cells

    PubMed Central

    YANG, HAI-BO; SONG, WEI; CHEN, LAN-YING; LI, QI-FU; SHI, SONG-LIN; KONG, HAI-YAN; CHEN, PU

    2014-01-01

    Prohibitin (PHB), also known as inhibin, is important in cell proliferation, differentiation and apoptosis. This protein localizes to the inner membrane of mitochondria, where it acts as a chaperone protein, and is also found in the nucleus, where it negatively regulates transcription. The tumor-suppressive role of PHB in cell proliferation appears to be contradictory. In this study, we investigated the existence, localization and alterations in the expression of PHB in the whole cell and nuclear matrix and analyzed its co-localization with the expression products of related genes. The western blot analysis results revealed that PHB exists in the composition of nuclear matrix proteins and that the expression level of PHB is significantly increased in the whole cell and markedly decreased in the nuclear matrix after curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) treatment. The laser confocal scanning microscope results demonstrated the co-localization of PHB with p53, c-Myc, Bax, and Fas in HaCaT cells, and this co-localization region was transferred as a result of curcumin treatment. In addition, the results of the GST pull-down assay demonstrated the direct interaction of PHB with p53, c-Myc and Bax but not Fas in vitro. Results of the present study confirmed that the expression and distribution of PHB, which is a nuclear matrix protein, affect the apoptosis of HaCaT cells and its co-localization with specific gene products connected with cell apoptosis. PMID:24402549

  15. Glutathione metabolism in the HaCaT cell line as a model for the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin.

    PubMed

    Jacquoilleot, Sandrine; Sheffield, David; Olayanju, Adedamola; Sison-Young, Rowena; Kitteringham, Neil R; Naisbitt, Dean J; Aleksic, Maja

    2015-08-19

    Glutathione (GSH) is the most prominent antioxidant in cells and the co-factor of an important set of enzymes involved in the skin metabolic clearance system, glutathione S-transferases (GST). Here, we describe an LC-MS (liquid chromatography-mass spectroscopy) method to measure GSH and its disulfide form (GSSG) in HaCaT cells and a 3D Reconstructed Human Epidermis (RHE) model. In our assay, the basal level of GSH in both systems was in the low nmol/mg soluble protein range, while the level of GSSG was systematically below our limit of quantification (0.1 μM). We found that 2,4-dinitrohalobenzenes deplete the GSH present in HaCaT cells within the first hour of exposure, in a dose dependent manner. The level of GSH in HaCaT cells treated with a single non-toxic dose of 10 μM of dinitrohalobenzene was also shown to increase after two hours. While cells treated with 1-chloro-2,4-dinitrobenzene (DNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB) repleted GSH to levels similar to untreated control cells within 24h, 1-bromo-2,4-dinitrobenzene (DNBB) seemed to prevent such a repletion and appeared to be the most toxic compound in all assays. A mathematical modelling of experimental results was performed to further rationalise the differences observed between test chemicals. For this purpose the biological phenomena observed were simplified into two sequential events: the initial depletion of the GSH stock after chemical treatment followed by the repletion of the GSH once the chemical was cleared. Activation of the nuclear factor E2-related factor 2 (Nrf2) pathway was observed with all compounds within two hours, and at concentrations less than 10 μM. These data show that GSH depletion and repletion occur rapidly in skin cells and emphasize the importance of conducting kinetic studies when performing in vitro experiments exploring skin sensitization.

  16. Anti-inflammatory effects of Perillae Herba ethanolic extract against TNF-α/IFN-γ-stimulated human keratinocyte HaCaT cells.

    PubMed

    Yang, Ju-Hye; Yoo, Jae-Myung; Lee, Esther; Lee, BoHyoung; Cho, Won-Kyung; Park, Kwang-Il; Yeul Ma, Jin

    2017-09-29

    Perillae Herba is a perennial plant that is widely distributed throughout Asia. The leaves of Perillae Herba have been widely used to treat various diseases, such as cold due to wind-cold, headache, cough, abdominal fullness, distention, and fish and crab poisoning. To assess the anti-inflammatory activity of Perillae Herba leaf ethanolic extract (PHE) in human keratinocytes, we measured the tumor necrosis factor (TNF)-α/interferon (IFN)-γ-induced mRNA expression and production of proinflammatory chemokines such as thymus and activation-regulated chemokines; regulated on activation, normal T cell expressed and secreted; interleukin (IL)-6; and IL-8 in HaCaT cells. We evaluated the ability of PHE to decrease the expression of proinflammatory marker proteins, such as mitogen-activated protein kinase (MAPK), STAT-1, and NK-κB, using western blot analysis and immunocytochemistry. PHE inhibited activation of p38, ERK, and JNK and suppressed the phosphorylation of STAT-1 and NK-κB in TNF-α/IFN-γ-stimulated HaCaT cells. PHE also suppressed chemokine mRNA and protein levels in TNF-α/IFN-γ-stimulated HaCaT cells. PHE appears to regulate chemokine formation by inhibiting activation of MAPK, as well as the STAT-1 and NK-κB pathways. PHE suppresses the expression and production of TNF-α/IFN-γ-stimulated proinflammatory chemokines by blocking NF-κB, STAT-1, and MAPK activation. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Arecoline suppresses HaCaT cell proliferation through cell cycle regulatory molecules.

    PubMed

    Zhou, Zhong-Su; Li, Ming; Gao, Feng; Peng, Jie-Ying; Xiao, Hai-Bo; Dai, Li-Xia; Lin, Shi-Rong; Zhang, Rui; Jin, Long-Yu

    2013-06-01

    Betel nut chewing is the most common cause of oral submucous fibrosis (OSF). Arecoline is the main component of the betel nut, and is associated with the occurrence and development of OSF through cytotoxicity, genotoxicity and DNA damage. Similar types of stimuli elicit differential responses in different cells. In the present study, we investigated the effects of arecoline on the HaCaT epithelial and Hel fibroblast cell lines. The data showed that arecoline affected HaCaT cell morphology. MTT assay revealed that arecoline suppressed HaCaT cell proliferation. Furthermore, we found that arecoline induced the cell cycle arrest of HaCaT cells. In comparison with the untreated control cells, following treatment with ≥75 µg/ml arecoline an increased percentage of HaCaT cells remained at the G0/G1 phase of the cell cycle, accompanied by a reduced percentage of cells in the S phase. However, arecoline treatment did not significantly alter Hel cell cycle distribution. In the HaCaT epithelial cells, arecoline downregulated expression of the G1/S phase regulatory proteins cyclin D1, CDK4, CDK2, E2F1 as determined by reverse transcription-PCR analysis and western blotting. In summary, arecoline inhibits HaCaT epithelial cell proliferation and survival, in a dose-dependent manner, and cell cycle arrest in the G1/S phase, while this is not obvious in the Hel fibroblast cells. Potentially, our findings may aid in the prevention of arecoline-associated human OSF.

  18. Extremely low frequency 7 Hz 100 microT electromagnetic radiation promotes differentiation in the human epithelial cell line HaCaT.

    PubMed

    Lisi, Antonella; Foletti, Alberto; Ledda, Mario; Rosola, Emanuela; Giuliani, Livio; D'Emilia, Enrico; Grimaldi, Settimio

    2006-01-01

    Electromagnetic therapy is a treatment method in which an electromagnetic or magnetic stimulus is used to achieve physiological changes in the body. The specific aim of the present work concerns the effectiveness of low frequency electromagnetic fields to modify the biochemical properties of human keratinocytes (HaCaT). Cells exposed to a 7 Hz 100 microT electromagnetic field for one hour (twice daily), indicated modification in shape and morphology. These modifications were also associated with different actin distribution as revealed by phalloidin fluorescence analysis. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-Catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-Catenin expression, supporting the conclusion that exposure to electromagnetic field carries keratinocytes to an upper differentiation level. This study confirms our previous observation and supports the hypothesis that 7 Hz electromagnetic field, may modify cell biochemistry interfering in the differentiation and cellular adhesion of normal keratinocytes.

  19. Interaction of Mycobacterium leprae with the HaCaT human keratinocyte cell line: new frontiers in the cellular immunology of leprosy.

    PubMed

    Lyrio, Eloah C D; Campos-Souza, Ivy C; Corrêa, Luiz C D; Lechuga, Guilherme C; Verícimo, Maurício; Castro, Helena C; Bourguignon, Saulo C; Côrte-Real, Suzana; Ratcliffe, Norman; Declercq, Wim; Santos, Dilvani O

    2015-07-01

    Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF-α and IL-1β production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF-α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy.

  20. Modulation of viability and apoptosis of UVB-exposed human keratinocyte HaCaT cells by aqueous methanol extract of laver (Porphyra yezoensis).

    PubMed

    Kim, Saerong; You, Dong Hun; Han, Taejun; Choi, Eun-Mi

    2014-12-01

    We investigated the effect of 80% methanol extract of laver (Porphyra yezoensis) on the UVB-exposed HaCaT cells, human keratinocytes. The laver extract showed absorbance spectrum characteristic of porphyra-334 or shinorine, major mycosporine-like amino acids (MAAs) in red algae, and contained phenolic compounds. UVB exposure decreased cell viability and increased apoptotic cell fractions, and it also decreased the ratio of reduced (GSH) to oxidized glutathione (GSSG) and the total glutathione content. Post-treatment with the laver extract significantly increased the net viability and also the apoptotic cell fractions of UVB-exposed cells. The extract caused increase in GSH/GSSG ratio, yet it exacerbated the decrease in glutathione content in the UVB-exposed cells. These effects of the laver extract were also manifested in the sham-exposed cells, suggesting that those effects might be general phenomena caused by the laver extract. The extract treatment enhanced the UVB-induced phosphorylation of JNK and ERK, affecting more the latter. Our results suggest that the post-treatment with laver extract may protect UVB-exposed skin cells not only by increasing overall cell proliferation but also by enhancing apoptosis of damaged cells, via activating JNK and ERK signaling pathways, in which modulation of the content and redox status of glutathione may take significant parts.

  1. Protective Effect of Cyanidin-3-O-Glucoside against Ultraviolet B Radiation-Induced Cell Damage in Human HaCaT Keratinocytes.

    PubMed

    Hu, Yunfeng; Ma, Yuetang; Wu, Shi; Chen, Tianfeng; He, Yong; Sun, Jianxia; Jiao, Rui; Jiang, Xinwei; Huang, Yadong; Deng, Liehua; Bai, Weibin

    2016-01-01

    Ultraviolet radiation is the major environmental harmful factor that has emotional impact on human skin. The aim of the present study was to determine the mechanism of protection of cyanidin-3-O-glucoside against ultraviolet B (UVB)-induced damage to human HaCaT keratinocytes. Our results show that cyanidin-3-O-glucoside decreased the levels of intracellular reactive oxygen species generated by UVB treatment. Cyanidin-3-O-glucoside also decreased the UVB-augmented levels of the DNA damage indicators phospho-p53 and phospho-ATM/ATR. In addition, cyanidin-3-O-glucoside protected keratinocytes from UVB-induced injury by overturning the disruption of mitochondrial membrane potential and reversing apoptosis. The expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) was attenuated in UVB-exposed cells but restored in UVB/cyanidin-3-O-glucoside-treated cells. Furthermore, expression of the proapoptotic proteins Bcl-2-associated X (Bax) and the key apoptosis executer cleaved caspase-3 were increased in UVB-irradiated cells and decreased in UVB/cyanidin-3-O-glucoside-treated cells. For these reasons, the results demonstrate that cyanidin-3-O-glucoside protects human keratinocytes against UVB-induced oxidative stress and apoptosis. Our study provides a theoretical basis for the use of cyanidin-3-O-glucoside in the fight against light damage.

  2. Protective Effect of Cyanidin-3-O-Glucoside against Ultraviolet B Radiation-Induced Cell Damage in Human HaCaT Keratinocytes

    PubMed Central

    Hu, Yunfeng; Ma, Yuetang; Wu, Shi; Chen, Tianfeng; He, Yong; Sun, Jianxia; Jiao, Rui; Jiang, Xinwei; Huang, Yadong; Deng, Liehua; Bai, Weibin

    2016-01-01

    Ultraviolet radiation is the major environmental harmful factor that has emotional impact on human skin. The aim of the present study was to determine the mechanism of protection of cyanidin-3-O-glucoside against ultraviolet B (UVB)-induced damage to human HaCaT keratinocytes. Our results show that cyanidin-3-O-glucoside decreased the levels of intracellular reactive oxygen species generated by UVB treatment. Cyanidin-3-O-glucoside also decreased the UVB-augmented levels of the DNA damage indicators phospho-p53 and phospho-ATM/ATR. In addition, cyanidin-3-O-glucoside protected keratinocytes from UVB-induced injury by overturning the disruption of mitochondrial membrane potential and reversing apoptosis. The expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) was attenuated in UVB-exposed cells but restored in UVB/cyanidin-3-O-glucoside-treated cells. Furthermore, expression of the proapoptotic proteins Bcl-2-associated X (Bax) and the key apoptosis executer cleaved caspase-3 were increased in UVB-irradiated cells and decreased in UVB/cyanidin-3-O-glucoside-treated cells. For these reasons, the results demonstrate that cyanidin-3-O-glucoside protects human keratinocytes against UVB-induced oxidative stress and apoptosis. Our study provides a theoretical basis for the use of cyanidin-3-O-glucoside in the fight against light damage. PMID:27656146

  3. [Effects of estrogen on epidermis growth of mice and proliferation of human epidermal cell line HaCaT and its mechanism].

    PubMed

    Zhou, Tao; Chen, Jing; Huang, Zongwei; Fang, Li; Chen, Yu; Chen, Yajie; Peng, Yizhi

    2016-05-01

    To observe the effects of estrogen on epidermis growth of mice and proliferation of keratinocytes (human epidermal cell line HaCaT), and to explore its mechanism. (1) Five adult C57BL/6 mice in estrus cycle were identified by vaginal exfoliative cytology diagnosis and set as estrus group, while another 5 adult C57BL/6 mice with ovary resected before sexual development were set as ovariectomized group. The full-thickness skin from the tail root of mice in two groups were collected. The thickness of epidermis was observed and measured after HE staining. The distribution of proliferating cell nuclear antigen (PCNA)-positive cells in epidermis was observed by immunohistochemical staining, the number of which was counted. (2) HaCaT cells in logarithmic growth phase were cultured with RPMI 1640 nutrient solution containing 10% fetal bovine serum, and they were divided into negative control group (NC), pure estradiol group (PE), protein kinase B (Akt) inhibitor group (AI), and extracellular signal-regulated kinase (ERK) inhibitor group (EI) according to the random number table, with 20 wells in each group. To nutrient solution of each group, 1 μL dimethyl sulfoxide, 1 μL 17β-estradiol (100 nmol/L), 1 μL LY294002 (10 μmol/L), and 1 μL PD98059 (30 μmol/L) were added in group NC, group PE, group AI, and group EI respectively, and the last two groups were added with 1 μL 17β-estradiol (100 nmol/L) in addition. At post culture hour (PCH) 0 (immediately after culture), 24, 48, 72, 5 wells of cells from each group were collected to detect the proliferation activity of cells by cell counting kit 8 and microplate reader. (3) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 wells in each group. At PCH 72, cell cycle distribution was detected by flow cytometer to calculate proliferation index (PI) of cells. (4) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above

  4. Induction of apoptosis and expression of cell cycle regulatory proteins in response to a phytosphingosine derivative in HaCaT human keratinocyte cells.

    PubMed

    Kim, Hye Jung; Kim, Ho Jin; Lim, Sung Cil; Kim, Sang Hoon; Kim, Tae-Yoon

    2003-12-31

    Ceramide, a compound derived from sphingomyelin, a sphingolipid precursor, affects cell functions such as growth, differentiation, cell division and apoptosis. We have shown that the phytosphingosine derivative, tetra-acetyl phytosphingosine (TAPS), inhibits the growth of HaCaT cells mainly by inducing apoptosis. In this study, we investigated its effect on the cell cycle and on cell cycle regulatory proteins. We showed by flow cytometry and staining for BrdU and phosphorylated histone H3 that the cells accumulated in S phase and arrested in G2 phase and did not divide before undergoing apoptosis. The level of the pro-apoptotic regulator Bax peaked after 6 h and then returned to normal, whereas the level of the anti-apoptotic regulator Bcl-xL, which is presumably induced in order to inhibit apoptosis, started to increase at 6 h, and remained high for 24 h. Phosphorylation of Cdc2 on Tyr-15 greatly increased while p21 rose to a plateau at 8 h. Levels of p53 and Mad2 proteins were unchanged. Our observations suggest that TAPS induces apoptosis of the HaCaT cells at least in part via transient G2 arrest.

  5. Basic Red 51, a permitted semi-permanent hair dye, is cytotoxic to human skin cells: Studies in monolayer and 3D skin model using human keratinocytes (HaCaT).

    PubMed

    Zanoni, Thalita B; Tiago, Manoela; Faião-Flores, Fernanda; de Moraes Barros, Silvia B; Bast, Aalt; Hageman, Geja; de Oliveira, Danielle Palma; Maria-Engler, Silvya S

    2014-06-05

    The use of hair dyes is closely associated with the increase of cancer, inflammation and other skin disorders. The recognition that human skin is not an impermeable barrier indicates that there is the possibility of human systemic exposure. The carcinogenic potential of hair dye ingredients has attracted the attention of toxicologists for many decades, mainly due to the fact that some ingredients belong to the large chemical family of aromatic amines. Herein, we investigated the cytotoxicity of Basic Red 51 (BR51) in immortalized human keratinocytes (HaCaT). BR51 is a temporary hair dye that belongs to the azo group (NN); the cleavage of this bond may result in the release of toxic aromatic amines. The half maximal effective concentration (EC50) in HaCaT cells is 13μg/mL. BR51 induced a significant decrease on expression of p21 in a dose dependent manner. p53 was not affected, whereas BR51 decreased procaspase 8 and cleaved procaspase 9. These results proved that caspase 3 is fully involved in BR51-induced apoptosis. The dye was also able to stop this cell cycle on G2 in sub-toxic doses. Moreover, we reconstructed a 3D artificial epidermis using HaCaT cells; using this model, we observed that BR51 induced cell injury and cells were undergoing apoptosis, considering the fragmented nuclei. Subsequently, BR51 induced reactive oxygen species (ROS) leading to an increase on the levels of 8-oxo-dG. In conclusion, we provide strong evidence that consumer and/or professional exposure to BR51 poses risk to human health. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. No adaptive response is induced by chronic low-dose radiation from Ra-226 in the CHSE/F fish embryonic cell line and the HaCaT human epithelial cell line.

    PubMed

    Shi, Xiaopei; Mothersill, Carmel; Seymour, Colin

    2016-11-01

    To determine whether chronic low-dose α-particle radiation from Ra-226 over multiple cell generations can lead to an adaptive response in CHSE/F fish embryonic cells or HaCaT human epithelial cells receiving subsequent acute high-dose γ-ray radiation. CHSE/F and HaCaT cells were exposed to very low doses of Ra-226 in medium for multiple generations prior to being challenged by a higher dose γ-ray radiation. The clonogenic assay was used to test the clonogenic survival of cells with or without being pretreated by radiation from Ra-226. In general, pretreatment with chronic radiation has no significant influence on the reaction of cells to the subsequent challenge radiation. Compared to unprimed cells, the change in clonogenic survival of primed cells after receiving challenge radiation is mainly due to the influence of the chronic exposure, and there's little adaptive response induced. However at several dose points, pretreatment of CHSE/F fish cells with chronic radiation resulted in a radiosensitive response to a challenge dose of γ-ray radiation, and pretreatment of HaCaT cells resulted in no effect except for a slightly radioresistant response to the challenge radiation which was not significant. The results suggest that chronic low-dose radiation is not effective enough to induce adaptive response. There was a difference between human and fish cells and it may be important to consider results from multiple species before making conclusions about effects of chronic or low doses of radiation in the environment. The term "radiosensitive" or "adaptive" make no judgment about whether such responses are ultimately beneficial or harmful. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. In contrast to their murine counterparts, normal human keratinocytes and human epidermoid cell lines A431 and HaCaT fail to express IL-10 mRNA and protein

    PubMed Central

    TEUNISSEN, M B M; KOOMEN, C W; JANSEN, J; DE WAAL MALEFYT, R; SCHMITT, E; VAN DEN WIJNGAARD, R M J G J; DAS, P K; BOS, J D

    1997-01-01

    In mice, keratinocyte-derived IL-10 is up-regulated by ultraviolet-B (UVB) radiation and plays a major role in UVB-induced immunosuppression. The present study was designed to examine whether a comparable phenomenon can be detected in man. Freshly isolated or cultured normal human keratinocytes (NHK) and keratinocyte cell lines A431 and HaCaT were stimulated with graded doses of UVB (up to 200 J/m2) or with a variety of other stimuli. RNA was extracted at various time points post-stimulation and analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) using four different IL-10-specific primer pairs and RNA from monocytes or T cells as positive controls. We failed to detect IL-10 mRNA in NHK from 40 different donors (breast, abdomen, leg, scalp, foreskin) and in A431 and HaCaT cells, irrespective of the stimulation used and despite successful stimulation. Supernatants of NHK, A431 and HaCaT cultures were negative for IL-10 protein, as tested by four different ELISAs and a bioassay. Murine keratinocytes, stimulated under comparable conditions and tested by the same techniques, displayed a strong expression of IL-10 mRNA and protein. Remarkably, an IL-10 mRNA signal could be detected in NHK after a second round of PCR amplification. Because NHK suspensions are contaminated with Langerhans cells, melanocytes and possibly fibroblasts, we tested pure populations of each individual cell type to determine the origin of this IL-10 mRNA. Our results clearly indicate that NHK, Langerhans cells and fibroblasts fail to express IL-10 and that melanocytes are the principal source of IL-10 mRNA in normal human epidermis. PMID:9010278

  8. [Effect of low-energy 633 nm red light stimulation on proliferation and reactive oxygen species level of human epidermal cell line HaCaT].

    PubMed

    Chen, Z Y; Li, D L; Duan, X D; Peng, D Z

    2016-09-20

    To investigate the changes of proliferative activity and reactive oxygen species level of human epidermal cell line HaCaT after being irradiated with low-energy 633 nm red light. Irradiation distance was determined through preliminary experiment. HaCaT cells were conventionally sub-cultured with RPMI 1640 culture medium containing 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells of the third passage were used in the following experiments. (1) Cells were divided into blank control group and 0.082, 0.164, 0.245, 0.491, 1.472, 2.453, 4.910, and 9.810 J/cm(2) irradiation groups according to the random number table, with 3 wells in each group. Cells in blank control group were not irradiated, while cells in the latter 8 irradiation groups were irradiated with 633 nm red light for 10, 20, 30, 60, 180, 300, 600, and 1 200 s in turn. Cells were reirradiated once every 8 hours. After being irradiated for 48 hours (6 times) in irradiation groups, the proliferative activity of cells in 9 groups was determined with cell counting kit 8 and microplate reader (denoted as absorbance value). (2) Another batch of cells were grouped and irradiated as in experiment (1). After being irradiated for once in irradiation groups, cells in 9 groups were conventionally cultured for 60 min with detection reagent of reactive oxygen species. At post culture minute (PCM) 0 (immediately), 30, 60, and 120, reactive oxygen species level of cells was determined with microplate reader (denoted as absorbance value). (3) Another batch of cells were divided into blank control group, 0.082, 0.491, 2.453, and 9.810 J/cm(2) irradiation groups, and positive control group. Cells in blank control group and positive control group were not irradiated (positive control reagent of reactive oxygen species was added to cells in positive control group), and cells in irradiation groups were irradiated as in experiment (1) for once. The expression of reactive oxygen species in cells of each

  9. Fisetin inhibits TNF-α-induced inflammatory action and hydrogen peroxide-induced oxidative damage in human keratinocyte HaCaT cells through PI3K/AKT/Nrf-2-mediated heme oxygenase-1 expression.

    PubMed

    Seo, Seung-Hee; Jeong, Gil-Saeng

    2015-12-01

    Oxidative skin damage and skin inflammation play key roles in the pathogenesis of skin-related diseases. Fisetin is a naturally occurring flavonoid abundantly found in several vegetables and fruits. Fisetin has been shown to exert various positive biological effects, such as anti-cancer, anti-proliferative, neuroprotective and anti-oxidative effects. In this study, we investigate the skin protective effects and anti-inflammatory properties of fisetin in hydrogen peroxide- and TNF-α-challenged human keratinocyte HaCaT cells. When HaCaT cells were treated with non-cytotoxic concentrations of fisetin (1-20μM), heme oxygenase (HO)-1 mRNA and protein expression increased in a dose-dependent manner. Furthermore, fisetin dose-dependently increased cell viability and reduced ROS production in hydrogen peroxide-treated HaCaT cells. Fisetin also inhibited the production of NO, PGE2 IL-1β, IL-6, expression of iNOS and COX-2, and activation of NF-κB in HaCaT cells treated with TNF-α. Fisetin induced Nrf2 translocation to the nuclei. HO-1 siRNA transient transfection reversed the effects of fisetin on cytoprotection, ROS reduction, NO, PGE2, IL-1β, IL-6, and TNF-α production, and NF-κB DNA-binding activity. Moreover, fisetin increased Akt phosphorylation and a PI3K pathway inhibitor (LY294002) abolished fisetin-induced cytoprotection and NO inhibition. Taken together, these results provide evidence for a beneficial role of fisetin in skin therapy. Copyright © 2015. Published by Elsevier B.V.

  10. Zinc inhibits apoptosis and maintains NEP downregulation, induced by ropivacaine, in HaCaT cells.

    PubMed

    Kontargiris, Evangelos; Vadalouka, Athina; Ragos, Vasilios; Kalfakakou, Vasiliki

    2012-12-01

    Zinc (Zn), a cell-protective metal against various toxic compounds, is the key agent for neutral endopeptidase (NEP) functional structure. NEP is a zinc metalloenzyme which degrades endogenous opioids and is expressed in human keratinocytes (HaCaT). Ropivacaine, a widely used opiate local anaesthetic, exerts cell toxic and apoptotic effects against HaCaT cells. The aim of the present study is to investigate whether zinc modulates the effects of ropivacaine on proliferation, viability, apoptosis and NEP expression in HaCaT cells. To investigate the role of ropivacaine in NEP function, HaCaT cells overexpressing NEP were generated via cell transfection with plasmids carrying NEP cDNA. Ropivacaine's anti-proliferative effect was tested by Neubauer's chamber cell counting, and induction of cell death was demonstrated by trypan blue exclusion assay. Apoptosis due to ropivacaine was tested via DNA fragmentation and poly-ADP-ribose-polymerase (PARP) cleavage. NEP and PARP expression was performed by western blot analysis. Results showed that zinc (15 μΜ) inhibited proliferation and cell death induction by ropivacaine (0.5, 1 and 2 mM) (p < 0.05) as well as apoptosis induced by the drug (0.5 and 1 mM) in HaCaT cells. Ropivacaine (1.0, 2.0 and 5.0 mM) downregulated NEP expression in the presence of zinc (15 μΜ) while NEP overexpression enhanced ropivacaine's apoptotic effect. In conclusion, the abilities of zinc to inhibit the toxic and apoptotic effects of ropivacaine, to maintain NEP downregulation induced by the drug and, consequently, to enhance its anaesthetic result suggest that zinc may have a significant role in pain management and tissue protection.

  11. The ethyl acetate fraction of Sargassum muticum attenuates ultraviolet B radiation-induced apoptotic cell death via regulation of MAPK- and caspase-dependent signaling pathways in human HaCaT keratinocytes.

    PubMed

    Piao, Mei Jing; Kim, Ki Cheon; Zheng, Jian; Yao, Cheng Wen; Cha, Ji Won; Boo, Sun Jin; Yoon, Weon Jong; Kang, Hee Kyoung; Yoo, Eun Sook; Koh, Young Sang; Ko, Mi Hee; Lee, Nam Ho; Hyun, Jin Won

    2014-09-01

    Our previous work demonstrated that an ethyl acetate extract derived from Sargassum muticum (Yendo) Fenshol (SME) protected human HaCaT keratinocytes against ultraviolet B (UVB)-induced oxidative stress by increasing antioxidant activity in the cells, thereby inhibiting apoptosis. The aim of the current study was to further elucidate the anti-apoptotic mechanism of SME against UVB-induced cell damage. The expression levels of several apoptotic-associated and mitogen-activated kinase (MAPK) signaling proteins were determined by western blot analysis of UVB-irradiated HaCaT cells with or without prior SME treatment. In addition, the loss of mitochondrial membrane potential (Δψm) was detected using flow cytometry or confocal microscopy and the mitochondria membrane-permeate dye, JC-1. Apoptosis was assessed by quantifying DNA fragmentation and apoptotic body formation. Furthermore, cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. SME absorbed electromagnetic radiation in the UVB range (280-320 nm) of the UV/visible light spectrum. SME also increased Bcl-2 and Mcl-1 expression in UVB-irradiated cells and decreased the Bax expression. Moreover, SME inhibited the UVB-induced disruption of mitochondrial membrane potential and prevented UVB-mediated increases in activated caspase-9 and caspase-3 (an apoptotic initiator and executor, respectively) levels. Notably, treatment with a pan-caspase inhibitor enhanced the anti-apoptotic effects of SME in UVB-irradiated cells. Finally, SME reduced the UVB-mediated phosphorylation of p38 MAPK and JNK, and prevented the UVB-mediated dephosphorylation of Erk1/2 and Akt. The present results indicate that SME safeguards HaCaT keratinocytes from UVB-mediated apoptosis by inhibiting a caspase-dependent signaling pathway.

  12. Anti-wrinkle effects of Sargassum muticum ethyl acetate fraction on ultraviolet B-irradiated hairless mouse skin and mechanistic evaluation in the human HaCaT keratinocyte cell line

    PubMed Central

    Song, Jae Hyoung; Piao, Mei Jing; Han, Xia; Kang, Kyoung Ah; Kang, Hee Kyoung; Yoon, Weon Jong; Ko, Mi Hee; Lee, Nam Ho; Lee, Mi Young; Chae, Sungwook; Hyun, Jin Won

    2016-01-01

    The present study investigated the photoprotective properties of the ethyl acetate fraction of Sargassum muticum (SME) against ultraviolet B (UVB)-induced skin damage and photoaging in a mouse model. HR-1 strain hairless male mice were divided into three groups: An untreated control group, a UVB-irradiated vehicle group and a UVB-irradiated SME group. The UVB-irradiated mice in the SME group were orally administered with SME (100 mg/kg body weight in 0.1 ml water per day) and then exposed to radiation at a dose of 60–120 mJ/cm2. Wrinkle formation and skin damage were evaluated by analysis of skin replicas, epidermal thickness and collagen fiber integrity in the dermal connective tissue. The mechanism underlying the action of SME was also investigated in the human HaCaT keratinocyte cell line following exposure of the cells to UVB at a dose of 30 mJ/cm2. The protein expression levels and activity of matrix metalloproteinase-1 (MMP-1), and the binding of activator protein-1 (AP-1) to the MMP-1 promoter were assessed in the HaCaT cells using western blot analysis, an MMP-1 fluorescent assay and a chromatin immune-precipitation assay, respectively. The results showed that the mean length and depth of the wrinkles in the UVB-exposed hairless mice were significantly improved by oral administration of SME, which also prevented the increase in epidermal thickness triggered by UVB irradiation. Furthermore, a marked increase in collagen bundle formation was observed in the UVB-treated mice with SME administration. SME pretreatment also significantly inhibited the UVB-induced upregulation in the expression and activity of MMP-1 in the cultured HaCaT keratinocytes, and the UVB-enhanced association of AP-1 with the MMP-1 promoter. These results suggested that SME may be useful as an anti-photoaging resource for the skin. PMID:27573915

  13. Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study

    PubMed Central

    Wang, Yao-wen; Ren, Ji-hao; Xia, Kun; Wang, Shu-hui; Yin, Tuan-fang; Xie, Ding-hua; Li, Li-hua

    2012-01-01

    Objective: To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell), particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast. Methods: The normal dermal fibroblast and HaCat cell were cultured in vitro. Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution, and serum-free culture medium was used as control. The cellular morphology change, growth characteristics, cell proliferation, and apoptosis were observed at different intervals. For the fibroblasts, the mRNA expression changes of transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), procollagen I, and III were detected by reverse transcription polymerase chain reaction (RT-PCR). Results: The cultured normal human skin fibroblast and HaCat cell grew exponentially. A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells. Cell morphology changed, cell density decreased, and the growth curves were without an exponential phase. The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml. Meanwhile, 5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis. The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05). A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-β1, procollagen I and III, and a marked increase in the mRNA production of bFGF. Conclusions: Mitomycin can inhibit fibroblast proliferation, induce fibroblast apoptosis, and regulate intracellular protein expression on mRNA levels. In additon, mitomycin can inhibit HaCat cell proliferation, so epithelial cell needs more protecting to avoid mitomycin’s side effect when it is applied clinically. PMID

  14. Cobalt toxicity: chemical and radiological combined effects on HaCaT keratinocyte cell line.

    PubMed

    Gault, N; Sandre, C; Poncy, J-L; Moulin, C; Lefaix, J-L; Bresson, C

    2010-02-01

    Cobalt (Co) is an essential trace element well known as a constituent of vitamin B(12), but different compounds of Co are also described as highly toxic and/or radiotoxic for individuals or the environment. In nuclear power plants, (58)Co and (60)Co are radioactive isotopes of cobalt present as activation products of stable Co and Ni used in alloys. Skin exposure is a current occupational risk in the hard metal and nuclear industries. As biochemical and molecular cobalt-induced toxicological mechanisms are not fully identified, we investigated cobalt toxicity in a model human keratinocyte cell line, HaCaT. In this study, we propose a model to determine the in vitro chemical impact on cell viability of a soluble form of cobalt (CoCl(2)) with or without gamma-ray doses to mimic contamination by (60)Co, to elucidate the mechanisms of cobalt intracellular chemical and radiological toxicity. Intracellular cobalt concentration was determined after HaCaT cell contamination and chemical toxicity was evaluated in terms of cellular viability and clonogenic survival. We investigated damage to DNA in HaCaT cells by combined treatment with chemical cobalt and a moderate gamma-ray dose. Additive effects of cobalt and irradiation were demonstrated. The underlying mechanism of cobalt toxicity is not clearly established, but our results seem to indicate that the toxicity of Co(II) and of irradiation arises from production of reactive oxygen species.

  15. Inter-Relationship between Low-Dose Hyper-Radiosensitivity and Radiation-Induced Bystander Effects in the Human T98G Glioma and the Epithelial HaCaT Cell Line.

    PubMed

    Fernandez-Palomo, Cristian; Seymour, Colin; Mothersill, Carmel

    2016-02-01

    Over the past several years, investigations in both low-dose hyper-radiosensitivity and increased radioresistance have been a focus of radiation oncology and biology research, since both conditions occur primarily in tumor cell lines. There has been significant progress in elucidating their signaling pathways, however uncertainties exist when they are studied together with radiation-induced bystander effects. Therefore, the aim of this work was to further investigate this relationship using the T98G glioma and HaCaT cell lines. T98G glioma cells have demonstrated a strong transition from hyper-radiosensitivity to induced radioresistance, and HaCaT cells do not show low-dose hypersensitivity. Both cell lines were paired using a mix-and-match protocol, which involved growing nonirradiated cells in culture media from irradiated cells and covering all possible combinations between them. The end points analyzed were clonogenic cell survival and live calcium measurements through the cellular membrane. Our data demonstrated that T98G cells produced bystander signals that decreased the survival of both reporter T98G and HaCaT cells. The bystander effect occurred only when T98G cells were exposed to doses below 1 Gy, which was corroborated by the induction of calcium fluxes. However, when bystander signals originated from HaCaT cells, the survival fraction increased in reporter T98G cells while it decreased in HaCaT cells. Moreover, the corresponding calcium data showed no calcium fluxes in T98G cells, while HaCaT cells displayed a biphasic calcium profile. In conclusion, our findings indicate a possible link between low-dose hyper-radiosensitivity and bystander effects. This relationship varies depending on which cell line functions as the source of bystander signals. This further suggests that the bystander mechanisms are more complex than previously expected and caution should be taken when extrapolating bystander results across all cell lines and all radiation doses.

  16. Photoprotection by tocopherol submicron emulsion against UV-mediated damage in HaCaT cells.

    PubMed

    Luo, Dan; Lin, X F; Min, W; Ma, Q H; Gu, N; Jin, S L; Wang, D G

    2007-04-01

    alpha-Tocopherol is a lipophilic vitamin E that shows antioxidative, antiaging and antiphotodamage activity. Nanometer biotechnology is more widely used in the entrainment system of drug carriers and the development for new pharmaceutical preparations. Ultraviolet irradiation to human skin in the long term can result in photoaging and photocarcinogenesis. The purpose of this study was to observe the biological features of tocopherol submicron emulsion (vitE SME) and to clarify the roles of vitE SME on UVB-induced photodamage in HaCaT keratinocytes (KC). VitE SME was prepared by high-pressure homogenization and microemulsion technique. HaCaT KC was incubated in the culture medium supplied with 1/200 and 1/400 of VitE SME prior to different dosages of UVB irradiation. The vitamin E amount in the culture medium was measured by high-performance liquid chromatography (HPLC). Cell growth and cellular viability was detected by MTT assay. The amount of vitamin E remaining in the culture medium significantly decreased during the first 8 h, and less than 10% can be detected by the terminal experiment (24 h). No cytotoxicity effect of tocopherol NM on HaCat KC was observed. In contrast to the control group, the cellular viability of VitE SME-treated group increased 44.22% by 24 h. Compared with irradiated groups without VitE SME, cell proliferation decreased by 17.77% and 40.42% when the HaCaT KC was irradiated with 30 mJ/cm(2) and 90 mJ/cm(2) UVB irradiation, respectively. VitE SME has no toxicity to cell culture system and is characterized by stable release and penetration. Pre-incubation with VitE SME can partly reduce UV-induced cell damage, and the photoprotective efficiency to UVB irradiation also shows time dependence.

  17. Progressive stages of "transdifferentiation" from epidermal to mesenchymal phenotype induced by MyoD1 transfection, 5-aza-2'- deoxycytidine treatment, and selection for reduced cell attachment in the human keratinocyte line HaCaT

    PubMed Central

    1992-01-01

    The ability of the myogenic determination gene (MyoD1) to convert differentiating human keratinocytes (HaCaT cell-line) to the myogenic pathway and the effect of MyoD1 on the epidermal phenotype was studied in culture and in surface transplants on nude mice. MyoD1 transfection induced the synthesis of myosin, desmin, and vimentin without substantially altering the epidermal differentiation properties (morphology, keratin profile) in vitro nor epidermal morphogenesis (formation of a complex stratified squamous epithelium) in surface transplants, demonstrating the stability of the keratinocyte phenotype. 5-Aza-CdR treatment of these MyoD1-transfected cells had little effect on the cultured cells but a morphologically unstructured epithelium was formed with no indications of typical cell layers including cornification. Since prevention of epidermal strata in transplants was not accompanied by blocked epidermal differentiation markers (keratins K1 and K10, involucrin, and filaggrin), the dissociation of morphogenesis and expression of these markers argues for independently controlled processes. A subpopulation of less adhesive cells, isolated from the 5-aza-CdR treated MyoD1-transfectants, had lost most epithelial characteristics in culture (epidermal keratins, desmosomal proteins, and surface-glycoprotein Gp90) and had shifted to a mesenchymal/myogenic phenotype (fibroblastic morphology, transactivation of Myf3 and myogenin, expression of myosin, desmin, vimentin, and Gp130). Moreover, the cells had lost the ability to stratify and remained as a monolayer of flat elongated cells in transplants. These subsequent changes from a fully differentiated keratinocyte to a mesenchymal/myogenic phenotype strongly argue for a complex "transdifferentiation" process which occurred in the original monoclonal human epidermal HaCaT cells. PMID:1371288

  18. Bitter apricot essential oil induces apoptosis of human HaCaT keratinocytes.

    PubMed

    Li, Keyou; Yang, Wenhua; Li, Zhe; Jia, Wangwang; Li, Jiazhou; Zhang, Pengfei; Xiao, Tiancun

    2016-05-01

    Psoriasis is a chronic skin disease that affects approximately 2% of the world's population. Conventional therapeutic approaches are not effective or necessarily safe for treating symptoms due to the serious side effects and resistance to currently prescribed drugs. Traditionally, in oriental medicine, apricot seed (Semen Armeniacae amarum) is used to treat skin diseases. However, the underlying mechanism of action has not been systematically elucidated. In the present study, the anti-proliferative effect of bitter apricot essential oil (BAEO) on cultured HaCaT cells was evaluated and the mechanism of action investigated. BAEO was isolated by hydrodistillation, and gas chromatography-mass spectrometry (GC-MS) analysis identified benzaldehyde (75.35%), benzoic acid (6.21%) and mandelonitrile (5.38%). HaCaT cell growth, measured by sulforhodamine B assay (SRB), was inhibited by BAEO with an IC50 value of 142.45 μg/ml. Apoptosis of HaCaT cells treated with BAEO was detected by cell cycle, flow cytometry, and western blot analyses. These measurements revealed G0/G1 cell cycle arrest, elevated numbers of early and late stage apoptotic cells, and caspases-3/8/9 and PARP activation. Z-VAD-FMK, a broad-spectrum caspase inhibitor, attenuated BAEO-induced apoptosis. Also, increased Bax and decreased Bcl-2 levels suggest that BAEO-induced apoptosis is mediated through both death receptor and mitochondrial pathways. Moreover, reduced Rel/NF-κB levels suggest that BAEO-mediated apoptosis is also associated with inhibition of the NF-κB pathway. These data suggest that BAEO is a naturally occurring material that functions as a potent pro-apoptotic factor for human keratinocytes. Thus, it is a promising candidate to treat psoriasis. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Expression of VEGFR-2 on HaCaT cells is regulated by VEGF and plays an active role in mediating VEGF induced effects

    SciTech Connect

    Yang Xiaohong; Man Xiaoyong; Cai Suiqing; Yao Yonggang; Bu Zhangyu; Zheng Min . E-mail: minz@zju.edu.cn

    2006-10-13

    Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 play important roles in mitogenesis and chemotaxis of endothelial cells. In normal human skin, VEGF is expressed and secreted by epidermal keratinocytes. Emerging data suggest that keratinocyte-derived VEGF targets other cell types besides the dermal endothelial cells. We have recently showed that keratinocytes from human normal skin expressed all five known VEGF receptors and co-receptors (neuropilin 1 and 2). To define the functional significance of VEGFR-2 in epidermis, we examined its role in a keratinocyte cell line, HaCaT cells, in response to VEGF treatment. Expression of VEGFR-2 on HaCaT cells was confirmed at both RNA and protein levels and was regulated by VEGF{sub 165} treatment. Treatment of HaCaT cells with VEGF{sub 165} induced tyrosine-autophosphorylation of VEGFR-2 and phosphorylation of PLC-{gamma} and p44/42 MAPK in a time-dependent manner. Preincubation with a neutralizing antibody for VEGFR-2 (MAB3571) completely abrogated these phosphorylation effects. Furthermore, VEGF{sub 165} stimulated proliferation and migration of HaCaT cells, and this effect was significantly blocked by a pretreatment with MAB3571. Neutralizing VEGFR-2 in HaCaT cells increased cell adhesion during culture. Our results suggest that VEGFR-2 expressed on HaCaT cells plays a crucial role in VEGF-mediated regulation of cell activity.

  20. Rosmarinic Acid Attenuates Cell Damage against UVB Radiation-Induced Oxidative Stress via Enhancing Antioxidant Effects in Human HaCaT Cells

    PubMed Central

    Fernando, Pattage Madushan Dilhara Jayatissa; Piao, Mei Jing; Kang, Kyoung Ah; Ryu, Yea Seong; Hewage, Susara Ruwan Kumara Madduma; Chae, Sung Wook; Hyun, Jin Won

    2016-01-01

    This study was designed to investigate the cytoprotective effect of rosmarinic acid (RA) on ultraviolet B (UVB)-induced oxidative stress in HaCaT keratinocytes. RA exerted a significant cytoprotective effect by scavenging intracellular ROS induced by UVB. RA also attenuated UVB-induced oxidative macromolecular damage, including protein carbonyl content, DNA strand breaks, and the level of 8-isoprostane. Furthermore, RA increased the expression and activity of superoxide dismutase, catalase, heme oxygenase-1, and their transcription factor Nrf2, which are decreased by UVB radiation. Collectively, these data indicate that RA can provide substantial cytoprotection against the adverse effects of UVB radiation by modulating cellular antioxidant systems, and has potential to be developed as a medical agent for ROS-induced skin diseases. PMID:26759705

  1. Paeonol attenuates aging MRC-5 cells and inhibits epithelial-mesenchymal transition of premalignant HaCaT cells induced by aging MRC-5 cell-conditioned medium.

    PubMed

    Yang, Lihua; Xing, Shangping; Wang, Kun; Yi, Hua; Du, Biaoyan

    2017-08-12

    Senescence-associated secretory phenotype (SASP) factors, such as IL-6 and IL-8, are extremely critical in tissue microenvironment. Senescent human fibroblasts facilitate epithelial-mesenchymal transition (EMT) in premalignant epithelial cells mainly through the secretion of SASP factors. Meanwhile, premalignant human HaCaT Keratinocyte (HaCaT) cells as immortal epithelial cells are susceptible to malignant transformation. Paeonol, an herbal phenolic component found in peonies, exerts anti-aging and anti-tumor efficacies, while the molecular mechanisms of paeonol on EMT in premalignant HaCaT cells induced by SASP factors are unclear. In this study, we first established a senescent human fetal lung fibroblast MRC-5 cell model using hydrogen peroxide evaluated by senescence-associated β-galactosidase assay. Upon paeonol treatment, intracellular reactive oxygen species levels in aging MRC-5 cells were significantly decreased via regulation of nuclear translocation of Nrf2. Then we curiously studied whether the aging MRC-5 cell-conditioned medium could induce EMT in premalignant HaCaT cells, and the results showed that paeonol significantly reduced the clonogenic, migratory, and invasive capacities of premalignant HaCaT cells potentially induced by IL-6 and IL-8. Moreover, we found that paeonol notably altered pluripotency of EMT-associated markers via the modulation of ERK and TGF-β1/Smad pathway in premalignant HaCaT cells. These findings suggest that paeonol may be used as an adjuvant therapy for SASP factor-mediated EMT in premalignant lesion.

  2. Anti-wrinkle effects of Sargassum muticum ethyl acetate fraction on ultraviolet B-irradiated hairless mouse skin and mechanistic evaluation in the human HaCaT keratinocyte cell line.

    PubMed

    Song, Jae Hyoung; Piao, Mei Jing; Han, Xia; Kang, Kyoung Ah; Kang, Hee Kyoung; Yoon, Weon Jong; Ko, Mi Hee; Lee, Nam Ho; Lee, Mi Young; Chae, Sungwook; Hyun, Jin Won

    2016-10-01

    The present study investigated the photoprotective properties of the ethyl acetate fraction of Sargassum muticum (SME) against ultraviolet B (UVB)‑induced skin damage and photoaging in a mouse model. HR‑1 strain hairless male mice were divided into three groups: An untreated control group, a UVB‑irradiated vehicle group and a UVB‑irradiated SME group. The UVB‑irradiated mice in the SME group were orally administered with SME (100 mg/kg body weight in 0.1 ml water per day) and then exposed to radiation at a dose of 60‑120 mJ/cm2. Wrinkle formation and skin damage were evaluated by analysis of skin replicas, epidermal thickness and collagen fiber integrity in the dermal connective tissue. The mechanism underlying the action of SME was also investigated in the human HaCaT keratinocyte cell line following exposure of the cells to UVB at a dose of 30 mJ/cm2. The protein expression levels and activity of matrix metalloproteinase‑1 (MMP‑1), and the binding of activator protein‑1 (AP‑1) to the MMP‑1 promoter were assessed in the HaCaT cells using western blot analysis, an MMP‑1 fluorescent assay and a chromatin immune‑precipitation assay, respectively. The results showed that the mean length and depth of the wrinkles in the UVB‑exposed hairless mice were significantly improved by oral administration of SME, which also prevented the increase in epidermal thickness triggered by UVB irradiation. Furthermore, a marked increase in collagen bundle formation was observed in the UVB‑treated mice with SME administration. SME pretreatment also significantly inhibited the UVB‑induced upregulation in the expression and activity of MMP‑1 in the cultured HaCaT keratinocytes, and the UVB‑enhanced association of AP‑1 with the MMP‑1 promoter. These results suggested that SME may be useful as an anti-photoaging resource for the skin.

  3. Resveratrol Couples Apoptosis with Autophagy in UVB-Irradiated HaCaT Cells

    PubMed Central

    Vitale, Nicoletta; Kisslinger, Annamaria; Paladino, Simona; Procaccini, Claudio; Matarese, Giuseppe; Pierantoni, Giovanna Maria; Mancini, Francesco Paolo; Tramontano, Donatella

    2013-01-01

    UVB radiation causes about 90% of non-melanoma skin cancers by damaging DNA either directly or indirectly by increasing levels of reactive oxygen species (ROS). Skin, chronically exposed to both endogenous and environmental pro-oxidant agents, contains a well-organised system of chemical and enzymatic antioxidants. However, increased or prolonged free radical action can overwhelm ROS defence mechanisms, contributing to the development of cutaneous diseases. Thus, new strategies for skin protection comprise the use of food antioxidants to counteract oxidative stress. Resveratrol, a phytoalexin from grape, has gained a great interest for its ability to influence several biological mechanisms like redox balance, cell proliferation, signal transduction pathways, immune and inflammatory response. Therefore, the potential of resveratrol to modify skin cell response to UVB exposure could turn out to be a useful option to protect skin from sunlight-induced degenerative diseases. To investigate into this matter, HaCaT cells, a largely used model for human skin keratinocytes, were treated with 25 or 100 µM resveratrol for 2 and 24 hours prior to UVB irradiation (10 to 100 mJ/cm2). Cell viability and molecular markers of proliferation, oxidative stress, apoptosis, and autophagy were analyzed. In HaCaT cells resveratrol pretreatment: reduces UVB-induced ROS formation, enhances the detrimental effect of UVB on HaCaT cell vitality, increases UVB-induced caspase 8, PARP cleavage, and induces autophagy. These findings suggest that resveratrol could exert photochemopreventive effects by enhancing UVB-induced apoptosis and by inducing autophagy, thus reducing the odds that damaged cells could escape programmed cell death and initiate malignant transformation. PMID:24260465

  4. Titanium Dioxide Nanoparticle Penetration into the Skin and Effects on HaCaT Cells

    PubMed Central

    Crosera, Matteo; Prodi, Andrea; Mauro, Marcella; Pelin, Marco; Florio, Chiara; Bellomo, Francesca; Adami, Gianpiero; Apostoli, Pietro; De Palma, Giuseppe; Bovenzi, Massimo; Campanini, Marco; Larese Filon, Francesca

    2015-01-01

    Titanium dioxide nanoparticles (TiO2NPs) suspensions (concentration 1.0 g/L) in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue® and propidium iodide, PI, uptake assays) was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact and damaged skin. Titanium was found in the epidermal layer after 24 h of exposure (0.47 ± 0.33 μg/cm2) while in the dermal layer, the concentration was below the limit of detection. Damaged skin, in its whole, has shown a similar concentration (0.53 ± 0.26 μg/cm2). Cytotoxicity studies on HaCaT cells demonstrated that TiO2NPs induced cytotoxic effects only at very high concentrations, reducing cell viability after seven days of exposure with EC50s of 8.8 × 10−4 M (MTT assay), 3.8 × 10−5 M (AlamarBlue® assay), and 7.6 × 10−4 M (PI uptake, index of a necrotic cell death). Our study demonstrated that TiO2NPs cannot permeate intact and damaged skin and can be found only in the stratum corneum and epidermis. Moreover, the low cytotoxic effect observed on human HaCaT keratinocytes suggests that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure. PMID:26262634

  5. Titanium Dioxide Nanoparticle Penetration into the Skin and Effects on HaCaT Cells.

    PubMed

    Crosera, Matteo; Prodi, Andrea; Mauro, Marcella; Pelin, Marco; Florio, Chiara; Bellomo, Francesca; Adami, Gianpiero; Apostoli, Pietro; De Palma, Giuseppe; Bovenzi, Massimo; Campanini, Marco; Filon, Francesca Larese

    2015-08-07

    Titanium dioxide nanoparticles (TiO2NPs) suspensions (concentration 1.0 g/L) in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue(®) and propidium iodide, PI, uptake assays) was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact and damaged skin. Titanium was found in the epidermal layer after 24 h of exposure (0.47 ± 0.33 μg/cm(2)) while in the dermal layer, the concentration was below the limit of detection. Damaged skin, in its whole, has shown a similar concentration (0.53 ± 0.26 μg/cm(2)). Cytotoxicity studies on HaCaT cells demonstrated that TiO2NPs induced cytotoxic effects only at very high concentrations, reducing cell viability after seven days of exposure with EC50s of 8.8 × 10(-4) M (MTT assay), 3.8 × 10(-5) M (AlamarBlue(®) assay), and 7.6 × 10(-4) M (PI uptake, index of a necrotic cell death). Our study demonstrated that TiO2NPs cannot permeate intact and damaged skin and can be found only in the stratum corneum and epidermis. Moreover, the low cytotoxic effect observed on human HaCaT keratinocytes suggests that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure.

  6. An in vitro study on the cytotoxicity of bismuth oxychloride nanosheets in human HaCaT keratinocytes.

    PubMed

    Gao, Xiaoya; Zhang, Xiaochao; Wang, Yawen; Wang, Yunfang; Peng, Shiqi; Fan, Caimei

    2015-06-01

    As an emerging nanomaterial, bismuth oxychloride (BiOCl) has attracted explosive interests in diverse areas. However, how it interfaces with biological systems, particularly its interaction with human cells and the resulting effects are completely unknown. In this paper, the cytotoxicity of BiOCl nanosheets (NSs) was investigated toward a human skin derived cell line (HaCaT). It was found that BiOCl-NSs had no cytotoxicity at low concentrations (<0.5 µg/mL), whereas higher concentrations (5-100 µg/mL) of BiOCl-NSs could trigger toxic effects on HaCaT cells, with changes in cell morphology and impairment of intracellular structures (mitochondria and cytoskeleton). BiOCl-NSs also led to cell apoptosis and cells cycle arrest in G0/G1 phase. Flow cytometric data showed that BiOCl-NSs were effectively incorporated into HaCaT cells. Transmission electron microscope (TEM) images further revealed that BiOCl-NSs sequestered in the lysosomes, mitochondria, nuclei, and vesicles. Results of DCFH-DA assay and nutritional antioxidant N-acetylcysteine (NAC) experiments suggested that an oxidative stress mechanism was involved in the cytotoxic effects of BiOCl-NSs. Taken together, this work represents the first study on the behavior of BiOCl-NSs on human cells, and constitutes the first and essential step for the risk assessment of BiOCl nanomaterials.

  7. Potential antipsoriatic agents: lapacho compounds as potent inhibitors of HaCaT cell growth.

    PubMed

    Müller, K; Sellmer, A; Wiegrebe, W

    1999-08-01

    A number of lapacho compounds, representing the most common constituents of the inner bark of Tabebuia impetiginosa, together with some synthetic analogues, were evaluated in vitro against the growth of the human keratinocyte cell line HaCaT. With an IC(50) value of 0.7 microM, beta-lapachone (4) displayed activity comparable to that of the antipsoriatic drug anthralin. 2-Acetyl-8-hydroxynaphtho[2,3-b]furan-4,9-dione (7), which was prepared in a four-step synthesis from 2,8-dihydroxy-1, 4-naphthoquinone, was the most potent inhibitor among the known lapacho-derived compounds and inhibited cell growth with an IC(50) value of 0.35 microM. Furthermore, other active constituents of lapacho inhibited keratinocyte growth, with IC(50) values in the range of 0.5-3.0 microM. However, as already observed with anthralin, treatment of HaCaT cells with these potent lapacho compounds also caused remarkable damage to the plasma membrane. This was documented by leakage of lactate dehydrogenase into the culture medium, which significantly exceeded that of the vehicle control. Because of their potent activity against the growth of human keratinocytes, some lapacho-derived compounds appear to be promising as effective antipsoriatic agents.

  8. Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes

    PubMed Central

    Kim, Hye Kyung

    2016-01-01

    Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes. PMID:27483310

  9. Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes.

    PubMed

    Kim, Hye Kyung

    2016-07-29

    Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes.

  10. Comparative Cytotoxicity of Glycyrrhiza glabra Roots from Different Geographical Origins Against Immortal Human Keratinocyte (HaCaT), Lung Adenocarcinoma (A549) and Liver Carcinoma (HepG2) Cells.

    PubMed

    Basar, Norazah; Oridupa, Olayinka Ayotunde; Ritchie, Kenneth J; Nahar, Lutfun; Osman, Nashwa Mostafa M; Stafford, Angela; Kushiev, Habibjon; Kan, Asuman; Sarker, Satyajit D

    2015-06-01

    Glycyrrhiza glabra L. (Fabaceae), commonly known as 'liquorice', is a well-known medicinal plant. Roots of this plant have long been used as a sweetening and flavouring agent in food and pharmaceutical products, and also as a traditional remedy for cough, upper and lower respiratory ailments, kidney stones, hepatitis C, skin disorder, cardiovascular diseases, diabetes, gastrointestinal ulcers and stomach ache. Previous pharmacological and clinical studies have revealed its antitussive, antiinflammatory, antiviral, antimicrobial, antioxidant, immunomodulatory, hepatoprotective and cardioprotective properties. While glycyrrhizin, a sweet-tasting triterpene saponin, is the principal bioactive compound, several bioactive flavonoids and isoflavonoids are also present in the roots of this plant. In the present study, the cytotoxicity of the methanol extracts of nine samples of the roots of G. glabra, collected from various geographical origins, was assessed against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cell lines using the in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide cell toxicity/viability assay. Considerable variations in levels of cytotoxicity were observed among various samples of G. glabra. Copyright © 2015 John Wiley & Sons, Ltd.

  11. The influence of Tribenoside on expression and deposition of epidermal laminins in HaCaT cells.

    PubMed

    Kikkawa, Yamato; Takaki, Shu; Matsuda, Yuji; Okabe, Koichi; Taniguchi, Masakazu; Oomachi, Kengo; Samejima, Teruyuki; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi

    2010-01-01

    Tribenoside has been used clinically for hemorrhoidal disease associated with coagulation, inflammation, and wounds. However, the pharmacological mechanism of tribenoside activity has never been clear. In this study we examined whether tribenoside affected expression and deposition of laminins that are required for reconstruction of basement membranes (BMs) during wound healing in hemorrhoidal disease. HaCaT cells, which are derived from human epidermis, were treated in growth media supplemented with tribenoside. Reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific for laminin chains showed that HaCaT cells constitutively expressed laminin alpha3, alpha5, beta1, beta3, gamma1, and gamma2 chains. Tribenoside treatment of HaCaT cells did not induce expression of other laminin chains. We also quantified the expression of laminin chains in tribenoside-treated cells using real-time PCR. The expression level of laminin alpha3, beta1, beta3, gamma1, and gamma2 chains was not affected. In contrast, the expression of laminin alpha5 in the tribenoside-treated cells was four times higher than that of control cells. Immunocytochemistry also showed that tribenoside accelerated the focal deposition of laminin-332 (alpha3, beta3, gamma2). These results suggest that tribenoside interacts with epidermal cells and regulates the expression and localization of laminins to help reconstruct BMs in wound healing of hemorrhoids.

  12. Ethanol Extract of Cirsium japonicum var. ussuriense Kitamura Exhibits the Activation of Nuclear Factor Erythroid 2-Related Factor 2-dependent Antioxidant Response Element and Protects Human Keratinocyte HaCaT Cells Against Oxidative DNA Damage

    PubMed Central

    Yoo, Ok-Kyung; Choi, Bu Young; Park, Jin-Oh; Lee, Ji-Won; Park, Byoung-Kwon; Joo, Chul Gue; Heo, Hyo-Jung; Keum, Young-Sam

    2016-01-01

    Keratinocytes are constantly exposed to extracellular insults, such as ultraviolet B, toxic chemicals and mechanical stress, all of which can facilitate the aging of keratinocytes via the generation of intracellular reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a critical role in protecting keratinocytes against oxidants and xenobiotics by binding to the antioxidant response element (ARE), a cis-acting element existing in the promoter of most phase II cytoprotective genes. In the present study, we have attempted to find novel ethanol extract(s) of indigenous plants of Jeju island, Korea that can activate the Nrf2/ARE-dependent gene expression in human keratinocyte HaCaT cells. As a result, we identified that ethanol extract of Cirsium japonicum var. ussuriense Kitamura (ECJUK) elicited strong stimulatory effect on the ARE-dependent gene expression. Supporting this observation, we found that ECJUK induced the expression of Nrf2, hemoxygenase-1, and NAD(P)H:quinone oxidoreductase-1 and this event was correlated with Akt1 phosphorylation. We also found that ECJUK increased the intracellular reduced glutathione level and suppressed 12-O-tetradecanoylphorbol acetate-induced 8-hydroxyguanosine formation without affecting the overall viability. Collectively, our results provide evidence that ECJUK can protect against oxidative stress-mediated damages through the activation of Nrf2/ARE-dependent phase II cytoprotective gene expression. PMID:27051652

  13. Metabolic activation of pyrrolizidine alkaloids leading to phototoxicity and photogenotoxicity in human HaCaT keratinocytes.

    PubMed

    Wang, Chia-Chi; Xia, Qingsu; Li, Meng; Wang, Shuguang; Zhao, Yuewei; Tolleson, William H; Yin, Jun-Jie; Fu, Peter P

    2014-01-01

    Pyrrolizidine alkaloids, produced by a large number of poisonous plants with wide global distribution, are associated with genotoxicity, tumorigenicity, and hepatotoxicity in animals and humans. Mammalian metabolism converts pyrrolizidine alkaloids to reactive pyrrolic metabolites (dehydropyrrolizidine alkaloids) that form covalent protein and DNA adducts. Although a mechanistic understanding is currently unclear, pyrrolizidine alkaloids can cause secondary (hepatogenous) photosensitization and induce skin cancer. In this study, the phototoxicity of monocrotaline, riddelliine, dehydromonocrotaline, dehydroriddelliine, and dehydroretronecine (DHR) in human HaCaT keratinocytes under ultraviolet A (UVA) irradiation was determined. UVA irradiation of HaCaT cells treated with dehydromonocrotaline, dehydroriddelline, and DHR resulted in increased release of lactate dehydrogenase and enhanced photocytotoxicity proportional to the UVA doses. UVA-induced photochemical DNA damage also increased proportionally with dehydromonocrotaline and dehydroriddelline. UVA treatment potentiated the formation of 8-hydroxy-2'-deoxyguanosine DNA adducts induced by dehydromonocrotaline in HaCaT skin keratinocytes. Using electron spin resistance trapping, we found that UVA irradiation of dehydromonocrotaline and dehydroriddelliine generates reactive oxygen species (ROS), including hydroxyl radical, singlet oxygen, and superoxide, and electron transfer reactions, indicating that cytotoxicity and genotoxicity of these compounds could be mediated by ROS. Our results suggest that dehydropyrrolizidine alkaloids formed or delivered to the skin cause pyrrolizidine alkaloid-induced secondary photosensitization and possible skin cancer.

  14. Basonuclin Regulates a Subset of Ribosomal RNA Genes in HaCaT Cells

    PubMed Central

    Zhang, Shengliang; Wang, Junwen; Tseng, Hung

    2007-01-01

    Basonuclin (Bnc1), a cell-type-specific ribosomal RNA (rRNA) gene regulator, is expressed mainly in keratinocytes of stratified epithelium and gametogenic cells of testis and ovary. Previously, basonuclin was shown in vitro to interact with rRNA gene (rDNA) promoter at three highly conserved sites. Basonuclin's high affinity binding site overlaps with the binding site of a dedicated and ubiquitous Pol I transcription regulator, UBF, suggesting that their binding might interfere with each other if they bind to the same promoter. Knocking-down basonuclin in mouse oocytes eliminated approximately one quarter of RNA polymerase I (Pol I) transcription foci, without affecting the BrU incorporation of the remaining ones, suggesting that basonuclin might regulate a subset of rDNA. Here we show, via chromatin immunoprecipitation (ChIP), that basonuclin is associated with rDNA promoters in HaCaT cells, a spontaneously established human keratinocyte line. Immunoprecipitation data suggest that basonuclin is in a complex that also contains the subunits of Pol I (RPA194, RPA116), but not UBF. Knocking-down basonuclin in HaCaT cells partially impairs the association of RPA194 to rDNA promoter, but not that of UBF. Basonuclin-deficiency also reduces the amount of 47S pre-rRNA, but this effect can be seen only after cell-proliferation related rRNA synthesis has subsided at a higher cell density. DNA sequence of basonuclin-bound rDNA promoters shows single nucleotide polymorphisms (SNPs) that differ from those associated with UBF-bound promoters, suggesting that basonuclin and UBF interact with different subsets of promoters. In conclusion, our results demonstrate basonuclin's functional association with rDNA promoters and its interaction with Pol I in vivo. Our data also suggest that basonuclin-Pol I complex transcribes a subset of rDNA. PMID:17878937

  15. Pepsin-solubilised collagen (PSC) from Red Sea cucumber (Stichopus japonicus) regulates cell cycle and the fibronectin synthesis in HaCaT cell migration.

    PubMed

    Park, Soo-Yeong; Lim, Hee Kyoung; Lee, Seogjae; Hwang, Hyeong Cheol; Cho, Somi K; Cho, Moonjae

    2012-05-01

    Pepsin-solubilised collagen (PSC) from Red Sea cucumber (Stichopus japonicus) was studied with respect to its wound-healing effects on a human keratinocyte (HaCaT) cell line. Disaggregated collagen fibres were treated with 0.1M NaOH for 24h and digested with pepsin for 72h to reach maximum yield of 26.6%. The results of an in vitro wound-healing test showed that migration of HaCaT cells was 1.5-fold faster on PSC-coated plates than on untreated plates. The migration rate of sea cucumber PSC was similar to that of rat PSC, but five times higher than that of bovine gelatin. HaCaT cells grown on PSC-coated plates revealed increased fibronectin synthesis (6-fold and 3-fold compared to gelatin and rat PSC, respectively). Additionally, sea cucumber PSCs induced HaCaT cell proliferation by decreasing the G1 phase by 5% and maintaining a larger population (8%) of cells in mitosis. Collagen from Red Sea cucumber might be useful as an alternative to mammalian collagen in the nutraceutical and pharmaceutical industries. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. The ETS Family Transcription Factor ELK-1 Regulates Induction of the Cell Cycle-regulatory Gene p21Waf1/Cip1 and the BAX Gene in Sodium Arsenite-exposed Human Keratinocyte HaCaT Cells*

    PubMed Central

    Shin, Soon Young; Kim, Chang Gun; Lim, Yoongho; Lee, Young Han

    2011-01-01

    Cyclin-dependent kinase inhibitor (CDKN1A), often referred to as p21Waf1/Cip1 (p21), is induced by a variety of environmental stresses. Transcription factor ELK-1 is a member of the ETS oncogene superfamily. Here, we show that ELK-1 directly trans-activates the p21 gene, independently of p53 and EGR-1, in sodium arsenite (NaASO2)-exposed HaCaT cells. Promoter deletion analysis and site-directed mutagenesis identified the presence of an ELK-1-binding core motif between −190 and −170 bp of the p21 promoter that confers inducibility by NaASO2. Chromatin immunoprecipitation and electrophoretic mobility shift analyses confirmed the specific binding of ELK-1 to its putative binding sequence within the p21 promoter. In addition, NaASO2-induced p21 promoter activity was enhanced by exogenous expression of ELK-1 and reduced by expression of siRNA targeted to ELK-1 mRNA. The importance of ELK-1 in response to NaASO2 was further confirmed by the observation that stable expression of ELK-1 siRNA in HaCaT cells resulted in the attenuation of NaASO2-induced p21 expression. Although ELK-1 was activated by ERK, JNK, and p38 MAPK in response to NaASO2, ELK-1-mediated activation of the p21 promoter was largely dependent on ERK. In addition, EGR-1 induced by ELK-1 seemed to be involved in NaASO2-induced expression of BAX. This supports the view that the ERK/ELK-1 cascade is involved in p53-independent induction of p21 and BAX gene expression. PMID:21642427

  17. The ETS family transcription factor ELK-1 regulates induction of the cell cycle-regulatory gene p21(Waf1/Cip1) and the BAX gene in sodium arsenite-exposed human keratinocyte HaCaT cells.

    PubMed

    Shin, Soon Young; Kim, Chang Gun; Lim, Yoongho; Lee, Young Han

    2011-07-29

    Cyclin-dependent kinase inhibitor (CDKN1A), often referred to as p21(Waf1/Cip1) (p21), is induced by a variety of environmental stresses. Transcription factor ELK-1 is a member of the ETS oncogene superfamily. Here, we show that ELK-1 directly trans-activates the p21 gene, independently of p53 and EGR-1, in sodium arsenite (NaASO(2))-exposed HaCaT cells. Promoter deletion analysis and site-directed mutagenesis identified the presence of an ELK-1-binding core motif between -190 and -170 bp of the p21 promoter that confers inducibility by NaASO(2). Chromatin immunoprecipitation and electrophoretic mobility shift analyses confirmed the specific binding of ELK-1 to its putative binding sequence within the p21 promoter. In addition, NaASO(2)-induced p21 promoter activity was enhanced by exogenous expression of ELK-1 and reduced by expression of siRNA targeted to ELK-1 mRNA. The importance of ELK-1 in response to NaASO(2) was further confirmed by the observation that stable expression of ELK-1 siRNA in HaCaT cells resulted in the attenuation of NaASO(2)-induced p21 expression. Although ELK-1 was activated by ERK, JNK, and p38 MAPK in response to NaASO(2), ELK-1-mediated activation of the p21 promoter was largely dependent on ERK. In addition, EGR-1 induced by ELK-1 seemed to be involved in NaASO(2)-induced expression of BAX. This supports the view that the ERK/ELK-1 cascade is involved in p53-independent induction of p21 and BAX gene expression.

  18. Protective Effects of Minor Components of Curcuminoids on Hydrogen Peroxide-Treated Human HaCaT Keratinocytes.

    PubMed

    Liu, Yuh-Hwa; Lin, Yin-Shiou; Huang, Yu-Wei; Fang, Sheng-Uei; Lin, Shyr-Yi; Hou, Wen-Chi

    2016-05-11

    Hydrogen peroxide, one of the reactive oxygen species (ROS), can cause intracellular oxidative stress associated with skin aging and/or photoaging. Curcumin, a polyphenol in turmeric, has been reported to exhibit biological activity. In this study, five naturally occurring curcuminoids [curcumin, demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), monohydroxy-DMC, and monohydroxy-BDMC] were used to investigate their protective roles against hydrogen peroxide-induced oxidative stress in the immortalized human keratinocyte cell lines (HaCaT cells). These five curcuminoids at 10 μM, but not at 5 μM, were shown to exhibit cytotoxicities toward HaCaT keratinocytes. Therefore, a 5 μM concentration of the five curcuminoids was selected for further investigations. Cells were pretreated with or without curcuminoids for 2.5 h before 24-h hydrogen peroxide (150 μM) treatments. Pretreatments with the minor components monohydroxy-DMC or monohydroxy-BDMC, but not curcumin, DMC, and BDMC, showed protective activity, elevating cell viability compared to cells with direct hydrogen peroxide treatments. Pretreatments with monohydroxy-DMC and monohydroxy-BDMC showed the best protective effects, reducing apoptotic cell populations and intracellular ROS, as demonstrated by flow cytometry, as well as reducing the changes of the mitochondrial membrane potential compared to cells with direct hydrogen peroxide treatments. The pretreatments with monohydroxy-DMC and monohydroxy-BDMC reduced c-jun and c-fos mRNA expression and p53 tumor suppressor protein expression and increased HO-1 protein expression and glutathione peroxidase (GPx) activity, respectively, compared to cells with direct hydrogen peroxide treatments. The five curcuminoids exhibited similar hydrogen peroxide-scavenging activity in vitro. It was proposed that monohydroxy-DMC and monohydroxy-BDMC could induce antioxidant defense systems better than curcumin, DMC, or BDMC could against hydrogen peroxide-induced oxidative

  19. Photoprotection by Punica granatum seed oil nanoemulsion entrapping polyphenol-rich ethyl acetate fraction against UVB-induced DNA damage in human keratinocyte (HaCaT) cell line.

    PubMed

    Baccarin, Thaisa; Mitjans, Montserrat; Ramos, David; Lemos-Senna, Elenara; Vinardell, Maria Pilar

    2015-12-01

    There has been an increase in the use of botanicals as skin photoprotective agents. Pomegranate (Punica granatum L.) is well known for its high concentration of polyphenolic compounds and for its antioxidant and anti-inflammatory properties. The aim of this study was to analyze the photoprotection provided by P. granatum seed oil nanoemulsion entrapping the polyphenol-rich ethyl acetate fraction against UVB-induced DNA damage in the keratinocyte HaCaT cell line. For this purpose, HaCaT cells were pretreated for 1h with nanoemulsions in a serum-free medium and then irradiated with UVB (90-200 mJ/cm(2)) rays. Fluorescence microscopy analysis provided information about the cellular internalization of the nanodroplets. We also determined the in vitro SPF of the nanoemulsions and evaluated their phototoxicity using the 3T3 Neutral Red Uptake Phototoxicity Test. The nanoemulsions were able to protect the cells' DNA against UVB-induced damage in a concentration dependent manner. Nanodroplets were internalized by the cells but a higher proportion was detected along the cell membrane. The SPF obtained (~25) depended on the concentration of the ethyl acetate fraction and pomegranate seed oil in the nanoemulsion. The photoprotective formulations were classified as non-phototoxic. In conclusion, nanoemulsions entrapping the polyphenol-rich ethyl acetate fraction show potential for use as a sunscreen product. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Cell wall mannoprotein of Candida albicans induces cell cycle alternation and inhibits apoptosis of HaCaT cells via NF-κB signal pathway.

    PubMed

    Han, Yang; Jiang, Hang-Hang; Zhang, Yu-Jing; Hao, Xing-Jia; Sun, Yu-Zhe; Qi, Rui-Qun; Chen, Hong-Duo; Gao, Xing-Hua

    2017-10-01

    Candida albicans (C. albicans) is a commensal organism in human and a well-known dimorphic opportunistic pathogenic fungus. Though plenty of researches on the pathogenesis of C. albicans have been performed, the mechanism is not fully understood. The cell wall components of C. albicans have been documented to play important roles in its pathogenic processes. To further study the infectious mechanism of C. albicans, we investigated the potential functional role of its cell wall mannoprotein in cell cycle and apoptosis of HaCaT cells. We found that mannoprotein could promote the transition of cell cycle from G1/G0 to S phase, in which Cyclin D1, CDK4 and p-Rb, the major regulators of the cell cycle progression, showed significant upregulation, and CDKN1A (cyclin dependent kinase inhibitor 1A (p21)) showed significant downregulation. Mannoprotein also could inhibit apoptosis of HaCaT cells, which was well associated with increased expression of BCL2 (Bcl-2). Moreover, mannoprotein could increase the phosphorylation levels of RELA (p65) and NFKBIA (IκBα), as the key factors of NF-κB signal pathway in HaCaT cells, suggesting the activation of NF-κB signal pathway. Additionally, a NF-κB specific inhibitor, PDTC, could rescue the effect of mannoprotein on cell cycle and apoptosis of HaCaT cells, which suggested that mannoprotein could activate NF-κB signal pathway to mediate cell cycle alternation and inhibit apoptosis. Copyright © 2017. Published by Elsevier Ltd.

  1. PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK)

    SciTech Connect

    Senthilkumar, P.K.; Robertson, L.W.; Ludewig, G.

    2012-02-15

    Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24 days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism. -- Highlights: ► Human immortal (HaCaT) and primary (NFK) keratinocytes were exposed to PCB153. ► PCB153 significantly reduced telomerase activity and telomere length in HaCaT. ► No effect on telomere length and

  2. Incoming human papillomavirus 16 genome is lost in PML protein-deficient HaCaT keratinocytes.

    PubMed

    Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Keiffer, Timothy R; Guion, Lucile G M; DiGiuseppe, Stephen; Scott, Rona S; Sapp, Martin

    2017-05-01

    Human papillomaviruses (HPVs) target promyelocytic leukemia (PML) nuclear bodies (NBs) during infectious entry and PML protein is important for efficient transcription of incoming viral genome. However, the transcriptional down regulation was shown to be promoter-independent in that heterologous promoters delivered by papillomavirus particles were also affected. To further investigate the role of PML protein in HPV entry, we used small hairpin RNA to knockdown PML protein in HaCaT keratinocytes. Confirming previous findings, PML knockdown in HaCaT cells reduced HPV16 transcript levels significantly following infectious entry without impairing binding and trafficking. However, when we quantified steady-state levels of pseudogenomes in interphase cells, we found strongly reduced genome levels compared with parental HaCaT cells. Because nuclear delivery was comparable in both cell lines, we conclude that viral pseudogenome must be removed after successful nuclear delivery. Transcriptome analysis by gene array revealed that PML knockdown in clonal HaCaT cells was associated with a constitutive interferon response. Abrogation of JAK1/2 signaling prevented genome loss, however, did not restore viral transcription. In contrast, knockdown of PML protein in HeLa cells did not affect HPV genome delivery and transcription. HeLa cells are transformed by HPV18 oncogenes E6 and E7, which have been shown to interfere with the JAK/Stat signaling pathway. Our data imply that PML NBs protect incoming HPV genomes. Furthermore, they provide evidence that PML NBs are key regulators of the innate immune response in keratinocytes. Promyelocytic leukemia nuclear bodies (PML NBs) are important for antiviral defense. Many DNA viruses target these subnuclear structures and reorganize them. Reorganization of PML NBs by viral proteins is important for establishment of infection. In contrast, HPVs require the presence of PML protein for efficient transcription of incoming viral genome. Our

  3. The hormesis effect of plasma-elevated intracellular ROS on HaCaT cells

    NASA Astrophysics Data System (ADS)

    Szili, Endre J.; Harding, Frances J.; Hong, Sung-Ha; Herrmann, Franziska; Voelcker, Nicolas H.; Short, Robert D.

    2015-12-01

    We have examined the link between ionized-gas plasma delivery of reactive oxygen species (ROS) to immortalized keratinocyte (HaCaT) cells and cell fate, defined in terms of cell viability versus death. Phospholipid vesicles were used as cell mimics to measure the possible intracellular ROS concentration, [ROSi], delivered by various plasma treatments. Cells were exposed to a helium cold atmospheric plasma (CAP) jet for different plasma exposure times (5-60 s) and gas flow rates (50-1000 ml min-1). Based upon the [ROSi] data we argue that plasma-generated ROS in the cell culture medium can readily diffuse into real cells. Plasma exposure that equated to an [ROSi] in the range of 3.81  ×  10-10-9.47  ×  10-8 M, measured at 1 h after the plasma exposure, resulted in increased cell viability at 72 h; whereas a higher [ROSi] at 1 h decreased cell viability after 72 h of culture. This may be because of the manner in which the ROS are delivered by the plasma: HaCaT cells better tolerate a low ROS flux over an extended plasma exposure period of 1 min, compared to a high flux delivered in a few seconds, although the final [ROSi] may be the same. Our results suggest that plasma stimulation of HaCaT cells follows the principle of hormesis.

  4. Protective Effects of Lindera coreana on UVB-induced Oxidative Stress in Human HaCaT Keratinocytes

    PubMed Central

    Song, Jia-Le; Gao, Yang

    2014-01-01

    The present study was designed to investigate the protective effect of ethanol extracts from Lindera coreana leaves (LCE) on UVB-induced oxidative stress in HaCaT keratinocytes. The HaCaT cells were pretreated with LCE for 24 h and then exposed to UVB (20 mJ/cm2) for 2 h. UVB significantly decreased the cell viability (p<0.05). LCE did not exhibit significantly cytotoxic effects and increased the viability of the HaCaT cells in a concentration-dependent manner. To further investigate the protective effects of LCE on UVB-induced oxidative stress in HaCaT cells, the cellular levels of reactive oxygen species (ROS), lipid peroxidation and endogenous antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-px), were analyzed. LCE decreased the intracellular levels of ROS and lipid peroxidation and increased the activity of antioxidant enzymes. These results suggest that LCE exerted cytoprotective activity against UVB-induced oxidative stress in HaCaT cells through the inhibition of lipid peroxidation, reduction of ROS levels and stimulation of antioxidant enzymes activities. In addition, LCE also decreased the TNF-α, IL-1β and IL-6 levels in UVB-irradiated HaCaT cells. PMID:25587326

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

    SciTech Connect

    Iwata, Masahiro; Kawahara, Ko-ichi; Kawabata, Hisashi; Ito, Takashi; Mera, Kentaro; Biswas, Kamal Krishna; Tancharoen, Salunya; Higashi, Yuko; Kikuchi, Kiyoshi; Hashiguchi, Teruto

    2008-12-12

    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 (10 mJ/cm{sup 2}) 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.

  6. PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK).

    PubMed

    Senthilkumar, P K; Robertson, L W; Ludewig, G

    2012-02-15

    Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism.

  7. PCB153 reduces Telomerase Activity and Telomere Length in Immortalized Human Skin Kerantinocytes (HaCaT) but not in Human Foreskin Keratinocytes (NFK)

    PubMed Central

    Senthilkumar, P.K.; Robertson, L.W.; Ludewig, G.

    2012-01-01

    Polychlorinated Biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24 days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism. PMID:22210444

  8. [Effect of silicon dioxide nanoparticles on expression and DNA methylation of PARP-1 gene in HaCaT cells].

    PubMed

    Gong, Chunmei; Yang, Linqing; Zhou, Jichang; Tao, Gonghua; Liu, Xiaoli; Zhuang, Zhixiong

    2015-07-01

    To study the effect of silicon dioxide nanoparticles on the expression and promoter region CpG islands methylation of (Poly [ADP-ribose] polymerase 1, PARP-1) gene in human HaCaT Cell. HaCaT Cells were treated with nm-SiO₂at 0, 2.5, 5 and 10 µg/mL and micro-SiO₂at 10 µg/ml for 24 h and DAC treatment was given at 10 µg/ml group for 48 h. Real-time PCR and western blot assay was used to detect the expression of PARP-1 mRNA and protein. BSP (Bisulfite Pyrosequence, BSP) assay was used to detect the promoter region CpG islands methylation status of PARP-1 gene. After exposure to nano-SiO₂particles, compared to CTRL group, the mRNA and protein expression of PARP-1 in micro-SiO₂and 2.5 µg/ml group unchanged, but he mRNA and protein expression of PARP-1 in 5, 10 µg/ml as well as DAC group was down-regulated and there are statistical significance between CTRL group and 5, 10 µg/ml as well as DAC group and the PARP-1 promoter region CpG islands showed methylation. nano-SiO₂can down-regulate PARP-1 expression in HaCaT Cell and this is associated with the change in the methylation of PARP-1 gene promoter region CpG islands induced by nano-SiO₂particles.

  9. Role of antioxidants in prevention of pyrimidine dimer formation in UVB irradiated human HaCaT keratinocytes.

    PubMed

    Hochberg, Malka; Kohen, Ron; Enk, Claes D

    2006-06-01

    The objective of the present study was to study the role of reactive oxygen species (ROS) in UVB induced cyclobutane pyrimidine dimer (CPD) formation in human keratinocytes, and to examine the modulating activity of low molecular weight antioxidants. To demonstrate the involvement of ROS, we examined the protective capacity of alpha-tocopherol, tempamine, and diethyldithiocarbamate (DDC) on CPD formation in intact cells and naked DNA. HaCaT cells and naked DNA in water solution were irradiated with UVB in the presence of the antioxidants and CPD was determined by ELISA. We found that all three antioxidants provided protection against UVB induced CPD formation. The protection was observed in intact cells only and not in naked DNA. Since some of the tested antioxidants do not possess UV absorbing qualities, our findings suggest that in a cellular environment ROS play a role in CPD formation.

  10. Succinate dehydrogenase activity regulates PCB3-quinone induced metabolic oxidative stress and toxicity in HaCaT human keratinocytes

    PubMed Central

    Xiao, Wusheng; Sarsour, Ehab H.; Wagner, Brett A.; Doskey, Claire M.; Buettner, Garry R.; Domann, Frederick E.; Goswami, Prabhat C.

    2015-01-01

    Polychlorinated biphenyls (PCBs) and their metabolites are environmental pollutants that are known to have adverse health effects. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone-metabolite of 4-Monochlorobiphenyl (PCB3, present in the environment and human blood) is toxic to human skin keratinocytes, and breast and prostate epithelial cells. This study investigates the hypothesis that 4-ClBQ-induced metabolic oxidative stress regulates toxicity in human keratinocytes. Results from Seahorse XF96 Analyzer showed that the 4-ClBQ treatment increased extracellular acidification rate, proton production rate, oxygen consumption rate and ATP content, indicative of metabolic oxidative stress. Results from a q-RT-PCR assay showed significant increases in the mRNA levels of hexokinase 2 (hk2), pyruvate kinase M2 (pkm2) and glucose-6-phosphate dehydrogenase (g6pd), and decreases in the mRNA levels of succinate dehydrogenase (complex II) subunit C and D (sdhc and sdhd). Pharmacological inhibition of G6PD-activity enhanced the toxicity of 4-ClBQ, suggesting that the protective function of the pentose phosphate pathway is functional in 4-ClBQ treated cells. The decrease in sdhc and sdhd expression was associated with a significant decrease in complex II activity and increase in mitochondrial levels of ROS. Overexpression of sdhc and sdhd suppressed 4-ClBQ-induced inhibition of complex II activity, increase in mitochondrial levels of ROS, and toxicity. These results suggest that the 4-ClBQ treatment induces metabolic oxidative stress in HaCaT cells, and while the protective function of the pentose phosphate pathway is active, inhibition of complex II activity sensitizes HaCaT cells to 4-ClBQ induced toxicity. PMID:25417049

  11. Loss of CAR promotes migration and proliferation of HaCaT cells, and accelerates wound healing in rats via Src-p38 MAPK pathway

    PubMed Central

    Su, Linlin; Fu, Lanqing; Li, Xiaodong; Zhang, Yue; Li, Zhenzhen; Wu, Xue; Li, Yan; Bai, Xiaozhi; Hu, Dahai

    2016-01-01

    The coxsackie and adenovirus receptor (CAR) is a cell adhesion molecule mostly localized to cell-cell contacts in epithelial and endothelial cells. CAR is known to regulate tumor progression, however, its physiological role in keratinocyte migration and proliferation, two essential steps in re-epithelialization during wound healing, has less been investigated. Here we showed that CAR was predominantly expressed in the epidermis of human skin, CAR knockdown by RNAi significantly accelerated HaCaT cell migration and proliferation. In addition, knockdown of CAR in vitro increased p-Src, p-p38, and p-JNK protein levels; however, Src inhibitor PP2 prevented the increase of p-Src and p-p38 induced by CAR RNAi, but not p-JNK, and decelerated cell migration and proliferation. More intriguingly, in vivo CAR RNAi on the skin area surrounding the wounds on rat back visually accelerated wound healing and re-epithelialization process, while treatment with PP2 or p38 inhibitor SB203580 obviously inhibited these effects. By contrast, overexpressing CAR in HaCaT cells significantly decelerated cell migration and proliferation. Above results demonstrate that suppression of CAR could accelerate HaCaT cell migration and proliferation, and promote wound healing in rat skin, probably via Src-p38 MAPK pathway. CAR thus might serve as a novel therapeutic target for facilitating wound healing. PMID:26804208

  12. Loss of CAR promotes migration and proliferation of HaCaT cells, and accelerates wound healing in rats via Src-p38 MAPK pathway.

    PubMed

    Su, Linlin; Fu, Lanqing; Li, Xiaodong; Zhang, Yue; Li, Zhenzhen; Wu, Xue; Li, Yan; Bai, Xiaozhi; Hu, Dahai

    2016-01-25

    The coxsackie and adenovirus receptor (CAR) is a cell adhesion molecule mostly localized to cell-cell contacts in epithelial and endothelial cells. CAR is known to regulate tumor progression, however, its physiological role in keratinocyte migration and proliferation, two essential steps in re-epithelialization during wound healing, has less been investigated. Here we showed that CAR was predominantly expressed in the epidermis of human skin, CAR knockdown by RNAi significantly accelerated HaCaT cell migration and proliferation. In addition, knockdown of CAR in vitro increased p-Src, p-p38, and p-JNK protein levels; however, Src inhibitor PP2 prevented the increase of p-Src and p-p38 induced by CAR RNAi, but not p-JNK, and decelerated cell migration and proliferation. More intriguingly, in vivo CAR RNAi on the skin area surrounding the wounds on rat back visually accelerated wound healing and re-epithelialization process, while treatment with PP2 or p38 inhibitor SB203580 obviously inhibited these effects. By contrast, overexpressing CAR in HaCaT cells significantly decelerated cell migration and proliferation. Above results demonstrate that suppression of CAR could accelerate HaCaT cell migration and proliferation, and promote wound healing in rat skin, probably via Src-p38 MAPK pathway. CAR thus might serve as a novel therapeutic target for facilitating wound healing.

  13. MicroRNA-184 Promotes Proliferation and Inhibits Apoptosis in HaCaT Cells: An In Vitro Study

    PubMed Central

    Bi, Xiaodong; Cao, Yu; Chen, Rixin; Liu, Chengyin; Chen, Jinghong; Min, Dongfang

    2016-01-01

    Background This study aimed to investigate the role of miR-184 in the proliferation and apoptosis of keratinocyte (HaCaT cells). Material/Methods HaCaT cells were cultured in a growth medium. The miR-184 was transfected with siRNA, then cell viability and apoptosis were assayed by MTT and flow cytometry, respectively. The colony-forming efficacy of HaCaT cells were detected as well. mRNA expressions of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-β1 were measured with RT-PCR. The expressions of apoptosis-related proteins caspase-3 and Bcl-x in HaCaT cells were determined by Western blot. Results After miR-184 was transfected with siRNA, cell viability and colony forming ability decreased significantly, and apoptosis was significantly increased. The expressions of growth factors TGF-β1 and bFGF mRNAs, as well as apoptosis-related proteins Bcl-x, in HaCaT cells declined significantly after miR-184 was transfected with siRNA. In addition, the expression of pro-apoptotic protein caspase-3 increased significantly. Conclusions Our results suggest distinct roles of miR-184 during the growth, proliferation, and apoptosis of keratinocytes. PMID:27571235

  14. Selenium Polysaccharide SPMP-2a from Pleurotus geesteranus Alleviates H2O2-Induced Oxidative Damage in HaCaT Cells

    PubMed Central

    Zhou, Cheng; Huang, Shoucheng

    2017-01-01

    Selenium- (Se-) enriched polysaccharide SPMP-2a was extracted and purified from Pleurotus geesteranus. SPMP-2a is a white flocculent polysaccharide and soluble in water, with a molecular weight of 3.32 × 104 Da. Fourier transform infrared spectroscopy spectral analysis indicated that it belongs to an acid Se polysaccharide with α-D-glucopyranoside bond. The effects of Se polysaccharide SPMP-2a in P. geesteranus against hydrogen peroxide- (H2O2-) induced oxidative damage in human keratinocytes (HaCaT) cells were evaluated further. Reduced cell viability and elevated apoptotic rates in H2O2-treated HaCaT cells were proven by MTT and flow cytometry assays. Hoechst 33342 staining revealed chromatin condensations in the nuclei of HaCaT cells. However, with the addition of SPMP-2a, cell viability improved, nuclear condensation declined, and cell apoptotic rates dropped significantly. Ultrastructural observation consistently revealed that treatments with SPMP-2a reduced the number of swollen and vacuolar mitochondria in the H2O2-treated cells compared with the controls. Furthermore, SPMP-2a increased the superoxide dismutase (SOD) and catalase (CAT) activities and reduced reactive oxygen species (ROS) content. Western blot analysis showed that SPMP-2a treatment effectively increased B-cell lymphoma 2 (Bcl-2) protein expression. Therefore, SPMP-2a could improve cellular antioxidant enzyme activities, reduce ROS levels, and increase Bcl-2 protein expression levels, thereby reducing cell apoptosis and protecting HaCaT cells from H2O2-induced oxidative damage. PMID:28293636

  15. ER signaling is activated to protect human HaCaT keratinocytes from ER stress induced by environmental doses of UVB

    SciTech Connect

    Mera, Kentaro; Kawahara, Ko-ichi; Tada, Ko-ichi; Kawai, Kazuhiro; Hashiguchi, Teruto; Maruyama, Ikuro; Kanekura, Takuro

    2010-06-25

    Proteins are folded properly in the endoplasmic reticulum (ER). Various stress such as hypoxia, ischemia and starvation interfere with the ER function, causing ER stress, which is defined by the accumulation of unfolded protein (UP) in the ER. ER stress is prevented by the UP response (UPR) and ER-associated degradation (ERAD). These signaling pathways are activated by three major ER molecules, ATF6, IRE-1 and PERK. Using HaCaT cells, we investigated ER signaling in human keratinocytes irradiated by environmental doses of ultraviolet B (UVB). The expression of Ero1-L{alpha}, an upstream signaling molecule of ER stress, decreased at 1-4 h after 10 mJ/cm{sup 2} irradiation, indicating that the environmental dose of UVB-induced ER stress in HaCaT cells, without growth retardation. Furthermore, expression of intact ATF6 was decreased and it was translocated to the nuclei. The expression of XBP-1, a downstream molecule of IRE-1, which is an ER chaperone whose expression is regulated by XBP-1, and UP ubiquitination were induced by 10 mJ/cm{sup 2} UVB at 4 h. PERK, which regulates apoptosis, was not phosphorylated. Our results demonstrate that UVB irradiation generates UP in HaCaT cells and that the UPR and ERAD systems are activated to protect cells from UVB-induced ER stress. This is the first report to show ER signaling in UVB-irradiated keratinocytes.

  16. Chromium III histidinate exposure modulates antioxidant gene expression in HaCaT human keratinocytes exposed to oxidative stress

    USDA-ARS?s Scientific Manuscript database

    While the toxicity of hexavalent chromium is well established, trivalent Cr (Cr(III)) is an essential nutrient involved in insulin and glucose homeostasis. Recently, antioxidant effects of chromium (III) histidinate (Cr(III)His) were reported in HaCaT human keratinocytes exposed to oxidative stress...

  17. Protective effects of kappa-ca3000+CP against ultraviolet-induced damage in HaCaT and MEF cells.

    PubMed

    Ren, Shu-Wen; Li, Jing; Wang, Wei; Guan, Hua-Shi

    2010-10-05

    In this study, the complex kappa-ca3000+CP combined collagen peptide with kappa-carrageenan oligosaccharide was tested for its ability to moderate UV-induced damage and investigated for its protective mechanism against UV radiation. Human keratinocytes (HaCaT) and mouse embryonic fibroblasts (MEF) were used to monitor the effects of kappa-ca3000+CP on cell viability, apoptosis, level of collagen I and MMP-1, MAPKs activation and intracellular ROS production after UV-irradiation. The results indicated that application of the kappa-ca3000+CP (100 microg/ml) could significantly attenuate UV-induced HaCaT and MEF death, as well as inhibit the UV-induced apoptosis of HaCaT cells. The decreased collagen I synthesis and the increased MMP-1 expression of MEF by UV radiation were almost restored back to normal level after treatment with kappa-ca3000+CP. Moreover, kappa-ca3000+CP could significantly suppress UV-induced MAPKs activation and intracellular ROS production. Taken together, these results showed that antioxidant property of kappa-ca3000+CP can effectively attenuate UV-caused cell damage and skin photoaging by suppressing cell apoptosis and expression of MMP-1 through the MAPKs signaling pathways. Thus, kappa-ca3000+CP has potential antiaging effects and prominent protective effects on UV-induced skin cell damages, which might be used in pharmaceutical and cosmetic industries.

  18. Deoxynivalenol induced mouse skin tumor initiation: Elucidation of molecular mechanisms in human HaCaT keratinocytes.

    PubMed

    Mishra, Sakshi; Tewari, Prachi; Chaudhari, Bhushan P; Dwivedi, Premendra D; Pandey, Haushila P; Das, Mukul

    2016-11-01

    Among food contaminants, mycotoxins are toxic to both human and animal health. Our prior studies suggest that Deoxynivalenol (DON), a mycotoxin, behaves as a tumor promoter by inducing edema, hyperplasia, ODC activity and activation of MAPK's in mouse skin. In this study, topical application of DON, 336 and 672 nmol significantly enhanced ROS levels, DNA damage and apoptosis with concomitant downregulation of Ki-67, cyclin D, cyclin E, cyclin A and cyclin-dependent kinases (CDK4 and CDK2) thereby resulting in tumor initiation in mouse skin. Further, the elucidation of molecular mechanisms of tumor initiation by DON (0.42-3.37 nmol/ml) in HaCaT keratinocytes, revealed (i) enhanced ROS generation with cell cycle phase arrest in G0/G1 phase, (ii) increase in levels of 8-OxoG (6-24 hr) and γH2AX protein, (iii) significant enhancement in oxidative stress marker enzymes LPO, GSH, GR with concomitant decrease in antioxidant enzymes catalase, GPx, GST, SOD and mitochondrial membrane potential after DON (1.68 nmol) treatment, (iv) suppression of Nrf2 translocation to nucleus, enhanced phosphorylation with subsequent activation ERK1/2, p38 and JNK MAPK's following DON (1.68 nmol) treatment, (v) overexpression of c-jun, c-fos proteins, upregulation of Bax along with downregulation of Bcl-2 proteins, (vi) increase in cytochrome-c, caspase-9, caspase-3 and poly ADP ribose polymerase levels leads to apoptosis. Pretreatment of superoxide dismutase, mannitol and ethanol to HaCaT cells resulted in significant reduction in ROS levels and apoptosis indicating the role of superoxide and hydroxyl radicals in DON induced apoptosis as an early event and skin tumor initiation as a late event.

  19. Polypeptides from the Skin of Rana chensinensis Exert the Antioxidant and Antiapoptotic Activities on HaCaT Cells.

    PubMed

    Zhang, Xin; Cheng, Yunyun; Yang, Yang; Liu, Songcai; Shi, Hui; Lu, Chao; Li, Siming; Nie, Linyan; Su, Dan; Deng, Xuming; Ding, Kexiang; Hao, Linlin

    2017-01-02

    Studies have shown that frog skin secretes many types of peptides that are good for human skin. In this study, acid and enzymatic extracts of Rana skin peptides (acid/enzymatic Rana skin peptides, ARPs/ERPs) were obtained. The chemical and physical properties of the ARPs and ERPs were identified through UV scanning, HGLC, FRIT, and MS. MTS and flow cytometry were used to test the proproliferative and antiapoptotic effects of the ARPs and ERPs on human immortalized keratinocytes (HaCaT cells). To elucidate the antiapoptotic mechanisms, the mRNA and protein levels of EGF (epidermal growth factor, which enhances stimulation of cellular proliferation in both cells and epithelial tissues) and caspase-3 were evaluated using quantitative RT-PCR. The results indicated that the ARPs and ERPs were extracted from the Rana skin with yields of 0.65% and 0.52%, respectively. Treatment with ARPs (1.6 g/L) and ERPs (0.8 g/L) showed a 1.66-fold (p < 0.001) and 2.1-fold (p < 0.001) enhancement in the proliferation rates of HaCaT cells. The rate of apoptosis decreased by 2.6 fold (p < 0.01) and 3.4 fold (p < 0.01) under the UVB stimulation, respectively, at the same time, the up-regulation of EGF and down-regulation of caspase-3 were found. These results suggested that we can dig into the potential value of ARPs/ERPs in a new field.

  20. Glycolic Acid Silences Inflammasome Complex Genes, NLRC4 and ASC, by Inducing DNA Methylation in HaCaT Cells.

    PubMed

    Tang, Sheau-Chung; Yeh, Jih-I; Hung, Sung-Jen; Hsiao, Yu-Ping; Liu, Fu-Tong; Yang, Jen-Hung

    2016-03-01

    AHAs (α-hydroxy acids), including glycolic acid (GA), have been widely used in cosmetic products and superficial chemical peels. Inflammasome complex has been shown to play critical roles in inflammatory pathways in human keratinocytes. However, the anti-inflammatory mechanism of GA is still unknown. The aim of this study is to investigate the relationship between the expression of the inflammasome complex and epigenetic modification to elucidate the molecular mechanism of the anti-inflammatory effect of GA in HaCaT cells. We evaluated NLRP3, NLRC4, AIM2, and ASC inflammasome complex gene expression on real-time polymerase chain reaction (PCR). Methylation changes were detected in these genes following treatment with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza) with or without the addition of GA using methylation-specific PCR (MSP). GA inhibited the expressions of these inflammasome complex genes, and the decreases in the expressions of mRNA were reversed by 5-Aza treatment. Methylation was detected in NLRC4 and ASC on MSP, but not in NLRP3 or AIM2. GA decreased NLRC4 and ASC gene expression by increasing not only DNA methyltransferase 3B (DNMT-3B) protein level, but also total DNMT activity. Furthermore, silencing of DNMT-3B (shDNMT-3B) increased the expressions of NLRC4 and ASC. Our data demonstrated that GA treatment induces hypermethylation of promoters of NLRC4 and ASC genes, which may subsequently lead to the hindering of the assembly of the inflammasome complex in HaCaT cells. These results highlight the anti-inflammatory potential of GA-containing cosmetic agents in human skin cells and demonstrate for the first time the role of aberrant hypermethylation in this process.

  1. An observed effect of ultraviolet radiation emitted from beta-irradiated HaCaT cells upon non-beta-irradiated bystander cells.

    PubMed

    Le, Michelle; McNeill, Fiona E; Seymour, Colin; Rainbow, Andrew J; Mothersill, Carmel E

    2015-03-01

    Previous research has shown that beta radiation can induce ultraviolet (UV) photon emission in human keratinocyte cells. Spectral analysis using a filter-based method in the ultraviolet range demonstrated that the strongest externally measureable photon emission was induced by beta radiation in the UVA range. In the current study, the potential biological implications of this UV photon emission from beta-irradiated cells were investigated. HaCaT human keratinocyte cells were irradiated with tritium ((3)H) and the photon emission induced was concurrently measured at the strongest externally measurable wavelength, 340 ± 5 nm, using a combination filter-photomultiplier tube system. Unirradiated reporter HaCaT cell cultures were also placed directly above (3)H-irradiated cells so that they would receive the induced secondary photons emitted from beta-irradiated cells, and the clonogenic survival in reporter cells was then assessed. Maximum photon emission (1207.04 ± 107.65 counts per second) was observed during irradiation of 2,000 cells/cm(2) with (3)H and the maximum reporter cell death (23.2 ± 0.9% reduction in survival) was observed under the same conditions. The measured photon emission from beta-irradiated cells and reporter cell death were strongly correlated (r = 0.977, P < 0.01). Placement of a polyethylene terephthalate filter, designed to eliminate >90% of UV wavelengths below 390 nm, between the directly irradiated and reporter cell layers was effective in nearly abolishing both 340 nm photon detection and reporter cell death in treated groups. Concurrent treatment of reporter cells with lomefloxacin during exposure to the secondary photons resulted in significantly increased cell killing, indicating a potential synergistic effect, while melanin treatment resulted in decreased reporter cell killing regardless of irradiation. These results suggest that secondary photons in the UV spectral range induced by beta irradiation play a role in inducing a

  2. The role of reactive oxygen species in silicon dioxide nanoparticle-induced cytotoxicity and DNA damage in HaCaT cells.

    PubMed

    Gong, Chunmei; Tao, Gonghua; Yang, Linqing; Liu, Jianjun; He, Haowei; Zhuang, Zhixiong

    2012-04-01

    The increasing applications of silicon dioxide (SiO(2)) nanomaterials have been widely concerned over their biological effects and potential hazard to human health. In this study, we explored the effects of SiO(2) nanoparticles (15, 30, and 100 nm) and their micro-sized counterpart on cultured human epidermal Keratinocyte (HaCaT) cells. Cell viability, cell morphology, reactive oxygen species (ROS), DNA damage (8-OHdG, γH2AX and comet assay) and apoptosis were assessed under control and SiO(2) nanoparticles exposed conditions. As observed in the Cell Counting Kit-8 (CCK-8) assay, exposure to 15, 30 or 100 nm SiO(2) nanoparticles at dosage levels between 0 and 100 μg/ml decreased cell viability in a concentration- and size dependent manner and the IC50 of 24 hour exposure was 19.4 ± 1.3, 27.7 ± 1.5 and 35.9 ± 1.6 μg/ml for 15, 30 and 100 nm SiO(2) nanoparticles, respectively. Morphological examination revealed cell shrinkage and cell wall missing after SiO(2) nanoparticle exposure. Increase in intracellular ROS level and DNA damage as well as apoptosis were also observed in SiO(2) nanoparticle-exposed HaCaT cells. Exposure to SiO(2) nanoparticles results in a concentration- and size-dependent cytotoxicity and DNA damage in cultural HaCaT cells which is closely correlated to increased oxidative stress.

  3. Ligand-independent activation of aryl hydrocarbon receptor signaling in PCB3-quinone treated HaCaT human keratinocytes.

    PubMed

    Xiao, Wusheng; Son, Jyungmean; Vorrink, Sabine U; Domann, Frederick E; Goswami, Prabhat C

    2015-03-18

    Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that plays a critical role in metabolism, cell proliferation, development, carcinogenesis, and xenobiotic response. In general, dioxin-like polychlorinated biphenyls (PCBs) exhibit a ligand-dependent activation of AhR-signaling. Results from this study show that a quinone-derivative (1-(4-Chlorophenyl)-benzo-2,5-quinone; 4-ClBQ) of a non-dioxin like PCB (PCB3) also activates AhR-signaling. Treatments of HaCaT human keratinocytes with 4-ClBQ and dioxin-like PCB126 significantly increased AhR-target gene expression, CYP1A1 mRNA and protein levels. 4-ClBQ-induced increase CYP1A1 expression was associated with an increase in the nuclear translocation of AhR protein as well as an increase in the luciferase-reporter activity of a human CYP1A1 xenobiotic response element (XRE). 6,2',4'-Trimethoxyflavone (TMF), a well-characterized AhR-ligand antagonist significantly suppressed PCB126-induced increase in CYP1A1 expression, while the same treatment did not suppress 4-ClBQ-induced increase in CYP1A1 expression. However, siRNA-mediated down-regulation of AhR significantly inhibited 4-ClBQ-induced increase in CYP1A1 expression, suggesting that AhR mediates 4-ClBQ-induced increase in CYP1A1 expression. Interestingly, treatment with the antioxidant N-acetyl-l-cysteine significantly suppressed 4-ClBQ-induced increase in CYP1A1 expression. Furthermore, CYP1A1 expression also increased in cells treated with hydrogen peroxide. These results demonstrate that a ligand-independent and oxidative stress dependent pathway activates AhR-signaling in 4-ClBQ treated HaCaT cells. Because AhR signaling is believed to mediate xenobiotics response, our results may provide a mechanistic rationale for the use of antioxidants as effective countermeasure to environmental pollutant-induced adverse health effects. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Ligand-independent activation of Aryl hydrocarbon receptor signaling in PCB3-quinone treated HaCaT human keratinocytes

    PubMed Central

    Xiao, Wusheng; Son, Jyungmean; Vorrink, Sabine U.; Domann, Frederick E.; Goswami, Prabhat C.

    2015-01-01

    Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that plays a critical role in metabolism, cell proliferation, development, carcinogenesis, and xenobiotic response. In general, dioxin-like polychlorinated biphenyls (PCBs) exhibit a ligand-dependent activation of AhR-signaling. Results from this study show that a quinone-derivative (1-(4-Chlorophenyl)-benzo-2,5-quinone; 4-ClBQ) of a non-dioxin like PCB (PCB3) also activates AhR-signaling. Treatments of HaCaT human keratinocytes with 4-ClBQ and dioxin-like PCB126 significantly increased AhR-target gene expression, CYP1A1 mRNA and protein levels. 4-ClBQ-induced increase CYP1A1 expression was associated with an increase in the nuclear translocation of AhR protein as well as an increase in the luciferase-reporter activity of a human CYP1A1 xenobiotic response element (XRE). 6,2′,4′-trimethoxyflavone (TMF), a well-characterized AhR-ligand antagonist significantly suppressed PCB126-induced increase in CYP1A1 expression, while the same treatment did not suppress 4-ClBQ-induced increase in CYP1A1 expression. However, siRNA-mediated down-regulation of AhR significantly inhibited 4-ClBQ-induced increase in CYP1A1 expression, suggesting that AhR mediates 4-ClBQ-induced increase in CYP1A1 expression. Interestingly, treatment with the antioxidant N-acetyl-L-cysteine significantly suppressed 4-ClBQ-induced increase in CYP1A1 expression. Furthermore, CYP1A1 expression also increased in cells treated with hydrogen peroxide. These results demonstrate that a ligand-independent and oxidative stress dependent pathway activates AhR-signaling in 4-ClBQ treated HaCaT cells. Because AhR signaling is believed to mediate xenobiotics response, our results may provide a mechanistic rationale for the use of antioxidants as effective countermeasure to environmental pollutant-induced adverse health effects. PMID:25668756

  5. Sodium arsenite-induced stress-related gene expression in normal human epidermal, HaCaT, and HEL30 keratinocytes.

    PubMed Central

    Trouba, Kevin J; Geisenhoffer, Kristen M; Germolec, Dori R

    2002-01-01

    Arsenic is a carcinogen that poses a significant health risk in humans. Based on evidence that arsenic has differential effects on human, rodent, normal, and transformed cells, these studies addressed the relative merits of using normal human epidermal keratinocytes (NHEK) and immortalized human (HaCaT) and mouse (HEL30) keratinocytes when examining stress-induced gene expression that may contribute to carcinogenesis. We hypothesize that redox-related gene expression is differentially modulated by arsenic in normal versus immortalized keratinocytes. To test the hypothesis, we exposed keratinocytes to sodium arsenite for 4 or 24 hr, at which time serine threonine kinase-25 (stk25) and nicotine adenine dinucleotide phosphate [nad(p)h] quinone oxidoreductase gene expression were measured. The effect of glutathione reduction on arsenite-induced cytotoxicity and gene expression in NHEK also was evaluated by addition of l-buthionine-[S,R]-sulfoximine (BSO) to culture media. Results indicate the term LC(50) for arsenite is approximately 10-15 microM in NHEK and HEL30 keratinocytes and 30 microM in HaCaT keratinocytes. Compared with HaCaT and HEL30 keratinocytes, a nontoxic concentration of arsenite (2.5 microM) increases stk25 and nad(p)h quinone oxidoreductase gene expression in NHEK, an effect partially attenuated by BSO. These data indicate that NHEK and HaCaT/HEL30 keratinocytes have similar sensitivities toward arsenite-induced cytotoxicity but unique gene expression responses. They also suggest that arsenite modulates gene expression in NHEK involved in cellular signaling and other aspects of intermediary metabolism that may contribute to the carcinogenic process. PMID:12426128

  6. Protective effects of Scutellaria baicalensis Georgi against hydrogen peroxide-induced DNA damage and apoptosis in HaCaT human skin keratinocytes.

    PubMed

    Yoon, Jung Jeh; Jeong, Jin-Woo; Choi, Eun Ok; Kim, Min Ju; Hwang-Bo, Hyun; Kim, Hong Jae; Hong, Su Hyun; Park, Cheol; Lee, Dong Hee; Choi, Yung Hyun

    2017-01-01

    Oxidative stress due to excessive accumulation of reactive oxygen species (ROS) is one of the risk factors for the development of several chronic diseases. In this study, we investigated the protective effects of Scutellaria baicalensis rhizome ethanol extract (SBRE) against oxidative stress-induced cellular damage and elucidated the underlying mechanisms in the HaCaT human skin keratinocyte cell line. Our results revealed that treatment with SBRE prior to hydrogen peroxide (H2O2) exposure significantly increased viability of HaCaT cells. SBRE also effectively attenuated H2O2-induced comet tail formation and inhibited the H2O2-induced phosphorylation levels of the histone γH2AX, as well as the number of apoptotic bodies and Annexin V-positive cells. In addition, SBRE exhibited scavenging activity against intracellular ROS generation and restored the mitochondrial membrane potential loss by H2O2. Moreover, H2O2 enhanced the cleavage of caspase-3 and degradation of poly (ADP-ribose)-polymerase, a typical substrate protein of activated caspase-3, as well as DNA fragmentation; however, these events were almost totally reversed by pretreatment with SBRE. Furthermore, SBRE increased the levels of heme oxygenase-1 (HO-1), which is a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). According to our data, SBRE is able to protect HaCaT cells from H2O2-induced DNA damage and apoptosis through blocking cellular damage related to oxidative stress through a mechanism that would affect ROS elimination and activating the Nrf2/HO-1 signaling pathway.

  7. Protective effects of Scutellaria baicalensis Georgi against hydrogen peroxide-induced DNA damage and apoptosis in HaCaT human skin keratinocytes

    PubMed Central

    Yoon, Jung Jeh; Jeong, Jin-Woo; Choi, Eun Ok; Kim, Min Ju; Hwang-Bo, Hyun; Kim, Hong Jae; Hong, Su Hyun; Park, Cheol; Lee, Dong Hee; Choi, Yung Hyun

    2017-01-01

    Oxidative stress due to excessive accumulation of reactive oxygen species (ROS) is one of the risk factors for the development of several chronic diseases. In this study, we investigated the protective effects of Scutellaria baicalensis rhizome ethanol extract (SBRE) against oxidative stress-induced cellular damage and elucidated the underlying mechanisms in the HaCaT human skin keratinocyte cell line. Our results revealed that treatment with SBRE prior to hydrogen peroxide (H2O2) exposure significantly increased viability of HaCaT cells. SBRE also effectively attenuated H2O2-induced comet tail formation and inhibited the H2O2-induced phosphorylation levels of the histone γH2AX, as well as the number of apoptotic bodies and Annexin V-positive cells. In addition, SBRE exhibited scavenging activity against intracellular ROS generation and restored the mitochondrial membrane potential loss by H2O2. Moreover, H2O2 enhanced the cleavage of caspase-3 and degradation of poly (ADP-ribose)-polymerase, a typical substrate protein of activated caspase-3, as well as DNA fragmentation; however, these events were almost totally reversed by pretreatment with SBRE. Furthermore, SBRE increased the levels of heme oxygenase-1 (HO-1), which is a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). According to our data, SBRE is able to protect HaCaT cells from H2O2-induced DNA damage and apoptosis through blocking cellular damage related to oxidative stress through a mechanism that would affect ROS elimination and activating the Nrf2/HO-1 signaling pathway. PMID:28694748

  8. Aberrant Cytokeratin Expression During Arsenic-induced Acquired Malignant Phenotype in Human HaCaT Keratinocytes Consistent with Epidermal Carcinogenesis

    PubMed Central

    Sun, Yang; Pi, Jingbo; Wang, Xueqian; Tokar, Erik J.; Liu, Jie; Waalkes, Michael P.

    2009-01-01

    Inorganic arsenic is a known human skin carcinogen. Chronic arsenic exposure results in various human skin lesions, including hyperkeratosis and squamous cell carcinoma (SCC), both characterized by distorted cytokeratin (CK) production. Prior work shows the human skin keratinocyte HaCaT cell line, when exposed chronically for >25 weeks to a low level of inorganic arsenite (100 nM) results in cells able to produce aggressive SCC upon inoculation into nude mice. In the present study, CK expression analysis was performed in arsenic-exposed HaCaT cells during the progressive acquisition of this malignant phenotype (0 to 20 weeks) to further validate this model as relevant to epidermal carcinogenesis induced by arsenic in humans. Indeed, we observed clear evidence of acquired cancer phenotype by 20 weeks of arsenite exposure including the formation of giant cells, a >4-fold increase in colony formation in soft agar and a ∼2.5-fold increase in matrix metalloproteinase-9 secretion, an enzyme often secreted by cancer cells to help invade through the local extra-cellular matrix. During this acquired malignant phenotype, various CK genes showed markedly altered expression at the transcript and protein levels in a time-dependent manner. For example, CK1, a marker of hyperkeratosis, increased up to 34-fold during arsenic-induced transformation, while CK13, a marker for dermal cancer progression, increased up to 45-fold. The stem cell marker, CK15, increased up to 7-fold, particularly during the later stages of arsenic exposure, indicating a potential emergence of cancer stem-like cells with arsenic-induced acquired malignant phenotype. The expression of involucrin and loricrin, markers for keratinocyte differentiation, increased up to 9-fold. Thus, during arsenic-induced acquired cancer phenotype in human keratinocytes, dramatic and dynamic alterations in CK expression occur which are consistent with the process of epidermal carcinogenesis helping validate this as an

  9. Aberrant cytokeratin expression during arsenic-induced acquired malignant phenotype in human HaCaT keratinocytes consistent with epidermal carcinogenesis.

    PubMed

    Sun, Yang; Pi, Jingbo; Wang, Xueqian; Tokar, Erik J; Liu, Jie; Waalkes, Michael P

    2009-08-03

    Inorganic arsenic is a known human skin carcinogen. Chronic arsenic exposure results in various human skin lesions, including hyperkeratosis and squamous cell carcinoma (SCC), both characterized by distorted cytokeratin (CK) production. Prior work shows the human skin keratinocyte HaCaT cell line, when exposed chronically for >25 weeks to a low level of inorganic arsenite (100nM) results in cells able to produce aggressive SCC upon inoculation into nude mice. In the present study, CK expression analysis was performed in arsenic-exposed HaCaT cells during the progressive acquisition of this malignant phenotype (0-20 weeks) to further validate this model as relevant to epidermal carcinogenesis induced by arsenic in humans. Indeed, we observed clear evidence of acquired cancer phenotype by 20 weeks of arsenite exposure including the formation of giant cells, a >4-fold increase in colony formation in soft agar and a approximately 2.5-fold increase in matrix metalloproteinase-9 secretion, an enzyme often secreted by cancer cells to help invade through the local extra-cellular matrix. During this acquired malignant phenotype, various CK genes showed markedly altered expression at the transcript and protein levels in a time-dependent manner. For example, CK1, a marker of hyperkeratosis, increased up to 34-fold during arsenic-induced transformation, while CK13, a marker for dermal cancer progression, increased up to 45-fold. The stem cell marker, CK15, increased up to 7-fold, particularly during the later stages of arsenic exposure, indicating a potential emergence of cancer stem-like cells with arsenic-induced acquired malignant phenotype. The expression of involucrin and loricrin, markers for keratinocyte differentiation, increased up to 9-fold. Thus, during arsenic-induced acquired cancer phenotype in human keratinocytes, dramatic and dynamic alterations in CK expression occur which are consistent with the process of epidermal carcinogenesis helping validate this as an

  10. Antidiabetics and diuretics show phototoxicity in HaCaT cells

    NASA Astrophysics Data System (ADS)

    Selvaag, Edgar; Petersen, Anita B.; Gniadecki, Robert; Thorn, Tine; Wulf, Hans Christian

    2001-10-01

    The antidiabetics tolbutamide, glibenclamide, and glipizide, and the diuretics bendroflumethiazide, butizide, furosemide, hydrochlorothiazide, and trichlormethiazide were investigated for potential phototoxicity in the HaCaT cell line. The cells were incubated with the drugs and then exposed to UVA1 irradiation. The effects of the antioxidants L-ascorbic acid, and (alpha) -tocopherol on oxidative DNA damage were assessed. Bendroflumethiazide, furosemide, hydrochlorothiazide, trichlormethiazide, or tolbutamide induced dose-dependent phototoxicity. Cells incubated with bendroflumethiazide, tolbutamide, and glibenclamide, and irradiated with UVA1 demonstrated an increased oxidative DNA damage. Pre-treatment with L-ascorbic acid, or (alpha) -tocopherol, suppressed the UVA-induced DNA damage in cells incubated with 1 mM of bendroflumethiazide, furosemide, glibenclamide, glipizide, tolbutamide, and trichloromethiazide, further implying the involvement of reactive oxygen species in the phototoxic DNA damage. These results may indicate a link between phototoxic and photocancerogenic potential of the sulfonamide-derived oral antidiabetic and diuretic drugs, as it has previously been recognized for psoralen, chlorpromazine, and fluoroquinolones. Excessive exposure to UV light may be deleterious for patients treated with these drugs.

  11. Saussurea lappa alleviates inflammatory chemokine production in HaCaT cells and house dust mite-induced atopic-like dermatitis in Nc/Nga mice.

    PubMed

    Lim, Hye-Sun; Ha, Hyekyung; Lee, Mee-Young; Jin, Seong-Eun; Jeong, Soo-Jin; Jeon, Woo-Young; Shin, Na-Ra; Sok, Dai-Eun; Shin, Hyeun-Kyoo

    2014-01-01

    Saussurea lappa is a traditional herbal medicine used for to treat various inflammatory diseases. In this study, we investigated the protective effects of S. lappa against atopic dermatitis using human keratinocyte HaCaT cells, murine mast cell line MC/9 cells, and a house dust mite-induced atopic dermatitis model of Nc/Nga mice. Treatment with the S. lappa caused a significant reduction in the mRNA levels and production of inflammatory chemokines and cytokine, including thymus- and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), regulated on activation, normal T-cell expressed and secreted (RANTES), and interleukin-8 (IL-8) in tumor necrosis factor-α/interferone-γ-stimulated HaCaT cells. S. lappa exhibited the significant reduction in histamine production in MC/9 cells. In the atopic dermatitis model, S. lappa significantly reduced the dermatitis score and serum IgE and TARC levels. In addition, the back skin and ears of S. lappa-treated Nc/Nga mice exhibited reduced histological manifestations of atopic skin lesions such as erosion, hyperplasia of the epidermis and dermis, and inflammatory cell infiltration. In conclusion, an extract of S. lappa effectively suppressed the development of atopic dermatitis, which was closely related to the reduction of chemokines and cytokine. Our study suggests that S. lappa may be a potential treatment for atopic dermatitis.

  12. Nicotinamide enhances repair of arsenic and ultraviolet radiation-induced DNA damage in HaCaT keratinocytes and ex vivo human skin.

    PubMed

    Thompson, Benjamin C; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Arsenic-induced skin cancer is a significant global health burden. In areas with arsenic contamination of water sources, such as China, Pakistan, Myanmar, Cambodia and especially Bangladesh and West Bengal, large populations are at risk of arsenic-induced skin cancer. Arsenic acts as a co-carcinogen with ultraviolet (UV) radiation and affects DNA damage and repair. Nicotinamide (vitamin B3) reduces premalignant keratoses in sun-damaged skin, likely by prevention of UV-induced cellular energy depletion and enhancement of DNA repair. We investigated whether nicotinamide modifies DNA repair following exposure to UV radiation and sodium arsenite. HaCaT keratinocytes and ex vivo human skin were exposed to 2μM sodium arsenite and low dose (2J/cm2) solar-simulated UV, with and without nicotinamide supplementation. DNA photolesions in the form of 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers were detected by immunofluorescence. Arsenic exposure significantly increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine in irradiated cells. Nicotinamide reduced both types of photolesions in HaCaT keratinocytes and in ex vivo human skin, likely by enhancing DNA repair. These results demonstrate a reduction of two different photolesions over time in two different models in UV and arsenic exposed cells. Nicotinamide is a nontoxic, inexpensive agent with potential for chemoprevention of arsenic induced skin cancer.

  13. Nicotinamide Enhances Repair of Arsenic and Ultraviolet Radiation-Induced DNA Damage in HaCaT Keratinocytes and Ex Vivo Human Skin

    PubMed Central

    Thompson, Benjamin C.; Halliday, Gary M.; Damian, Diona L.

    2015-01-01

    Arsenic-induced skin cancer is a significant global health burden. In areas with arsenic contamination of water sources, such as China, Pakistan, Myanmar, Cambodia and especially Bangladesh and West Bengal, large populations are at risk of arsenic-induced skin cancer. Arsenic acts as a co-carcinogen with ultraviolet (UV) radiation and affects DNA damage and repair. Nicotinamide (vitamin B3) reduces premalignant keratoses in sun-damaged skin, likely by prevention of UV-induced cellular energy depletion and enhancement of DNA repair. We investigated whether nicotinamide modifies DNA repair following exposure to UV radiation and sodium arsenite. HaCaT keratinocytes and ex vivo human skin were exposed to 2μM sodium arsenite and low dose (2J/cm2) solar-simulated UV, with and without nicotinamide supplementation. DNA photolesions in the form of 8-oxo-7,8-dihydro-2′-deoxyguanosine and cyclobutane pyrimidine dimers were detected by immunofluorescence. Arsenic exposure significantly increased levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine in irradiated cells. Nicotinamide reduced both types of photolesions in HaCaT keratinocytes and in ex vivo human skin, likely by enhancing DNA repair. These results demonstrate a reduction of two different photolesions over time in two different models in UV and arsenic exposed cells. Nicotinamide is a nontoxic, inexpensive agent with potential for chemoprevention of arsenic induced skin cancer. PMID:25658450

  14. Barium Promotes Anchorage-Independent Growth and Invasion of Human HaCaT Keratinocytes via Activation of c-SRC Kinase

    PubMed Central

    Thang, Nguyen Dinh; Yajima, Ichiro; Kumasaka, Mayuko Y.; Ohnuma, Shoko; Yanagishita, Takeshi; Hayashi, Rumiko; Shekhar, Hossain U.; Watanabe, Daisuke; Kato, Masashi

    2011-01-01

    Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl2) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5–50 µM) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 µM) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5–5 µM) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro. PMID:22022425

  15. Barium promotes anchorage-independent growth and invasion of human HaCaT keratinocytes via activation of c-SRC kinase.

    PubMed

    Thang, Nguyen Dinh; Yajima, Ichiro; Kumasaka, Mayuko Y; Ohnuma, Shoko; Yanagishita, Takeshi; Hayashi, Rumiko; Shekhar, Hossain U; Watanabe, Daisuke; Kato, Masashi

    2011-01-01

    Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl(2)) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5-50 µM) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 µM) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5-5 µM) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro.

  16. Polypeptide from Chlamys farreri restores endoplasmic reticulum (ER) redox homeostasis, suppresses ER stress, and inhibits ER stress-induced apoptosis in ultraviolet B-irradiated HaCaT cells

    PubMed Central

    Xie, Jing; Zhong, Feng; Han, Yantao; Gao, Hui; Wang, Chunbo; Peng, Jianjun

    2015-01-01

    Objective: To investigate the effects of polypeptide from Chlamys farreri (PCF) on ultraviolet B (UVB)-induced apoptosis in human keratinocyte HaCaT cells. Methods: In HaCaT cells at 4 h or 18 h after UVB irradiation, the cell viability was measured by MTT assay. Cellular apoptosis was detected with annexin V-FITC/PI staining by flow cytometry. The expression levels of PDI, Ero-1α, GRP78, and CHOP were assessed by Western blot analysis. Mitochondrial membrane potential (MMP) was measured by fluorescent probe JC-1. Caspase activities were detected with fluorogenic substrates. Results: PCF alleviated cell viability loss and inhibited apoptosis in HaCaT cells after UVB irradiation. Moreover, PCF increased the expression levels of PDI and Ero-1α, which were related with the ER redox homeostasis. Furthermore, PCF treatment inhibited the expression of GRP78 at 4 h after UVB irradiation, and suppressed CHOP expression at 18 h post-irradiation, indicating that PCF could inhibit UVB-evoked ER stress in the early stage post-irradiation, and suppress the ER stress-induced apoptosis in the late stage. In addition, PCF alleviated UVB-induced MMP loss, and inhibited the activation of caspase-9/-3, in HaCaT cells after UVB irradiation. On the other hand, MMP loss and caspase-9/-3 activation could be partly blocked by the ER stress inhibitor 4-PBA. Conclusions: PCF inhibits UVB-induced apoptosis through restoring ER redox homeostasis, suppressing ER stress, and inhibiting ER stress-induced mitochondrial apoptosis in HaCaT cells. These findings provide evidence for the mechanism underlying UVB-induced skin damages, and support the promising role of PCF in treatment of the diseases. PMID:26175857

  17. Induction of apoptosis in HaCaT cells by photodynamic therapy with chlorin e6 or pheophorbide a.

    PubMed

    Radestock, A; Elsner, P; Gitter, B; Hipler, U-C

    2007-01-01

    The two photosensitizers, chlorin e6 and pheophorbide a, were tested in an in vitro model of topical photodynamic therapy (PDT). Both dyes accumulate in HaCaT keratinocytes as verified by fluorescence measurement but pheophorbide a is enriched fivefold more strongly than chlorin e6 after 24 h. HaCaT cells are susceptible to PDT with both dyes. The phototoxicity measured by ATP bioluminescence is caused by necrosis and apoptosis depending on the photosensitizer used and the treatment modality. Chlorin e6 shows higher toxic potential because it elicits nearly 90% cell mortality 24 h after PDT comparable to pheophorbide a but with a fivefold lower rate of accumulation. These results implicate caution with topical PDT of oncologic diseases due to the risk of serious side effects on healthy skin in the course of topical photodynamic treatment. But the lack of dark toxicity and the time-dependent enrichment of both dyes in HaCaT cells are arguments for the application of these sensitizers in topical PDT of non-malign skin disorders. Further studies are necessary to discover appropriate lower doses and mechanisms of action of topical PDT with both compounds.

  18. Protective Effect of Inositol Hexaphosphate Against UVB Damage in HaCaT Cells and Skin Carcinogenesis in SKH1 Hairless Mice

    PubMed Central

    Williams, Kendra A; Kolappaswamy, Krishnan; DeTolla, Louis J; Vucenik, Ivana

    2011-01-01

    UVB radiation damages keratinocytes, potentially inducing chronic skin damage, cutaneous malignancy, and suppression of the immune system. Naturally occurring agents have been considered for prevention and treatment of various kinds of cancer, including skin cancer. Inositol hexaphosphate (IP6), an antioxidant, is a naturally occurring polyphosphorylated carbohydrate that has shown a strong anticancer activity in several experimental models. We assessed the protective effects of IP6 against UVB irradiation-induced injury and photocarcinogenesis by using HaCaT cells (human immortalized keratinocytes) and SKH1 hairless mice. We found that IP6 counteracts the harmful effects of UVB irradiation and increases the viability and survival of UVB-exposed cells. Treatment with IP6 after UVB irradiation (30 mJ/cm2) arrested cells in the G1 and G2M phases while decreasing the S phase of the cell cycle. Treatment with IP6 also decreased UVB-induced apoptosis and caspase 3 activation. Topical application of IP6 followed by exposure to UVB irradiation in SKH1 hairless mice decreased tumor incidence and multiplicity as compared with control mice. Our results suggest that IP6 protects HaCaT cells from UVB-induced apoptosis and mice from UVB-induced tumors. PMID:21819680

  19. Protective effect of inositol hexaphosphate against UVB damage in HaCaT cells and skin carcinogenesis in SKH1 hairless mice.

    PubMed

    Williams, Kendra A; Kolappaswamy, Krishnan; Detolla, Louis J; Vucenik, Ivana

    2011-02-01

    UVB radiation damages keratinocytes, potentially inducing chronic skin damage, cutaneous malignancy, and suppression of the immune system. Naturally occurring agents have been considered for prevention and treatment of various kinds of cancer, including skin cancer. Inositol hexaphosphate (IP6), an antioxidant, is a naturally occurring polyphosphorylated carbohydrate that has shown a strong anticancer activity in several experimental models. We assessed the protective effects of IP6 against UVB irradiationinduced injury and photocarcinogenesis by using HaCaT cells (human immortalized keratinocytes) and SKH1 hairless mice. We found that IP6 counteracts the harmful effects of UVB irradiation and increases the viability and survival of UVB-exposed cells. Treatment with IP6 after UVB irradiation (30 mJ/cm(2)) arrested cells in the G(1) and G(2) M phases while decreasing the S phase of the cell cycle. Treatment with IP6 also decreased UVB-induced apoptosis and caspase 3 activation. Topical application of IP6 followed by exposure to UVB irradiation in SKH1 hairless mice decreased tumor incidence and multiplicity as compared with control mice. Our results suggest that IP6 protects HaCaT cells from UVB-induced apoptosis and mice from UVB-induced tumors.

  20. Effects of macelignan isolated from Myristica fragrans Houtt. on UVB-induced matrix metalloproteinase-9 and cyclooxygenase-2 in HaCaT cells.

    PubMed

    Anggakusuma; Yanti; Hwang, Jae-Kwan

    2010-02-01

    UVB irradiation (290-320 nm) is the most damaging component of the UV spectrum and causes both direct and indirect damage to the basal cell layer of the epidermis; this results in the activation of a number of signaling pathways involved in pathophysiological processes in the skin, such as photoaging and inflammation. In photoaging UVB irradiation promotes degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and, in inflammation, UVB irradiation promotes the expression of inducible cyclooxygenase (COX-2), leading to overproduction of inflammatory mediators. We first investigated the protective effects of macelignan from Myristica fragrans Houtt. on immortalized human keratinocytes (HaCaT) against UVB damage. We then explored the inhibitory effects of macelignan on UVB-induced MMP-9 and COX-2 and investigated the molecular mechanism underlying those effects. HaCaT cells were treated with macelignan for the indicated times followed by irradiation with UVB. Secretion of MMP-9 was measured by gelatin zymography. Expression of COX-2, mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase/Akt (PI3K/Akt), c-Fos, c-Jun, and CREB were assayed by western analysis. Macelignan at a concentration of 0.1-1 microM increased the viability of HaCaT cells following UVB irradiation and inhibited MMP-9 secretion and COX-2 expression in a concentration-dependent manner. An inhibitory effect was also seen in the signal transduction network, where macelignan treatment reduced the activation of UVB-induced MAPKs, PI3K/Akt, and their downstream transcription factors. These results suggest that macelignan protects skin keratinocytes from UVB-induced damage and inhibits MMP-9 and COX-2 expression by attenuating the activation of MAPKs and PI3K/Akt. Copyright 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  1. Polypeptide from Chlamys farreri suppresses ultraviolet-B irradiation-induced apoptosis through restoring ER redox homeostasis, scavenging ROS generation, and suppressing the PERK-eIF2a-CHOP pathway in HaCaT cells.

    PubMed

    Zhong, Feng; Xie, Jing; Zhang, Di; Han, Yantao; Wang, Chunbo

    2015-10-01

    The aim of this study was to investigate the effect of polypeptide from Chlamys farreri (PCF) on ultraviolet B (UVB) irradiation-induced apoptosis in human keratinocyte HaCaT cells. HaCaT cells were treated with 20 mJ/cm(2) UVB irradiation for 18 h. The cell viability was measured by MTT assay, and apoptosis was detected with Hoechst 33258 staining and caspase-3 activity detection. Protein expression levels were assessed by Western blot analysis, and the intracellular ROS levels were also measured. Our results from the MTT assay showed that UVB irradiation significantly declined the viability of HaCaT cells, which could be restored by PCF treatment. PCF decreased the apoptosis rate in HaCaT cells treated with UVB irradiation. Moreover, PCF increased the expression levels of PDI and Ero-1a, and scavenged the intracellular ROS. Furthermore, PCF inhibited the expressions of GRP78, p-PERK, p-eIF2a, and CHOP, and suppressed the ER stress-induced apoptosis, in UVB-irradiated HaCaT cells. In addition, the ROS scavenging effect of 4-PBA was less potent than PCF, indicating that ER stress-related ROS production contribute partially to the total ROS level, and ER was not the only target of PCF treatment. Our results indicate that PCF inhibits UVB irradiation-induced apoptosis through restoring ER redox homeostasis and suppressing the PERK-eIF2a-CHOP pathway. These findings provide evidence for the application of PCF in the protection of skin from UV irradiation. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Atmospheric pressure plasma jet treatment evokes transient oxidative stress in HaCaT keratinocytes and influences cell physiology.

    PubMed

    Wende, Kristian; Straßenburg, Susanne; Haertel, Beate; Harms, Manuela; Holtz, Sarah; Barton, Annemarie; Masur, Kai; von Woedtke, Thomas; Lindequist, Ulrike

    2014-04-01

    Modern non-thermal atmospheric pressure plasma sources enable controllable interaction with biological systems. Their future applications - e.g. wound management - are based on their unique mixture of reactive components sparking both stimulatory as well as inhibitory processes. To gain detailed understanding of plasma-cell interaction and with respect to risk awareness, key mechanisms need to be identified. This study focuses on the impact of an argon non-thermal atmospheric pressure plasma jet (kINPen 09) on human HaCaT keratinocytes. With increasing duration, cell viability decreased. In accordance, cells accumulated in G2/M phase within the following 24 h. DNA single-strand breaks were detected immediately after treatment and receded in the aftermath, returning to control levels after 24 h. No directly plasma-related DNA double-strand breaks were detected over the same time. Concurrently, DNA synthesis decreased. Coincident with treatment time, an increase in intracellular 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) conversion increased reactive oxygen species (ROS) levels. The radical scavenging activity of culture medium crucially influenced these effects. Thus, ROS changed DNA integrity, and the effectiveness of cellular defence mechanisms characterises the interaction of non-thermal plasma and eukaryotic cells. Effects were time-dependent, indicating an active response of the eukaryotic cells. Hence, a stimulation of eukaryotic cells using short-term non-thermal plasma treatment seems possible, eg in the context of chronic wound care. Long-term plasma treatments stopped in cell proliferation and apoptosis, which might be relevant in controlling neoplastic conditions.

  3. (+)-Nootkatone inhibits tumor necrosis factor α/interferon γ-induced production of chemokines in HaCaT cells

    SciTech Connect

    Choi, Hyeon-Jae; Lee, Jin-Hwee; Jung, Yi-Sook

    2014-05-02

    Highlights: • (+)-Nootkatone inhibits TNF-α/IFN-γ-induced TARC and MDC expression in HaCaT cells. • PKCζ, p38 MAPK, or NF-κB mediate TNF-α/IFN-γ-induced TARC and MDC expression. • (+)-Nootkatone inhibits TNF-α/IFN-γ-induced activation of PKCζ, p38 MAPK, or NF-κB. • (+)-Nootkatone suppresses chemokine expression by inhibiting of PKCζ and p38 pathways. - Abstract: Chemokines are important mediators of cell migration, and thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are well-known typical inflammatory chemokines involved in atopic dermatitis (AD). (+)-Nootkatone is the major component of Cyperus rotundus. (+)-Nootkatone has antiallergic, anti-inflammatory, and antiplatelet activities. The purpose of this study was to investigate the effect of (+)-nootkatone on tumor necrosis factor α (TNF-α)/interferon γ (IFN-γ)-induced expression of Th2 chemokines in HaCaT cells. We found that (+)-nootkatone inhibited the TNF-α/IFN-γ-induced expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells. It also significantly inhibited TNF-α/IFN-γ-induced activation of nuclear factor kappa B (NF-κB), p38 mitogen-activated protein kinase (MAPK), and protein kinase Cζ (PKCζ). Furthermore, we showed that PKCζ and p38 MAPK contributed to the inhibition of TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression by blocking IκBα degradation in HaCaT cells. Taken together, these results suggest that (+)-nootkatone may suppress TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression in HaCaT cells by inhibiting of PKCζ and p38 MAPK signaling pathways that lead to activation of NF-κB. We propose that (+)-nootkatone may be a useful therapeutic candidate for inflammatory skin diseases such as AD.

  4. Microenvironment Induced Spheroid to Sheeting Transition of Immortalized Human Keratinocytes (HaCaT) Cultured in Microbubbles Formed in Polydimethylsiloxane

    PubMed Central

    Chandrasekaran, Siddarth; Giang, Ut-Binh; King, Michael R.; DeLouise, Lisa A

    2011-01-01

    The in vivo cellular microenvironment is regulated by a complex interplay of soluble factors and signaling molecules secreted by cells and it plays a critical role in the growth and development of normal and diseased tissues. In vitro systems that can recapitulate the microenvironment at the cellular level are needed to investigate the influence of autocrine signaling and extracellular matrix effects on tissue homeostasis, regeneration, and disease development and progression. In this study we report the use of microbubble technology as a means to culture cells in a controlled microenvironment in which cells can influence their function through autocrine signaling. Microbubbles (MB) are small spherical cavities about 100–300 µm in diameter formed in hydrophobic polymer polydimethylsiloxane (PDMS) with ~60–100 µm circular openings and aspect ratio ~3.5. We demonstrate that the unique architecture of the microbubble compartment is advantaged for cell culture using HaCaT cells, an immortalized keratinocyte cell line. We observe that HaCaT cells, seeded in microbubbles (15–20 cells / MB) and cultured under standard conditions, adopt a compact 3-D spheroidal morphology. Within 2–3 days, the cells transition to a sheeting morphology. Through experimentation and simulation we show that this transition in morphology is due to the unique architecture of the microbubble compartment which enables cells to condition their local microenvironment. The small media volume per cell and the development of shallow concentration gradients allow factors secreted by the cells to rise to bioactive levels. The kinetics of the morphology transition depends on the number of cells seeded per microbubble; higher cell seeding induces a more rapid transition. HaCaT cells seeded onto PDMS cured in 96-well plates also form compact spheroids but they do not transition to a sheeting morphology even after several weeks of culture. The importance of soluble factor accumulation in driving

  5. Increased 5-hydroxymethylcytosine and Ten-eleven Translocation Protein Expression in Ultraviolet B-irradiated HaCaT Cells

    PubMed Central

    Wang, Dan; Huang, Jin-Hua; Zeng, Qing-Hai; Gu, Can; Ding, Shu; Lu, Jian-Yun; Chen, Jing; Yang, Sheng-Bo

    2017-01-01

    Background: DNA hydroxymethylation refers to a chemical modification process in which 5-methylcytosine (5mC) is catalyzed to 5- hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) family proteins. Recent studies have revealed that aberrant TETs expression or 5hmC level may play important roles in the occurrence and development of various pathological and physiological processes including cancer and aging. This study aimed to explore the relation between aberrant DNA hydroxymethylation with skin photoaging and to investigate the levels of TETs, 5mC, and 5hmC expression 24 h after 40 mJ/cm2 and 80 mJ/cm2 doses of ultraviolet B (UVB) irradiation to HaCaT cells. Methods: To explore whether aberrant DNA hydroxymethylation is also related to skin photoaging, 40 mJ/cm2 and 80 mJ/cm2 doses of UVB were chosen to treat keratinocytes (HaCaT cells). After 24 h of UVB irradiation, 5mC and 5hmC levels were determined by immunohistochemistry (IHC) and immunofluorescence (IF), and at the same time, the expression levels of matrix metalloproteinase 1 (MMP-1) and TETs were assessed by reverse transcription-polymerase chain reaction or Western blot analysis. Results: After 40 mJ/cm2 and 80 mJ/cm2 doses of UVB exposure, both IHC and IF results showed that 5hmC levels increased significantly, while the 5mC levels did not exhibit significant changes in HaCaT cells, compared with HaCat cells without UVB exposure. Moreover, compared with HaCat cells without UVB exposure, the levels of TET1, TET2, and TET3 mRNA and protein expression were significantly upregulated (mRNA: P = 0.0022 and 0.0043 for TET1; all P < 0.0001 for TET2; all P = 0.0006 for TET3; protein: P = 0.0012 and 0.0006 for TET1; all P = 0.0022 for TET2; and all P = 0.0002 for TET3), and the levels of MMP-1 mRNA expression increased dose dependently in 40 mJ/cm2 and 80 mJ/cm2 UVB-irradiated groups. Conclusion: UVB radiation could cause increased 5hmC and TET expression, which might become a novel biomarker in UVB

  6. Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes

    SciTech Connect

    Seo, MiRan; Juhnn, Yong-Sung

    2010-03-05

    Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the {alpha} subunit of Gq protein (G{alpha}q) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active G{alpha}q (G{alpha}qQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of G{alpha}q with shRNA in HaCaT human keratinocytes. G{alpha}q was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipase C (PLC), protein kinase C{delta} (PKC{delta}), and matrix metaloprotease-2 (MMP-2). Moreover, G{alpha}qQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that G{alpha}q mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKC{delta} and MMP-2 in HaCaT cells.

  7. Hesperidin ameliorates UV radiation-induced skin damage by abrogation of oxidative stress and inflammatory in HaCaT cells.

    PubMed

    Li, Min; Lin, Xiang-Fei; Lu, Jie; Zhou, Bing-Rong; Luo, Dan

    2016-12-01

    Ultraviolet A (UVA) radiation contributes to skin photoaging. Hesperidin which is a flavanone glycoside found in citrus fruit peels, have been intensively studied for their UVA-protective activity, but its effects and mechanisms on UVA irradiation-induced inflammation and oxidative stress have never been described. Thus, the purpose of this study was to evaluate the effects of hesperidin in skin oxidative stress and inflammation induced by UVA irradiation. In this study, we firstly examined whether hesperidin may exert direct protective effects on the UVA-induced in human keratinocytes (HaCaT) cell injury in vitro. Cell viability was determined by MTT assay. The levels of superoxide dismutase (SOD), malondialdehyde (MDA) and total antioxidative capacity (T-AOC) were measured by using a commercially available kits. Quantitative reverse transcriptase PCR (qRT-PCR) and ELISA were used to determine messenger RNA (mRNA) and protein levels of the tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6. UVA significantly decreased the cell viability (P<0.05). In our study, hesperidin (220μg/ml) significantly reduced UVA-induced oxidative stress and inflammatory response. In conclusion, hesperidin treatment effectively protected HaCaT keratinocytes from these UVA radiation-induced skin injuries, suggesting that the underlying mechanism involves the anti-oxidative and anti-inflammatory capacities, it is possible to be used as a sunscreen agent. Copyright © 2016. Published by Elsevier B.V.

  8. INDUCTION OF CELL PROLIFERATION AND APOPTOSIS IN HL60 AND HACAT CELLS BY ARSENIC, ARSENATE, AND ARSENIC-CONTAMINATED DRINKING WATER

    EPA Science Inventory

    Induction of cell proliferation and apoptosis in HL-60 and HaCaT cells by arsenite, arsenate and arsenic-contaminated drinking water. T-C. Zhang, M. Schmitt, J. L. Mumford National Research Council, Washington DC and U.S. Environmental Protection Agency, NHEERL, Research Triangle...

  9. INDUCTION OF CELL PROLIFERATION AND APOPTOSIS IN HL60 AND HACAT CELLS BY ARSENIC, ARSENATE, AND ARSENIC-CONTAMINATED DRINKING WATER

    EPA Science Inventory

    Induction of cell proliferation and apoptosis in HL-60 and HaCaT cells by arsenite, arsenate and arsenic-contaminated drinking water. T-C. Zhang, M. Schmitt, J. L. Mumford National Research Council, Washington DC and U.S. Environmental Protection Agency, NHEERL, Research Triangle...

  10. Carbon Nanotubes and Human Cells?

    ERIC Educational Resources Information Center

    King, G. Angela

    2005-01-01

    Single-walled carbon nanotubes that were chemically altered to be water soluble are shown to enter fibroblasts, T cells, and HL60 cells. Nanoparticles adversely affect immortalized HaCaT human keratinocyte cultures, indicating that they may enter cells.

  11. Carbon Nanotubes and Human Cells?

    ERIC Educational Resources Information Center

    King, G. Angela

    2005-01-01

    Single-walled carbon nanotubes that were chemically altered to be water soluble are shown to enter fibroblasts, T cells, and HL60 cells. Nanoparticles adversely affect immortalized HaCaT human keratinocyte cultures, indicating that they may enter cells.

  12. The Protecting Effect of Deoxyschisandrin and Schisandrin B on HaCaT Cells against UVB-Induced Damage

    PubMed Central

    Hou, Wei; Gao, Wei; Wang, Datao; Liu, Qingxiu; Zheng, Siwen; Wang, Yingping

    2015-01-01

    Schisandra chinensis is a traditional Chinese medicine that has multiple biological activities, including antioxidant, anticancer, tonic, and anti-aging effects. Deoxyschisandrin (SA) and schisandrin B (SB), the two major lignans isolated from S. chinensis, exert high antioxidant activities in vitro and in vivo by scavenging free radicals, such as reactive oxygen species (ROS). Ultraviolet B-ray (UVB) radiation induces the production of ROS and DNA damage, which eventually leads to cell death by apoptosis. However, it is unknown whether SA or SB protects cells against UVB-induced cellular DNA damage. Our study showed that both SA and SB effectively protected HaCaT cells from UVB-induced cell death by antagonizing UVB-mediated production of ROS and induction of DNA damage. Our results showed that both SA and SB significantly prevented UVB-induced loss of cell viability using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assays showed that the production of ROS following UVB exposure was inhibited by treatment with SA and SB. Moreover, SA and SB decreased the UVB-induced DNA damage in HaCaT cells by comet assays. In addition, SA and SB also prevented UVB-induced cell apoptosis and the cleavage of caspase-3, caspase-8 and caspase-9. In a word, our results imply that the antioxidants SA and SB could protect cells from UVB-induced cell damage via scavenging ROS. PMID:25978330

  13. Down-regulation of peroxisome proliferator activated receptor γ coactivator 1α induces oxidative stress and toxicity of 1-(4-Chlorophenyl)-benzo-2,5-quinone in HaCaT human keratinocytes

    PubMed Central

    Xiao, Wusheng; Goswami, Prabhat C.

    2015-01-01

    Peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) is a transcriptional coactivator that is known to regulate oxidative stress response by enhancing the expression of antioxidant genes. We have shown previously that 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone-metabolite of 4-monochlorobiphenyl (PCB3) induces oxidative stress and toxicity in human skin keratinocytes, and breast and prostate epithelial cells. In this study, we investigate whether PGC-1α regulates oxidative stress and toxicity in 4-ClBQ treated HaCaT human keratinocytes. Results showed significant down-regulation in the expression of PGC-1α and catalase in 4-ClBQ treated HaCaT cells. Down-regulation of PGC-1α expression was associated with 4-ClBQ induced increase in the steady-state levels of cellular reactive oxygen species (ROS) and toxicity. Overexpression of pgc-1α enhanced the expression of catalase and suppressed 4-ClBQ induced increase in cellular ROS levels and toxicity. These results suggest that pgc-1α mediates 4-ClBQ induced oxidative stress and toxicity in HaCaT cells presumably by regulating catalase expression. PMID:26004620

  14. A novel method to generate monocyte-derived dendritic cells during coculture with HaCaT facilitates detection of weak contact allergens in cosmetics.

    PubMed

    Frombach, Janna; Sonnenburg, Anna; Krapohl, Björn-Dirk; Zuberbier, Torsten; Stahlmann, Ralf; Schreiner, Maximilian

    2017-01-01

    The in vitro sensitization assay LCSA (Loose-fit Coculture-based Sensitization Assay) has proved reliable for the detection of contact sensitizers in the past. However, the coculture of human monocyte-derived dendritic cells (DCs) with primary human keratinocytes (KCs) in serum-free medium is relatively complex compared to other sensitization assays which use continuous cell lines. To facilitate high-throughput screening of chemicals, we replaced KCs with the HaCaT cell line under various culture conditions. Coculture of HaCaT with peripheral blood mononuclear cells in serum-supplemented medium leads to generation of CD1a(+)/CD1c(+) DCs after addition of GM-CSF, IL-4, and TGF-β1 (as opposed to CD1a(-)/CD1c(-) DCs which arise in the "classic" LCSA coculture). These cells resemble monocyte-derived DCs generated in monoculture, but, unlike those, they show a marked upregulation CD86 after treatment with contact allergens. All of the nine sensitizers in this study were correctly identified by CD1a(+)/CD1c(+) DCs in coculture with HaCaT. Among the substances were weak contact allergens such as propylparaben (which is false negative in the local lymph node assay in mice) and resorcinol (which was not detected by CD1a(-)/CD1c(-) DCs in the "classic" LCSA). The level of CD86 upregulation on CD1a(+)/CD1c(+) DCs was higher for most allergens compared to CD1a(-)/CD1c(-) DCs, thus improving the assay's discriminatory power. Three out of four non-sensitizers were also correctly assessed by the coculture assay. A false-positive reaction to caprylic (octanoic) acid confirms earlier results that some fatty acids are able to induce CD86 on DC in vitro. In conclusion, change of the LCSA protocol led to reduction of time and cost while even increasing the assay's sensitivity and discriminatory power.

  15. Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells

    PubMed Central

    Lee, Su Jeong; Park, Jeen-Woo

    2014-01-01

    Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells. [BMB Reports 2014; 47(4): 209-214] PMID:24286310

  16. Effects of Ginger Phenylpropanoids and Quercetin on Nrf2-ARE Pathway in Human BJ Fibroblasts and HaCaT Keratinocytes

    PubMed Central

    Schadich, Ermin; Hlaváč, Jan; Volná, Tereza; Varanasi, Lakshman; Hajdúch, Marián; Džubák, Petr

    2016-01-01

    Quercetin and phenylpropanoids are well known chemoprotective compounds identified in many plants. This study was aimed at determining their effects on activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response element (Nrf2-ARE) signalling pathway and expression of its important downstream effector phase II detoxification enzyme glutathione-S-transferase P1 (GSTP1) in BJ foreskin fibroblasts and skin HaCaT keratinocytes. Cell lines and their corresponding Nrf2-ARE luciferase reporter cells were treated by ginger phenylpropanoids and quercetin for 10 h and the level of Nrf2 activity was subsequently determined. Both, ginger phenylpropanoids and quercetin, significantly increased the level of Nrf2 activity. Subsequent western blot analyses of proteins showed the increased expression level of glutathione-S-transferase P1 (GSTP1) in BJ cells but not in HaCaT cells. Such phenomenon of unresponsive downstream target expression in HaCaT cells was consistent with previous studies showing a constitutive expression of their GSTP1. Thus, while both ginger phenylpropanoids and quercetin have the property of increasing the level of Nrf2 both in HaCaT and in BJ cells, their effects on its downstream signalling were mediated only in BJ cells. PMID:26942188

  17. Effects of Ginger Phenylpropanoids and Quercetin on Nrf2-ARE Pathway in Human BJ Fibroblasts and HaCaT Keratinocytes.

    PubMed

    Schadich, Ermin; Hlaváč, Jan; Volná, Tereza; Varanasi, Lakshman; Hajdúch, Marián; Džubák, Petr

    2016-01-01

    Quercetin and phenylpropanoids are well known chemoprotective compounds identified in many plants. This study was aimed at determining their effects on activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response element (Nrf2-ARE) signalling pathway and expression of its important downstream effector phase II detoxification enzyme glutathione-S-transferase P1 (GSTP1) in BJ foreskin fibroblasts and skin HaCaT keratinocytes. Cell lines and their corresponding Nrf2-ARE luciferase reporter cells were treated by ginger phenylpropanoids and quercetin for 10 h and the level of Nrf2 activity was subsequently determined. Both, ginger phenylpropanoids and quercetin, significantly increased the level of Nrf2 activity. Subsequent western blot analyses of proteins showed the increased expression level of glutathione-S-transferase P1 (GSTP1) in BJ cells but not in HaCaT cells. Such phenomenon of unresponsive downstream target expression in HaCaT cells was consistent with previous studies showing a constitutive expression of their GSTP1. Thus, while both ginger phenylpropanoids and quercetin have the property of increasing the level of Nrf2 both in HaCaT and in BJ cells, their effects on its downstream signalling were mediated only in BJ cells.

  18. The effect of a phytosphingosine-like substance isolated from Asterina pectinifera on involucrin expression in mite antigen-stimulated HaCaT cells.

    PubMed

    Choi, Gui Hyang; Wahid, Fazli; Kim, You Young

    2010-07-01

    The aim of the present study was to investigate the effect of phytosphingosine (PS) on mite antigen-induced terminal differentiation abnormalities in HaCaT cells. For this purpose, a PS-like substance was isolated from Asterina pectinifera (starfish PS) using high-performance liquid chromatography and was partially characterized through 1H NMR analysis. The level of involucrin expression in HaCaT cell was measured by immunoblotting assay. Our results showed that PS treatments remarkably up-regulated the involucrin expression, which is known as a terminal differentiation marker in the epidermal mite antigen-treated HaCaT cells. This indicates that starfish PS could regulate mite antigen-induced terminal differentiation fluctuation in the epidermis. Taken together, the results suggest that starfish PS might be a useful therapeutic agent for atopic dermatitis.

  19. Rhodomyrtone as a potential anti-proliferative and apoptosis inducing agent in HaCaT keratinocyte cells.

    PubMed

    Chorachoo, Julalak; Saeloh, Dennapa; Srichana, Teerapol; Amnuaikit, Thanaporn; Musthafa, Khadar Syed; Sretrirutchai, Somporn; Voravuthikunchai, Supayang P

    2016-02-05

    Psoriasis is a skin disease associated with hyperproliferation and abnormal differentiation of keratinocytes. Available approaches using synthetic drugs for the treatment of severe psoriasis may cause side effects. Alternatively, plant-derived compounds are now receiving much attention as alternative candidates for the treatment of psoriasis. In this study, the effects of rhodomyrtone, a bioactive plant extract isolated from Rhodomyrtus tomentosa leaves on the proliferation, growth arrest, and apoptosis of HaCaT keratinocytes were investigated. Percentage anti-proliferative activity of rhodomyrtone on HaCaT cells at concentrations of 2-32µg/ml after 24, 48, and 72h ranged from 13.62-61.61%, 50.59-80.16%, and 61.82-85.34%, respectively. In a scratch assay, rhodomyrtone at 2 and 4µg/ml significantly delayed closure of a wound by up to 61.78%, and 71.65%, respectively, after 24h incubation. HaCaT keratinocytes treated with rhodomyrtone showed chromatin condensation and fragmentation of nuclei when stained with Hoechst 33342. This indicated that rhodomyrtone induced apoptosis in the keratinocytes. In addition, flow cytometric analysis demonstrated an increase in the percentage of apoptosis of keratinocytes after treatment with rhodomyrtone at 2-32µg/ml from 1.2-10%, 8.2-35.4%, and 21.0-77.8% after 24, 48, and 72h, respectively, compared with the control. To further develop the compound as a potential anti-psoriasis agent, a rhodomyrtone formulation was prepared and subjected to skin irritation tests in rabbits. The formulation caused no skin irritation including such as erythema and edema. The results indicated that rhodomyrtone had the potential as a promising candidate for further development as a natural anti-psoriasis agent.

  20. Wild carrot pentane-based fractions suppress proliferation of human HaCaT keratinocytes and protect against chemically-induced skin cancer.

    PubMed

    Shebaby, Wassim N; Mroueh, Mohamad A; Boukamp, Petra; Taleb, Robin I; Bodman-Smith, Kikki; El-Sibai, Mirvat; Daher, Costantine F

    2017-01-10

    Previous studies in our laboratory showed that the Lebanese Daucus carota ssp. carota (wild carrot) oil extract possesses in vitro and in vivo anticancer activities. The present study aims to examine the cytotoxic effect of Daucus carota oil fractions on human epidermal keratinocytes and evaluate the chemopreventive activity of the pentane diethyl ether fraction on DMBA/TPA induced skin carcinogenesis in mice. Wild carrot oil extract was chromatographed to yield four fractions (F1, 100% pentane; F2, 50:50 pentane:diethyl ether; F3, 100% diethyl ether; F4 93:7 chloroform:methanol). The cytotoxic effect of fractions (10, 25, 50 and 100 μg/mL) was tested on human epidermal keratinocytes (non-tumorigenic HaCaT cells and tumorigenic HaCaT-ras variants) using WST a ssay. Cell cycle phase distribution of tumorigenic HaCaT-ras variants was determined by flow cytometry post-treatment with F2 fraction. Apoptosis related proteins were also assessed using western blot. The antitumor activity of F2 fraction was also evaluated using a DMBA/TPA induced skin carcinoma in Balb/c mice. All fractions exhibited significant cytotoxicity, with HaCaT cells being 2.4-3 times less sensitive than HaCaT-ras A5 (benign tumorigenic), and HaCaT-ras II4 (malignant) cells. GC-MS analysis revealed the presence of a major compound (around 60%) in the pentane/diethylether fraction (F2), identified as 2-himachalen-6-ol. Treatment of HaCaT-ras A5 and HaCaT-ras II4 cells with F2 fraction resulted in the accumulation of cells in the sub-G1 apoptotic phase and decreased the population of cells in the S and G2/M phases. Additionally, F2 fraction treatment caused an up-regulation of the expression of pro-apoptotic (Bax) and down-regulation of the expression of anti-apoptotic (Bcl2) proteins. A decrease in the phosphorylation of AKT and ERK was also observed. Intraperitoneal treatment with F2 fraction (50 or 200 mg/kg) in the DMBA/TPA skin carcinogenesis mouse model showed a significant inhibition of

  1. Research Note Mesenchymal stem cells from skin lesions of psoriasis patients promote proliferation and inhibit apoptosis of HaCaT cells.

    PubMed

    Liu, R F; Wang, F; Wang, Q; Zhao, X C; Zhang, K M

    2015-12-22

    Psoriasis is an inflammatory skin disease characterized by excessive proliferation and abnormal differentiation and apoptosis of keratinocytes (KCs). Mesenchymal stem cells (MSCs) from skin lesions of psoriasis patients demonstrate abnormal cytokine secretion, which may affect KC proliferation and apoptosis. Here, we explored how MSCs from skin lesions of psoriasis patients affect HaCaT cell proliferation and apoptosis. First, flow cytometry and multipotent differentiation methods were used to identify skin MSCs, which were then co-cultured with HaCaT cells. HaCaT cell proliferation was analyzed in real-time, and cell cycle progression and apoptosis were assessed by flow cytometry. Cell morphologies and multipotencies of skin MSCs were similar between the psoriasis group and healthy control group, with high levels of CD29, CD44, CD73, CD90, and CD105 and limited expression of CD34, CD45, and HLA-DR. MSCs from skin lesions of psoriasis patients promote KC proliferation more potently and are less capable of inducing KC apoptosis. This may underlie KC proliferation and abnormal apoptosis in psoriasis skin lesions, which results in abnormal thickening of the epidermis.

  2. Protective Effect of Diphlorethohydroxycarmalol against Ultraviolet B Radiation-Induced DNA Damage by Inducing the Nucleotide Excision Repair System in HaCaT Human Keratinocytes

    PubMed Central

    Piao, Mei Jing; Madduma Hewage, Susara Ruwan Kumara; Han, Xia; Kang, Kyoung Ah; Kang, Hee Kyoung; Lee, Nam Ho; Hyun, Jin Won

    2015-01-01

    We investigated the protective properties of diphlorethohydroxycarmalol (DPHC), a phlorotannin, against ultraviolet B (UVB) radiation-induced cyclobutane pyrimidine dimers (CPDs) in HaCaT human keratinocytes. The nucleotide excision repair (NER) system is the pathway by which cells identify and repair bulky, helix-distorting DNA lesions such as ultraviolet (UV) radiation-induced CPDs and 6-4 photoproducts. CPDs levels were elevated in UVB-exposed cells; however, this increase was reduced by DPHC. Expression levels of xeroderma pigmentosum complementation group C (XPC) and excision repair cross-complementing 1 (ERCC1), which are essential components of the NER pathway, were induced in DPHC-treated cells. Expression of XPC and ERCC1 were reduced following UVB exposure, whereas DPHC treatment partially restored the levels of both proteins. DPHC also increased expression of transcription factor specificity protein 1 (SP1) and sirtuin 1, an up-regulator of XPC, in UVB-exposed cells. DPHC restored binding of the SP1 to the XPC promoter, which is reduced in UVB-exposed cells. These results indicate that DPHC can protect cells against UVB-induced DNA damage by inducing the NER system. PMID:26404324

  3. Protective Effect of Diphlorethohydroxycarmalol against Ultraviolet B Radiation-Induced DNA Damage by Inducing the Nucleotide Excision Repair System in HaCaT Human Keratinocytes.

    PubMed

    Piao, Mei Jing; Hewage, Susara Ruwan Kumara Madduma; Han, Xia; Kang, Kyoung Ah; Kang, Hee Kyoung; Lee, Nam Ho; Hyun, Jin Won

    2015-09-02

    We investigated the protective properties of diphlorethohydroxycarmalol (DPHC), a phlorotannin, against ultraviolet B (UVB) radiation-induced cyclobutane pyrimidine dimers (CPDs) in HaCaT human keratinocytes. The nucleotide excision repair (NER) system is the pathway by which cells identify and repair bulky, helix-distorting DNA lesions such as ultraviolet (UV) radiation-induced CPDs and 6-4 photoproducts. CPDs levels were elevated in UVB-exposed cells; however, this increase was reduced by DPHC. Expression levels of xeroderma pigmentosum complementation group C (XPC) and excision repair cross-complementing 1 (ERCC1), which are essential components of the NER pathway, were induced in DPHC-treated cells. Expression of XPC and ERCC1 were reduced following UVB exposure, whereas DPHC treatment partially restored the levels of both proteins. DPHC also increased expression of transcription factor specificity protein 1 (SP1) and sirtuin 1, an up-regulator of XPC, in UVB-exposed cells. DPHC restored binding of the SP1 to the XPC promoter, which is reduced in UVB-exposed cells. These results indicate that DPHC can protect cells against UVB-induced DNA damage by inducing the NER system.

  4. 4-O-Methylhonokiol Protects HaCaT Cells from TGF-β1-Induced Cell Cycle Arrest by Regulating Canonical and Non-Canonical Pathways of TGF-β Signaling

    PubMed Central

    Kim, Sang-Cheol; Kang, Jung-Il; Hyun, Jin-Won; Kang, Ji-Hoon; Koh, Young-Sang; Kim, Young-Heui; Kim, Ki-Ho; Ko, Ji-Hee; Yoo, Eun-Sook; Kang, Hee-Kyoung

    2017-01-01

    4-O-methylhonokiol, a neolignan compound from Magnolia Officinalis, has been reported to have various biological activities including hair growth promoting effect. However, although transforming growth factor-β (TGF-β) signal pathway has an essential role in the regression induction of hair growth, the effect of 4-O-methylhonokiol on the TGF-β signal pathway has not yet been elucidated. We thus examined the effect of 4-O-methylhonokiol on TGF-β-induced canonical and noncanonical pathways in HaCaT human keratinocytes. When HaCaT cells were pretreated with 4-O-methylhonokiol, TGF-β1-induced G1/G0 phase arrest and TGF-β1-induced p21 expression were decreased. Moreover, 4-O-methylhonokiol inhibited nuclear translocation of Smad2/3, Smad4 and Sp1 in TGF-β1-induced canonical pathway. We observed that ERK phosphorylation by TGF-β1 was significantly attenuated by treatment with 4-O-methylhonokiol. 4-O-methylhonokiol inhibited TGF-β1-induced reactive oxygen species (ROS) production and reduced the increase of NADPH oxidase 4 (NOX4) mRNA level in TGF-β1-induced noncanonical pathway. These results indicate that 4-O-methylhonokiol could inhibit TGF-β1-induced cell cycle arrest through inhibition of canonical and noncanonical pathways in human keratinocyte HaCaT cell and that 4-O-methylhonokiol might have protective action on TGF-β1-induced cell cycle arrest. PMID:28190316

  5. 4-O-Methylhonokiol Protects HaCaT Cells from TGF-β1-Induced Cell Cycle Arrest by Regulating of Canonical and Non-Canonical Pathways of TGF-β Signaling.

    PubMed

    Kim, Sang-Cheol; Kang, Jung-Il; Hyun, Jin-Won; Kang, Ji-Hoon; Koh, Young-Sang; Kim, Young-Heui; Kim, Ki-Ho; Ko, Ji-Hee; Yoo, Eun-Sook; Kang, Hee-Kyoung

    2017-02-13

    4-O-methylhonokiol, a neolignan compound from Magnolia Officinalis, has been reported to have various biological activities including hair growth promoting effect. However, although transforming growth factor-β (TGF-β) signal pathway has an essential role in the regression induction of hair growth, the effect of 4-O-methylhonokiol on the TGF-β signal pathwayhas not yet been elucidated. We thus examined the effect of 4-O-methylhonokiol on TGF-β-induced canonical and noncanonical pathways in HaCaT human keratinocytes. When HaCaT cells were pretreated with 4-O-methylhonokiol, TGF-β1-induced G1/G0 phase arrest and TGF- β1-induced p21 expression were decreased. Moreover, 4-O-methylhonokiol inhibited nuclear translocation of Smad2/3, Smad4 and Sp1 in TGF-β1-induced canonical pathway. We observed that ERK phosphorylation by TGF-β1 was significantly attenuated by treatment with 4-O-methylhonokiol. 4-O-methylhonokiol inhibited TGF-β1-induced reactive oxygen species (ROS) production and reduced the increase of NADPH oxidase 4 (NOX4) mRNA level in TGF-β1-induced noncanonical pathway. These results indicate that 4-O-methylhonokiol could inhibit TGF-β1-induced cell cycle arrest through inhibition of canonical and noncanonical pathways in human keratinocyte HaCaT cell and that 4-O-methylhonokiol might have protective action on TGF-β1-induced cell cycle arrest.

  6. Potentiation of UVB-induced apoptosis by novel phytosphingosine derivative, tetraacetyl phytosphingosine in HaCaT cell and mouse skin.

    PubMed

    Kim, H J; Kang, S-Y; Kim, S J; Kim, S H; Kim, T-Y

    2004-07-01

    Inappropriate apoptosis results in the epidermal hyperplasia as in psoriasis and UVB irradiation has been successfully used to treat this kind of skin disorders. Previously, we reported that the novel phytosphingosine derivative, tetraacetyl phytosphingosine (TAPS) induced apoptosis in HaCaT cells. This study examined the effect of UVB irradiation and/or TAPS on the induction of apoptosis in HaCaT. 10 mJ/cm2 of UVB irradiation or 10 microM of TAPS alone exhibited weak cytotoxicity but co-treatment of UVB and TAPS synergistically enhanced the cytotoxicity and apoptosis in HaCaT. The cells treated with UVB and TAPS showed much higher levels of cleaved caspase-3, -8, -9 and Bax than with UVB or TAPS alone, whereas Bcl-2 level was decreased by co-administration of UVB and TAPS. In hairless mice, co-treatment of UVB and TAPS synergistically increased apoptosis, as shown in the HaCaT co-treated with UVB and TAPS. Furthermore, UVB irradiation caused an increase of apoptotic cells in the epidermis and the TAPS-treated mice showed an increase of apoptotic cells in the dermis as well as in the epidermis. These results suggest that the TAPS co-treatment synergistically increases the level of UVB-induced apoptosis via caspase activation by regulating the level of pro-apoptotic Bax and anti-apoptotic Bcl-2. Copyright 2004 Kluwer Academic Publishers

  7. Differential Influence of Components Resulting from Atmospheric-Pressure Plasma on Integrin Expression of Human HaCaT Keratinocytes

    PubMed Central

    Haertel, Beate; Straßenburg, Susanne; Wende, Kristian; von Woedtke, Thomas

    2013-01-01

    Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells. PMID:23936843

  8. Differential influence of components resulting from atmospheric-pressure plasma on integrin expression of human HaCaT keratinocytes.

    PubMed

    Haertel, Beate; Straßenburg, Susanne; Oehmigen, Katrin; Wende, Kristian; von Woedtke, Thomas; Lindequist, Ulrike

    2013-01-01

    Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells.

  9. Exploring the mode of action of dithranol therapy for psoriasis: a metabolomic analysis using HaCaT cells.

    PubMed

    Hollywood, Katherine A; Winder, Catherine L; Dunn, Warwick B; Xu, Yun; Broadhurst, David; Griffiths, Christopher E M; Goodacre, Royston

    2015-08-01

    Psoriasis is a common, immune-mediated inflammatory skin disease characterized by red, heavily scaled plaques. The disease affects over one million people in the UK and causes significant physical, psychological and societal impact. There is limited understanding regarding the exact pathogenesis of the disease although it is believed to be a consequence of genetic predisposition and environmental triggers. Treatments vary from topical therapies, such as dithranol, for disease of limited extent (<5% body surface area) to the new immune-targeted biologic therapies for severe psoriasis. Dithranol (also known as anthralin) is a topical therapy for psoriasis believed to work by inhibiting keratinocyte proliferation. To date there have been no metabolomic-based investigations into psoriasis. The HaCaT cell line is a model system for the epidermal keratinocyte proliferation characteristic of psoriasis and was thus chosen for study. Dithranol was applied at therapeutically relevant doses to HaCaT cells. Following the optimisation of enzyme inactivation and metabolite extraction, gas chromatography-mass spectrometry was employed for metabolomics as this addresses central metabolism. Cells were challenged with 0-0.5 μg mL(-1) in 0.1 μg mL(-1) steps and this quantitative perturbation generated data that were highly amenable to correlation analysis. Thus, we used a combination of traditional principal components analysis, hierarchical cluster analysis, along with correlation networks. All methods highlighted distinct metabolite groups, which had different metabolite trajectories with respect to drug concentration and the interpretation of these data established that cellular metabolism had been altered significantly and provided further clarification of the proposed mechanism of action of the drug.

  10. Dihydroxyacetone, the active browning ingredient in sunless tanning lotions, induces DNA damage, cell-cycle block and apoptosis in cultured HaCaT keratinocytes.

    PubMed

    Petersen, Anita B; Wulf, Hans Christian; Gniadecki, Robert; Gajkowska, Barbara

    2004-06-13

    Dihydroxyacetone (DHA), the active substance in sunless tanning lotions reacts with the amino groups of proteins to form a brown-colored complex. This non-enzymatic glycation, known as the Maillard reaction, can also occur with free amino groups in DNA, raising the possibility that DHA may be genotoxic. To address this issue we investigated the effects of DHA on cell survival and proliferation of a human keratinocyte cell line, HaCaT. Dose- and time-dependent morphological changes, chromatin condensation, cytoplasmic budding and cell detachment were seen in cells treated with DHA. Several dead cells were observed after long-time (24 h) incubation with 25 mM DHA or more. Furthermore, an extensive decline in proliferation was observed 1 day after DHA exposure for 24 h. When applied in different concentrations (5-50 mM) and for different time periods (1, 3 or 24 h) DHA caused a G(2)/M block after the cyclin B(1) restriction point. Exit from this cell-cycle block was associated with massive apoptosis, as revealed by a clonogenic assay, TUNEL staining and electron microscopy. Furthermore, DHA caused DNA damage as revealed by the alkaline comet assay. Preincubation with antioxidants prevented the formation of DNA strand breaks. The DHA toxicity may be caused by direct redox reactions, with formation of ROS as the crucial intermediates. The genotoxic capacity of DHA raises a question about the long-term clinical consequences of treatment of the skin with this commonly used compound.

  11. Wavelength-dependent photodynamic inactivation of HaCaT human keratinocytes after preincubation with 5-aminolevulinic acid

    NASA Astrophysics Data System (ADS)

    Szeimies, Rolf-Markus; Baeumler, Wolfgang; Karrer, Sigrid; Landthaler, Michael

    1995-03-01

    To determine the optimum wavelength for irradiation with coherent light after 5-ALA-induced protoporphyrin IX-sensitization, HaCaT cells were allowed to take up 5-ALA (30 (mu) g/ml) for 24 h. A tunable dye laser was used for PDT (40 mW/cm2, 30 J/cm2, wavelengths between 622 - 649 nm in steps of 3 nm). Afterwards, an MTT assay was performed to check cell viability (CV). Four groups (n equals 8) were formed: A equals controls, B equals irradiation only, C equals 5-ALA only, D equals 5-ALA and irradiation. CV of groups B, C, D were compared to those of group A (100%). Group B showed no significant difference (92% range at different wavelengths between 72% and 100%). Group D showed a significant decrease of CV (61%, range at different wavelengths between 35% and 86%). Lowest values for CV were detected at 634 and 637 nm. We believe therefore, that PDT with 5-ALA as sensitizer shows better cytotoxicity when wavelengths at 634 - 637 nm are used.

  12. Enhancement of Protective Effects of Radix Scutellariae on UVB-induced Photo Damage in Human HaCaT Keratinocytes.

    PubMed

    Wang, Yu-Shuai; Cho, Jin-Gyeong; Hwang, Eun-Son; Yang, Jung-Eun; Gao, Wei; Fang, Min-Zhe; Zheng, Sheng-Dao; Yi, Tae-Hoo

    2017-09-26

    Radix Scutellariae (RS) has long been used in the treatment of inflammatory and allergic diseases. Its main flavonoids, baicalin (BG) and wogonoside (WG), can be hydrolyzed into their corresponding aglycones, baicalein (B) and wogonin (W). In this study, we developed a safe and effective method of transforming these glycosides using Peclyve PR. The transformation rate of BG and WG reached 98.5 and 98.1%, respectively, with 10% enzyme at 40 °C for 60 h. Furthermore, we compared the anti-photoaging activity of RS before and after enzyme treatment, as well as their respective main components, in UVB-irradiated HaCaT cells. Results found that enzyme-treated RS (ERS) appeared to be much better at preventing UVB-induced photoaging than RS. ERS significantly inhibited the upregulation of matrix metalloproteinase-1 and IL-6 caused by UVB radiation by inactivating the MAPK/AP-1 and NF-κB/IκB-α signaling pathways. ERS treatment also recovered UVB-induced reduction of procollagen type I by activating the TGF-β/Smad pathway. In addition, ERS exhibited an excellent antioxidant activity, which could increase the expression of cytoprotective antioxidants such as HO-1 and NQ-O1, by facilitating Nrf2 nuclear transfer. These findings demonstrated that the photoprotective effects of RS were significantly improved by enzyme-modified biotransformation.

  13. Anti-inflammatory effects of Sanguisorbae Radix water extract on the suppression of mast cell degranulation and STAT-1/Jak-2 activation in BMMCs and HaCaT keratinocytes.

    PubMed

    Yang, Ju-Hye; Yoo, Jae-Myung; Cho, Won-Kyung; Ma, Jin Yeul

    2016-09-06

    Sanguisorbae Radix (SR) is a well-known herbal medicine used to treat inflammatory disease and skin burns in Asia. In addition, it is used to treat many types of allergic skin diseases, including urticaria, eczema, and allergic dermatitis. SR has been reported to exhibit anti-wrinkle, anti-oxidant, and anti-contact dermatitis bioactivities. In this study, we investigated the mechanism underlying the anti-inflammatory effects of SR water extract (WSR) using human keratinocyte (HaCaT) cells and BALB/c mouse bone marrow-derived mast cells (BMMCs). Viability assays were used to evaluate non-cytotoxic concentrations of WSR in both BMMCs and HaCaT cells. To investigate the effect of WSR treatment on the degranulation of IgE/Ag-activated BMMCs, we measured the release of β-hexosaminidase (β-HEX). We determined the production of pro-inflammatory chemokines including thymus and activation regulated chemokine (TARC; CCL17), regulated on activation, normal T-cell expressed and secreted (RANTES; CCL5), macrophage-derived chemokine (MDC; CCL22), and interleukin 8 (IL-8; CXCL8) in stimulated human keratinocytes. The ability of WSR to reduce the expression of pro-inflammatory marker proteins was evaluated by Western blotting in HaCaT cells stimulated with tumor necrosis factor (TNF)-α/interferon (IFN)-γ. WSR inhibited IgE/Ag-activated mast cell degranulation in BMMCs. Treatment with various concentrations of WSR decreased β-HEX release in a dose-dependent manner with an IC50 of 27.5 μg/mL. In keratinocytes, WSR suppressed TNF-α/IFN-γ-induced chemokine production and pro-inflammatory molecules via a blockade STAT-1, Jak-2, p38, and JNK activation. This results demonstrate that WSR inhibits degranulation of IgE/Ag-activated mast cells and inhibits the production of pro-inflammatory chemokines by suppressing the phosphorylation of p38 and JNK in HaCaT cells.

  14. The role of peroxisome proliferator-activated receptor-{beta}/{delta} in epidermal growth factor-induced HaCaT cell proliferation

    SciTech Connect

    Liang Pengfei; Jiang Bimei; Yang Xinghua; Xiao Xianzhong Huang Xu; Long Jianhong; Zhang Pihong; Zhang Minghua; Xiao Muzhang; Xie Tinghong; Huang Xiaoyuan

    2008-10-15

    Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) expression and activation is involved in the cell proliferation. However, little is known about the role of PPAR{beta}/{delta} in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPAR{beta}/{delta} mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPAR{beta}/{delta} protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPAR{beta}/{delta} binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPAR{beta}/{delta} caused selectively inhibition of PPAR{beta}/{delta} protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPAR{beta}/{delta}, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPAR{beta}/{delta} up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPAR{beta}/{delta} promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPAR{beta}/{delta} expression in a c-Jun-dependent manner and PPAR{beta}/{delta} plays a vital role in EGF-stimulated proliferation of HaCaT cells.

  15. Alantolactone from Saussurea lappa Exerts Antiinflammatory Effects by Inhibiting Chemokine Production and STAT1 Phosphorylation in TNF-α and IFN-γ-induced in HaCaT cells.

    PubMed

    Lim, Hye-Sun; Jin, Sung-Eun; Kim, Ohn-Soon; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

    2015-07-01

    Skin inflammation is the most common condition seen in dermatology practice and can be caused by various allergic reactions and certain toxins or chemicals. In the present study, we investigated the antiinflammatory effects of Saussurea lappa, a medicinal herb, and its marker compounds alantolactone, caryophyllene, costic acid, costunolide, and dehydrocostuslactone in the HaCaT human keratinocyte cell line. HaCaT cells were stimulated with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), and treated with S. lappa or each of five marker compounds. Chemokine production and expression were analyzed by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Phosphorylation of signal transducer and activator of transcription (STAT) 1 was determined by immunoblotting. Stimulation with TNF-α and IFN-γ significantly increased the production of the following chemokines: thymus-regulated and activation-regulated chemokine (TARC): regulated on activation, normal T-cell expressed and secreted (RANTES): macrophage-derived chemokine (MDC): and interleukin-8 (IL-8). By contrast, S. lappa and the five marker compounds significantly reduced the production of these chemokines by TNF-α and IFN-γ-treated cells. S. lappa and alantolactone suppressed the TNF-α and IFN-γ-stimulated increase in the phosphorylation of STAT1. Our results demonstrate that alantolactone from S. lappa suppresses TNF-α and IFN-γ-induced production of RANTES and IL-8 by blocking STAT1 phosphorylation in HaCaT cells.

  16. The role of lipid raft translocation of prohibitin in regulation of Akt and Raf-protected apoptosis of HaCaT cells upon ultraviolet B irradiation.

    PubMed

    Wu, Qiong; Wu, Shiyong

    2017-02-20

    Prohibitin (PHB) plays a role in regulation of ultraviolet B light (UVB)-induced apoptosis of human keratinocytes, HaCaT cells. The regulatory function of PHB appears to be associated with its lipid raft translocation. However, the detailed mechanism for PHB-mediated apoptosis of these keratinocytes upon UVB irradiation is not clear. In this report, we determined the role of lipid raft translocation of PHB in regulation of UVB-induced apoptosis. Our data show that upon UVB irradiation PHB is translocated from the non-raft membrane to the lipid rafts, which is correlated with a release of both Akt and Raf from membrane. Overexpression of Akt and/or Raf impedes UVB-induced lipid raft translocation of PHB. Immunoprecipitation analysis indicates that UVB alters the interactions among PHB, Akt, and Raf. Reduced expression of PHB leads to a decreased phosphorylation of Akt and ERK, as well as a decreased activity of Akt, and increased apoptosis of the cells upon UVB irradiation. These results suggest that PHB regulates UVB-induced apoptosis of keratinocytes via a mechanism that involves detachment from Akt and Raf on the plasma membrane, and sequential lipid raft translocation.

  17. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

    PubMed Central

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

    2016-01-01

    Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

  18. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells.

    PubMed

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan; Ahn, Kyu Joong

    2016-08-01

    We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation.

  19. Polypeptide from Chlamys farreri inhibits UVB-induced apoptosis of HaCaT cells via iNOS/NO and HSP90

    NASA Astrophysics Data System (ADS)

    Zhang, Zhengyang; Liu, Xiaojin; Liu, Tuo; Yan, Lin; Wang, Yuejun; Wang, Chunbo

    2009-09-01

    Polypeptide from Chlamys farreri (PCF) is a novel marine bioactive product that was isolated from the gonochoric Chinese scallop Chlamys farreri, and was found to be an effective antioxidant in our recent studies. In this study, we investigated the effect of PCF on ultraviolet B (UVB)-induced apoptosis of HaCaT cells and the intracellular signaling pathways involved. Pretreatment with the inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea sulfate inhibited UVB-induced apoptosis, indicating that iNOS and NO play important roles in apoptosis. On the other hand, the inhibition of UVB-induced apoptosis in the immortalized keratinocyte (HaCaT) cells by PCF was estimated using a DNA ladder. PCF treatment inhibited UVB-induced iNOS activation, as determined by RT-PCR, NO production, as determined by ESR, and up-regulated heat shock protein (HSP) 90 activation, as determined by Western blotting. Our results indicate that iNOS and NO are involved in UVB-induced apoptosis of HaCaT cells and the protective effect of PCF against UVB irradiation is exerted by suppressing the expression of iNOS, followed by inhibition of NO release and enhanced activation of HSP90.

  20. Light-Induced Cytotoxicity of 16 Polycyclic Aromatic Hydrocarbons on the US EPA Priority Pollutant List in Human Skin HaCaT Keratinocytes: Relationship Between Phototoxicity and Excited State Properties

    PubMed Central

    Wang, Shuguang; Sheng, Yinghong; Feng, Manliang; Leszczynski, Jerzy; Wang, Lei; Tachikawa, Hiroyasu; Yu, Hongtao

    2013-01-01

    The photocytotoxicity of 16 polycyclic aromatic hydrocarbons (PAHs) on the priority pollutant list of the United States Environmental Protection Agency (US EPA) were tested in human skin HaCaT keratinocytes. A selected PAH was mixed with HaCaT cells and irradiated with a solar simulator lamp for a dose equivalent to 5 min of outdoor sunlight and the cell viability was determined immediately and also after 24 h of incubation. For the cells without incubation after the treatments, it is found that all PAHs with three rings or less, except anthracene, are not photocytotoxic, while the four or five-ring PAHs (except chrysene), benz[a]anthracene, dibenzo[a,h]anthracene, benzo[ghi]perylene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, benzo[b]fluorenthene, fluorenthene, and pyrene, are photocytotoxic to the human skin HaCaT keratinocytes. If the cells were incubated for 24 h after the treatments, the photocytotoxic effect of the PAHs was greatly amplified in comparison to the nonincubated cells. For the 24 h incubated cells, all PAHs except naphthalene exhibit photocytotoxicity to some extent. Exposure to 5 μM of the 4- and 5-ring PAHs (except chrysene) and 3-ring anthracene more than 80% of the cells lose viability. The photocytotoxicity of the PAHs correlates well with several of their excited state properties: light absorption, excited singlet-state energy, excited triplet-state energy, and HOMO-LUMO energy gap. All the photocytotoxic PAHs absorb light at >300 nm, in the solar UVB and UVA region. There is a threshold for each of the three excited state descriptors of a photocytotoxic PAH: singlet energy <355 kJ/mol (corresponding to 337 nm light), triplet energy <230 kJ/mol (corresponding to 520 nm light), HOMO-LUMO gap <3.6 eV (corresponding to 344 nm light) obtained at the Density Functional Theory B3LYP/6-31G(d) level. PMID:17497637

  1. A comparative study on the enhancement efficacy of specific and non-specific iron chelators for protoporphyrin IX production and photosensitization in HaCat cells.

    PubMed

    Xia, Yumin; Huang, Yingying; Lin, Longde; Liu, Xiaoming; Jiang, Shan; Xiong, Layuan

    2009-12-01

    The iron chelators can be utilized in target cells to improve 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT). The purpose of this study is to compare the effect of two kinds of iron chelators, desferrioxamine (DFO) and ethylenediaminetetraacetic acid (EDTA) on the enhancement of ALA-PDT. HaCat cells were cultured in medium containing 2.0 mmol/L of ALA and 0.5 mmol/L of DFO or EDTA. After 3-h incubation in the dark, the concentration of cellular protoporphyrin IX (PpIX) was detected by high performance liquid chromatography (HPLC), and the fluorescence of PpIX was observed at 630 nm emission under confocal laser scanning microscope. For PDT, HaCat cells were irradiated using 632.8 nm laser, and the fractions of apoptotic and necrotic cells were flow cytometrically assayed. Related differences in morphology and ultrastructure of Ha-Cat cells were observed using optical microscope or transmission electron microscope. Compared to incubation with ALA alone, the addition of DFO or EDTA increased the concentration of cellular PpIX and the fluorescent density of PpIX, and also increased cell death ratio after PDT. PDT using ALA plus DFO produced the highest cellular PpIX level, greatest cell death ratio and most severe structural damage to the cells. It was concluded that both DFO and EDTA could enhance ALA-based PpIX production and PDT. Compared to the non-specific iron chelator of EDTA, the specific chelator, DFO, showed more potential for the enhancement.

  2. Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation

    SciTech Connect

    Jeon, Youngsic; Jung, Yujung; Kim, Min Cheol; Kwon, Hak Cheol; Kang, Ki Sung; Kim, Yong Kee; Kim, Su-Nam

    2014-08-08

    Highlights: • SHQA increases PPARα/γ transactivation and inhibits MMP-2/-9 expression. • SHQA inhibits TNFα-induced AP-1 and MAPK signaling. • SHQA inhibits TNFα-induced p65 translocation and IκBα phosphorylation. • SHQA inhibits TNFα-induced AP-1 and NF-κB signaling via PPARα. - Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-aging and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ’s role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα.

  3. The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21)NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

    PubMed Central

    Di Grazia, Antonio; Cappiello, Floriana; Imanishi, Akiko; Mastrofrancesco, Arianna; Picardo, Mauro; Paus, Ralf; Mangoni, Maria Luisa

    2015-01-01

    One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs) produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21)NH2, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells) over a wide range of peptide concentrations (0.025–4 μM), and this notably more efficiently than human cathelicidin (LL-37). This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21)NH2 now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21)NH2 to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers. PMID:26068861

  4. The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21)NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

    PubMed

    Di Grazia, Antonio; Cappiello, Floriana; Imanishi, Akiko; Mastrofrancesco, Arianna; Picardo, Mauro; Paus, Ralf; Mangoni, Maria Luisa

    2015-01-01

    One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs) produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21)NH2, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells) over a wide range of peptide concentrations (0.025-4 μM), and this notably more efficiently than human cathelicidin (LL-37). This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21)NH2 now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21)NH2 to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.

  5. Fraxetin Induces Heme Oxygenase-1 Expression by Activation of Akt/Nrf2 or AMP-activated Protein Kinase α/Nrf2 Pathway in HaCaT Cells

    PubMed Central

    Kundu, Juthika; Chae, In Gyeong; Chun, Kyung-Soo

    2016-01-01

    Background Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a coumarin derivative, has been reported to possess antioxidative, anti-inflammatory and neuroprotective effects. A number of recent observations suggest that the induction of heme oxygenase-1 (HO-1) inhibits inflammation and tumorigenesis. In the present study, we determined the effect of fraxetin on HO-1 expression in HaCaT human keratinocytes and investigated its underlying molecular mechanisms. Methods Reverse transcriptase-PCR and Western blot analysis were performed to detect HO-1 mRNA and protein expression, respectively. Cell viability was measured by the MTS test. The induction of intracellular reactive oxygen species (ROS) by fraxetin was evaluated by 2′,7′-dichlorofluorescin diacetate staining. Results Fraxetin upregulated mRNA and protein expression of HO-1. Incubation with fraxetin induced the localization of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus and increased the antioxidant response element-reporter gene activity. Fraxetin also induced the phosphorylation of Akt and AMP-activated protein kinase (AMPK)α and diminished the expression of phosphatase and tensin homolog, a negative regulator of Akt. Pharmacological inhibition of Akt and AMPKα abrogated fraxetin-induced expression of HO-1 and nuclear localization of Nrf2. Furthermore, fraxetin generated ROS in a concentration-dependent manner. Conclusions Fraxetin induces HO-1 expression through activation of Akt/Nrf2 or AMPKα/Nrf2 pathway in HaCaT cells. PMID:27722139

  6. Exploiting the Innate Antitumor Activity of Human Gamma-Delta T-Cells for the Treatment of Prostate Cancer

    DTIC Science & Technology

    2005-04-01

    Manassas, Vir- that the majority of ex vivo, expanded, apoptosis resistant ginia) and the normal human keratinocyte cell line HaCat ŗ ’y-T cells expressed...provided the HaCat cell line . Blood, 102: 200, 2003 10. Ottones, F., Liautard, J., Gross, A., Rabenoelina, F., Liautard, REFERENCES J. P. and Favero...against human prostate cancer cell lines . Purpose and scope: The aims of this project are, 1) to more precisely characterize the extent and breadth

  7. Lipoteichoic acid isolated from Staphylococcus aureus induces both epithelial-mesenchymal transition and wound healing in HaCaT cells.

    PubMed

    Kim, Seongjae; Kim, Hyeoung-Eun; Kang, Boyeon; Lee, Youn-Woo; Kim, Hangeun; Chung, Dae Kyun

    2017-08-03

    Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is recognized by toll-like receptor (TLR) 2, expressed on certain mammalian cell surfaces, initiating signaling cascades that include nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK). There are many structural and functional varieties of LTA, which vary according to the different species of gram-positive bacteria which produce them. In this study, we examined whether LTA isolated from Staphylococcus aureus (aLTA) affects the expression of junction proteins in keratinocytes. In HaCaT cells, tight junction-related gene expression was not affected by aLTA, while adherens junction-related gene expression was modified. High doses of aLTA induced the phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), which in turn induced the epithelial-mesenchymal transition (EMT) of HaCaT cells. When cells were given a low dose of aLTA, however, NF-κB was activated and the total cell population increased. Taken together, our study suggests that LTA from S. aureus infections in the skin may contribute both to the outbreak of EMT-mediated carcinogenesis and to the genesis of wound healing in a dose-dependent manner.

  8. Mitochondrial membrane potential (DeltaPsi) and Ca(2+)-induced differentiation in HaCaT keratinocytes.

    PubMed

    Savignan, F; Ballion, B; Odessa, M F; Charveron, M; Bordat, P; Dufy, B

    2004-01-01

    We have used the human calcium- and temperature-dependent (HaCaT) keratinocyte cell line to elucidate mechanisms of switching from a proliferating to a differentiating state. When grown in low calcium medium (<0.1 mM) HaCaT cells proliferate. However, an increase in the calcium concentration of the culture medium, [Ca(2+)](0), induces growth arrest and the cells start to differentiate. Numerous studies have already shown that the increase in [Ca(2+)](0) results in acute and sustained increases in intracellular calcium concentration, [Ca(2+)](i). We find that the Ca(2+)-induced cell differentiation of HaCaT cells is also accompanied by a significant decrease in mitochondrial membrane potential, DeltaPsi. By combining patch-clamp electrophysiological recordings and microspectrofluorimetric measurements of DeltaPsi on single cells, we show that the increase in [Ca(2+)](i) led to DeltaPsi depolarization. In addition, we report that tetraethylammonium (TEA), a blocker of plasma membrane K(+) channels, which is known to inhibit cell proliferation, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a blocker of plasma membrane Cl(-) channels, also affect DeltaPsi. Both these agents stimulate HaCaT cell differentiation. These data therefore strongly suggest a direct causal relationship between depolarization of DeltaPsi and the inhibition of proliferation and induction of differentiation in HaCaT keratinocytes.

  9. Differential cellular metabolite alterations in HaCaT cells caused by exposure to the aryl hydrocarbon receptor-binding polycyclic aromatic hydrocarbons chrysene, benzo[a]pyrene and dibenzo[a,l]pyrene.

    PubMed

    Potratz, Sarah; Jungnickel, Harald; Grabiger, Stefan; Tarnow, Patrick; Otto, Wolfgang; Fritsche, Ellen; von Bergen, Martin; Luch, Andreas

    2016-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the human environment. Since they are present in crude oilfractions used for the production of rubber and plastics, consumers may come into direct dermal contacts with these compounds (e.g., via tool handles) on a daily basis. Some individual PAHs are identified as genotoxic mutagens thereby prompting particular toxicological and environmental concern. Among this group, benzo[a]pyrene (BAP) constitutes a model carcinogen which is also used as reference compound for risk assessment purposes. It acts as a strong agonist of the aryl hydrocarbon receptor (AHR) and becomes metabolically activated toward mutagenic and carcinogenic intermediates by cytochrome P450-dependent monooxygenases (CYPs). While BAP has been exhaustively characterized with regard to its toxicological properties, there is much less information available for other PAHs. We treated an AHR-proficient immortal human keratinocyte cell line (i.e., HaCaT) with three selected PAHs: BAP, chrysene (CRY) and dibenzo[a,l]pyrene (DALP). Compound-mediated alterations of endogenous metabolites were investigated by an LC-MS/MS-based targeted approach. To examine AHR-dependent changes of the measured metabolites, AHR-deficient HaCaT knockdown cells (AHR-KD) were used for comparison. Our results reveal that 24 metabolites are sufficient to separate the PAH-exposed cells from untreated controls by application of a multivariate model. Alterations in the metabolomics profiles caused by each PAH show influences on the energy and lipid metabolism of the cells indicating reduced tricarboxylic acid (TCA) cycle activity and β-oxidation. Up-regulation of sphingomyelin levels after exposure to BAP and DALP point to pro-apoptotic processes caused by these two potent PAHs. Our results suggest that in vitro metabolomics can serve as tool to develop bioassays for application in hazard assessment.

  10. Influence of USP laser radiation on cell morphology: HaCat and MG-63 cell lines for bone and soft tissue modelling in dentistry

    NASA Astrophysics Data System (ADS)

    Meister, Joerg; Schelle, Florian; Beier, Imke; Bourauel, Christoph; Frentzen, Matthias; Kraus, Dominik

    Due to the high intensities of USP laser radiation, the interaction with matter is always attended with a plasma formation. Therefore the surrounding tissue can be influenced by heat generation and additional light emission from the UV up to the near and mid infrared. In dentistry it is of importance that the treatment of bone and soft tissues, i.e. oral mucosa, with a USP laser should not cause any kind of morphological changes on the cell level leading to a delayed wound healing or cell mutation. HaCaT keratinocyte cells were used for epidermal (soft tissue) and MG-63 osteoblast-like cells for hard tissue (bone) modelling. Cell growing was realized on glas cover slips. Irradiation was carried out with a USP Nd:YVO4 laser having a center wavelength at 1064 nm. Based on the pulse duration of 8 ps and a pulse repetition rate of 500 kHz the laser emits an average power of 9 W. For efficiency testing of cell removal on glas cover slips, 1, 5, 25, 50 and 75 repetitions of the scanning pattern (scan loops) were used. Heat distribution during laser irradiation was measured with an infrared camera system. Subsequently haematoxylin staining and SEM investigations were used to analyse the morphological changes. Differences of cell removal efficiency were observed with repetitions <=25. Irradiated areas with repetitions >=50 were cell-free. Additionally, repetitions >=25 showed side effects for both cell lines. Cell destruction in both cell lines could be verified using the haematoxylin staining and the SEM pictures.

  11. Ethanol Extract of Peanut Sprout Exhibits a Potent Anti-Inflammatory Activity in Both an Oxazolone-Induced Contact Dermatitis Mouse Model and Compound 48/80-Treated HaCaT Cells

    PubMed Central

    Choi, Da-In; Choi, Jee-Young; Kim, Young Jee; Lee, Jee-Bum; Kim, Sun-Ouck; Shin, Hyong-Taek

    2015-01-01

    Background We developed an ethanol extract of peanut sprouts (EPS), a peanut sprout-derived natural product, which contains a high level of trans-resveratrol (176.75 µg/ml) and was shown to have potent antioxidant activity. Objective We evaluated the potential anti-inflammatory activity of EPS by measuring its antioxidant potential in skin. Methods The anti-inflammatory activity of EPS was tested using two models of skin inflammation: oxazolone (OX)-induced contact dermatitis in mice and compound 48/80-treated HaCaT cells. As biomarkers of skin inflammation, cyclooxygenase-2 (COX-2) and nerve growth factor (NGF) levels were measured. Results OX-induced contact dermatitis was suppressed markedly in mice that were treated with an ointment containing 5% EPS as evidenced by a decrease in the extent of scaling and thickening (p<0.05) and supported by a histological study. COX-2 (messenger RNA [mRNA] and protein) and NGF (mRNA) levels, which were upregulated in the skin of OX-treated mice, were suppressed markedly in the skin of OX+EPS-treated mice. Consistent with this, compound 48/80-induced expression of COX-2 (mRNA and protein) and NGF (mRNA) in HaCaT cells were suppressed by EPS treatment in a dose-dependent manner. As an inhibitor of NF-κB, IκB protein levels were dose-dependently upregulated by EPS. Fluorescence-activated cell sorting (FACS) analysis revealed that EPS scavenged compound 48/80-induced reactive oxygen species (ROS) in HaCaT cells. Conclusion EPS exerts a potent anti-inflammatory activity via its anti-oxidant activity in both mouse skin and compound 48/80-treated HaCaT cells in vitro. Compound 48/80-treated HaCaT cells are a useful new in vitro model of skin inflammation. PMID:25834352

  12. Identification of daidzein as a ligand of retinoic acid receptor that suppresses expression of matrix metalloproteinase-9 in HaCaT cells.

    PubMed

    Oh, Hyeon-Jeong; Kang, Young-Gyu; Na, Tae-Young; Kim, Hyeon-Ji; Park, Jun Seong; Cho, Won-Jea; Lee, Mi-Ock

    2013-08-25

    Retinoids have been used as therapeutics for diverse skin diseases, but their side effects limit clinical usage. Here, we report that extracts of two soybeans, Glycine max and Rhynchosia nulubilis, and their ethyl acetate fractions increased the transcriptional activity of retinoic acid receptors (RARs), and that daidzin and genistin were the major constituents of the active fractions. Daidzin and its aglycone, daidzein, induced transcriptional activity of RAR and RARγ. FRET analysis demonstrated that daidzein, but not daidzin, bound both RAR and RARγ with EC50 values of 28μM and 40μM, respectively. Daidzein increased expression of mRNA of RARγ through direct binding of RAR and recruitment of p300 to the RARγ2 promoter. Further, mRNA and gelatinolytic activity of matrix metalloproteinase-9 were decreased by daidzein in HaCaT cells. Together, these results indicate that daidzein functions as a ligand of RAR that could be a candidate therapeutic for skin diseases.

  13. MicroRNA138 regulates keratin 17 protein expression to affect HaCaT cell proliferation and apoptosis by targeting hTERT in psoriasis vulgaris.

    PubMed

    Feng, Shi-Jun; Chu, Rui-Qi; Ma, Jing; Wang, Zheng-Xiang; Zhang, Guang-Jing; Yang, Xiu-Fang; Song, Zhi; Ma, Yun-Yi

    2017-01-01

    The purpose of this study is to explore the how microRNA-138 (miR-138) affects the expression of keratin 17 (K17) and psoriasis development. Twenty-eight skin lesions from patients with psoriasis vulgaris and twenty-four normal skin tissues from healthy controls were collected. The HaCaT cells were assigned into blank, negative control (NC), miR-138 mimic, miR-138 inhibitor, hTERT siRNA and miR-138 inhibitor+hTERT siRNA groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the miR-138 expression. The hTERT and K17 protein expression were testified by Western Blotting. MTT assay, flow cytometry with PI single staining and Annexin V/PI double staining were performed to detect the cell proliferation activity, cell cycle and apoptosis, respectively. Compared with the healthy skin, the expression of miR-138 decreased in the psoriatic skin, but hTERT and K17 protein expressions increased. The miR-138 mimic and hTERT siRNA groups showed significantly decreased hTERT and K17 protein expressions, inhibited cell proliferation, increased number of cells at G1 phase and elevated apoptosis rate in comparison to the rest three groups. The hTERT and K17 protein expressions in the miR-138 inhibitor group were up-regulated with promoted cell proliferation and reduced apoptosis rate as compared with the other four groups. In the miR-138 inhibitor+hTERT siRNA group, the hTERT and K17 protein expressions, cell proliferation and apoptosis were intermediate between the miR-138 inhibitor and hTERT siRNA groups. These findings indicated that the expression of miR-138 was lower in the psoriatic skin, which was negatively correlated to K17 expression. MiR-138 may regulate K17 protein expression to affect HaCaT cell proliferation and apoptosis by targeting hTERT gene.

  14. Phototoxicity of nano titanium dioxides in HaCaT keratinocytes--generation of reactive oxygen species and cell damage.

    PubMed

    Yin, Jun-Jie; Liu, Jun; Ehrenshaft, Marilyn; Roberts, Joan E; Fu, Peter P; Mason, Ronald P; Zhao, Baozhong

    2012-08-15

    Nano-sized titanium dioxide (TiO(2)) is among the top five widely used nanomaterials for various applications. In this study, we determine the phototoxicity of TiO(2) nanoparticles (nano-TiO(2)) with different molecular sizes and crystal forms (anatase and rutile) in human skin keratinocytes under UVA irradiation. Our results show that all nano-TiO(2) particles caused phototoxicity, as determined by the MTS assay and by cell membrane damage measured by the lactate dehydrogenase (LDH) assay, both of which were UVA dose- and nano-TiO(2) dose-dependent. The smaller the particle size of the nano-TiO(2) the higher the cell damage. The rutile form of nano-TiO(2) showed less phototoxicity than anatase nano-TiO(2). The level of photocytotoxicity and cell membrane damage is mainly dependent on the level of reactive oxygen species (ROS) production. Using polyunsaturated lipids in plasma membranes and human serum albumin as model targets, and employing electron spin resonance (ESR) oximetry and immuno-spin trapping as unique probing methods, we demonstrated that UVA irradiation of nano-TiO(2) can induce significant cell damage, mediated by lipid and protein peroxidation. These overall results suggest that nano-TiO(2) is phototoxic to human skin keratinocytes, and that this phototoxicity is mediated by ROS generated during UVA irradiation. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Application of platelet-rich plasma accelerates the wound healing process in acute and chronic ulcers through rapid migration and upregulation of cyclin A and CDK4 in HaCaT cells.

    PubMed

    Kim, Sung-Ae; Ryu, Han-Won; Lee, Kyu-Suk; Cho, Jae-We

    2013-02-01

    Application of autologous platelet-rich plasma (PRP) has been used for chronic wound healing. The aim of this study was to evaluate the effect of PRP on the wound healing processes of both acute and chronic ulcers and the underlying molecular mechanisms involved. We treated 16 patients affected by various acute and chronic ulcers with PRP. We performed molecular studies of cell proliferation, migration assays, immunoblotting and chloramphenicol acetyltransferase (CAT) assays in PRP-treated HaCaT keratinocyte cells. PRP treatment induced increased rates of cell proliferation and cell migration of HaCaT cells. In addition, the expression of cyclin A and cyclin dependent kinase (CDK) 4 proteins was markedly increased with a low concentration (0.5%) of PRP treatment in HaCaT cells. In 11 patients with chronic ulcers, including stasis ulcers, diabetic ulcers, venous leg ulcers, livedoid vasculitis, claw foot and traumatic ulcers, 9 patients showed 90-100% epithelization after 15.18 days. In 5 patients with acute ulcers, such as dehiscence, open wound and burn wound, 80-100% epithelization was achieved between 4 to 20 days. Topical application of PRP to acute and chronic skin ulcers significantly accelerated the epithelization process, likely through upregulation of the cell cycle regulatory proteins cyclin A and CDK4.

  16. The modulatory effect of ellagic acid and rosmarinic acid on ultraviolet-B-induced cytokine/chemokine gene expression in skin keratinocyte (HaCaT) cells.

    PubMed

    Lembo, Serena; Balato, Anna; Di Caprio, Roberta; Cirillo, Teresa; Giannini, Valentina; Gasparri, Franco; Monfrecola, Giuseppe

    2014-01-01

    Ultraviolet radiation (UV) induces an increase in multiple cutaneous inflammatory mediators. Ellagic acid (EA) and rosmarinic acid (RA) are natural anti-inflammatory and immunomodulatory compounds found in many plants, fruits, and nuts. We assessed the ability of EA and RA to modulate IL-1β, IL-6, IL-8, IL-10, MCP-1, and TNF-α gene expression in HaCaT cells after UVB irradiation. Cells were treated with UVB (100 mJ/cm(2)) and simultaneously with EA (5 μM in 0.1% DMSO) or RA (2.7 μM in 0.5% DMSO). Moreover, these substances were added to the UVB-irradiated cells 1 h or 6 h before harvesting, depending on the established UVB-induced cytokine expression peak. Cytokine gene expression was examined using quantitative real time polymerase chain reaction. RA produced a significant reduction in UVB-induced expression of IL-6, IL-8, MCP-1, and TNF-α when applied at the same time as irradiation. EA showed milder effects compared with RA, except for TNF-α. Both substances decreased IL-6 expression, also when applied 5 h after irradiation, and always produced a significant increase in UVB-induced IL-10 expression. Our findings suggest that EA and RA are able to prevent and/or limit the UVB-induced inflammatory cascade, through a reduction in proinflammatory mediators and the enhancement of IL-10, with its protective function.

  17. Baicalein Protects Human Skin Cells against Ultraviolet B-Induced Oxidative Stress

    PubMed Central

    Oh, Min Chang; Piao, Mei Jing; Fernando, Pattage Madushan Dilhara Jayatissa; Han, Xia; Hewage, Susara Ruwan Kumara Madduma; Park, Jeong Eon; Ko, Mi Sung; Jung, Uhee; Kim, In Gyu; Hyun, Jin Won

    2016-01-01

    Baicalein (5,6,7-trihydroxy-2-phenyl-chromen-4-one) is a flavone, a type of flavonoid, originally isolated from the roots of Scutellaria baicalensis. This study evaluated the protective effects of baicalein against oxidative damage-mediated apoptosis induced by ultraviolet B (UVB) radiation in a human keratinocyte cell line (HaCaT). Baicalein absorbed light within the wavelength range of UVB. In addition, baicalein decreased the level of intracellular reactive oxygen species (ROS) in response to UVB radiation. Baicalein protected cells against UVB radiation-induced DNA breaks, 8-isoprostane generation and protein modification in HaCaT cells. Furthermore, baicalein suppressed the apoptotic cell death by UVB radiation. These findings suggest that baicalein protected HaCaT cells against UVB radiation-induced cell damage and apoptosis by absorbing UVB radiation and scavenging ROS. PMID:27257012

  18. Phototoxicity of nano titanium dioxides in HaCaT keratinocytes—Generation of reactive oxygen species and cell damage

    SciTech Connect

    Yin, Jun-Jie; Liu, Jun; Ehrenshaft, Marilyn; Roberts, Joan E.; Fu, Peter P.; Mason, Ronald P.; Zhao, Baozhong

    2012-08-15

    Nano-sized titanium dioxide (TiO{sub 2}) is among the top five widely used nanomaterials for various applications. In this study, we determine the phototoxicity of TiO{sub 2} nanoparticles (nano-TiO{sub 2}) with different molecular sizes and crystal forms (anatase and rutile) in human skin keratinocytes under UVA irradiation. Our results show that all nano-TiO{sub 2} particles caused phototoxicity, as determined by the MTS assay and by cell membrane damage measured by the lactate dehydrogenase (LDH) assay, both of which were UVA dose- and nano-TiO{sub 2} dose-dependent. The smaller the particle size of the nano-TiO{sub 2} the higher the cell damage. The rutile form of nano-TiO{sub 2} showed less phototoxicity than anatase nano-TiO{sub 2}. The level of photocytotoxicity and cell membrane damage is mainly dependent on the level of reactive oxygen species (ROS) production. Using polyunsaturated lipids in plasma membranes and human serum albumin as model targets, and employing electron spin resonance (ESR) oximetry and immuno-spin trapping as unique probing methods, we demonstrated that UVA irradiation of nano-TiO{sub 2} can induce significant cell damage, mediated by lipid and protein peroxidation. These overall results suggest that nano-TiO{sub 2} is phototoxic to human skin keratinocytes, and that this phototoxicity is mediated by ROS generated during UVA irradiation. Highlights: ► We evaluate the phototoxicity of nano-TiO{sub 2} with different sizes and crystal forms. ► The smaller the particle size of the nano-TiO{sub 2} the higher the cell damage. ► The rutile form of nano-TiO{sub 2} showed less phototoxicity than anatase nano-TiO{sub 2}. ► ESR oximetry and immuno-spin trapping techniques confirm UVA-induced cell damage. ► Phototoxicity is mediated by ROS generated during UVA irradiation of nano-TiO{sub 2}.

  19. Redox Mechanisms of AVS022, an Oriental Polyherbal Formula, and Its Component Herbs in Protection against Induction of Matrix Metalloproteinase-1 in UVA-Irradiated Keratinocyte HaCaT Cells

    PubMed Central

    Pluemsamran, Thanyawan; Tripatara, Pinpat; Phadungrakwittaya, Rattana; Akarasereenont, Pravit; Laohapand, Tawee

    2013-01-01

    Ayurved Siriraj HaRak (AVS022) formula has been used for topical remedy of dermatologic disorders. Oxidative stress induced by ultraviolet (UV) A irradiation could be implicated in photoaged skin through triggering matrix metalloproteinase-1 (MMP-1). We, therefore, explored the antioxidant mechanisms by which AVS022 formulation and its individual components protected against UVA-dependent MMP-1 upregulation in keratinocyte HaCaT cells. TLC analysis revealed the presence of multiple phenolics including gallic acid (GA) in the AVS022 extracts. We demonstrated that pretreatment with the whole formula and individual herbal components except T. triandra protected against increased MMP-1 activity in irradiated HaCaT cells. Moreover, all herbal extracts and GA, used as the reference compound, were able to reverse cytotoxicity, oxidant production, glutathione (GSH) loss, and inactivation of catalase and glutathione peroxidase (GPx). F. racemosa was observed to yield the strongest abilities to abolish UVA-mediated induction of MMP-1 and impairment of antioxidant defenses including GSH and catalase. Our observations suggest that upregulation of endogenous antioxidants could be the mechanisms by which AVS022 and its herbal components suppressed UVA-stimulated MMP-1 in HaCaT cells. In addition, pharmacological actions of AVS022 formula may be attributed to the antioxidant potential of its components, in particular F. racemosa, and several phenolics including GA. PMID:24171043

  20. Saussurea tridactyla Sch. Bip.-derived polysaccharides and flavones reduce oxidative damage in ultraviolet B-irradiated HaCaT cells via a p38MAPK-independent mechanism

    PubMed Central

    Guo, Yan; Sun, Juan; Ye, Juan; Ma, Wenyu; Yan, Hualing; Wang, Gang

    2016-01-01

    Objective To investigate whether Saussurea tridactyla Sch. Bip.-derived polysaccharides and flavones exert apoptosis-inhibiting effects in ultraviolet B (UVB)-irradiated HaCaT cells. Methods We divided HaCaT cells into low radiation UVB and high radiation UVB groups. Low radiation UVB and high radiation UVB groups were further divided into a control group, UVB radiation group (UVB group), S. tridactyla Sch. Bip.-derived polysaccharides and flavones low-dose group, and S. tridactyla Sch. Bip.-derived polysaccharides and flavones high-dose group. Cell viability and morphology were assayed by MTT and trypan blue staining. Superoxide dismutase activity, glutathione content, malondialdehyde content, and catalase activity test kits were used to detect superoxide dismutase activity, glutathione content, malondialdehyde content, and catalase activity, respectively. Cell apoptosis, intracellular Ca2+ levels, and mitochondrial membrane potential (Δψ) were detected by flow cytometry. Protein levels were analyzed by Western blotting and immunofluorescence. Results S. tridactyla Sch. Bip.-derived polysaccharides and flavones were found to increase the absorbance of MTT, decrease cell death, alleviate the degree of cell edema, restore the cell morphology, reduce cell death fragments and chip phenomenon, increase superoxide dismutase activity, glutathione content, and catalase activity while decreasing the content of malondialdehyde, lowering the population of apoptotic cells, reducing the intracellular Ca2+ fluorescence, increasing the mitochondrial membrane potential (Δψ), increasing the expressions of p-38, p-53, Bcl-2, and decreasing the expressions of Bax and active-caspase-3. Conclusion S. tridactyla Sch. Bip.-derived polysaccharides and flavones can reduce cell apoptosis to protect HaCaT cells from oxidative damage after UVB irradiation; however, this effect does not occur via the p38MAPK pathway. PMID:26855564

  1. The Modulatory Effect of Ellagic Acid and Rosmarinic Acid on Ultraviolet-B-Induced Cytokine/Chemokine Gene Expression in Skin Keratinocyte (HaCaT) Cells

    PubMed Central

    Lembo, Serena; Balato, Anna; Di Caprio, Roberta; Cirillo, Teresa; Giannini, Valentina; Gasparri, Franco; Monfrecola, Giuseppe

    2014-01-01

    Ultraviolet radiation (UV) induces an increase in multiple cutaneous inflammatory mediators. Ellagic acid (EA) and rosmarinic acid (RA) are natural anti-inflammatory and immunomodulatory compounds found in many plants, fruits, and nuts. We assessed the ability of EA and RA to modulate IL-1β, IL-6, IL-8, IL-10, MCP-1, and TNF-α gene expression in HaCaT cells after UVB irradiation. Cells were treated with UVB (100 mJ/cm2) and simultaneously with EA (5 μM in 0.1% DMSO) or RA (2.7 μM in 0.5% DMSO). Moreover, these substances were added to the UVB-irradiated cells 1 h or 6 h before harvesting, depending on the established UVB-induced cytokine expression peak. Cytokine gene expression was examined using quantitative real time polymerase chain reaction. RA produced a significant reduction in UVB-induced expression of IL-6, IL-8, MCP-1, and TNF-α when applied at the same time as irradiation. EA showed milder effects compared with RA, except for TNF-α. Both substances decreased IL-6 expression, also when applied 5 h after irradiation, and always produced a significant increase in UVB-induced IL-10 expression. Our findings suggest that EA and RA are able to prevent and/or limit the UVB-induced inflammatory cascade, through a reduction in proinflammatory mediators and the enhancement of IL-10, with its protective function. PMID:25162011

  2. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line

    PubMed Central

    1988-01-01

    In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation- specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long- term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation. PMID:2450098

  3. The Role of DCT in HPV16 Infection of HaCaTs.

    PubMed

    Aksoy, Pınar; Meneses, Patricio I

    2017-01-01

    Persistent infection with high-risk human papillomavirus (HPV) genotype is a major factor leading to many human cancers. Mechanisms of HPV entry into host cells and genome trafficking towards the nucleus are incompletely understood. Dopachrome tautomerase (DCT) was identified as a cellular gene required for HPV infection in HeLa cells on a siRNA screen study. Here, we confirm that DCT knockdown significantly decreases HPV infection in the human keratinocyte HaCaT cells as was observed in HeLas. We investigated the effects of DCT knockdown and found that DCT depletion caused increased reactive oxygen species (ROS) levels, DNA damage and altered cell cycle in HaCaT cells. We observed increased viral DNA localization at the endoplasmic reticulum but an overall decrease in infection in DCT knockdown cells. This observation suggests that viral DNA might be retained in the ER due to altered cell cycle, and viral particles are incapable of further movement towards the nucleus in DCT knockdown cells.

  4. The Role of DCT in HPV16 Infection of HaCaTs

    PubMed Central

    Aksoy, Pınar; Meneses, Patricio I.

    2017-01-01

    Persistent infection with high-risk human papillomavirus (HPV) genotype is a major factor leading to many human cancers. Mechanisms of HPV entry into host cells and genome trafficking towards the nucleus are incompletely understood. Dopachrome tautomerase (DCT) was identified as a cellular gene required for HPV infection in HeLa cells on a siRNA screen study. Here, we confirm that DCT knockdown significantly decreases HPV infection in the human keratinocyte HaCaT cells as was observed in HeLas. We investigated the effects of DCT knockdown and found that DCT depletion caused increased reactive oxygen species (ROS) levels, DNA damage and altered cell cycle in HaCaT cells. We observed increased viral DNA localization at the endoplasmic reticulum but an overall decrease in infection in DCT knockdown cells. This observation suggests that viral DNA might be retained in the ER due to altered cell cycle, and viral particles are incapable of further movement towards the nucleus in DCT knockdown cells. PMID:28095444

  5. Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNF{alpha} in human epidermal keratinocytes (HaCaT)

    SciTech Connect

    Onda, Kenji . E-mail: knjond@ps.toyaku.ac.jp; Nagashima, Masahiro; Kawakubo, Yo; Inoue, Shota; Hirano, Toshihiko; Oka, Kitaro

    2006-12-08

    Glucocorticoids (GCs) are essential drugs administered topically or systematically for the treatment of autoimmune skin diseases such as pemphigus. However, a certain proportion of patients does not respond well to GCs. Although studies on the relationship between cytokines and GC insensitivity in local tissues have attracted attention recently, little is known about the underlying mechanism(s) for GC insensitivity in epidermal keratinocytes. Here, we report that tumor necrosis factor (TNF) {alpha} reduces GC-induced transactivation of endogenous genes as well as a reporter plasmid which contains GC responsive element (GRE) in human epidermal keratinocyte cells (HaCaT). The GC insensitivity by TNF{alpha} was not accompanied by changes in mRNA expressions of GR isoforms ({alpha} or {beta}). However, we observed that mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors (PD98059 and U0126) significantly sensitized the GC-induced transactivation of anti-inflammatory genes (glucocorticoid-induced leucine zipper (GILZ) and mitogen-activated protein kinase phosphatase (MKP)-1) and FK506 binding protein (FKBP) 51 gene in the presence of TNF{alpha}. Additionally, we observed that TNF{alpha} reduced prednisolone (PSL)-dependent nuclear translocation of GR, which was restored by pre-treatment of MEK-1 inhibitors. This is the first study demonstrating a role of the MEK-1/ERK cascade in TNF{alpha}-mediated GC insensitivity. Our data suggest that overexpression of TNF{alpha} leads to topical GC insensitivity by reducing GR nuclear translocation in keratinocytes, and our findings also suggest that inhibiting the MEK-1/ERK cascade may offer a therapeutic potential for increasing GC efficacy in epidermis where sufficient inflammatory suppression is required.

  6. p38 Mitogen-activated protein kinase and c-Jun NH2-terminal protein kinase regulate the accumulation of a tight junction protein, ZO-1, in cell-cell contacts in HaCaT cells.

    PubMed

    Minakami, Masahiko; Kitagawa, Norio; Iida, Hiroshi; Anan, Hisashi; Inai, Tetsuichiro

    2015-02-01

    To investigate the involvement of stress-activated protein kinases, JNK and p38 MAPK, in the assembly of tight junctions in keratinocytes, we treated HaCaT cells with various combinations of SP600125 (an inhibitor of JNK), SB202190 (an inhibitor of p38 MAPK) and anisomycin (an activator of both JNK and p38 MAPK) and examined the localization of ZO-1, an undercoat constitutive protein of the tight junction. Short-term (8h) incubation with SP600125, SB202190 or anisomycin induced the accumulation of ZO-1 in the cell-cell contacts, with reduced ZO-1 staining in the cytoplasm, while only long-term (24h) incubation with SP600125 induced the accumulation of ZO-1. SP600125, SB202190 or SP600125 plus SB202190 treatment induced thin linear staining for ZO-1 in the cell-cell contacts. Anisomycin treatment induced thick and irregular linear staining for ZO-1, while anisomycin plus SP600125 treatment induced zipper-like staining for ZO-1. Anisomycin plus SB202190 treatment or anisomycin plus both SP600125 and SB202190 treatment for 8h failed to lead to the accumulation of ZO-1 in cell-cell contacts, but induced thin linear staining with several gaps 16 h after removal of these agents. These results suggest that the localization of ZO-1 in cell-cell contacts is differently regulated by activation and inhibition of JNK and/or p38 MAPK depending on the incubation period.

  7. MiR-26a inhibits proliferation and migration of HaCaT keratinocytes through regulating PTEN expression.

    PubMed

    Yu, Nanze; Yang, Yang; Li, Xiongwei; Zhang, Mingzi; Huang, Jiuzuo; Wang, Xiaojun; Long, Xiao

    2016-12-05

    MicroRNAs (miRNAs) have been shown to be associated with differentiation, migration and apoptosis in keratinocyte. Although it has been reported that microRNA-26a (miR-26a) plays important roles in tumor cells, its biological functions in keratinocytes are still not well elucidated. In this study, we confirmed expression of miR-26a in human keratinocytes using RT-PCR and further studied the role of miR-26a in cell proliferation and cell migration. Ectopic expression of MiR-26a mimic or inhibitor increased or decreased miR-26a expression respectively in HaCaT cells. Proliferation of HaCaT keratinocyte can be suppressed or promoted by overexpression or down-expression of miR-26a. In scratch wound-healing assay and Boyden chamber cell migration assay, upregulating miR-26a expression blocked cell migration, while downregulating miR-26a expression enhanced the migration. Using quantitative RT-PCR (qRT-PCR) and western blot, we further discovered that both mRNA and protein level of phosphatase and tensin homolog deleted from chromosome 10(PTEN) were regulated by miR-26a in HaCaT cells. Meanwhile the level of active form of AKT was also regulated by the miR-26a. In rescue experiment, knockdown of PTEN in the miR-26a mimic transduced cells recovered the migration ability of HaCaT cells. Together these results suggest that miR-26a modulates the proliferation and migration of keratinocytes via regulating PTEN/AKT signaling pathway.

  8. The effects of chronic, low doses of Ra-226 on cultured fish and human cells.

    PubMed

    Shi, Xiaopei; Seymour, Colin; Mothersill, Carmel

    2016-07-01

    To determine the chronic low-dose radiation effects caused by α-particle radiation from (226)Ra over multiple cell generations in CHSE/F fish cells and HaCaT human cells. CHSE/F cells and HaCaT cells were cultured in medium containing (226)Ra to deliver the chronic low-dose α-particle radiation. Clonogenic assay was used to test the clonogenic survival fractions of cells with or without being exposed to radiation from (226)Ra. The chronic low-dose radiation from (226)Ra does have effects on the clonogenic survival of CHSE/F cells and HaCaT cells. When CHSE/F cells were cultured in (226)Ra-medium over 9 passages for about 134 days, the clonogenic surviving fractions for cells irradiated at dose rates ranging from 0.00066 to 0.66mGy/d were significantly lower than that of cells sham irradiated. For HaCaT cells grown in medium containing the same range of (226)Ra activity, the clonogenic surviving fraction decreased at first and reached the lowest value at about 42 days (8 passages). After that, the clonogenic survival began to increase, and was significantly higher than that of control cells by the end of the experimental period. The chronic, low-dose high LET radiation from (226)Ra can influence the clonogenic survival of irradiated cells. CHSE/F cells were sensitized by the radiation, and HaCaT cells were initially sensitized but later appeared to be adapted. The results could have implications for determining risk from chronic versus acute exposures to radium. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. HPV16 infection of HaCaTs is dependent on β4 integrin, and α6 integrin processing.

    PubMed

    Aksoy, Pınar; Abban, Cynthia Y; Kiyashka, Elizabeth; Qiang, Weitao; Meneses, Patricio I

    2014-01-20

    Our understanding of human papillomavirus (HPV) is still evolving. To further study the field, our laboratory has focused on determining the role of integrins in the initial steps of viral endocytosis into HaCaT cells. Our and others' previous findings have shown that α6 is necessary for infection. Here we show that α3 and β1 were dispensable, and we identified integrin α6β4 complex as necessary for infection in HaCaTs. β4 knock down resulted in a significant decrease in HPV16 PsV infection and perhaps most importantly resulted in defective post-translational α6 processing. We showed that the unprocessed α6 does not localize to the cell surface. We propose that the α6β4 complex is necessary for the formation of an endocytic complex that results in the signaling transduction events necessary for initial endocytosis.

  10. Sodium alginate-cross-linked polymyxin B sulphate-loaded solid lipid nanoparticles: Antibiotic resistance tests and HaCat and NIH/3T3 cell viability studies.

    PubMed

    Severino, Patrícia; Chaud, Marco V; Shimojo, Andrea; Antonini, Danilo; Lancelloti, Marcelo; Santana, Maria Helena A; Souto, Eliana B

    2015-05-01

    Polymyxins are a group of antibiotics with a common structure of a cyclic peptide with a long hydrophobic tail. Polymyxin B sulphate (PLX) has cationic charge, which is an obstacle for the efficient loading into Solid Lipid Nanoparticles (SLN). In the present paper, we describe an innovative method to load PLX into SLN to achieve the sustained release of the drug. PLX was firstly cross-linked with sodium alginate (SA) at different ratios (1:1, 1:2 and 1:3 SA/PLX), and loaded into SLN produced by high pressure homogenization (HPH). Optimized SLN were produced applying 500bar pressure and 5 homogenization cycles. The best results were obtained with SA/PLX (1:1), recording 99.08±1.2% for the association efficiency of the drug with SA, 0.99±10g for the loading capacity and 212.07±5.84% degree of swelling. The rheological profile of aqueous SA solution followed the typical behaviour of concentrated polymeric solutions, whereas aqueous SA/PLX solution exhibited a gel-like dynamic behaviour. Micrographs show that SA/PLX depicted a porous and discontinuous amorphous phase in different ratios. The encapsulation efficiency of SA/PLX (1:1) in SLN, the mean particle diameter, polydispersity index and zeta potential were, respectively, 82.7±5.5%; 439.5±20.42nm, 0.241±0.050 and -34.8±0.55mV. The effect of SLN on cell viability was checked in HaCat and NIH/3T3 cell lines, and the minimal inhibitory concentrations (MIC) were determined in Pseudomonas aeruginosa strains. SA/PLX-loaded SLN were shown to be less toxic than free PLX. Minimal inhibitory concentrations (MIC) showed the presence of the cross-linker polymer-drug complex, and SLN were shown to enhance MIC in the evaluated strains. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Factors affecting ultraviolet-A photon emission from β-irradiated human keratinocyte cells.

    PubMed

    Le, M; Mothersill, C E; Seymour, C B; Ahmad, S B; Armstrong, A; Rainbow, A J; McNeill, F E

    2015-08-21

    The luminescence intensity of 340±5 nm photons emitted from HaCaT (human keratinocyte) cells was investigated using a single-photon-counting system during cellular exposure to (90)Y β-particles. Multiple factors were assessed to determine their influence upon the quantity and pattern of photon emission from β-irradiated cells. Exposure of 1 x 10(4) cells/5 mL to 703 μCi resulted in maximum UVA photoemission at 44.8 x 10(3)±2.5 x 10(3) counts per second (cps) from live HaCaT cells (background: 1-5 cps); a 16-fold increase above cell-free controls. Significant biophoton emission was achieved only upon stimulation and was also dependent upon presence of cells. UVA luminescence was measured for (90)Y activities 14 to 703 μCi where a positive relationship between photoemission and (90)Y activity was observed. Irradiation of live HaCaT cells plated at various densities produced a distinct pattern of emission whereby luminescence increased up to a maximum at 1 x 10(4) cells/5 mL and thereafter decreased. However, this result was not observed in the dead cell population. Both live and dead HaCaT cells were irradiated and were found to demonstrate different rates of photon emission at low β activities (⩽400 μCi). Dead cells exhibited greater photon emission rates than live cells which may be attributable to metabolic processes taking place to modulate the photoemissive effect. The results indicate that photon emission from HaCaT cells is perturbed by external stimulation, is dependent upon the activity of radiation delivered, the density of irradiated cells, and cell viability. It is postulated that biophoton emission may be modulated by a biological or metabolic process.

  12. Factors affecting ultraviolet-A photon emission from β-irradiated human keratinocyte cells

    NASA Astrophysics Data System (ADS)

    Le, M.; Mothersill, C. E.; Seymour, C. B.; Ahmad, S. B.; Armstrong, A.; Rainbow, A. J.; McNeill, F. E.

    2015-08-01

    The luminescence intensity of 340+/- 5 nm photons emitted from HaCaT (human keratinocyte) cells was investigated using a single-photon-counting system during cellular exposure to 90Y β-particles. Multiple factors were assessed to determine their influence upon the quantity and pattern of photon emission from β-irradiated cells. Exposure of 1× {{10}4} cells/5 mL to 703 μCi resulted in maximum UVA photoemission at 44.8× {{10}3}+/- 2.5× {{10}3} counts per second (cps) from live HaCaT cells (background: 1-5 cps); a 16-fold increase above cell-free controls. Significant biophoton emission was achieved only upon stimulation and was also dependent upon presence of cells. UVA luminescence was measured for 90Y activities 14 to 703 μCi where a positive relationship between photoemission and 90Y activity was observed. Irradiation of live HaCaT cells plated at various densities produced a distinct pattern of emission whereby luminescence increased up to a maximum at 1× {{10}4} cells/5 mL and thereafter decreased. However, this result was not observed in the dead cell population. Both live and dead HaCaT cells were irradiated and were found to demonstrate different rates of photon emission at low β activities (⩽400 μCi). Dead cells exhibited greater photon emission rates than live cells which may be attributable to metabolic processes taking place to modulate the photoemissive effect. The results indicate that photon emission from HaCaT cells is perturbed by external stimulation, is dependent upon the activity of radiation delivered, the density of irradiated cells, and cell viability. It is postulated that biophoton emission may be modulated by a biological or metabolic process.

  13. Comparison of the cellular transport mechanism of cationic, star-shaped polymers and liposomes in HaCat cells

    PubMed Central

    Luo, Heng-Cong; Li, Na; Yan, Li; Mai, Kai-jin; Sun, Kan; Wang, Wei; Lao, Guo-Juan; Yang, Chuan; Zhang, Li-Ming; Ren, Meng

    2017-01-01

    Several biological barriers must be overcome to achieve efficient nonviral gene delivery. These barriers include target cell uptake, lysosomal degradation, and dissociation from the carrier. In this study, we compared the differences in the uptake mechanism of cationic, star-shaped polymer/MMP-9siRNA complexes (β-CD-(D3)7/MMP-9siRNA complexes: polyplexes) and commercial liposome/MMP-9siRNA complexes (Lipofectamine® 2000/MMP-9siRNA complexes: liposomes). The uptake pathway and transfection efficiency of the polyplexes and liposomes were determined by fluorescence microscopy, flow cytometry, and reverse transcriptase-polymerase chain reaction. The occurrence of intracellular processing was assessed by confocal laser scanning microscopy. Endosomal acidification inhibitors were used to explore the endosomal escape mechanisms of the polyplexes and lysosomes. We concluded that the polyplexes were internalized by non-caveolae- and non-clathrin-mediated pathways, with no lysosomal trafficking, thereby inducing successful transfection, while the majority of liposomes were internalized by clathrin-dependent endocytosis (CDE), caveolae-mediated endocytosis, and macropinocytosis, and only CDE induced successful transfection. Liposomes might escape more quickly than polyplexes, and the digestion effect of acidic organelles on liposomes was faint compared to the polyplexes, although both complexes escaped from endolysosomes via the proton sponge mechanism. This may be the key aspect that leads to the lower transfection efficiency of the β-CD-(D3)7/MMP-9siRNA complexes. The present study may offer some insights for the rational design of novel delivery systems with increased transfection efficiency but decreased toxicity. PMID:28223800

  14. Comparison of the cellular transport mechanism of cationic, star-shaped polymers and liposomes in HaCat cells.

    PubMed

    Luo, Heng-Cong; Li, Na; Yan, Li; Mai, Kai-Jin; Sun, Kan; Wang, Wei; Lao, Guo-Juan; Yang, Chuan; Zhang, Li-Ming; Ren, Meng

    2017-01-01

    Several biological barriers must be overcome to achieve efficient nonviral gene delivery. These barriers include target cell uptake, lysosomal degradation, and dissociation from the carrier. In this study, we compared the differences in the uptake mechanism of cationic, star-shaped polymer/MMP-9siRNA complexes (β-CD-(D3)7/MMP-9siRNA complexes: polyplexes) and commercial liposome/MMP-9siRNA complexes (Lipofectamine(®) 2000/MMP-9siRNA complexes: liposomes). The uptake pathway and transfection efficiency of the polyplexes and liposomes were determined by fluorescence microscopy, flow cytometry, and reverse transcriptase-polymerase chain reaction. The occurrence of intracellular processing was assessed by confocal laser scanning microscopy. Endosomal acidification inhibitors were used to explore the endosomal escape mechanisms of the polyplexes and lysosomes. We concluded that the polyplexes were internalized by non-caveolae- and non-clathrin-mediated pathways, with no lysosomal trafficking, thereby inducing successful transfection, while the majority of liposomes were internalized by clathrin-dependent endocytosis (CDE), caveolae-mediated endocytosis, and macropinocytosis, and only CDE induced successful transfection. Liposomes might escape more quickly than polyplexes, and the digestion effect of acidic organelles on liposomes was faint compared to the polyplexes, although both complexes escaped from endolysosomes via the proton sponge mechanism. This may be the key aspect that leads to the lower transfection efficiency of the β-CD-(D3)7/MMP-9siRNA complexes. The present study may offer some insights for the rational design of novel delivery systems with increased transfection efficiency but decreased toxicity.

  15. In Vitro Toxicity of Aluminum Nanoparticles in Human Keratinocytes

    DTIC Science & Technology

    2008-03-01

    Cell Line The HaCaT cell line consists of immortalized adult human keratinocytes developed by the German Cancer Research Center (DKFZ, Heidelberg...IL-8 using the R & D Systems®, Inc. assay kits would help validate the results of this research. The HaCaT cell line used in this study did not... HaCaT cell line . 1. Vacuum excess liquid from cell culture flasks. 2. Wash cells with 5 mL of 1X PBS. 3. Vacuum liquid from flask.

  16. Effect of recombinant erythropoietin on functional activity of cultured human cells.

    PubMed

    Emel'yanova, E A; Kosykh, A V; Sukhanov, Yu V; Vorotelyak, E A; Vasil'ev, A V

    2012-08-01

    We studied the effect of recombinant human erythropoietin on functional activity of skin cells in vitro. It was found that erythropoietin stimulated proliferation of mesenchymal and epithelial cells and effectively protected epidermal HaCaT cells from apoptosis. Insignificant effect of erythropoietin on contraction of collagen gel by mesenchymal cells was revealed. These findings suggest that erythropoietin can be a promising component of wound-healing preparations.

  17. Expression of IL-10, TGF-β1 and TNF-α in Cultured Keratinocytes (HaCaT Cells) after IPL Treatment or ALA-IPL Photodynamic Treatment

    PubMed Central

    Byun, Ji Yeon; Choi, Hae Young; Myung, Ki Bum

    2009-01-01

    Background Depending on the light dose and concentration of photosensitizer for photodynamic treatment (PDT), a multitude of dose-related events are demonstrable in PDT-treated cells. Sublethal doses may result in the alteration of cytokine release and consequently modify immune actions, rather than cause cell death. Objective The purpose of this study was to investigate cytokine expression in cultured HaCaT cells after intense pulse light (IPL) treatment or PDT utilizing 5-aminolevulinic acid (ALA) and IPL at sublethal doses. Methods Cultured HaCaT cells were treated with either IPL only (4, 8 and 12 J/cm2) or ALA-IPL PDT (100µmol/L of ALA; 0, 4, 8, and 12 J/cm2 of IPL). The expression of IL-10, TGF-β1 and TNF-α was investigated by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay. Results IL-10 protein increased up to 5.95-fold after IPL treatment and up to 2.85-fold after PDT. TGF-β1 mRNA and protein showed slight increases after both IPL treatment and PDT, of which the latter induced slightly larger increases. TNF-α mRNA and protein showed no induction or reduction after PDT. Conclusion Increased expressions of IL-10 and TGF-β1 was observed after PDT. The induction of IL-10 may contribute to the anti-inflammatory effect, which explains the therapeutic benefit of PDT for inflammatory dermatoses, and that of TGF-β1 may be related to the therapeutic effect for psoriasis. The finding that IL-10 induction was more marked after IPL treatment than after PDT suggests that other mechanisms than IL-10 induction in keratinocytes after PDT may participate in the anti-inflammatory effect of PDT. PMID:20548849

  18. Identification of protein phosphatase interacting proteins from normal and UVA-irradiated HaCaT cell lysates by surface plasmon resonance based binding technique using biotin-microcystin-LR as phosphatase capturing molecule.

    PubMed

    Bécsi, Bálint; Dedinszki, Dóra; Gyémánt, Gyöngyi; Máthé, Csaba; Vasas, Gábor; Lontay, Beáta; Erdődi, Ferenc

    2014-09-05

    Identification of the interacting proteins of protein phosphatases is crucial to understand the cellular roles of these enzymes. Microcystin-LR (MC-LR), a potent inhibitor of protein phosphatase-1 (PP1), -2A (PP2A), PP4, PP5 and PP6, was biotinylated, immobilized to streptavidin-coupled sensorchip surface and used in surface plasmon resonance (SPR) based binding experiments to isolate phosphatase binding proteins. Biotin-MC-LR captured PP1 catalytic subunit (PP1c) stably and the biotin-MC-LR-PP1c complex was able to further interact with the regulatory subunit (MYPT1) of myosin phosphatase. Increased biotin-MC-LR coated sensorchip surface in the Surface Prep unit of Biacore 3000 captured PP1c, PP2Ac and their regulatory proteins including MYPT1, MYPT family TIMAP, inhibitor-2 as well as PP2A-A and -Bα-subunits from normal and UVA-irradiated HaCaT cell lysates as revealed by dot blot analysis of the recovered proteins. Biotin-MC-LR was used for the subcellular localization of protein phosphatases in HaCaT cells by identification of phosphatase-bound biotin-MC-LR with fluorescent streptavidin conjugates. Partial colocalization of the biotin-MC-LR signals with those obtained using anti-PP1c and anti-PP2Ac antibodies was apparent as judged by confocal microscopy. Our results imply that biotin-MC-LR is a suitable capture molecule in SPR for isolation of protein phosphatase interacting proteins from cell lysates in sufficient amounts for immunological detection.

  19. Cultivated ginseng inhibits 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in NC/Nga mice and TNF-α/IFN-γ-induced TARC activation in HaCaT cells.

    PubMed

    Choi, Jae Ho; Jin, Sun Woo; Park, Bong Hwan; Kim, Hyung Gyun; Khanal, Tilak; Han, Hwa Jeong; Hwang, Yong Pil; Choi, Jun Min; Chung, Young Chul; Hwang, Sang Kyu; Jeong, Tae Cheon; Jeong, Hye Gwang

    2013-06-01

    Ginseng contains many bioactive constituents, including various ginsenosides that are believed to have anti-allergic, anti-oxidant, and immunostimulatory activities; however, its effects on atopic dermatitis (AD) remain unclear. In the current study, we hypothesized that cultivated ginseng (CG) would inhibit 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin lesions in NC/Nga mice by regulating the T helper (Th)1/Th2 balance. Also, CG inhibits TNF-α/IFN-γ-induced thymus- and activation-regulated chemokine (TARC) expression through nuclear factor-kappa B (NF-κB)-dependent signaling in HaCaT cells. CG ameliorated DNCB-induced dermatitis severity, serum levels of IgE and TARC, and mRNA expression of TARC, TNF-α, IFN-γ, IL-4, IL-5, and IL-13 in mice. Histopathological examination showed reduced thickness of the epidermis/dermis and dermal infiltration of inflammatory cells in the ears. Furthermore, CG suppressed the TNF-α/IFN-γ-induced mRNA expression of TARC in HaCaT cells. CG inhibited TNF-α/IFN-γ-induced NF-κB activation. These results suggest that CG inhibited the development of the AD-like skin symptoms by modulating Th1 and Th2 responses in the skin lesions in mice and TARC expression by suppressing TNF-α/IFN-γ-induced NF-κB activation in keratinocytes, and so may be a useful tool in the therapy of AD-like skin symptoms.

  20. Protective effects of polyvinylpyrrolidone-wrapped fullerene against intermittent ultraviolet-A irradiation-induced cell injury in HaCaT cells.

    PubMed

    Saitoh, Yasukazu; Ohta, Hiroaki; Hyodo, Sayuri

    2016-10-01

    To identify compounds that suppress UV irradiation-induced oxidative stress in the skin, various types of antioxidants have been studied. Polyvinylpyrrolidone-entrapped fullerene (C60/PVP) is known as a powerful antioxidant that exerts a cytoprotective effect against UV irradiation-induced cell injury in human skin cells and skin models. However, the effects of the alternate attractive C60/PVP feature, persistent antioxidant ability, on cytoprotection have rarely been ascertained. In this study we therefore investigated the efficacies of C60/PVP using an intermittently repeated UVA irradiation model wherein human keratinocytes were repeatedly exposed to UVA five times every 1h and compared the cytoprotective effects with those provided by ascorbic acid-2-O-phosphate-disodium salt (APS) and α-tocopherol (α-Toc). Our results demonstrated that C60/PVP yielded prominent cytoprotective effects against intermittently repeated UVA irradiation-induced injuries in a dose-dependent manner and suppressed intracellular superoxide anion radical (O2(-)) generation both during and after the repeated UVA irradiation. Additionally, C60/PVP also repressed the intermittent UVA irradiation-induced apoptosis via suppression of chromatin condensation and caspase-3/7 activation. Furthermore, the observed cytoprotective effects were superior to the effects of the typical antioxidants APS and α-Toc. These data suggest that C60/PVP might function as a potent cosmetic antioxidant against the effects of repeated and prolonged UVA irradiation through its persistent antioxidative property.

  1. The analysis of scalp irritation by coacervates produced in hair shampoo via FTIR with focal plane array detector, X-ray photoelectron microscopy and HaCaT cells.

    PubMed

    Jung, I K; Park, S C; Kim, S H; Kim, J H; Cha, N R; Bae, W R; Kim, H N; Cho, S A; Yoo, J W; Kim, B M; Lee, J H

    2017-04-01

    Coacervates are inevitably formed on scalp on using hair washing products. Our goal was to analyse the coacervates in detail to identify the part responsible for scalp stimulation. Shampoo that increases coacervate formation was applied to in vitro skin and was washed. The residue was then analysed using Fourier transform infrared spectroscopy-focal plane array (FTIR-FPA) and X-ray photoelectron microscopy (XPS). And HaCaT cells were used for irritant test of coacervate. Through this research, it was confirmed that the coacervate was a macromolecule structurally similar to a cationic polymer and contains an anionic surfactant. Its anionic surfactant was structurally semi-stable so that it released onto scalp when it absorbs moisture. Coacervate releases sulphate bonding into the matrix when it is exposed to water. Thus, the scalp stimulation would be expected. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  2. Ma Huang Tang Suppresses the Production and Expression of Inflammatory Chemokines via Downregulating STAT1 Phosphorylation in HaCaT Keratinocytes

    PubMed Central

    Jin, Seong-Eun; Lee, Mee-Young

    2016-01-01

    Ma huang tang (MHT) is a traditional herbal medicine comprising six medicinal herbs and is used to treat influenza-like illness. However, the effects of MHT on inflammatory skin diseases have not been verified scientifically. We investigated determining the inhibitory effects of MHT against inflammation responses in skin using HaCaT human keratinocyte cells. We found that MHT suppressed production of thymus and activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22), regulated on activation of normal T-cell expressed and secreted (RANTES/CCL5), and interleukin-8 (IL-8) in tumor necrosis factor-α (TNF-α) and interferon-γ- (IFN-γ-) stimulated HaCaT cells. Consistently, MHT suppressed the mRNA expression of TARC, MDC, RANTES, and IL-8 in TNF-α and IFN-γ-stimulated cells. Additionally, MHT inhibited TNF-α and IFN-γ-stimulated signal transducer and activator of transcription 1 (STAT1) phosphorylation in a dose-dependent manner and nuclear translocation in HaCaT cells. Our finding indicates that MHT inhibits production and expression of inflammatory chemokines in the stimulated keratinocytes by downregulating STAT1 phosphorylation, suggesting that MHT may be a possible therapeutic agent for inflammatory skin diseases. PMID:27847527

  3. Ginsenoside Rb1, Rg1 and three extracts of traditional Chinese medicine attenuate ultraviolet B-induced G1 growth arrest in HaCaT cells and dermal fibroblasts involve down-regulating the expression of p16, p21 and p53.

    PubMed

    Wang, Xiao-Yong; Wang, Yun-Gui; Wang, Yan-Fei

    2011-08-01

    The aims of this study were to confirm whether traditional Chinese medicine ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1), polygonum multiflorum (PM), ginkgo extract (GE) and lycium barbarum polysaccharide (LBP) can attenuate G1 growth arrest of HaCaT cells and dermal fibroblasts induced by 10 subcytotoxic ultraviolet B (UVB) exposures, and to explore the possible mechanism in terms of the expression of cell-cycle regulatory proteins p16, p21 and p53. Ten subcytotoxic exposures to UVB induced G1 growth arrest of HaCaT cells and dermal fibroblasts. Cell-cycle analysis was performed using flow cytometry, and mRNA levels of p16, p21 and p53 were detected by a reverse transcription-polymerase chain reaction (RT-PCR), and protein levels were detected using Western blot analysis. Five types of traditional Chinese medicine attenuated UVB-induced G1 growth arrest. The mRNA and protein levels of p16, p21 and p53 in HaCaT cells and dermal fibroblasts increased after UVB irradiation, but pretreatment with five types of traditional Chinese medicine decreased the expression of p16, p21 and p53. These results indicated that five types of traditional Chinese medicine can attenuate G1 growth arrest of HaCaT cells and dermal fibroblasts induced by UVB exposures, which was caused by down-regulating the expression of cell-cycle regulatory proteins p16, p21 and p53. © 2011 John Wiley & Sons A/S.

  4. Exposure to Carbon Nanotube Material: Assessment of Nanotube Cytotoxicity Using Human Keratinocyte Cells

    NASA Technical Reports Server (NTRS)

    Shvedova, Anna A.; Castranova, Vincent; Kisin, Elena R.; Schwegler-Berry, Diane; Murray, Ashley R.; Gandelsman, Vadim Z.; Maynard, Andrew; Baron, Paul

    2003-01-01

    Carbon nanotubes are new members of carbon allotropes similar to fullerenes and graphite. Because of their unique electrical, mechanical, and thermal properties, carbon nanotubes are important for novel applications in the electronics, aerospace, and computer industries. Exposure to graphite and carbon materials has been associated with increased incidence of skin diseases, such as carbon fiber dermatitis, hyperkeratosis, and naevi. We investigated adverse effects of single-wall carbon nanotubes (SWCNT) using a cell culture of immortalized human epidermal keratinocytes (HaCaT). After 18 h of exposure of HaCaT to SWCNT, oxidative stress and cellular toxicity were indicated by formation of free radicals, accumulation of peroxidative products, antioxidant depletion, and loss of cell viability. Exposure to SWCNT also resulted in ultrastructural and morphological changes in cultured skin cells. These data indicate that dermal exposure to unrefined SWCNT may lead to dermal toxicity due to accelerated oxidative stress in the skin of exposed workers.

  5. Exposure to Carbon Nanotube Material: Assessment of Nanotube Cytotoxicity Using Human Keratinocyte Cells

    NASA Technical Reports Server (NTRS)

    Shvedova, Anna A.; Castranova, Vincent; Kisin, Elena R.; Schwegler-Berry, Diane; Murray, Ashley R.; Gandelsman, Vadim Z.; Maynard, Andrew; Baron, Paul

    2003-01-01

    Carbon nanotubes are new members of carbon allotropes similar to fullerenes and graphite. Because of their unique electrical, mechanical, and thermal properties, carbon nanotubes are important for novel applications in the electronics, aerospace, and computer industries. Exposure to graphite and carbon materials has been associated with increased incidence of skin diseases, such as carbon fiber dermatitis, hyperkeratosis, and naevi. We investigated adverse effects of single-wall carbon nanotubes (SWCNT) using a cell culture of immortalized human epidermal keratinocytes (HaCaT). After 18 h of exposure of HaCaT to SWCNT, oxidative stress and cellular toxicity were indicated by formation of free radicals, accumulation of peroxidative products, antioxidant depletion, and loss of cell viability. Exposure to SWCNT also resulted in ultrastructural and morphological changes in cultured skin cells. These data indicate that dermal exposure to unrefined SWCNT may lead to dermal toxicity due to accelerated oxidative stress in the skin of exposed workers.

  6. Real-time Visualization of Photochemically Induced Fluorescence of 8-Halogenated Quinolones: Lomefloxacin, Clinafloxacin and Bay3118 in Live Human HaCaT Keratinocytes†

    PubMed Central

    Koker, Edmond B.; Bilski, Piotr J.; Motten, Ann G.; Zhao, Baozhong; Chignell, Colin F.; He, Yu-Ying

    2010-01-01

    Halogenoquinolones are potent and widely used antimicrobials blocking microbial DNA synthesis. However, they induce adverse photoresponses through the absorption of UV light, including phototoxicity and photocarcinogenicity. The phototoxic responses may be the result of photosensitization of singlet oxygen, production of free radicals and/or other reactive species resulting from photodehalogenation. Here, we report the use of laser scanning confocal microscopy to detect and to follow the fluorescence changes of one monohalogenated and three di-halogenated quinolones in live human epidermal keratinocyte cells during in situ irradiation by confocal laser in real time. Fluorescence image analysis and co-staining with the LysoTracker probe showed that lysosomes are a preferential site of drug localization and phototransformations. As the lysosomal environment is relatively acidic, we also determined how low pH may affect the dehalogenation and concomitant fluorescence. With continued UV irradiation, fluorescence increased in the photoproducts from BAY y3118 and clinafloxacin, whereas it decreased for lomefloxacin and moxifloxacin. Our images not only help to localize these phototoxic agents in the cell, but also provide means for dynamic monitoring of their phototransformations in the cellular environment. PMID:20492567

  7. Mitochondrial tolerance to single and repeat exposure to simulated sunlight in human epidermal and dermal skin cells.

    PubMed

    Kelly, J; Murphy, J E J

    2016-12-01

    Sunlight represents the primary threat to mitochondrial integrity in skin given the unique nature of the mitochondrial genome and its proximity to the electron transport chain. The accumulation of mitochondrial DNA (mtDNA) mutations is a key factor in many human pathologies and this is linked to key roles of mitochondrial function in terms of energy production and cell regulation. The main objective of this study was to evaluate solar radiation induced changes in mitochondrial integrity, function and dynamics in human skin cells using a Q-Sun solar simulator to deliver a close match to the intensity of summer sunlight. Spontaneously immortalised human skin epidermal keratinocytes (HaCaT) and Human Dermal Fibroblasts (HDFn) were divided into two groups. Group A were irradiated once and Group B twice 7days apart and evaluated using cell survival, viability and mitochondrial membrane potential (MMP) and mass at 1, 4 and 7days post one exposure for Group A and 1, 4, 7 and 14days post second exposure for Group B. Viability and survival of HaCaT and HDFn cells decreased after repeat exposure to Simulated Sunlight Irradiation (SSI) with no recovery. HDFn cells showed no loss in MMP after one or two exposures to SSI compared to HaCaT cells which showed a periodic loss of MMP after one exposure with a repeat exposure causing a dramatic decrease from which cells did not recover. Mitochondrial Mass in exposed HDFn cells was consistent with control after one or two exposures to SSI; however mitochondrial mass was significantly decreased in HaCaT cells. Data presented here suggests that mitochondria in epidermal cells are more sensitive to sunlight damage compared to mitochondria in dermal cells, despite their origin, confirming a skin layer specific sensitivity to sunlight, but not as expected. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Human ACAT inhibitory effects of shikonin derivatives from Lithospermum erythrorhizon.

    PubMed

    An, Sojin; Park, Yong-Dae; Paik, Young-Ki; Jeong, Tae-Sook; Lee, Woo Song

    2007-02-15

    Three naphthoquinones were isolated by bioassay-guided fractionation from the CHCl(3) extracts of roots of Lithospermum erythrorhizon. They were identified as acetylshikonin (1), isobutyrylshikonin (2), and beta-hydroxyisovalerylshikonin (3) on the basis of their spectroscopic analyses. The compounds 1-3 were tested for their inhibitory activities against human ACAT-1 (hACAT-1) or human ACAT-2 (hACAT-2). Compound 2 preferentially inhibited hACAT-2 (IC(50)=57.5microM) than hACAT-1 (32% at 120microM), whereas compounds 1 and 3 showed weak inhibitory activities in both hACAT-1 and -2. To develop more potent hACAT inhibitor, shikonin derivatives (5-11) were synthesized by semi-synthesis of shikonin (4), which was prepared by hydrolysis of 1-3. Among them, compounds 5 and 7 exhibited the strong inhibitory activities against hACAT-1 and -2. Furthermore, we demonstrated that compound 7 behaved as a potent ACAT inhibitor in not only in vitro assay system but also cell-based assay system.

  9. Orange peel extract, containing high levels of polymethoxyflavonoid, suppressed UVB-induced COX-2 expression and PGE2 production in HaCaT cells through PPAR-γ activation.

    PubMed

    Yoshizaki, Norihiro; Fujii, Takahiro; Masaki, Hitoshi; Okubo, Takeshi; Shimada, Kunio; Hashizume, Ron

    2014-10-01

    Ultraviolet light (UV) induces an inflammatory response in the skin by cyclooxygenase (COX)-2 expression and prostaglandin (PG) E2 production. Citrus peel has been used as a natural medicine. It contains polymethoxyflavonoids (PMFs) as a major ingredient, which have anti-inflammatory activity. We obtained orange peel extract containing high levels of PMFs. The extract suppressed UVB-induced COX-2 expression and PGE2 production in HaCaT cells. Furthermore, it was found that this extract acted as a peroxisome proliferator-activated receptor (PPAR)-γ agonist. The suppression of UVB-induced COX-2 expression by this extract was inhibited by GW 9662 and T0070907, which are both PPAR-γ antagonists. It is therefore suggested that orange peel extract, containing high levels of PMFs, suppresses UVB-induced COX-2 expression and PGE2 production through PPAR-γ. Hence, these extracts could provide useful protection against or alleviation of UV damage. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Fucoidan reduces oxidative stress by regulating the gene expression of HO‑1 and SOD‑1 through the Nrf2/ERK signaling pathway in HaCaT cells.

    PubMed

    Ryu, Min Ju; Chung, Ha Sook

    2016-10-01

    Fucoidan, a sulfated polysaccharide, is found in edible brown algae. In the present study, the molecular mechanisms of fucoidan against mild oxidative stress in human keratinocytes were investigated. The current study indicated that fucoidan significantly augmented the antioxidants heme oxygenase‑1 (HO‑1) and superoxide dismutase‑1 (SOD‑1) via the upregulation of nuclear factor erythroid 2‑related factor 2 (Nrf2) and markedly reduced the cytoplasmic stability of kelch‑like ECH‑associated protein 1. The upregulation of HO‑1 and SOD‑1 detected in the fucoidan‑treated cells may be responsible for the increased resistance to mild oxidative stress, indicating that fucoidan may augment the activities of antioxidant enzymes via stimulating Nrf2. This is the first report, to the best of our knowledge, to demonstrate that fucoidan attenuates oxidative stress by regulating the gene expression of SOD‑1 and HO‑1 via the Nrf2/extracellular signal‑regulated kinase signaling pathway.

  11. Allopurinol suppresses expression of the regulatory T-cell migration factors TARC/CCL17 and MDC/CCL22 in HaCaT keratinocytes via restriction of nuclear factor-κB activation.

    PubMed

    Osabe, Makoto; Tajika, Toshiyuki; Tohkin, Masahiro

    2017-09-26

    Recent studies have shown that sparse distribution of regulatory T cells (Tregs) in the skin might be involved in the onset of severe cutaneous adverse drug reactions such as Stevens-Johnson syndrome and toxic epidermal necrolysis. Treg migration toward epithelial cells is regulated by certain chemokines, including TARC/CCL17 and MDC/CCL22. In this study, we analyzed the effect of allopurinol (APN), a drug known to cause severe adverse reactions, on the expression of factors affecting Treg migration and the mechanisms involved. APN inhibited the tumor necrosis factor (TNF)-α- and interferon (IFN)-γ-associated expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells in a dose-dependent manner. Consistent with this, APN also suppressed TNF-α- and IFN-γ-induced production of TARC/CCL17 and MDC/CCL22 proteins and the migration of C-C chemokine receptor type 4-positive cells. Activity of the transcription factors NF-κB and STAT1, which are involved in TARC/CCL17 and MDC/CCL22 expression, was also investigated. APN inhibited activation of NF-κB, but not that of STAT1. Furthermore, it restricted p38 MAPK phosphorylation. These results suggest that APN inhibits TNF-α- and IFN-γ-induced TARC/CCL17 and MDC/CCL22 production through downregulation of p38 MAPK and NF-κB signaling, resulting in the sparse distribution of Tregs in the skin of patients with APN-associated Stevens-Johnson syndrome/toxic epidermal necrolysis. Copyright © 2017 John Wiley & Sons, Ltd.

  12. Centella asiatica protects against UVB-induced HaCaT keratinocyte damage through microRNA expression changes.

    PubMed

    An, In-Sook; An, Sungkwan; Choe, Tae-Βoo; Kang, Sang-Μo; Lee, Jae Ho; Park, In-Chul; Jin, Young-Woo; Lee, Su-Jae; Bae, Seunghee

    2012-12-01

    This study aimed to evaluate the protective effects of Centella asiatica (C. asiatica) against ultraviolet B (UVB) damage in human keratinocytes using microRNA (miRNA) expression profiling analysis. Titrated extract of C. asiatica (TECA) demonstrated low cytotoxicity in normal human HaCaT keratinocytes only at low doses (<5 µg/ml). UVB (50 mJ/cm2) irradiation significantly decreased cell viability, and TECA treatment decreased the UVB toxicity. By using miRNA microarrays, we determined that 72 miRNAs had an altered expression following TECA treatment in UVB-irradiated keratinocytes (46 upregulated and 26 downregulated). Using an miRNA target gene prediction tool and Gene Ontology (GO) analysis, we determined that miRNAs with altered expression were functionally related with the inhibition of apoptosis and cell proliferation. Overall, these results provide meaningful information to facilitate the understanding of TECA-mediated UVB protection in human keratinocytes.

  13. Bifunctional Alkylating Agent-Induced p53 and Nonclassical Nuclear Factor kB Responses and Cell Death are Altered by Caffeic Acid Phenethyl Ester: A Potential Role for Antioxidant/Electrophilic Response-Element Signaling

    DTIC Science & Technology

    2007-01-01

    CAPE disrupted BFA-induced phosphoryla- talized HaCaT keratinocytes, a p53-mutated cell line , to delin- tion of p53 and p90 ribosomal S6 kinase (p90RSK...Exposure of an NF-KB reporter gene-expressing HaCaT anisms of action of BFA-induced inflammation and cell death cell line to 12.5, 50, or 100 AM SM activated...were examined BFA-induced loss of membrane integrity by 24 h in HaCaT cells in normal human epidermal keratinocytes (NHEK) and immor- but not in NHEK

  14. Circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis.

    PubMed

    Xue, Junchao; Liu, Yang; Luo, Fei; Lu, Xiaolin; Xu, Hui; Liu, Xinlu; Lu, Lu; Yang, Qianlei; Chen, Chao; Fan, Weimin; Liu, Qizhan

    2017-03-01

    Circular RNAs (circRNAs), a class of noncoding RNAs generated from pre-mRNAs, participate in regulation of genes. The mechanism for regulation, however, is unknown. Here, to determine if, in human keratinocyte (HaCaT) cells, circular RNAs are involved in arsenite-induced acceleration of the cell cycle, a circRNA microarray was performed to analyze the variability of circRNAs in arsenite-treated HaCaT (As-HaCaT) cells and in arsenite-transformed (T-HaCaT) cells in comparison to control HaCaT cells. Among the circRNAs up-regulated in both As-HaCaT cells and T-HaCaT cells, hsa:circRNA_100284 (circ100284) had the greatest increase and was chosen for further research. The presence of circ100284 was confirmed in HaCaT cells. In these cells, arsenite induced increases of EZH2 and cyclin D1 and accelerated the cell cycle. MicroRNA (miR)-217 suppressed the expression of EZH2 was involved in regulation of the cell cycle. Further, in HaCaT cells exposed to arsenite, EZH2 regulated the cell cycle by binding to the promoter of CCND1, which codes for cyclin D1. Moreover, knockdown of circ100284 with siRNA inhibited the cell cycle acceleration induced by arsenite, but this inhibition was reversed by co-transfection with circ100284 siRNA and by a miR-217 inhibitor. Knockdown of circ100284 with siRNA or transfected with miR-217 mimic inhibited the capacity of T-HaCaT cells for colony formation, invasion, and migration, effects that were reversed by co-transfection with a miR-217 inhibitor or by epigenetic expression of EZH2. These results suggest that, in HaCaT cells, arsenite increases circ100284 levels, which act as a sponge for miR-217 and up-regulate the miR-217 target, EZH2, which, in turn, up-regulates cyclin D1and CDK4, and thus accelerates the cell cycle and leads to malignant transformation. Thus, circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis. This establishes a previously unknown

  15. Effects of lunar and mars dust simulants on HaCaT keratinocytes and CHO-K1 fibroblasts

    NASA Astrophysics Data System (ADS)

    Rehders, Maren; Grosshäuser, Bianka B.; Smarandache, Anita; Sadhukhan, Annapurna; Mirastschijski, Ursula; Kempf, Jürgen; Dünne, Matthias; Slenzka, Klaus; Brix, Klaudia

    2011-04-01

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respiratory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of extraterrestrial lunar dusts on human health is required to best support future missions to moon, mars or other destinations. In this study, we used several methods to assess the specific effects of extraterrestrial dusts onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and because a well orchestrated program ensures proper wound healing. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology and viability of the cells were determined. Cytotoxicity was measured using the MTT assay and by monitoring culture impedance, while phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells which was also investigated by propidium iodide intake. It was found that the effects of the two types of dust simulants on the different features of both cell lines varied to a considerable extent. Moreover, proliferation of HaCaT keratinocytes, as analyzed by Ki67 labeling, was suppressed in sub-confluent cultures exposed to lunar dust simulant. Furthermore, experimental evidence is provided for a delay in regeneration of keratinocyte monolayers from scratch-wounding when exposed to lunar dust simulant. The obtained results will facilitate further investigations of dust exposure during wound healing and will ease risk assessment studies e.g., for lunar lander approaches. The investigations will help to determine safety measures to be taken during

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

    PubMed

    Pérez-Sánchez, A; Barrajón-Catalán, E; Caturla, N; Castillo, J; Benavente-García, O; Alcaraz, M; Micol, V

    2014-07-05

    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 250 mg 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 12 weeks (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.

  17. A Sensitive Sensor Cell Line for the Detection of Oxidative Stress Responses in Cultured Human Keratinocytes

    PubMed Central

    Hofmann, Ute; Priem, Melanie; Bartzsch, Christine; Winckler, Thomas; Feller, Karl-Heinz

    2014-01-01

    In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells) expressing green fluorescent protein (GFP) under the control of the stress-inducible HSP70B' promoter were constructed. Exposure of HaCaT sensor cells to 25 μM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ∼300-fold induction of transcript level of the gene coding for heat shock protein HSP70B'. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B' gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B' promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 μM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (μTAS) that may be utilized in dermatology, toxicology, pharmacology and drug screenings. PMID:24967604

  18. A sensitive sensor cell line for the detection of oxidative stress responses in cultured human keratinocytes.

    PubMed

    Hofmann, Ute; Priem, Melanie; Bartzsch, Christine; Winckler, Thomas; Feller, Karl-Heinz

    2014-06-25

    In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells) expressing green fluorescent protein (GFP) under the control of the stress-inducible HSP70B' promoter were constructed. Exposure of HaCaT sensor cells to 25 µM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ~300-fold induction of transcript level of the gene coding for heat shock protein HSP70B'. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B' gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B' promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 µM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (µTAS) that may be utilized in dermatology, toxicology, pharmacology and drug screenings.

  19. Cell death pathways in directly irradiated cells and cells exposed to medium from irradiated cells.

    PubMed

    Jella, Kishore Kumar; Garcia, Amaya; McClean, Brendan; Byrne, Hugh J; Lyng, Fiona M

    2013-03-01

    The aim of this study was to compare levels of apoptosis, necrosis, mitotic cell death and senescence after treatment with both direct radiation and irradiated cell conditioned medium. Human keratinocytes (HaCaT cell line) were irradiated (0.005, 0.05 and 0.5 Gy) using a cobalt 60 teletherapy unit. For bystander experiments, the medium was harvested from donor HaCaT cells 1 hour after irradiation and transferred to recipient HaCaT cells. Clonogenic assay, apoptosis, necrosis, mitotic cell death, senescence and cell cycle analysis were measured in both directly irradiated cells and bystander cells A reduction in cell survival was observed for both directly irradiated cells and irradiated cell conditioned medium (ICCM)-treated cells. Early apoptosis and necrosis was observed predominantly after direct irradiation. An increase in the number of cells in G2/M phase was observed at 6 and 12 h which led to mitotic cell death after 72 h following direct irradiation and ICCM treatment. No senescence was observed in the HaCaT cell line following either direct irradiation or treatment with ICCM. This study has shown that directly irradiated cells undergo apoptosis, necrosis and mitotic cell death whereas ICCM-treated cells predominantly undergo mitotic cell death.

  20. Tamarind Seed Xyloglucans Promote Proliferation and Migration of Human Skin Cells through Internalization via Stimulation of Proproliferative Signal Transduction Pathways

    PubMed Central

    Nie, W.; Deters, A. M.

    2013-01-01

    Xyloglucans (XGs) of Tamarindus indica L. Fabaceae are used as drug vehicles or as ingredients of cosmetics. Two xyloglucans were extracted from T. indica seed with cold water (TSw) and copper complex precipitation (TSc). Both were analyzed in regard to composition and influence on cell viability, proliferation, cell cycle progression, migration, MAPK phosphorylation, and gene expression of human skin keratinocytes (NHEK and HaCaT) and fibroblasts (NHDF) in vitro. TSw and TSc differed in molecular weight, rhamnose content, and ratios of xylose, arabinose, galactose, and glucose. Both XGs improved keratinocytes and fibroblast proliferation, promoted the cell cycle, and stimulated migration and intracellular enzyme activity of NHDF after endosomal uptake. Only TSw significantly enhanced HaCaT migration and extracellular enzyme activity of NHDF and HaCaT. TSw and TSc predominantly enhanced the phosphorylation of molecules that referred to Erk signaling in NHEK. In NHDF parts of the integrin signaling and SAPK/JNK pathway were affected. Independent of cell type TSw marginally regulated the expression of genes, which referred to membrane proteins, cytoskeleton, cytokine signaling, and ECM as well as to processes of metabolism and transcription. Results show that T. indica xyloglucans promote skin regeneration by a direct influence on cell proliferation and migration. PMID:24106497

  1. Enhanced constitutive invasion activity in human nontumorigenic keratinocytes exposed to a low level of barium for a long time.

    PubMed

    Thang, Nguyen D; Yajima, Ichiro; Ohnuma, Shoko; Ohgami, Nobutaka; Kumasaka, Mayuko Y; Ichihara, Gaku; Kato, Masashi

    2015-02-01

    We have recently demonstrated that exposure to barium for a short time (≤4 days) and at a low level (5 µM = 687 µg/L) promotes invasion of human nontumorigenic HaCaT cells, which have characteristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time enhances malignant characteristics. Here we examined the effect of exposure to low level of barium for a long time, a condition mimicking the exposure to barium through well water, on malignant characteristics of HaCaT keratinocytes. Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma cells were augmented following an increase in malignancy grade of the cells. Constitutive invasion activity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with 5 µM barium for 4 months were significantly higher than those of control untreated HaCaT keratinocytes. Taken together, our results suggest that exposure to a low level of barium for a long time enhances constitutive malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion.

  2. Aronia melanocarpa Concentrate Ameliorates Pro-Inflammatory Responses in HaCaT Keratinocytes and 12-O-Tetradecanoylphorbol-13-Acetate-Induced Ear Edema in Mice.

    PubMed

    Goh, Ah Ra; Youn, Gi Soo; Yoo, Ki-Yeon; Won, Moo Ho; Han, Sang-Zin; Lim, Soon Sung; Lee, Keun Wook; Choi, Soo Young; Park, Jinseu

    2016-07-01

    Abnormal expression of pro-inflammatory mediators such as cell adhesion molecules and cytokines has been implicated in various inflammatory skin diseases, including atopic dermatitis. In this study, we investigated the anti-inflammatory activity of Aronia melanocarpa concentrate (AC) and its action mechanisms using in vivo and in vitro skin inflammation models. Topical application of AC on mouse ears significantly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema formation, as judged by measuring ear thickness and weight, and histological analysis. Topical administration of AC also reduced the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in TPA-stimulated mouse ears. Pretreatment with AC suppressed TNF-α-induced ICAM-I expression and subsequent monocyte adhesiveness in human keratinocyte cell line HaCaT. In addition, AC significantly decreased intracellular reactive oxygen species (ROS) generation as well as mitogen-activated protein kinase (MAPK) activation in TNF-α-stimulated HaCaT cells. AC and its constituent cyanidin 3-glucoside also attenuated TNF-α-induced IKK activation, IκB degradation, p65 phosphorylation/nuclear translocation, and p65 DNA binding activity in HaCaT cells. Overall, our results indicate that AC exerts anti-inflammatory activities by inhibiting expression of pro-inflammatory mediators in vitro and in vivo possibly through suppression of ROS-MAPK-NF-κB signaling pathways. Therefore, AC may be developed as a therapeutic agent to treat various inflammatory skin diseases.

  3. Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells.

    PubMed

    Huang, Li-Wen; Hsieh, Bau-Shan; Cheng, Hsiao-Ling; Hu, Yu-Chen; Chang, Wen-Tsan; Chang, Kee-Lung

    2012-01-15

    Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24h exposure, arecoline inhibited BCC-1/KMC cell growth and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis.

  4. alpha-Melanocyte-stimulating hormone, MSH 11-13 KPV and adrenocorticotropic hormone signalling in human keratinocyte cells.

    PubMed

    Elliott, Richard J; Szabo, Marika; Wagner, Mark J; Kemp, E Helen; MacNeil, Sheila; Haycock, John W

    2004-04-01

    alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of alpha-MSH (KPV / KP-D-V) are the smallest minimal sequences reported to prevent inflammation but it is not known if they operate via MC-1R or cyclic AMP. The aim of this study was to examine the intracellular signalling of key MSH and ACTH peptides in human keratinotocytes. No elevation in cyclic AMP was detected in either HaCaT or normal human keratinocytes in response to alpha-MSH, KPV or ACTH peptides. Rapid and acute intracellular calcium, however, were observed in HaCaT keratinocytes in response to alpha-MSH (10(-15)-10(-7) M), KPV (10(-15)-10(-7) M), KP-D-V (10(-15)-10(-7) M) and ACTH (10(-15)-10(-7) M), but only in the presence of PIA, an adenosine agonist that inhibits the cyclic AMP pathway. Normal keratinocytes responded to all the above peptides but in addition responded to ACTH 1-17 (10(-13)-10(-7) M) in contrast to the HaCaT keratinocytes. Stable transfection of Chinese hamster ovary cells with the MC-1 receptor showed that alpha-MSH and the KPV peptides elevated intracellular calcium.

  5. Human papillomavirus 16 E5 induces bi-nucleated cell formation by cell-cell fusion

    SciTech Connect

    Hu Lulin; Plafker, Kendra; Vorozhko, Valeriya; Zuna, Rosemary E.; Hanigan, Marie H.; Gorbsky, Gary J.; Plafker, Scott M.; Angeletti, Peter C.; Ceresa, Brian P.

    2009-02-05

    Human papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes - E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis.

  6. Influence of chronic low-dose/dose-rate high-LET irradiation from radium-226 in a human colorectal carcinoma cell line.

    PubMed

    Vo, Nguyen T K; Sokeechand, Bibi S H; Seymour, Colin B; Mothersill, Carmel E

    2017-07-01

    To evaluate potential damages of chronic environmentally relevant low-dose/dose-rate high-LET irradiation from a naturally occurring alpha-emitting radionuclide (radium-226, (226)Ra) on a human colorectal carcinoma HCT116 p53(+/+) cell line. Clonogenic survival assays and mitochondrial membrane potential (MMP) measurement with a sensitive fluorescent MMP probe JC-1 were performed in HCT116 p53(+/+) cells chronically exposure to low doses/dose rates of (226)Ra with high-LET. Comparisons were made with the human non-transformed keratinocyte HaCaT cell line and acute low-dose direct low-LET gamma radiation. The chronic low-dose/dose-rate alpha radiation (CLD/DRAR) did not reduce the clonogenic survival of HCT116 p53(+/+) cells over the period of 70 days of exposure. Only one significant reduction in the HCT116 p53(+/+) cells' clonogenic survival was when cells were grown with 10,000mBq/mL (226)Ra for 40 days and progeny cells were clonogenically assessed in the presence of 10,000mBq/mL (226)Ra. The cumulative doses that cells received during this period ranged from 0.05 to 46.2mGy. The mitochondrial membrane potential (MMP) dropped initially in both HCT116 p53(+/+) and HaCaT cells in response to CLD/DRAR. The MMP in HCT116 p53(+/+) cells recovered more quickly at all dose points than and that in HaCaT cells until the end of the exposure period. The highest dose rate of 0.66mGy/day depolarized the HaCaT's mitochondria more consistently during the exposure period. The faster recovery status of the MMP in HCT116 p53(+/+) cells than that in HaCaT cells was also observed after exposure to acute low-dose gamma rays. Overall, it was found that CLD/DRAR had little impact on the MMP of human colorectal cancer and keratinocyte cell lines. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Antioxidant Biomarkers from Vanda coerulea Stems Reduce Irradiated HaCaT PGE-2 Production as a Result of COX-2 Inhibition

    PubMed Central

    Simmler, Charlotte; Antheaume, Cyril; Lobstein, Annelise

    2010-01-01

    Background In our investigations towards the isolation of potentially biologically active constituents from Orchidaceae, we carried out phytochemical and biological analyses of Vanda species. A preliminary biological screening revealed that Vanda coerulea (Griff. ex. Lindl) crude hydro-alcoholic stem extract displayed the best DPPH /•OH radical scavenging activity and in vitro inhibition of type 2 prostaglandin (PGE-2) release from UVB (60 mJ/cm2) irradiated HaCaT keratinocytes. Principal Findings Bio-guided fractionation and phytochemical analysis led to the isolation of five stilbenoids: imbricatin (1) methoxycoelonin (2) gigantol (3) flavidin (4) and coelonin (5). Stilbenoids (1–3) were the most concentrated in crude hydro-alcoholic stem extract and were considered as Vanda coerulea stem biomarkers. Dihydro-phenanthropyran (1) and dihydro-phenanthrene (2) displayed the best DPPH/•OH radical scavenging activities as well as HaCaT intracellular antioxidant properties (using DCFH-DA probe: IC50 8.8 µM and 9.4 µM, respectively) compared to bibenzyle (3) (IC50 20.6 µM). In turn, the latter showed a constant inhibition of PGE-2 production, stronger than stilbenoids (1) and (2) (IC50 12.2 µM and 19.3 µM, respectively). Western blot analysis revealed that stilbenoids (1–3) inhibited COX-2 expression at 23 µM. Interestingly, stilbenoids (1) and (2) but not (3) were able to inhibit human recombinant COX-2 activity. Conclusions Major antioxidant stilbenoids (1–3) from Vanda coerulea stems displayed an inhibition of UVB-induced COX-2 expression. Imbricatin (1) and methoxycoelonin (2) were also able to inhibit COX-2 activity in a concentration-dependent manner thereby reducing PGE-2 production from irradiated HaCaT cells. Our studies suggest that stilbenoids (1–3) could be potentially used for skin protection against the damage caused by UVB exposure. PMID:21060890

  8. Topical treatments of Saussurea costus root and Thuja orientalis L. synergistically alleviate atopic dermatitis-like skin lesions by inhibiting protease-activated receptor-2 and NF-κB signaling in HaCaT cells and Nc/Nga mice.

    PubMed

    Yang, Hye Jeong; Kim, Min Jung; Kang, Suna; Moon, Na Rang; Kim, Da Sol; Lee, Na Ra; Kim, Kang Sung; Park, Sunmin

    2017-03-06

    The root of Saussurea costus (Aucklandia lappa Decne, Aucklandiae Radix, SC) and Thuja orientalis L. (TOL) have been traditionally used as anti-inflammatory agents in Korea. However, they have not been studied for the efficacy of atopic dermatitis (AD) treatment, a chronic inflammatory skin disease. We investigated the efficacy of topical applications with 1,3-butyleneglycol extracts of SC and TOL to alleviate the symptoms of AD. HaCaT cells and the dorsal skin of Nc/Nga mice had a local exposure of house mite extracts and 2,4-dinitrochlorobenzene (DNCB), respectively. After lesions developed, we topically applied 1,3-butylen glycol (vehicle; control), SC (30%), TOL (30%), or SC (15%)+TOL (15%) to the skin lesions for 5 weeks. The normal-control was not exposed to DNCB. The skin thickness, mast cell infiltration, serum immunoglobulin E (IgE) and IgG1 and gene expressions of interleukin (IL)-4, IL-13, and IFN-γ in the dorsal skin and HaCaT cells were measured. Chlorogenic acid (129.6±10.2μg/g) for SC and catechin and apigenin (93.4±13.2 and 16.9±1.3μg/g, respectively) for TOL were used as indicator compounds for the strength of the extracts. SC+TOL decreased the expression of protease-activated receptor-2 and ICAM-1 and the release of TNF-α and IL-6 in HaCaT cells activated by 3μg/mL house mite extracts in comparison to either of SC or TOL alone. In Nc/Nga mice challenged with DNCB, SC+TOL synergistically attenuated clinical symptoms of AD such as erythema, hemorrhage, edema, excoriation and dryness in the dorsal skin better than either SC or TOL alone. Histological analysis of the dorsal skin also showed that SC+TOL treatment significantly and additively decreased the inflammatory cellular infiltrate, including mast cells and eosinophils in comparison to either of SC or TOL. SC+TOL also decreased serum IgE and IgG1 levels and the expression of IFN-γ, IL-4, and IL-13 mRNA in dorsal skin in DNCB-treated Nc/Nga mice. SC+TOL relieved the symptoms of AD by

  9. Comparison of liposomal and 2-hydroxypropyl-β-cyclodextrin-lidocaine on cell viability and inflammatory response in human keratinocytes and gingival fibroblasts.

    PubMed

    Ferreira, Luiz Eduardo Nunes; Muniz, Bruno Vilela; Dos Santos, Cleiton Pita; Volpato, Maria Cristina; de Paula, Eneida; Groppo, Francisco Carlos

    2016-06-01

    The aim of this study was to observe the effect multilamellar liposomes (MLV) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in the in-vitro effects of lidocaine in cell viability, pro-inflammatory cytokines and prostaglandin E2 release of both human keratinocytes (HaCaT) and gingival fibroblasts (HGF) cells. HaCaT and HGF cells were exposed to lidocaine 100-1 μm in plain, MLV and HP-β-CD formulations for 6 h or 24 h. The formulation effects in cell viability were measured by XTT assay and by fluorescent labelling. Cytokines (IL-8, IL-6 and TNF-α) and PGE2 release were quantified by ELISA. MLV and HP-β-CD formulations did not affect the HaCaT viability, which was significantly decreased by plain lidocaine after 24 h of exposure. Both drug carriers increased all cytokines released by HGF after 24-h exposure, and none of the carriers was able to reduce the PGE2 release induced by lidocaine. The effect of drug carrier in the lidocaine effects was dependent on the cell type, concentration and time of exposure. MLV and HP-β-CD showed benefits in improving cell viability; however, both of them showed a tendency to increase cytokine release when compared to the plain solution. © 2016 Royal Pharmaceutical Society.

  10. Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines

    PubMed Central

    Souza, Raquel P.; Gimenes, Fabrícia; Ratti, Bianca A.; Kaplum, Vanessa; Bruschi, Marcos L.; Nakamura, Celso V.; Maria-Engler, Silvya S.

    2017-01-01

    Recently, the cytotoxic effects of apigenin (4′,5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes. PMID:28191273

  11. Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines.

    PubMed

    Souza, Raquel P; Bonfim-Mendonça, Patrícia de S; Gimenes, Fabrícia; Ratti, Bianca A; Kaplum, Vanessa; Bruschi, Marcos L; Nakamura, Celso V; Silva, Sueli O; Maria-Engler, Silvya S; Consolaro, Marcia E L

    2017-01-01

    Recently, the cytotoxic effects of apigenin (4',5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes.

  12. Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes.

    PubMed

    Xie, Xin; Dai, Hui; Zhuang, Binyu; Chai, Li; Xie, Yanguang; Li, Yuzhen

    2016-04-08

    The effects and the underlying mechanisms of hydrogen sulfide (H2S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H2S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H2S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H2S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation. Copyright © 2016. Published by Elsevier Inc.

  13. The influence of Citrate or PEG coating on silver nanoparticle toxicity to a human keratinocyte cell line.

    PubMed

    Bastos, V; Ferreira de Oliveira, J M P; Brown, D; Jonhston, H; Malheiro, E; Daniel-da-Silva, A L; Duarte, I F; Santos, C; Oliveira, H

    2016-05-13

    Surface coating of silver nanoparticles may influence their toxicity, in a way yet to decipher. In this study, human keratinocytes (HaCaT cells) were exposed for 24 and 48h to well-characterized 30nm AgNPs coated either with citrate (Cit30 AgNPs) or with poly(ethylene glycol) (PEG30 AgNPs), and assessed for cell viability, reactive oxygen species (ROS), cytokine release, apoptosis and cell cycle dynamics. The results showed that Cit30 AgNPs and PEG30 AgNPs decreased cell proliferation and viability, the former being more cytotoxic. The coating molecules per se were not cytotoxic. Moreover, Ag(+) release and ROS production were similar for both AgNP types. Cit30 AgNPs clearly induced apoptotic death, while cells exposed to PEG30 AgNPs appeared to be at an earlier phase of apoptosis, supported by changes in BAX, BCL2 and CASP-3 expressions. Concerning the impact on cell cycle dynamics, both Cit30 and PEG30 AgNPs affected cell cycle regulation of HaCaT cells, but, again, citrate-coating induced more drastic effects, showing earlier downregulation of cyclin B1 gene and cellular arrest at the G2 phase. Overall, this study has shown that the surface coating of AgNPs influences their toxicity by differently regulating cell-cycle and cell death mechanisms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells

    SciTech Connect

    Huang, Li-Wen; Hsieh, Bau-Shan; Cheng, Hsiao-Ling; Hu, Yu-Chen; Chang, Wen-Tsan; Chang, Kee-Lung

    2012-01-15

    Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100 μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24 h exposure, arecoline inhibited BCC-1/KMC cell growth and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis. Highlights: ► Arecoline has potential to prevent against basal cell carcinoma tumorigenesis. ► It has more effectiveness on BCC as compared with a human keratinocyte cell line. ► Mechanisms involved including reducing tumor cells’ survival factor IL-6, ► Decreasing Cdc25C phosphatase, enhancing tumor suppressor factor p53, ► Eliciting G2/M

  15. Purinoceptor-mediated calcium mobilization and proliferation in HaCaT keratinocytes.

    PubMed

    Lee, W K; Choi, S W; Lee, H R; Lee, E J; Lee, K H; Kim, H O

    2001-02-01

    To investigate the effect of nucleotides on cytosolic free calcium mobilization and proliferation activity in HaCaT keratinocytes, nucleotides-induced intracellular free calcium concentration ([Ca(2+)](i)) and cell proliferation observed. [Ca(2+)](i) to the extracellular nucleotides was determined using Ca(2+) sensitive indicator, Fura-2/AM with digital video fluorescence imaging microscopy, and cell proliferation was evaluated by counting of cell number. An adenosine 5'-triphosphate (ATP)-induced [Ca(2+)](i) increase was observed from the concentration of 10(-8) M and was more conspicuous at higher concentrations in a concentration-dependent manner. Additionally, other nucleotides such as ADP, UTP, and 2-me-S-ATP also induced a [Ca(2+)](i) increase in a concentration-dependent manner. However, adenosine induced a slight increase of [Ca(2+)](i) only at 10(-3) M. alpha,-methylene-ATP did not evoke any rise in [Ca(2+)](i). The maximal response observed occurred with ATP and UTP at a concentration of 10(-4) M. The ATP-induced transient [Ca(2+)](i) increase was attenuated by the pretreatment with phospholipase C (PLC) inhibitor, U-73122 (10 microM) for 30 min. ATP-induced [Ca(2+)](i) increase and cell proliferation were inhibited by putative P2Y receptor antagonist, suramin (10(-4) M). When the HaCaT cells were stimulated with nucleotides on a concentration of 10(-4) M and cultured for 5 days, the order of effect on cell proliferation was observed to be ATP>UTP>ADP>2-me-S-ATP. Based on these results, we suggest that extracellular ATP stimulate HaCaT keratinocytes proliferation via purinoceptor-mediated [Ca(2+)](i) mobilization

  16. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage.

    PubMed

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-07-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death.

  17. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage

    PubMed Central

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-01-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death. PMID:26157553

  18. Wound healing potential of a dimeric InlB variant analyzed by in vitro experiments on re-epithelialization of human skin models.

    PubMed

    Kolditz, F; Krausze, J; Heinz, D W; Niemann, H H; Müller-Goymann, C C

    2014-02-01

    A constitutively dimeric truncated variant of internalin B (InlB321-CD), acting as stimulator of the receptor tyrosine kinase MET, was tested for dermal wound-healing potential. Due to a lack of the endogenous MET agonist HGF/SF in chronic wounds, HGF/SF substitution by an InlB321-CD-loaded hydrogel might be beneficial in chronic wound therapy. In this study, InlB321-CD in solution and incorporated in a hydrogel was tested for mitogenic effects on immortalized human dermal keratinocytes (HaCaT) with an MTT assay. Cell migration was investigated with a scratch assay on primary keratinocytes (PHK) and on HaCaT. For the latter, scratching needed to be mitomycin C-controlled. InlB321-CD effects on a model of human skin were analyzed histologically with respect to viability. InlB321-CD led to dose-dependent proliferative effects on HaCaT cells whereas the equimolar dose of monomeric InlB321 did not. Upon hydrogel incorporation of InlB321-CD its mitogenic activity for HaCaT cells was maintained thus confirming the hydrogel as a promising drug delivery system. Motogenic effects were shown on both HaCaT and PHK cells. InlB321-CD neither possesses cytotoxic effects on the viability of a human skin model nor alters its organotypic cell morphology.

  19. Intermediate heparan sulfate binding during HPV-16 infection in HaCaTs.

    PubMed

    Kumar, Annandita; Jacob, Taylor; Abban, Cynthia Y; Meneses, Patricio I

    2014-01-01

    Human papillomavirus (HPV) is the most prevalent sexually transmitted disease in the United States and can cause cancer with persistent infection. The most common cancer caused by HPV is cervical carcinoma with an average of 12,000 cases reported every year in the United States. Worldwide, over 500,000 cases of cervical cancer are reported yearly with over 250,000 deaths attributed to the disease. Although much is known about the serious health risks associated with HPV infection, there is still much to be discovered about how HPV binds and enters target cells. Understanding is required on how HPV infections will lead to strategies and therapies for reducing the number of infections and HPV-related diseases, including cancers. The HPV viral particle is composed of 2 viral proteins, L1 and L2. Data suggest that binding of the viral capsid to cells is dependent on the L1 protein. We hypothesize that this initial binding to a heparan sulfate is composed of 2 independent events: the first results in a structural change that exposes a hidden portion of the L1 protein leading to a second binding event on the heparan sulfate. Our experiments tested if this "hidden" portion of L1 is necessary for infection and explored the nature of this binding. We generated a peptide with the sequence of the "hidden" portion of L1. Infection of HaCaT cells in the presence of this peptide is highly reduced. Our results suggest that the binding of the L1 C-terminal domain is dependent on amino acid sequence and is necessary for infection.

  20. Formaldehyde solutions in simulated sweat increase human melanoma but not normal human keratinocyte cells proliferation.

    PubMed

    Rizzi, M; Cravello, B; Tonello, S; Renò, F

    2016-12-01

    Our skin is in close contact with clothes most of the time thus risking potentially noxious chemicals contact. One of the potentially harmful manufacturing by-products that can be released by textiles when sweating is formaldehyde, used as an anti-crease treatment. As it is known to be carcinogenic to humans and a potent skin sensitizer, the aim of this study was to investigate its effects on both normal human keratinocytes (HaCaT cells) and on a highly invasive malignant melanoma cell line (SK-MEL-28) in order to contribute to the definition of safety cut-off to be applied to the production processes. Formaldehyde concentrations below the commonly accepted limits (10-50μM) were obtained by diluting formaldehyde in simulated sweat (UNI EN ISO 105-E04). The effects on cell proliferation were evaluated by cell counting, while ERK pathway activation was evaluated by western blot. Low concentrations of formaldehyde (10μM) in both acidic and alkaline simulated sweat were able to increase malignant melanoma cell proliferation, while not affecting normal keratinocytes. Melanoma proliferation increase was greater in acidic (pH=5.5) than in alkaline (pH=8) conditions. Moreover, formaldehyde stimulation was able to induce ERK pathway activation. The data obtained suggest the need for an even increasing attention to the potentially harmful effects of textile manufacturing by-products. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Proliferation and motility of HaCaT keratinocyte derivatives is enhanced by fibroblast nemosis

    SciTech Connect

    Raesaenen, Kati; Vaheri, Antti

    2010-06-10

    The role of paracrine tumor-stroma regulation in the progression of cancer is under intense investigation. Activated fibroblasts are key components of the tumor microenvironment providing the soluble factors mediating the regulation. Nemosis is an experimental model to study these parameters: formation of a multicellular spheroid activates fibroblasts and leads to increased production of soluble factors involved in the promotion of growth and motility. Role of nemosis was investigated in the tumorigenesis of HaCaT derivatives representing skin carcinoma progression. Conditioned medium from fibroblast spheroids increased proliferation rate of HaCaT derivatives. Expression of proliferation marker Ki-67 increased significantly in benign A5 and low-grade malignant II-4 cells, but did not further increase in the metastatic RT3 cells. Expression of p63, keratinocyte stem cell marker linked to cancer progression, was augmented by medium from nemotic fibroblasts; this increase was also seen in RT3 cells. Scratch-wound healing of the keratinocytes was enhanced in response to fibroblast nemosis. Neutralizing antibodies against growth factors inhibited wound healing to some extent; the response varied between benign and malignant keratinocytes. Migration and invasion were enhanced by conditioned medium from nemotic fibroblasts in benign and low-grade malignant cells. RT3 keratinocyte migration was further augmented, but invasion was not, indicating their intrinsic capacity to invade. Our data demonstrate that fibroblast nemosis increases proliferation and motility of HaCaT keratinocyte derivatives, and thus nemosis can be used as a model to study the role of soluble factors secreted by fibroblasts in tumor progression.

  2. [Effect of cetirizine hydrochloride on the expression of substance P receptor and cytokines production in human epidermal keratinocytes and dermal fibroblasts].

    PubMed

    Liu, Ji-Yong; Zhao, Yong-Zhe; Peng, Cheng; Li, Feng-Qian; Zhu, Quan-Gang; Hu, Jin-Hong

    2008-04-01

    To investigate the effect of cetirizine hydrochloride on the expression of neurokinin 1 receptor (NK-1R) and cytokines production induced by substance P (SP) in HaCaT cells (a human epidermal keratinocyte cell line) and dermal fibroblasts. The effect of cetirizine on the expression of NK-1R protein was detected by flow cytometry and Western blotting analysis. The modulation of cetirizine on the production of interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6 and IL-8 in HaCaT cells and fibroblasts was measured by ELISA. The results showed that cetirizine significantly inhibited the expression of NK-1R in HaCaT cells and fibroblasts. SP induced the production of IFN-gamma, IL-1beta and IL-8 in both cell types. Cetirizine 1-100 micromol x L(-1) inhibited SP-induced IL-1beta and IL-8 production in HaCaT cells and fibroblasts, while had no effect on the production of IFN-gamma in both cells. Both SP and cetirizine had no effect on the secretion of IL-6 in HaCaT cells and fibroblasts. These findings suggest that cetirizine may be involved in the treatment of SP-induced skin inflammation by inhibiting the expression of substance P receptor and regulation the production of IL-1beta and IL-8 in epidermal keratinocyte and dermal fibroblasts.

  3. Human memory B cells.

    PubMed

    Seifert, M; Küppers, R

    2016-12-01

    A key feature of the adaptive immune system is the generation of memory B and T cells and long-lived plasma cells, providing protective immunity against recurring infectious agents. Memory B cells are generated in germinal center (GC) reactions in the course of T cell-dependent immune responses and are distinguished from naive B cells by an increased lifespan, faster and stronger response to stimulation and expression of somatically mutated and affinity matured immunoglobulin (Ig) genes. Approximately 40% of human B cells in adults are memory B cells, and several subsets were identified. Besides IgG(+) and IgA(+) memory B cells, ∼50% of peripheral blood memory B cells express IgM with or without IgD. Further smaller subpopulations have additionally been described. These various subsets share typical memory B cell features, but likely also fulfill distinct functions. IgM memory B cells appear to have the propensity for refined adaptation upon restimulation in additional GC reactions, whereas reactivated IgG B cells rather differentiate directly into plasma cells. The human memory B-cell pool is characterized by (sometimes amazingly large) clonal expansions, often showing extensive intraclonal IgV gene diversity. Moreover, memory B-cell clones are frequently composed of members of various subsets, showing that from a single GC B-cell clone a variety of memory B cells with distinct functions is generated. Thus, the human memory B-cell compartment is highly diverse and flexible. Several B-cell malignancies display features suggesting a derivation from memory B cells. This includes a subset of chronic lymphocytic leukemia, hairy cell leukemia and marginal zone lymphomas. The exposure of memory B cells to oncogenic events during their generation in the GC, the longevity of these B cells and the ease to activate them may be key determinants for their malignant transformation.

  4. Inflammatory responses of a human keratinocyte cell line to 10 nm citrate- and PEG-coated silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Bastos, V.; Brown, D.; Johnston, H.; Daniel-da-Silva, A. L.; Duarte, I. F.; Santos, C.; Oliveira, H.

    2016-07-01

    Silver nanoparticles (AgNPs) are among the most commonly used engineered NPs and various commercially available products are designed to come in direct contact with the skin (wound dressings, textiles, creams, among others). Currently, there is limited understanding of the influence of coatings on the toxicity of AgNPs and in particular their ability to impact on AgNP's mediated inflammatory responses. As AgNPs are often stabilized by different coatings, including citrate and polyethyleneglycol (PEG), in this study we investigate the influence of citrate (Cit10) or PEG (PEG10) coatings to 10 nm AgNP on skin, using human HaCaT keratinocytes. AgNPs cytotoxicity and inflammatory response (nuclear factor (NF)-κB induction and cytokine production) of HaCaT were assessed after in vitro exposure to 10 and 40 µg/mL after 4, 24, and 48 h. Results showed that although both types of coated AgNPs decreased cell proliferation and viability, Cit10 AgNPs were more toxic. NF-κB inhibition was observed for the highest concentration (40 µg/mL) of PEG10 AgNPs, and the putative link to early apoptotic pathways observed in these cells is discussed. No production of IL-1β, IL-6, IL-10, and TNFα was stimulated by AgNPs. Furthermore, Cit10 and PEG10 AgNPs decreased the release of MCP-1 by HaCaT cells after 48 h of exposure. As cytokines are vital for the immunologic regulation in the human body, and it is demonstrated that they may interfere with NPs, more research is needed to understand how different AgNPs affect the immune system.

  5. [The influence of AMPs from HaCaT keratinocytes on some bacteria strains derived from skin changes].

    PubMed

    Wiechuła, Barbara; Wilk, Iwona; Ekiel, Alicja; Tustanowski, Jerzy; Martirosian, Gayane

    2006-01-01

    The aim of this study was to evaluate antimicrobial activity of protein extracts from HaCaT cell line against bacterial strains, isolated from clinical materials, obtained from patients with clinical symptoms of acne (Propionibacterium acnes) and gas gangrene (Clostridium perfringens and Sterptococcus pyogenes). Reference strain of Staphylococcus aureus ATCC 25923 also was used. Protein extracts from cultured HaCaT cells were obtained by 3-fold freezing/defreezing cells in dry ice following by centrifugation and incubated with appropriate bacterial suspension (0.5 McFarland scale) during 6 and 24 hours. We observed time-depending and strain-depending activity of HaCaT--protein extract. Interestingly, high activity was demonstrated against strains of S. pyogenes and C. perfringens. Because of increasing bacterial resistance to antibiotics further studies in the field of antimicrobial peptides are required.

  6. Effects of lunar and mars dust on HaCaT keratinocytes and CHO-K1 fibroblasts

    NASA Astrophysics Data System (ADS)

    Brix, Klaudia; Slenzka, Klaus; Rehders, Maren; Sadhukhan, Annapurna; Mistry, Rima; Duenne, Matthias; Kempf, Juergen

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respira-tory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of lunar dust on human health is required to best support future missions to moon. In this study, we used different methods to assess the specific effects of lunar dust onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and since a well orchestrated program ensures proper repair in cases of wounding. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology, metabolic state, survival and proliferation of the cells were determined. Cytotoxi-city and proliferation were measured using the MTT assay, metabolic activity was analyzed by vital staining of mitochondria, and phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells. It was found that the effects of the two types of soils on the different features of both cell lines varied to considerable extent, and that lunar and mars dust were specific in their effects. The obtained results will facilitate detailed inves-tigations of dust exposure during wound healing and will ease risk assessment studies for e.g. lunar lander approaches. The investigations will help to assess the risks and to determine safety measures to be taken during extraterrestrial expeditions in order to minimize risks to human health associated with exposure of human skin to dust contaminants.

  7. Overabundance of Putative Cancer Stem Cells in Human Skin Keratinocyte Cells Malignantly Transformed by Arsenic

    PubMed Central

    Sun, Yang; Tokar, Erik J.; Waalkes, Michael P.

    2012-01-01

    Arsenic is a human skin carcinogen. Cancer is probably a disease driven by stem cells (SCs), and SCs are likely a key target during arsenic oncogenesis. In utero arsenic exposure predisposes mice to skin cancers that overproduce cancer SCs (CSCs) and have distorted CSC signaling and population dynamics. Therefore, we hypothesized CSC accumulation may occur during arsenic-induced malignant transformation in vitro of human skin keratinocytes. Thus, the HaCaT cell line, malignantly transformed by arsenite (100nM, 30 weeks; termed As-TM cells) in prior work, was further studied for the quantity and nature of SCs after this transformation. SCs were isolated from passage-matched control and As-TM cells by a magnetic bead system that enriches for CD34-positive cells. There were 2.5 times more SCs isolated from As-TM cells than control. Holoclone production from As-TM putative CSCs was 2.5-fold higher by 1 week and 3.5-fold higher by 2 weeks than control SCs. Potential malignant phenotype was assessed in isolated SC/CSCs. Transcript level of SC/CSC markers were elevated in both isolated As-TM CSCs and control SCs compared with parental cells, but compared with control SCs, As-TM putative CSCs had elevated CD34, K5, K14, K15, and K19 transcripts and dramatically stronger staining for p63, Rac1, K5, Notch1, and K19. As-TM putative CSCs also showed markedly elevated MMP-9 secretion and colony formation, indicators of cancer phenotype, even compared with total population of As-TM cells. Thus, malignant phenotype is particularly pronounced in CSCs after arsenic-induced transformation of human skin cells and occurs concurrently with a potential overproduction of these cells. PMID:22011395

  8. Potassium channel in the mitochondria of human keratinocytes.

    PubMed

    Toczyłowska-Mamińska, Renata; Olszewska, Anna; Laskowski, Michał; Bednarczyk, Piotr; Skowronek, Krzysztof; Szewczyk, Adam

    2014-03-01

    The activation of mitochondrial potassium channels induces cytoprotection in various cell types. Hence, the identification of ion channels present in the inner mitochondrial membrane of keratinocytes is important in distinguishing possible protective mechanisms in these cells. In this paper, inner membrane mitochondrial ion channels of the human keratinocyte HaCaT cell line were investigated using a patch-clamp technique. We observed potassium-selective channel activity with a conductance of 83 pS at positive voltages. The I-V curve indicates that the observed channel has rectifying properties. Moreover, the channel activity was inhibited by acidic pH and 1 mM lidocaine. Using reverse transcriptase-PCR, we found an mRNA transcript for the TASK-3 (tandem pore domain acid-sensitive K channels) channel. We observed co-localization of the TASK-3 protein and a mitochondrial marker in the mitochondria of HaCaT cells. Additionally, we showed that TASK-3 knockdown HaCaT cells markedly decreased viability after UVB radiation exposure compared with control cells. In summary, the single-channel activity and properties of a mitochondrial potassium channel in a keratinocyte HaCaT cell line have been described.

  9. Chitosan-shelled oxygen-loaded nanodroplets abrogate hypoxia dysregulation of human keratinocyte gelatinases and inhibitors: New insights for chronic wound healing

    SciTech Connect

    Khadjavi, Amina; Magnetto, Chiara; Panariti, Alice; Argenziano, Monica; Gulino, Giulia Rossana; Rivolta, Ilaria; Cavalli, Roberta; Giribaldi, Giuliana; Guiot, Caterina; Prato, Mauro

    2015-08-01

    Background: : In chronic wounds, efficient epithelial tissue repair is hampered by hypoxia, and balances between the molecules involved in matrix turn-over such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are seriously impaired. Intriguingly, new oxygenating nanocarriers such as 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNs) might effectively target chronic wounds. Objective: : To investigate hypoxia and chitosan-shelled OLN effects on MMP/TIMP production by human keratinocytes. Methods: : HaCaT cells were treated for 24 h with 10% v/v OLNs both in normoxia or hypoxia. Cytotoxicity and cell viability were measured through biochemical assays; cellular uptake by confocal microscopy; and MMP and TIMP production by enzyme-linked immunosorbent assay or gelatin zymography. Results: : Normoxic HaCaT cells constitutively released MMP-2, MMP-9, TIMP-1 and TIMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. After cellular uptake by keratinocytes, nontoxic OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring physiological balances. OLN abilities were specifically dependent on time-sustained oxygen diffusion from OLN core. Conclusion: : Chitosan-shelled OLNs effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Therefore, topical administration of exogenous oxygen, properly encapsulated in nanodroplet formulations, might be a promising adjuvant approach to promote healing processes in hypoxic wounds. - Highlights: • Hypoxia impairs MMP9/TIMP1 and MMP2/TIMP2 balances in HaCaT human keratinocytes. • Chitosan-shelled oxygen-loaded nanodroplets (OLNs) are internalised by HaCaT cells. • OLNs are not toxic to HaCaT cells. • OLNs effectively counteract hypoxia effects on MMP/TIMP balances in HaCaT cells. • OLNs appear as promising and cost-effective therapeutic tools for hypoxic

  10. Does shape matter? Bioeffects of gold nanomaterials in a human skin cell model.

    PubMed

    Schaeublin, Nicole M; Braydich-Stolle, Laura K; Maurer, Elizabeth I; Park, Kyoungweon; MacCuspie, Robert I; Afrooz, A R M Nabiul; Vaia, Richard A; Saleh, Navid B; Hussain, Saber M

    2012-02-14

    Gold nanomaterials (AuNMs) have distinctive electronic and optical properties, making them ideal candidates for biological, medical, and defense applications. Therefore, it is imperative to evaluate the potential biological impact of AuNMs before employing them in any application. This study investigates two AuNMs with different aspect ratios (AR) on mediation of biological responses in the human keratinocyte cell line (HaCaT) to model potential skin exposure to these AuNMs. The cellular responses were evaluated by cell viability, reactive oxygen species (ROS) generation, alteration in gene and protein expression, and inflammatory response. Gold nanospheres, nominally 20 nm in diameter and coated with mercaptopropane sulfonate (AuNS-MPS), formed agglomerates when dispersed in cell culture media, had a large fractal dimension (D(f) = 2.57 ± 0.4) (i.e., tightly bound and densely packed) and were found to be nontoxic even at the highest dose of 100 μg/mL. Highly uniform, 16.7 nm diameter, and 43.8 nm long polyethylene glycol-capped gold nanorods (AuNR-PEG) also formed agglomerates when dispersed into the cell culture media. However, the agglomerates had a smaller fractal dimension (D(f) = 1.28 ± 0.08) (i.e., loosely bound) and were found to be cytotoxic to the HaCaT cells, with a significant decrease in cell viability occurring at 25 μg/mL and higher. Moreover, AuNR-PEG caused significant ROS production and up-regulated several genes involved in cellular stress and toxicity. These results, combined with increased levels of inflammatory and apoptotic proteins, demonstrated that the AuNR-PEG induced apoptosis. Exposure to AuNS-MPS, however, did not show any of the detrimental effects observed from the AuNR-PEG. Therefore, we conclude that shape appears to play a key role in mediating the cellular response to AuNMs.

  11. Palmitic acid induces production of proinflammatory cytokines interleukin-6, interleukin-1β, and tumor necrosis factor-α via a NF-κB-dependent mechanism in HaCaT keratinocytes.

    PubMed

    Zhou, Bing-rong; Zhang, Jia-an; Zhang, Qian; Permatasari, Felicia; Xu, Yang; Wu, Di; Yin, Zhi-qiang; Luo, Dan

    2013-01-01

    To investigate whether palmitic acid can be responsible for the induction of inflammatory processes, HaCaT keratinocytes were treated with palmitic acid at pathophysiologically relevant concentrations. Secretion levels of interleukin-6 (IL-6), tumor necrosis factor- α (TNF- α), interleukin-1 β (IL-1 β), NF- κ B nuclear translocation, NF- κ B activation, Stat3 phosphorylation, and peroxisome proliferator-activated receptor alpha (PPAR α) mRNA and protein levels, as well as the cell proliferation ability were measured at the end of the treatment and after 24 hours of recovery. Pyrrolidine dithiocarbamate (PDTC, a selective chemical inhibitor of NF- κ B) and goat anti-human IL-6 polyclonal neutralizing antibody were used to inhibit NF- κ B activation and IL-6 production, respectively. Our results showed that palmitic acid induced an upregulation of IL-6, TNF- α , IL-1 β secretions, accompanied by NF- κ B nuclear translocation and activation. Moreover, the effect of palmitic acid was accompanied by PPAR α activation and Stat3 phosphorylation. Palmitic acid-induced IL-6, TNF- α , IL-1 β productions were attenuated by NF- κ B inhibitor PDTC. Palmitic acid was administered in amounts able to elicit significant hyperproliferation and can be attenuated by IL-6 blockage. These data demonstrate for the first time that palmitic acid can stimulate IL-6, TNF- α , IL-1 β productions in HaCaT keratinocytes and cell proliferation, thereby potentially contributing to acne inflammation and pilosebaceous duct hyperkeratinization.

  12. Role for Heat Shock Protein 90α in the Proliferation and Migration of HaCaT Cells and in the Deep Second-Degree Burn Wound Healing in Mice

    PubMed Central

    Li, Na; Li, Xiaoqiang; Han, Fei; Su, Linlin; Hu, Dahai

    2014-01-01

    Inflammation, proliferation, and tissue remodeling are essential steps for wound healing. The hypoxic wound microenvironment promotes cell migration through a hypoxia—heat shock protein 90 alpha (Hsp90α)—low density lipoprotein receptor-related protein-1 (LRP-1) autocrine loop. To elucidate the role of this autocrine loop on burn wound healing, we investigated the expression profile of Hsp90α at the edge of burn wounds and found a transient increase in both mRNA and protein levels. Experiments performed with a human keratinocyte cell line—HaCaT also confirmed above results. 17-dimethylaminoethylamino-17demethoxygeldanamycin hydrochloride (17-DMAG), an Hsp90α inhibitor, was used to further evaluate the function of Hsp90α in wound healing. Consistently, topical application of Hsp90α in the early stage of deep second-degree burn wounds led to reduced inflammation and increased tissue granulation, with a concomitant reduction in the size of the wound at each time point tested (p<0.05). Consequently, epidermal cells at the wound margin progressed more rapidly causing an expedited healing process. In conclusion, these results provided a rationale for the therapeutic effect of Hsp90α on the burn wound management. PMID:25111496

  13. Hsp40 regulates the amount of keratin proteins via ubiquitin-proteasome pathway in cultured human cells.

    PubMed

    Yamazaki, Shota; Uchiumi, Asato; Katagata, Yohtaro

    2012-02-01

    Keratins represent important structural components of intermediate filament proteins. Their expression profiles are remarkably tissue-specific. Recent data have shown that keratins associate with many proteins including heat shock proteins (HSP). We recently identified cell-specific keratin and HSP expression. We aimed to gain further insight into the regulation of keratins by specific inhibition through knockdown of Hsp40 in human keratinocyte cells. Keratin-HSP interaction in HaCaT cell lysate was evaluated by immunoprecipitation followed by Western blotting. Immunofluorescence, was used to examine the co-localization of keratins and Hsp40. Hsp40 depletion led to an increase in the levels of keratin proteins (K5, K14, K10) and a decrease in keratin ubiquitination without influencing keratin gene expression. Our results demonstrate direct or indirectly association of Hsp40 and imply that expressed keratin proteins were regulated by Hsp40 depending on the ubiquitin-proteasome pathway in HaCaT. Furthermore, the K10 differentiation marker was increased by knockdown of Hsp40. The results presented in this study indicate that Hsp40 is related to the differentiation exchange of keratin pairs.

  14. Freezing human ES cells.

    PubMed

    Trish, Erin; Dimos, John; Eggan, Kevin

    2006-10-12

    Here we demonstrate how our lab freezes HuES human embryonic stem cell lines. A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction. Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature. During this time cells can be observed rounding, and colonies lifting off the plate surface. Gentle repeated pipetting will remove cells and colonies from the plate surface. Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun. Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10 cm plate. After cells are adequately resuspended, ice cold freezing media is added at equal volume. Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80 C over 24 hours. Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

  15. Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes

    SciTech Connect

    Xie, Xin; Dai, Hui; Zhuang, Binyu; Chai, Li; Xie, Yanguang; Li, Yuzhen

    2016-04-08

    The effects and the underlying mechanisms of hydrogen sulfide (H{sub 2}S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H{sub 2}S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H{sub 2}S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H{sub 2}S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation. - Highlights: • Exogenous H{sub 2}S promotes keratinocyte proliferation and differentiation. • The effects of H{sub 2}S on proliferation and differentiation is modulated by autophagy. • Exogenous H{sub 2}S has no effect on keratinocyte apoptosis.

  16. Metformin inhibits proliferation of human keratinocytes through a mechanism associated with activation of the MAPK signaling pathway

    PubMed Central

    LI, WEINING; MA, WEIYUAN; ZHONG, HUA; LIU, WENBIN; SUN, QING

    2014-01-01

    In the present study, the effects of metformin on the proliferation of human immortalized keratinocytes (HaCaTs) and the underlying mechanisms were investigated. HaCaT cells in the logarithmic growth phase were treated with 50 mM metformin for 24, 48 and 72 h. Cell morphology after 24 h of treatment was observed under a microscope. Cell proliferation was detected using a colorimetric cell proliferation and cytotoxicity assay kit. Western blot analyses were performed to detect the protein phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) and extracellular signal-related kinase 1/2 (ERK1/2). Metformin treatment resulted in morphological changes of the HaCaT cells. The survival rates of HaCaT cells treated with metformin were 36.18, 12.70 and 10.12% at 24, 48 and 72 h, respectively. As the treatment time extended, the survival rates of HaCaT cells decreased. Western blot analysis results showed that the mean level of phosphorylated (p)-AMPK in the HaCaT cells without metformin treatment was 2.856±0.323. However, the mean p-AMPK level following metformin treatment for 24 h increased to 5.198±0.625, indicating a significant difference between these two groups (P<0.05). The mean absorbance ratio of p-ERK1/2 was 7.550±1.087 for the untreated cells, but the levels in cells following metformin treatment for 24 h increased to 10.430±1.217, indicating a significant difference between the two groups (P<0.05). In conclusion, metformin treatment upregulated the levels of p-AMPK and p-ERK1/2 in HaCaT cells, and significantly inhibited HaCaT cell proliferation in vitro by a mechanism associated with activation of the mitogen-activated protein kinase signaling pathway. PMID:24396411

  17. Human innate lymphoid cells.

    PubMed

    Mjösberg, Jenny; Spits, Hergen

    2016-11-01

    Innate lymphoid cells (ILCs) are increasingly acknowledged as important mediators of immune homeostasis and pathology. ILCs act as early orchestrators of immunity, responding to epithelium-derived signals by expressing an array of cytokines and cell-surface receptors, which shape subsequent immune responses. As such, ILCs make up interesting therapeutic targets for several diseases. In patients with allergy and asthma, group 2 innate lymphoid cells produce high amounts of IL-5 and IL-13, thereby contributing to type 2-mediated inflammation. Group 3 innate lymphoid cells are implicated in intestinal homeostasis and psoriasis pathology through abundant IL-22 production, whereas group 1 innate lymphoid cells are accumulated in chronic inflammation of the gut (inflammatory bowel disease) and lung (chronic obstructive pulmonary disease), where they contribute to IFN-γ-mediated inflammation. Although the ontogeny of mouse ILCs is slowly unraveling, the development of human ILCs is far from understood. In addition, the growing complexity of the human ILC family in terms of previously unrecognized functional heterogeneity and plasticity has generated confusion within the field. Here we provide an updated view on the function and plasticity of human ILCs in tissue homeostasis and disease. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  18. Effects of titanium dioxide nanoparticles on human keratinocytes.

    PubMed

    Wright, Clayton; Iyer, Anand Krishnan V; Wang, Liying; Wu, Nianqiang; Yakisich, Juan S; Rojanasakul, Yon; Azad, Neelam

    2017-01-01

    Titanium dioxide (TiO2) is a ubiquitous whitening compound widely used in topical products such as sunscreens, lotions and facial creams. The damaging health effects of TiO2 inhalation has been widely studied in rats, mice and humans showing oxidative stress increase, DNA damage, cell death and inflammatory gene upregulation in lung and throat cells; however, the effects on skin cells from long-term topical use of various products remain largely unknown. In this study, we assessed the effect of specific TiO2 nanoparticles (H2TiO7) on a human keratinocyte cell line (HaCaT). We performed a comparative analysis using three TiO2 particles varying in size (Fine, Ultrafine and H2TiO7) and analyzed their effects on HaCaTs. There is a clear dose-dependent increase in superoxide production, caspase 8 and 9 activity, and apoptosis in HaCaTs after treatment with all three forms of TiO2; however, there is no consistent effect on cell viability and proliferation with either of these TiO2 particles. While there is data suggesting UV exposure can enhance the carcinogenic effects of TiO2, we did not observe any significant effect of UV-C exposure combined with TiO2 treatment on HaCaTs. Furthermore, TiO2-treated cells showed minimal effects on VEGF upregulation and Wnt signaling pathway thereby showing no potential effect on angiogenesis and malignant transformation. Overall, we report here an increase in apoptosis, which may be caspase 8/Fas-dependent, and that the H2TiO7 nanoparticles, despite their smaller particle size, had no significant enhanced effect on HaCaT cells as compared to Fine and Ultrafine forms of TiO2.

  19. Effects of titanium dioxide nanoparticles on human keratinocytes

    PubMed Central

    Wright, Clayton; Iyer, Anand Krishnan V.; Wang, Liying; Wu, Nianqiang; Yakisich, Juan S.; Rojanasakul, Yon; Azad, Neelam

    2016-01-01

    Titanium dioxide (TiO2) is a ubiquitous whitening compound widely used in topical products such as sunscreens, lotions and facial creams. The damaging health effects of TiO2 inhalation has been widely studied in rats, mice and humans showing oxidative stress increase, DNA damage, cell death and inflammatory gene upregulation in lung and throat cells; however, the effects on skin cells from long-term topical use of various products remain largely unknown. In this study, we assessed the effect of specific TiO2 nanoparticles (H2TiO7) on a human keratinocyte cell line (HaCaT). We performed a comparative analysis using three TiO2 particles varying in size (Fine, Ultrafine and H2TiO7) and analyzed their effects on HaCaTs. There is a clear dose-dependent increase in superoxide production, caspase 8 and 9 activity, and apoptosis in HaCaTs after treatment with all three forms of TiO2; however, there is no consistent effect on cell viability and proliferation with either of these TiO2 particles. While there is data suggesting UV exposure can enhance the carcinogenic effects of TiO2, we did not observe any significant effect of UV-C exposure combined with TiO2 treatment on HaCaTs. Furthermore, TiO2-treated cells showed minimal effects on VEGF upregulation and Wnt signaling pathway thereby showing no potential effect on angiogenesis and malignant transformation. Overall, we report here an increase in apoptosis, which may be caspase 8/Fas-dependent, and that the H2TiO7 nanoparticles, despite their smaller particle size, had no significant enhanced effect on HaCaT cells as compared to Fine and Ultrafine forms of TiO2. PMID:27310834

  20. Photoprotective efficiency of PLGA-curcumin nanoparticles versus curcumin through the involvement of ERK/AKT pathway under ambient UV-R exposure in HaCaT cell line.

    PubMed

    Chopra, Deepti; Ray, Lipika; Dwivedi, Ashish; Tiwari, Shashi Kant; Singh, Jyoti; Singh, Krishna P; Kushwaha, Hari Narayan; Jahan, Sadaf; Pandey, Ankita; Gupta, Shailendra K; Chaturvedi, Rajnish Kumar; Pant, Aditya Bhushan; Ray, Ratan Singh; Gupta, Kailash Chand

    2016-04-01

    Curcumin (Cur) has been demonstrated to have wide pharmacological window including anti-oxidant and anti-inflammatory properties. However, phototoxicity under sunlight exposure and poor biological availability limits its applicability. We have synthesized biodegradable and non-toxic polymer-poly (lactic-co-glycolic) acid (PLGA) encapsulated formulation of curcumin (PLGA-Cur-NPs) of 150 nm size range. Photochemically free curcumin generates ROS, lipid peroxidation and induces significant UVA and UVB mediated impaired mitochondrial functions leading to apoptosis/necrosis and cell injury in two different origin cell lines viz., mouse fibroblasts-NIH-3T3 and human keratinocytes-HaCaT as compared to PLGA-Cur-NPs. Molecular docking studies suggested that intact curcumin from nanoparticles, bind with BAX in BIM SAHB site and attenuate it to undergo apoptosis while upregulating anti-apoptotic genes like BCL2. Real time studies and western blot analysis with specific phosphorylation inhibitor of ERK1 and AKT1/2/3 confirm the involvement of ERK/AKT signaling molecules to trigger the survival cascade in case of PLGA-Cur-NPs. Our finding demonstrates that low level sustained release of curcumin from PLGA-Cur-NPs could be a promising way to protect the adverse biological interactions of photo-degradation products of curcumin upon the exposure of UVA and UVB. Hence, the applicability of PLGA-Cur-NPs could be suggested as prolonged radical scavenging ingredient in curcumin containing products.

  1. Induction of dental epithelial cell differentiation marker gene expression in non-odontogenic human keratinocytes by transfection with thymosin beta 4.

    PubMed

    Kiyoshima, Tamotsu; Fujiwara, Hiroaki; Nagata, Kengo; Wada, Hiroko; Ookuma, Yukiko F; Shiotsuka, Maho; Kihara, Makiko; Hasegawa, Kana; Someya, Hirotaka; Sakai, Hidetaka

    2014-01-01

    Previous studies have shown that the recombination of cells liberated from developing tooth germs develop into teeth. However, it is difficult to use human developing tooth germ as a source of cells because of ethical issues. Previous studies have reported that thymosin beta 4 (Tmsb4x) is closely related to the initiation and development of the tooth germ. We herein attempted to establish odontogenic epithelial cells from non-odontogenic HaCaT cells by transfection with TMSB4X. TMSB4X-transfected cells formed nodules that were positive for Alizarin-red S (ALZ) and von Kossa staining (calcium phosphate deposits) when cultured in calcification-inducing medium. Three selected clones showing larger amounts of calcium deposits than the other clones, expressed PITX2, Cytokeratin 14, and Sonic Hedgehog. The upregulation of odontogenesis-related genes, such as runt-related transcription factor 2 (RUNX2), Amelogenin (AMELX), Ameloblastin (AMBN) and Enamelin (ENAM) was also detected. These proteins were immunohistochemically observed in nodules positive for the ALZ and von Kossa staining. RUNX2-positive selected TMSB4X-transfected cells implanted into the dorsal subcutaneous tissue of nude mice formed matrix deposits. Immunohistochemically, AMELX, AMBN and ENAM were observed in the matrix deposits. This study demonstrated the possibility of induction of dental epithelial cell differentiation marker gene expression in non-odontogenic HaCaT cells by TMSB4X. Copyright © 2013. Published by Elsevier B.V.

  2. Hydrogen water intake via tube-feeding for patients with pressure ulcer and its reconstructive effects on normal human skin cells in vitro

    PubMed Central

    2013-01-01

    Background Pressure ulcer (PU) is common in immobile elderly patients, and there are some research works to investigate a preventive and curative method, but not to find sufficient effectiveness. The aim of this study is to clarify the clinical effectiveness on wound healing in patients with PU by hydrogen-dissolved water (HW) intake via tube-feeding (TF). Furthermore, normal human dermal fibroblasts OUMS-36 and normal human epidermis-derived cell line HaCaT keratinocytes were examined in vitro to explore the mechanisms relating to whether hydrogen plays a role in wound-healing at the cellular level. Methods Twenty-two severely hospitalized elderly Japanese patients with PU were recruited in the present study, and their ages ranged from 71.0 to 101.0 (86.7 ± 8.2) years old, 12 male and 10 female patients, all suffering from eating disorder and bedridden syndrome as the secondary results of various underlying diseases. All patients received routine care treatments for PU in combination with HW intake via TF for 600 mL per day, in place of partial moisture replenishment. On the other hand, HW was prepared with a hydrogen-bubbling apparatus which produces HW with 0.8-1.3 ppm of dissolved hydrogen concentration (DH) and −602 mV to −583 mV of oxidation-reduction potential (ORP), in contrast to reversed osmotic ultra-pure water (RW), as the reference, with DH of < 0.018 ppm and ORP of +184 mV for use in the in vitro experimental research. In in vitro experiments, OUMS-36 fibroblasts and HaCaT keratinocytes were respectively cultured in medium prepared with HW and/or RW. Immunostain was used for detecting type-I collagen reconstruction in OUMS-36 cells. And intracellular reactive oxygen species (ROS) were quantified by NBT assay, and cell viability of HaCaT cells was examined by WST-1 assay, respectively. Results Twenty-two patients were retrospectively divided into an effective group (EG, n = 12) and a less effective group (LG, n = 10) according to

  3. Human dendritic cell subsets

    PubMed Central

    Collin, Matthew; McGovern, Naomi; Haniffa, Muzlifah

    2013-01-01

    Summary Dendritic cells are highly adapted to their role of presenting antigen and directing immune responses. Developmental studies indicate that DCs originate independently from monocytes and tissue macrophages. Emerging evidence also suggests that distinct subsets of DCs have intrinsic differences that lead to functional specialisation in the generation of immunity. Comparative studies are now allowing many of these properties to be more fully understood in the context of human immunology. PMID:23621371

  4. Human VAT-1: a calcium-regulated activation marker of human epithelial cells.

    PubMed

    Koch, Judith; Foekens, John; Timmermans, Mieke; Fink, Wolfram; Wirzbach, Alexander; Kramer, Michael D; Schaefer, Birgit M

    2003-09-01

    Human VAT-1 (hVAT-1) is a homologue of the synaptic vesicle membrane protein of Torpedo californica. Its coding gene is located near the BRCA1 locus and thus hVAT-1 may be linked to an inherited predisposition to breast and ovary cancer. However, the hVAT-1 protein expression pattern in normal epithelial tissues such as skin, mammary gland and ovary, as well as in tumours of the mammary gland and ovary, has not been studied. To address this issue, an immunohistological analysis of biopsies of normal epidermis and lesional epidermis of bullous pemphigoid and pemphigus vulgaris patients was undertaken. hVAT-1-expression was observed in basal keratinocytes of lesional epidermis of bullous pemphigoid patients but not in normal epidermis or in lesional epidermis of pemphigus vulgaris patients. Moreover, hVAT-1 expression in HaCaT cells was found to be calcium-dependent. Normal and malignant mammary and ovary epithelium were found to be hVAT-1-negative. Our results indicate that hVAT-1 exerts a specific function in keratinocyte physiology, in particular in calcium-regulated processes, with no evident deregulation in malignancies of the breast and ovary.

  5. Photoprotective Activity of Vulpinic and Gyrophoric Acids Toward Ultraviolet B-Induced Damage in Human Keratinocytes.

    PubMed

    Varol, Mehmet; Türk, Ayşen; Candan, Mehmet; Tay, Turgay; Koparal, Ayşe Tansu

    2016-01-01

    Vulpinic and gyrophoric acids are known as ultraviolet filters for natural lichen populations because of their chemical structures. However, to the best of our knowledge, there has been no reference to their cosmetic potential for skin protection against ultraviolet B (UVB)-induced damage and, consequently, we propose to highlight their photoprotective profiles in human keratinocytes (HaCaT). Therefore, vulpinic acid and gyrophoric acid were isolated from acetone extracts of Letharia vulpina and Xanthoparmelia pokornyi, respectively. Their photoprotective activities on irradiated HaCaT cells and destructive effects on non-irradiated HaCaT cells were compared through in vitro experimentation: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, 4',6-diamino-2-phenylindole and tetramethylrhodamine B isothiocyanate-phalloidin staining protocols. Both of the lichen substances effectively prevented cytotoxic, apoptotic and cytoskeleton alterative activities of 2.5 J/cm(2) UVB in a dose-dependent manner. Moreover, vulpinic and gyrophoric acids showed no toxic, apoptotic or cytoskeleton alterative effects on non-irradiated HaCaT cells, except at high doses (≥400 μM) of gyrophoric acid. The findings suggest that vulpinic and gyrophoric acids can be promising cosmetic ingredients to photo-protect human skin cells and should therefore be further investigated by in vitro and in vivo multiple bioassays.

  6. Human mast cell transcriptome project.

    PubMed

    Saito, H; Nakajima, T; Matsumoto, K

    2001-05-01

    After draft reading of the human genome sequence, systemic analysis of the transcriptome (the whole transcripts present in a cell) is progressing especially in commonly available cell types. Until recently, human mast cells were not commonly available. We have succeeded to generate a substantial number of human mast cells from umbilical cord blood and from adult peripheral blood progenitors. Then, we have examined messenger RNA selectively transcribed in these mast cells using high-density oligonucleotide probe arrays. Many unexpected but important transcripts were selectively expressed in human mast cells. We discuss the results obtained from transcriptome screening by introducing our data regarding mast-cell-specific genes.

  7. In vitro evaluation of the effects of yttria-alumina-silica microspheres on human keratinocyte cells.

    PubMed

    Radu, T; Chiriac, M T; Popescu, O; Simon, V; Simon, S

    2013-02-01

    The behavior of yttria-alumina-silica spray-dried microspheres was investigated in vitro on a human keratinocyte cell line, first to exclude their cytotoxicity. The HaCaT cells were chosen due to their well-characterized phenotype and their phagocytic ability. Microscopic analysis and cell viability tests showed no negative effect of the microspheres on cells morphology and behavior. Scanning electron microscopy and transmission electron microscopy results evidenced the cellular internalization of the microspheres at 48 h after their incubation with cultured cells. The shape, size distribution, structure, composition, and chemical states of the elements on samples surface were analyzed by SEM, transmission electron microscopy, x-ray diffraction, and x-ray photoelectron spectroscopy, because these properties could influence their internalization by cells. The yttrium distribution on the microspheres surface was indicated by fluorescence microscopy imaging. The microspheres dimension and shape inside the cells was in accordance with their dimension and shape before incubation. The microspheres seemed captured and engulfed by the cells in native form and appeared resistant to degradation over the first 48 h. Most of the analyzed cells took up more microspheres, suggesting that the microspheres were actively phagocytosed by the cells and accumulated within the cytoplasm. X-ray photoelectron spectroscopy results on Al and Si atomic environments denoted Al-O-Si crosslinks, which improve the surface protection to corrosion. Copyright © 2012 Wiley Periodicals, Inc.

  8. Human embryonic stem cells derived keratinocyte as an in vitro research model for the study of immune response.

    PubMed

    Kidwai, Fahad Karim; Jokhun, Doorgesh Sharma; Movahednia, Mohammad Mehdi; Yeo, Jin Fei; Tan, Kai Soo; Cao, Tong

    2013-09-01

    The innate immune response (IMR) is critical for the oral mucosa due to their continuous exposure to various oral pathogens. Keratinocytes play important role in IMR. Therefore, to date, keratinocytes from different sources have been used as in vitro research model for the study of IMR. However, current keratinocyte research models are hampered by the limited supply, patients' dependency and batch to batch variation. Therefore, in this study, we demonstrated the use of human embryonic stem cells (hESCs) derived keratinocytes (H9-Kert) as an alternative research model for the study of IMR. The expression kinetics of toll-like receptor (TLR) 2, TLR 4, interleukin (IL) -6, IL-8, inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-α), in H9-Kert and immortalized human keratinocyte cell line (HaCaT) were analysed at mRNA levels by both reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The activation of the inflammatory transcription factor nuclear factor kappa-b (NFĸB) was assayed in these cells by transiently transfecting the cells with NFĸB reporter plasmid. Activation of NFĸB following treatment with heat-killed Porphyromonas gingivalis (P. gingivalis), an oral pathogen, was determined by assaying for the reporter, secreted alkaline phosphatase activity. The expression of TLRs, cytokines and activation of NFĸB following bacterial stimulation showed in both H9-Kert and the widely used HaCaT keratinocyte cell line was similar. Overall, our results support the potential application of hESCs as an alternative limitless cell source for primary keratinocytes which can be used as consistent and dependable research tool with minimum variations and no donor's dependency. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Enhancement of P53-Mutant Human Colorectal Cancer Cells Radiosensitivity by Flavonoid Fisetin

    SciTech Connect

    Chen Wenshu; Lee Yijang; Yu Yichu; Hsaio Chinghui

    2010-08-01

    Purpose: The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. Methods and Materials: Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of {gamma}-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. Results: Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells but not human keratocyte HaCaT cells; it also prolonged radiation-induced G{sub 2}/M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. Conclusions: To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.

  10. In vitro sensitivity of human gastric cancer cells (HGC-27) to Helicobacter pylori cytotoxin.

    PubMed

    Pessina, A; Bayo, M; Croera, C; Meringolo, F; Neri, M G; Montesissa, L; Raimondi, A

    2003-12-01

    The VacA cytotoxin produced by Helicobacter pylori is considered an important co-factor in the pathogenesis of chronic gastritis, peptic ulcer and gastric carcinoma. The toxin remains partly bound on the bacterial surface, but a certain amount is secreted and can bind receptors on gastric epithelium. The vacuolizing activity of this toxin is related to alteration of endo-lysosomic function and pore formation into plasmatic membrane. We investigated the 'in vitro' effect of filtrates obtained from two broth cultures of H. pylori with different genotype (vacA+ and vacA-) as verified by PCR. The effect was studied on three cell lines of epithelial origin: HeLa cells (reference strain for testing vacuolization), human transformed keratinocytes HaCaT, human gastric carcinoma cells HGC-27, and on a murine leukaemia WEHI-3B. The filtrate concentrations capable of giving vacuolization (NRU test), antiproliferative and cytotoxic effects (MTT test) were determined. The modulating effect of filtrates on drug toxicity was investigated on HeLa and HGC-27 cells by testing topoisomerase inhibitors (Ciprofloxacin and Camptothecin) and non-steroidal anti-inflammatory molecules (Aspirin and Indomethacin). Our results confirm that vacuolizing activity is present only in VacA+ filtrate and that HaCaT and HeLa cells show a similar sensitivity, whereas gastric HGC-27 cells appear significantly resistant to VacA+ activity. Although VacA filtrate does not produce vacuolisation, it affects the cell proliferation and is cytotoxic to the four cell lines. Both the VacA+ and VacA- filtrates (at non-cytotoxic concentrations) produce a decrease in drug toxicity with the unique exception of Ciprofloxacin to gastric HGC-27 cells, which in the presence of VacA+ and VacA- produces a significant increase in toxicity. These data suggest that products from H. pylori (other than those that have antiproliferative and toxic activity) may modulate the sensitivity of cells to drugs 'in vitro'. If this also

  11. Regulation of Mcl-1 by constitutive activation of NF-kappaB contributes to cell viability in human esophageal squamous cell carcinoma cells

    PubMed Central

    2014-01-01

    Background Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies with a 5-year survival rate less than 15%. Understanding of the molecular mechanisms involved in the pathogenesis of ESCC becomes critical to develop more effective treatments. Methods Mcl-1 expression was measured by reverse transcription (RT)-PCR and Western blotting. Human Mcl-1 promoter activity was evaluated by reporter gene assay. The interactions between DNA and transcription factors were confirmed by electrophoretic mobility shift assay (EMSA) in vitro and by chromatin immunoprecipitation (ChIP) assay in cells. Results Four human ESCC cell lines, TE-1, Eca109, KYSE150 and KYSE510, are revealed increased levels of Mcl-1 mRNA and protein compare with HaCaT, an immortal non-tumorigenic cell line. Results of reporter gene assays demonstrate that human Mcl-1 promoter activity is decreased by mutation of kappaB binding site, specific NF-kappaB inhibitor Bay11-7082 or dominant inhibitory molecule DNMIkappaBalpha in TE-1 and KYSE150 cell lines. Mcl-1 protein level is also attenuated by Bay11-7082 treatment or co-transfection of DNMIkappaBalpha in TE-1 and KYSE150 cells. EMSA results indicate that NF-kappaB subunits p50 and p65 bind to human Mcl-1-kappaB probe in vitro. ChIP assay further confirm p50 and p65 directly bind to human Mcl-1 promoter in intact cells, by which regulates Mcl-1 expression and contributes to the viability of TE-1 cells. Conclusions Our data provided evidence that one of the mechanisms of Mcl-1 expression in human ESCC is regulated by the activation of NF-kappaB signaling. The newly identified mechanism might provide a scientific basis for developing effective approaches to treatment human ESCC. PMID:24529193

  12. Remediation of textile azo dye acid red 114 by hairy roots of Ipomoea carnea Jacq. and assessment of degraded dye toxicity with human keratinocyte cell line.

    PubMed

    Jha, Pamela; Jobby, Renitta; Desai, N S

    2016-07-05

    Bioremediation has proven to be the most desirable and cost effective method to counter textile dye pollution. Hairy roots (HRs) of Ipomoea carnea J. were tested for decolourization of 25 textile azo dyes, out of which >90% decolourization was observed in 15 dyes. A diazo dye, Acid Red 114 was decolourized to >98% and hence, was chosen as the model dye. A significant increase in the activities of oxidoreductive enzymes was observed during decolourization of AR114. The phytodegradation of AR114 was confirmed by HPLC, UV-vis and FTIR spectroscopy. The possible metabolites were identified by GCMS as 4- aminobenzene sulfonic acid 2-methylaniline and 4- aminophenyl 4-ethyl benzene sulfonate and a probable pathway for the biodegradation of AR114 has been proposed. The nontoxic nature of the metabolites and toxicity of AR114 was confirmed by cytotoxicity tests on human keratinocyte cell line (HaCaT). When HaCaT cells were treated separately with 150 μg mL(-1) of AR114 and metabolites, MTT assay showed 50% and ≈100% viability respectively. Furthermore, flow cytometry data showed that, as compared to control, the cells in G2-M and death phase increased by 2.4 and 3.6 folds respectively on treatment with AR114 but remained unaltered in cells treated with metabolites.

  13. Metabolism of melatonin and biological activity of intermediates of melatoninergic pathway in human skin cells

    PubMed Central

    Kim, Tae-Kang; Kleszczyński, Konrad; Janjetovic, Zorica; Sweatman, Trevor; Lin, Zongtao; Li, Wei; Reiter, Russel J.; Fischer, Tobias W.; Slominski, Andrzej T.

    2013-01-01

    Indolic and kynuric pathways of skin melatonin metabolism were monitored by liquid chromatography mass spectrometry in human keratinocytes, melanocytes, dermal fibroblasts, and melanoma cells. Production of 6-hydroxymelatonin [6(OH)M], N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and 5-methoxytryptamine (5-MT) was detected in a cell type-dependent fashion. The major metabolites, 6(OH)M and AFMK, were produced in all cells. Thus, in immortalized epidermal (HaCaT) keratinocytes, 6(OH)M was the major product with Vmax = 63.7 ng/106 cells and Km = 10.2 μM, with lower production of AFMK and 5-MT. Melanocytes, keratinocytes, and fibroblasts transformed melatonin primarily into 6(OH)M and AFMK. In melanoma cells, 6(OH)M and AFMK were produced endogenously, a process accelerated by exogenous melatonin in the case of AFMK. In addition, N-acetylserotonin was endogenously produced by normal and malignant melanocytes. Metabolites showed selective antiproliferative effects on human primary epidermal keratinocytes in vitro. In ex vivo human skin, both melatonin and AFMK-stimulated expression of involucrin and keratins-10 and keratins-14 in the epidermis, indicating their stimulatory role in building and maintaining the epidermal barrier. In summary, the metabolism of melatonin and its endogenous production is cell type-dependent and expressed in all three main cell populations of human skin. Furthermore, melatonin and its metabolite AFMK stimulate differentiation in human epidermis, indicating their key role in building the skin barrier.—Kim, T.-K., Kleszczyński, K., Janjetovic, Z., Sweatman, T., Lin, Z., Li, W., Reiter, R. J., Fischer, T. W., Slominski, A. T. Metabolism of melatonin and biological activity of intermediates of melatoninergic pathway in human skin cells. PMID:23620527

  14. Expression of heparanase in basal cell carcinoma and squamous cell carcinoma*

    PubMed Central

    Pinhal, Maria Aparecida Silva; Almeida, Maria Carolina Leal; Costa, Alessandra Scorse; Theodoro, Thérèse Rachell; Serrano, Rodrigo Lorenzetti; Machado Filho, Carlos D'Apparecida Santos

    2016-01-01

    Background Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Objectives Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Methods Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). Results The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. Conclusion The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment. PMID:27828631

  15. Expression of heparanase in basal cell carcinoma and squamous cell carcinoma.

    PubMed

    Pinhal, Maria Aparecida Silva; Almeida, Maria Carolina Leal; Costa, Alessandra Scorse; Theodoro, Thérèse Rachell; Serrano, Rodrigo Lorenzetti; Machado, Carlos D'Apparecida Santos

    2016-01-01

    Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

  16. Extract from Periostracum cicadae Inhibits Oxidative Stress and Inflammation Induced by Ultraviolet B Irradiation on HaCaT Keratinocytes

    PubMed Central

    Tsen, Jen-Horng; Yen, Hsuan; Yang, Ting-Ya

    2017-01-01

    Periostracum cicadae is widely used for the treatment of skin diseases such as eczema, pruritus, and itching. The current study sought to evaluate the effect of P. cicadae extract on ultraviolet B (UVB) irradiation and identify the mechanisms involved. Photodamage-protective activity of P. cicadae extracts against oxidative challenge was screened using HaCaT keratinocytes. P. cicadae extracts did not affect cell viability but decreased reactive oxygen species (ROS) production. The extract attenuates the expression of interleukin-6 (IL-6), matrix metalloproteinase-2 (MMP-2), and MMP-9 in UVB-treated HaCaT cells. Also, P. cicadae abrogated UVB-induced activation of NF-κB, p53, and activator protein-1 (AP-1). The downmodulation of IL-6 by P. cicadae was inhibited by the p38 inhibitor (SB203580) or JNK inhibitor (SP600125). Moreover, the extract attenuated the expression of NF-κB and induced thrombomodulin in keratinocytes and thereby effectively downregulated inflammatory responses in the skin. The nuclear accumulation and expression of NF-E2-related factor (Nrf2) were increased by P. cicadae treatment. Furthermore, treatment with P. cicadae remarkably ameliorated the skin's structural damage induced by irradiation. This study demonstrates that P. cicadae may protect skin cells against oxidative insult by modulating ROS concentration, IL-6, MMPs generation, antioxidant enzymes activity, and cell signaling pathways. PMID:28465707

  17. Expression of membrane type 1 matrix metalloproteinase in papillomavirus-positive cells: role of the human papillomavirus (HPV) 16 and HPV8 E7 gene products.

    PubMed

    Smola-Hess, Sigrun; Pahne, Jenny; Mauch, Cornelia; Zigrino, Paola; Smola, Hans; Pfister, Herbert J

    2005-05-01

    Matrix metalloproteinases (MMPs) degrade extracellular matrix. They are involved in cellular proliferation, migration, angiogenesis, invasion and metastasis. MT-1 MMP, a membrane-bound MMP, is expressed in carcinomas of the uterine cervix in vivo. This type of cancer is associated with human papillomavirus (HPV) infection. Here it was shown that keratinocytes transformed with HPV16 or HPV18 in vitro, and HPV-positive cervical carcinoma cell lines, constitutively expressed MT-1 MMP. Expression of the E7 protein from the mucosal and cutaneous high-risk types HPV16 and HPV8, but not from the cutaneous low-risk type HPV1, was sufficient to induce MT-1 MMP expression in primary human keratinocytes and HaCaT cells. As a consequence, MMP-2 was activated. MT-1 MMP expression might play a role in the HPV life cycle by promoting proliferation of host cells and might contribute to their invasive phenotype during malignant progression.

  18. Genome engineering in human cells.

    PubMed

    Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

    2014-01-01

    Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

  19. Cinnamoyl-based Nrf2-Activators Targeting Human Skin Cell Photo-oxidative Stress

    PubMed Central

    Wondrak, Georg T.; Cabello, Chris M.; Villeneuve, Nicole F.; Zhang, Shirley; Ley, Stephanie; Li, Yanjie; Sun, Zheng; Zhang, Donna D.

    2011-01-01

    Strong experimental evidence suggests the involvement of photo-oxidative stress mediated by reactive oxygen species as a crucial mechanism of solar damage relevant to human skin photoaging and photocarcinogenesis. Based on the established role of antioxidant response element (ARE)-mediated gene expression in cancer chemoprevention, we tested the hypothesis that small molecule Nrf2-activators may serve a photo-chemopreventive role by targeting skin cell photo-oxidative stress. A luciferase-based reporter gene assay was used as a primary screen for the identification of novel agents that modulate the Nrf2-Keap1 signaling pathway. A series of cinnamoyl-based electrophilic Michael acceptors including cinnamic aldehyde and methyl-1-cinnamoyl-5-oxo-2-pyrrolidine-carboxylate was identified as potent Nrf2-activators. Hit confirmation was performed in a secondary screen, based on immunodetection of Nrf2 protein upregulation in human Hs27 skin fibroblasts, HaCaT keratinocytes, and primary skin keratinocytes. Bioefficacy profiling of positive test compounds in skin cells demonstrated compound-induced upregulation of hemeoxygenase I and NAD(P)H-quinone oxidoreductase, two Nrf2 target genes involved in the cellular antioxidant response. Pretreatment with cinnamoyl-based Nrf2-activators suppressed intracellular oxidative stress and protected against photo-oxidative induction of apoptosis in skin cells exposed to high doses of singlet oxygen. Our pilot studies suggest feasibility of developing cinnamoyl-based Nrf2-activators as novel photo-chemopreventive agents targeting skin cell photo-oxidative stress. PMID:18482591

  20. Traditional Herbal Formula Banhasasim-tang Exerts Anti-Inflammatory Effects in RAW 264.7 Macrophages and HaCaT Keratinocytes

    PubMed Central

    Lim, Hye-Sun; Kim, Yeji; Seo, Chang-Seob; Shin, Hyeun-Kyoo

    2015-01-01

    Banhasasim-tang (BHSST) is a Korean traditional herbal formula comprising eight medicinal herbs. The aim of the present study was to investigate the anti-inflammatory effect of BHSST using macrophage and keratinocyte cell lines. First, we evaluated the effects of BHSST on inflammatory mediator and cytokine production in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. BHSST markedly inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin- (IL-) 6. BHSST significantly suppressed the protein expression of toll-like receptor 4 (TLR4) and phosphorylated nuclear factor-kappa B (NF-κB) p65 in RAW 264.7 cells. Second, we examined whether BHSST influences the production of chemokines and STAT1 phosphorylation in tumor necrosis factor-α/interferon-γ TI-stimulated HaCaT keratinocytes. BHSST significantly suppressed the production of RANTES/CCL5, TARC/CCL17, MDC/CCL22, and IL-8 in TI-stimulated HaCaT cells. BHSST also suppressed TI-induced phosphorylation of STAT1 in HaCaT cells. These results suggest that BHSST may be useful as an anti-inflammatory agent, especially for inflammatory skin diseases. PMID:25838833

  1. Constitutive expression of MC1R in HaCaT keratinocytes inhibits basal and UVB-induced TNF-alpha production.

    PubMed

    Garcin, Geneviève; Le Gallic, Lionel; Stoebner, Pierre-Emmanuel; Guezennec, Anne; Guesnet, Joelle; Lavabre-Bertrand, Thierry; Martinez, Jean; Meunier, Laurent

    2009-01-01

    Alpha-melanocyte stimulating hormone (alpha-MSH) binds to melanocortin-1 receptor (MC1R) on melanocytes to stimulate pigmentation and modulate various cutaneous inflammatory responses. MC1R expression is not restricted to melanocytic cells and may be induced in keratinocytes after UVB exposure. We hypothesized that MC1R signaling in keratinocytes, wherein basal conditions are barely expressed, may modulate mediators of inflammation, such as nuclear factor-kappa B (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha). Therefore, we generated HaCaT cells that stably express human MC1R or the Arg151Cys (R151C) nonfunctional variant. We demonstrate that: (1) the constitutive activity of MC1R results in elevated intracellular cAMP level, reduced NF-kappaB activity and decreased TNF-alpha transcription; (2) binding of alpha-MSH to MC1R and the subsequent increase in cAMP production do not inhibit TNFalpha-mediated NF-kappaB activation; (3) MC1R signaling is sufficient to strongly inhibit UVB-induced TNF-alpha expression and this inhibitory effect is further enhanced by alpha-MSH stimulation. Our findings suggest that the constitutive activity of the G-protein-coupled MC1R in keratinocytes may contribute to the modulation of inflammatory events and immune response induced by UV light.

  2. HMGB1/RAGE axis promotes autophagy and protects keratinocytes from ultraviolet radiation-induced cell death.

    PubMed

    Mou, Kuanhou; Liu, Wei; Han, Dan; Li, Pan

    2017-03-01

    The primary cause of skin cancer is ultraviolet (UV) light from the sun. Keratinocytes are the predominant cell type in the epidermis and form a barrier against environmental damage, especially from UV light irradiation. Autophagy is a self-digestion mechanism for energy homeostasis at critical times during development and as a response to stress. High-mobility group protein 1 (HMGB1) is a highly conserved nuclear protein that is associated with cell autophagy. We investigated the role of HMGB1 in keratinocytes exposed to UV irradiation and its regulation of keratinocyte autophagy. Specimens of UV-exposed human skin were assayed immunohistochemically for HMGB1. HaCaT immortalized human keratinocytes were used to investigate the mechanism of HMGB1 translocation induced by UV irradiation. Levels of cytosolic reactive oxygen species (ROS) were determined by H2DCF assay, apoptosis was assayed by flow cytometry and western-blot after lentivirus-mediated shRNA targeting of HMGB1 in keratinocytes by UV irradiation. Phosphorylated-Erk1/2 expression was assayed by western blotting. HMGB1 and its receptor (receptor for advanced glycation end products, RAGE) were both expressed by HaCaT cells, and HMGB1 was transferred from the nucleus to the cytoplasm after UV irradiation in both HaCaT and human skin keratinocytes. Knockdown of HMGB1 expression by lentivirus-mediated shRNA limited UV-induced autophagy and led to increased apoptosis of HaCaT cells. Pharmacological inhibition of HMGB1 cytoplasmic translocation by agents such as ethyl pyruvate limits starvation-induced autophagy. UV irradiation led to phosphorylation of Erk1/2 in HaCaT cells. Inhibition of RAGE and Erk1/2 limited HaCaT cell autophagy. Autocrine HMGB1 modulated HaCaT autophagy via a RAGE/HMGB1/extracellular signal-regulated Erk1/2-dependent pathway to protect keratinocytes from apoptosis during UV irradiation. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All

  3. Investigation of dermal toxicity of ionic liquids in monolayer-cultured skin cells and 3D reconstructed human skin models.

    PubMed

    Hwang, Jee-Hyun; Park, Hyeonji; Choi, Dal Woong; Nam, Ki Taek; Lim, Kyung-Min

    2017-09-25

    Ionic liquids have gained increasing attention in the chemical industry as potential green substitutes for traditional solvents. However, little is known about toxicity of ionic liquids on the skin, a major exposure portal to toxic substances. Here, we evaluated dermal toxicity of ionic liquids using human keratinocyte and fibroblast cell line, 3D reconstructed human epidermis, and full-thickness model to investigate underlying mechanisms. Cytotoxicity of ionic liquids was evaluated for representative anions, [TFSI], [PF6], [BF4], and [DCA], as well as for cations, [EMIM], [BMPY], [TBA] and [Zn], in human keratinocyte cell line, HaCaT, and human dermal fibroblasts. In our results, significant cytotoxicity was induced by ionic liquids with [TFSI] in both cell lines. Notably, cytotoxicity of [TFSI] containing ionic liquids was comparable to xylene, a toxic conventional organic solvent. Fluorescent and flow cytometric analysis revealed that [TFSI]-exposed cells underwent necrotic cell death. Reactive oxygen species (ROS) was increased while the amount of glutathione was decreased by [TFSI] in dose-dependent manner, which was reversed by antioxidant, N-acetylcysteine. In 3D reconstructed human epidermis and full-thickness model, a single application of [TFSI] induced toxicity although it was minimal and largely limited to epidermal layer. Collectively, these results demonstrated potential dermal toxicity of ionic liquids. Copyright © 2017. Published by Elsevier Ltd.

  4. An ethanol extract derived from Bonnemaisonia hamifera scavenges ultraviolet B (UVB) radiation-induced reactive oxygen species and attenuates UVB-induced cell damage in human keratinocytes.

    PubMed

    Piao, Mei Jing; Hyun, Yu Jae; Cho, Suk Ju; Kang, Hee Kyoung; Yoo, Eun Sook; Koh, Young Sang; Lee, Nam Ho; Ko, Mi Hee; Hyun, Jin Won

    2012-12-14

    The present study investigated the photoprotective properties of an ethanol extract derived from the red alga Bonnemaisonia hamifera against ultraviolet B (UVB)-induced cell damage in human HaCaT keratinocytes. The Bonnemaisonia hamifera ethanol extract (BHE) scavenged the superoxide anion generated by the xanthine/xanthine oxidase system and the hydroxyl radical generated by the Fenton reaction (FeSO₄ + H₂O₂), both of which were detected by using electron spin resonance spectrometry. In addition, BHE exhibited scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl radical and intracellular reactive oxygen species (ROS) that were induced by either hydrogen peroxide or UVB radiation. BHE reduced UVB-induced apoptosis, as shown by decreased apoptotic body formation and DNA fragmentation. BHE also attenuated DNA damage and the elevated levels of 8-isoprostane and protein carbonyls resulting from UVB-mediated oxidative stress. Furthermore, BHE absorbed electromagnetic radiation in the UVB range (280-320 nm). These results suggest that BHE protects human HaCaT keratinocytes against UVB-induced oxidative damage by scavenging ROS and absorbing UVB photons, thereby reducing injury to cellular components.

  5. Wnt-3a-activated human fibroblasts promote human keratinocyte proliferation and matrix destruction.

    PubMed

    Sobel, Katrin; Tham, Marius; Stark, Hans-Jürgen; Stammer, Hermann; Prätzel-Wunder, Silke; Bickenbach, Jackie R; Boukamp, Petra

    2015-06-15

    Aberrant Wnt regulation, detectable by nuclear translocation of beta-catenin, is a hallmark of many cancers including skin squamous cell carcinomas (SCCs). By analyzing primary human skin SCCs, we demonstrate that nuclear beta-catenin is not restricted to SCC cells but also detected in stromal fibroblasts, suggesting an important role for aberrant Wnt regulation also in the tumor microenvironment. When human keratinocytes and fibroblasts were treated with Wnt-3a, fibroblasts proved to be more responsive. Accordingly, Wnt-3a did not alter HaCaT cell functions in a cell-autonomous manner. However, when organotypic cultures (OTCs) were treated with Wnt-3a, HaCaT keratinocytes responded with increased proliferation. As nuclear beta-catenin was induced only in the fibroblasts, this argued for a Wnt-dependent, paracrine keratinocyte stimulation. Global gene expression analysis of Wnt-3a-stimulated fibroblasts identified genes encoding interleukin-8 (IL-8) and C-C motif chemokine 2 (CCL-2) as well as matrix metalloproteinase-1 (MMP-1) as Wnt-3a targets. In agreement, we show that IL-8 and CCL-2 were secreted in high amounts by Wnt-3a-stimulated fibroblasts also in OTCs. The functional role of IL-8 and CCL-2 as keratinocyte growth regulators was confirmed by directly stimulating HaCaT cell proliferation in conventional cultures. Most important, neutralizing antibodies against IL-8 and CCL-2 abolished the Wnt-dependent HaCaT cell hyperproliferation in OTCs. Additionally, MMP-1 was expressed in high amounts in Wnt-3a-stimulated OTCs and degraded the stromal matrix. Thus, our data show that Wnt-3a stimulates fibroblasts to secrete both keratinocyte proliferation-inducing cytokines and stroma-degrading metalloproteinases, thereby providing evidence for a novel Wnt deregulation in the tumor-stroma directly contributing to skin cancer progression.

  6. Quantitative assessment of the relative antineoplastic potential of the n-butanolic leaf extract of Annona muricata Linn. in normal and immortalized human cell lines.

    PubMed

    George, V Cijo; Kumar, D R Naveen; Rajkumar, V; Suresh, P K; Kumar, R Ashok

    2012-01-01

    Natural products have been the target for cancer therapy for several years but there is still a dearth of information on potent compounds that may protect normal cells and selectively destroy cancerous cells. The present study was aimed to evaluate the cytotoxic potential of n-butanolic leaf extract of Annona muricata L. on WRL-68 (normal human hepatic cells), MDA-MB-435S (human breast carcinoma cells) and HaCaT (human immortalized keratinocyte cells) lines by XTT assay. Prior to cytotoxicity testing, the extract was subjected to phytochemical screening for detecting the presence of compounds with therapeutic potential. Their relative antioxidant properties were evaluated using the reducing power and DPPH* radical scavenging assay. Since most of the observed chemo-preventive potential invariably correlated with the amount of total phenolics present in the extract, their levels were quantified and identified by HPLC analysis. Correlation studies indicated a strong and significant (P<0.05) positive correlation of phenolic compounds with free radical scavenging potential. The results revealed that the extract was moderately cytotoxic to normal cells with a mean IC50 value of 52.4 μg when compared with those obtained for cancerous cells (IC50 values of 29.2 μg for MDA-MB-435S and 30.1 μg for HaCaT respectively). The study confirms the presence of therapeutically active antineoplastic compounds in the n-butanolic leaf extract of Annona muricata. Isolation of the active metabolites from the extract is in prospect.

  7. Light-induced cytotoxicity and genotoxicity of a sunscreen agent, 2-phenylbenzimidazole in Salmonella typhimurium TA 102 and HaCaT keratinocytes.

    PubMed

    Mosley, Charity N; Wang, Lei; Gilley, Stephanie; Wang, Shuguang; Yu, Hongtao

    2007-06-01

    2-phenylbenzimidazole (PBI) is an ingredient found in sunscreen agents. PBI can absorb the UV portion of the solar light and undergo a series of light-induced reactions to cause adverse effects in humans. Therefore, chemical and photochemical toxicity of PBI were investigated in the bacteria Salmonella typhimurium TA 102 and human skin keratinocyte cells. There is no appreciable bacteria death due to the exposure to PBI alone, indicating that PBI is not chemically toxic to the bacteria at a dose as high as 625 microM. However, exposure to PBI and a solar simulator light (300-W Xe/Hg lamp, 30 min, 18.6 J/cm2, equivalent to 30 min outdoor sunlight) causes significant bacteria death: 35% at 25 microM and 55% at 625 microM PBI. Exposure of the bacteria to light and PBI at doses 5-25 microM causes the bacteria to revert, an indication of mutation. In the presence of PBI but without light irradiation, the number of revertant bacteria colonies is around 200 due to spontaneous mutation. Combination of light irradiation and PBI causes the number of revertant TA 102 colonies to increase in a dose dependent manner, reaching a maximum of around 1700 revertant colonies at 25 microM PBI. At higher PBI concentrations, the number of revertant colonies remains constant. This result clearly indicates that PBI is photomutagenic in TA 102. Exposure of the human skin HaCaT keratinocytes in aqueous solution in the presence of PBI causes the cell to lose its viability with or without light irradiation. There is no significant difference in cell viability for the light irradiated or non-irradiated groups, indication PBI is not photocytotoxic. However, exposure of the cells to both PBI and light irradiation causes cellular DNA damage, while exposure to PBI alone does not cause DNA damage.

  8. 1,25-dihydroxyvitamin D3 modulates effects of ionizing radiation (IR) on human keratinocytes: in vitro analysis of cell viability/proliferation, DNA-damage and -repair.

    PubMed

    Trémezaygues, Lea; Seifert, Markus; Vogt, Thomas; Tilgen, Wolfgang; Reichrath, Jörg

    2010-07-01

    We investigated the capacity of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to protect spontaneously immortalized human keratinocytes (HaCaT) and cutaneous squamous cell carcinoma cells (SCL-1) against the hazardous effects of ionizing radiation (IR). We pretreated HaCaT and SCL-1 cells in vitro with 1,25(OH)2D3 (10(-7) M) over 48 h and then irradiated them once with IR (1 Gy, 2 Gy, and 5 Gy). Using WST-1-assay and crystal violet (CV) assay, we compared viability/proliferation in 1,25(OH)2D3-pretreated cells with controls that were pretreated with the carrier substance ethanol alone. Additionally, we analyzed the effects of 1,25(OH)2D3 on the presence of IR-induced DNA-damage by immunocytochemical detection of gamma-H2AX-foci in HaCaT-keratinocytes. We demonstrate that 1,25(OH)2D3 (10(-7) M) inhibits proliferation of human keratinocytes and that IR (1-5 Gy) has no significant effect on proliferation and viability of HaCaT-keratinocytes and SCL-1 cells. Moreover, we show that IR modulates dose-dependently the number of gammaH2AX-foci in HaCaT-keratinocytes. Pretreatment of the cells with 1,25(OH)2D3 reduces the number of IR-induced gammaH2AX-foci after irradiation with 1 Gy and 2 Gy and increases it after irradiation with 5 Gy. To put it in a nutshell, our data support the hypothesis that 1,25(OH)2D3 modulates the effects of low-dose IR (1-5 Gy) on cultured human keratinocytes.

  9. Photodynamic effects of 31 different phthalocyanines on a human keratinocyte cell line.

    PubMed

    Jančula, Daniel; Maršálek, Blahoslav; Babica, Pavel

    2013-10-01

    Phthalocyanines (Pcs, colored macromolecular compounds with the ability to generate singlet oxygen) represent a promising group of photosensitizers due to their intense absorption in the red and UV portion of the spectrum which leads to their excitation. In order to characterize possible toxic effects associated with eventual practical use and application of these chemicals, we employed an in vitro cell culture model to evaluate cytotoxic effects of 31 different phthalocyanines using neutral red uptake assay. An immortalized human keratinocyte cell line HaCaT was exposed to the tested chemicals for 2 or 24h, either with or without illumination in the last 60 min of the exposure period. After 2- or 24-h exposure without illumination, no cytotoxic effects or weak cytotoxic effects were induced by any Pc under the study and EC50 values could not be obtained within the tested concentration ranges (1.25-20 mg L(-1) or 0.625-10 mg L(-1)). On the other hand, exposure to phthalocyanines under illumination induced a significant cytotoxic effect. The most pronounced cytotoxicity was elicited by Pcs previously shown to have high positive charge densities at peripheral parts of substituent groups, which is most likely the factor responsible for the binding of Pc to negatively charged membranes on the cell surface and thus guaranteeing the tight connection necessary for the singlet oxygen attack on the cell surface.

  10. Phenotypic and genotypic profile of human tympanic membrane derived cultured cells.

    PubMed

    Redmond, Sharon L; Levin, Brett; Heel, Kathryn A; Atlas, Marcus D; Marano, Robert J

    2011-02-01

    The human tympanic membrane (hTM), known more commonly as the eardrum, is a thin, multi-layered membrane that is unique in the body as it is suspended in air. When perforated, the hTM's primary function of sound-pressure transmission is compromised. For the purposes of TM reconstruction, we investigated the phenotype and genotype of cultured primary cells derived from hTM tissue explants, compared to epithelial (HaCaT cells) and mesenchymal (human dermal fibroblasts (HDF)) reference cells. Epithelium-specific ets-1 (ESE-1), E-cadherin, keratinocyte growth factor-1 (KGF-1/FGF-7), keratinocyte growth factor-2 (KGF-2/FGF10), fibroblast growth factor receptor 1 (FGFR1), variants of fibroblast growth factor receptor 2 (FGFR2), fibroblast surface protein (FSP), and vimentin proteins were used to assess the phenotypes of all cultured cells. Wholemount and paraffin-embedded hTM tissues were stained with ESE-1 and E-cadherin proteins to establish normal epithelial-specific expression patterns within the epithelial layers. Immunofluorescent (IF) cell staining of hTM epithelial cells (hTMk) demonstrated co-expression of both epithelial- and mesenchymal-specific proteins. Flow cytometry (FCM) analysis further demonstrated co-expression of these epithelial and mesenchymal-specific proteins, indicating the subcultured hTMk cells possessed a transitional phenotype. Gene transcript analysis of hTMk cells by reverse transcriptase polymerase chain reaction (RT-PCR) revealed a down regulation of ESE-1, E-cadherin, FGFR2, variant 1 and variant 2 (FGFR2v1 and FGFR2v2) between low and high passages, and up-regulation of KGF-1, KGF-2, and FGFR1. All results indicate a gradual shift in cell phenotype of hTMk-derived cells from epithelial to mesenchymal.

  11. The crucial role of IL-22 and its receptor in thymus and activation regulated chemokine production and T-cell migration by house dust mite extract.

    PubMed

    Jang, Mirim; Kim, Hyemin; Kim, Yejin; Choi, Jiyea; Jeon, Jane; Hwang, Youngil; Kang, Jae Seung; Lee, Wang Jae

    2016-08-01

    House dust mite (HDM) is known as one of the factors that causes atopic dermatitis (AD). Interleukin (IL)-22 and thymus and activation regulated chemokine (TARC) are related to skin inflammatory disease and highly expressed in AD lesions. However, the effects of HDM on IL-22 production in T cells and on TARC production and IL-22Rα receptor expression in keratinocytes are unknown. To identify the role of HDM in keratinocytes and T cells, we investigated IL-22Rα expression and TARC production in the human keratinocyte cell line HaCaT and IL-22 production in T cells treated with HDM extract as well as their roles in HDM-induced skin inflammation. HDM extract not only increased IL-22Rα expression and TARC production in HaCaT but also enhanced IL-22, tumor necrosis factor (TNF)-α and interferon (IFN)-γ production in T cells. The HDM extract-induced IL-22 from T cells significantly increased the production of IL-1α, IL-6 and TARC in HaCaT cells. In addition, we found that TARC produced in HDM extract-treated HaCaT induced T-cell recruitment. These results suggest that there is a direct involvement of HDM extract-induced IL-22 in TARC production and T-cell migration. Taken together, TARC production in HaCaT through the interaction between IL-22 and IL-22Rα facilitates T-cell migration. These data show one of the reasons for inflammation in the skin lesions of AD patients. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Human natural killer cell development.

    PubMed

    Freud, Aharon G; Caligiuri, Michael A

    2006-12-01

    Our understanding of human natural killer (NK) cell development lags far behind that of human B- or T-cell development. Much of our recent knowledge of this incomplete picture comes from experimental animal models that have aided in identifying fundamental in vivo processes, including those controlling NK cell homeostasis, self-tolerance, and the generation of a diverse NK cell repertoire. However, it has been difficult to fully understand the mechanistic details of NK cell development in humans, primarily because the in vivo cellular intermediates and microenvironments of this developmental pathway have remained elusive. Although there is general consensus that NK cell development occurs primarily within the bone marrow (BM), recent data implicate secondary lymphoid tissues as principal sites of NK cell development in humans. The strongest evidence stems from the observation that the newly described stages of human NK cell development are naturally and selectively enriched within lymph nodes and tonsils compared with blood and BM. In the current review, we provide an overview of these recent findings and discuss these in the context of existing tenets in the field of lymphocyte development.

  13. Responses of human cells to ZnO nanoparticles: a gene transcription study.

    PubMed

    Moos, Philip J; Olszewski, Kyle; Honeggar, Matthew; Cassidy, Pamela; Leachman, Sancy; Woessner, David; Cutler, N Shane; Veranth, John M

    2011-11-01

    The gene transcript profile responses to metal oxide nanoparticles was studied using human cell lines derived from the colon and skin tumors. Much of the research on nanoparticle toxicology has focused on models of inhalation and intact skin exposure, and effects of ingestion exposure and application to diseased skin are relatively unknown. Powders of nominally nanosized SiO2, TiO2, ZnO and Fe2O3 were chosen because these substances are widely used in consumer products. The four oxides were evaluated using colon-derived cell lines, RKO and CaCo-2, and ZnO and TiO2 were evaluated further using skin-derived cell lines HaCaT and SK Mel-28. ZnO induced the most notable gene transcription changes, even though this material was applied at the lowest concentration. Nano-sized and conventional ZnO induced similar responses suggesting common mechanisms of action. The results showed neither a non-specific response pattern common to all substances nor synergy of the particles with TNF-α cotreatment. The response to ZnO was not consistent with a pronounced proinflammatory signature, but involved changes in metal metabolism, chaperonin proteins, and protein folding genes. This response was observed in all cell lines when ZnO was in contact with the human cells. When the cells were exposed to soluble Zn, the genes involved in metal metabolism were induced but the genes involved in protein refoldling were unaffected. This provides some of the first data on the effects of commercial metal oxide nanoparticles on human colon-derived and skin-derived cells.

  14. Comparative toxicities of bismuth oxybromide and titanium dioxide exposure on human skin keratinocyte cells.

    PubMed

    Gao, Xiaoya; Wang, Yawen; Peng, Shiqi; Yue, Bin; Fan, Caimei; Chen, Weiyi; Li, Xiaona

    2015-09-01

    Nano-sized bismuth oxybromide (BiOBr) particles are being considered for applications within the semiconductor industry. However, little is known about their potential impact on human health. In this study, we comparatively investigated the cytotoxicity of BiOBr and titanium dioxide (TiO2) nanoparticles (NPs) using human skin keratinocyte cell line (HaCaT) as a research model. Results indicate that lamellar-shaped BiOBr (length: 200 nm, width: 150 nm, and an average thickness: around 15 nm) has less toxic effects on cell viability and intracellular organelles than TiO2 (P25) NPs. BiOBr mainly induced late cell apoptosis, while for TiO2, both early apoptosis and late apoptosis were involved. Cell cycle arrest was found in cells on both NPs exposure, and more prominent in TiO2-treated cells. More cellular uptake was achieved after TiO2 exposure, particularly at 10 μg mL(-1), presence of TiO2 resulted in more than 2-fold increase in cellular granularity compared with BiOBr. Furthermore, TiO2 had a high potential to generate intracellular reactive oxygen species (ROS) in cells, where a 2.7-fold increase in TiO2 group and 2.0-fold increase in BiOBr group at the same concentration of 25 μg mL(-1). Higher cellular uptake and ROS stimulation should contribute to the more hazards of TiO2 than BiOBr NPs. This knowledge is a crucial component in the environmental and human hazard assessment of BiOBr and TiO2 NPs.

  15. MHC class I-related chain A and B ligands are differentially expressed in human cervical cancer cell lines

    PubMed Central

    2011-01-01

    Background Natural killer (NK) cells are an important resource of the innate immune system directly involved in the spontaneous recognition and lysis of virus-infected and tumor cells. An exquisite balance of inhibitory and activating receptors tightly controls the NK cell activity. At present, one of the best-characterized activating receptors is NKG2D, which promotes the NK-mediated lysis of target cells by binding to a family of cell surface ligands encoded by the MHC class I chain-related (MIC) genes, among others. The goal of this study was to describe the expression pattern of MICA and MICB at the molecular and cellular levels in human cervical cancer cell lines infected or not with human papillomavirus, as well as in a non-tumorigenic keratinocyte cell line. Results Here we show that MICA and MICB exhibit differential expression patterns among HPV-infected (SiHa and HeLa) and non-infected cell lines (C33-A, a tumor cell line, and HaCaT, an immortalized keratinocyte cell line). Cell surface expression of MICA was higher than cell surface expression of MICB in the HPV-positive cell lines; in contrast, HPV-negative cells expressed lower levels of MICA. Interestingly, the MICA levels observed in C33-A cells were overcome by significantly higher MICB expression. Also, all cell lines released higher amounts of soluble MICB than of soluble MICA into the cell culture supernatant, although this was most pronounced in C33-A cells. Additionally, Real-Time PCR analysis demonstrated that MICA was strongly upregulated after genotoxic stress. Conclusions This study provides evidence that even when MICA and MICB share a high degree of homology at both genomic and protein levels, differential regulation of their expression and cell surface appearance might be occurring in cervical cancer-derived cells. PMID:21631944

  16. Gold Nano Popcorn Attached SWCNT Hybrid Nanomaterial for Targeted Diagnosis and Photothermal Therapy of Human Breast Cancer Cells

    PubMed Central

    Beqa, Lule; Fan, Zhen; Singh, Anant Kumar; Senapati, Dulal; Ray, Paresh Chandra

    2011-01-01

    Breast cancer presents greatest challenge in health care in today’s world. The key to ultimately successful treatment of breast cancer disease is an early and accurate diagnosis. Current breast cancer treatments are often associated with severe side effects. Driven by the need, we report the design of novel hybrid nanomaterial using gold nano popcorn-attached single wall carbon nanotube for targeted diagnosis and selective photothermal treatment. Targeted SK-BR-3 human breast cancer cell sensing have been performed in 10 cancer cells/mL level, using surface enhanced Raman scattering of single walls carbon nanotube’s D and G bands. Our data show that S6 aptamer attached hybrid nanomaterial based SERS assay is highly sensitive to targeted human breast cancer SK-BR-3 cell line and it will be able to distinguish it from other non targeted MDA-MB breast cancer cell line and HaCaT normal skin cell line. Our results also show that 10 minutes of photothermal therapy treatment by 1.5 W/cm2 power, 785 nm laser is enough to kill cancer cells very effectively using S6 aptamer attached hybrid nanomaterials. Possible mechanisms for targeted sensing and operating principle for highly efficient photothermal therapy have been discussed. Our experimental results reported here open up a new possibility for using aptamers modified hybrid nanomaterial for reliable diagnosis and targeted therapy of cancer cell lines quickly. PMID:21842867

  17. Gold nano-popcorn attached SWCNT hybrid nanomaterial for targeted diagnosis and photothermal therapy of human breast cancer cells.

    PubMed

    Beqa, Lule; Fan, Zhen; Singh, Anant Kumar; Senapati, Dulal; Ray, Paresh Chandra

    2011-09-01

    Breast cancer presents greatest challenge in health care in today's world. The key to ultimately successful treatment of breast cancer disease is an early and accurate diagnosis. Current breast cancer treatments are often associated with severe side effects. Driven by the need, we report the design of novel hybrid nanomaterial using gold nano popcorn-attached single wall carbon nanotube for targeted diagnosis and selective photothermal treatment. Targeted SK-BR-3 human breast cancer cell sensing have been performed in 10 cancer cells/mL level, using surface enhanced Raman scattering of single walls carbon nanotube's D and G bands. Our data show that S6 aptamer attached hybrid nanomaterial based SERS assay is highly sensitive to targeted human breast cancer SK-BR-3 cell line and it will be able to distinguish it from other non targeted MDA-MB breast cancer cell line and HaCaT normal skin cell line. Our results also show that 10 min of photothermal therapy treatment by 1.5 W/cm(2) power, 785 nm laser is enough to kill cancer cells very effectively using S6 aptamer attached hybrid nanomaterials. Possible mechanisms for targeted sensing and operating principle for highly efficient photothermal therapy have been discussed. Our experimental results reported here open up a new possibility for using aptamers modified hybrid nanomaterial for reliable diagnosis and targeted therapy of cancer cell lines quickly.

  18. Paracrine Activity from Adipose-Derived Stem Cells on In Vitro Wound Healing in Human Tympanic Membrane Keratinocytes.

    PubMed

    Ong, Huan Ting; Redmond, Sharon L; Marano, Robert J; Atlas, Marcus D; von Unge, Magnus; Aabel, Peder; Dilley, Rodney J

    2017-03-15

    Stem cell therapies for tympanic membrane repair have shown initial experimental success using mesenchymal stem cells in rat models to promote healing; however, the mechanisms providing this benefit are not known. We investigated in vitro the paracrine effects of human adipose-derived stem cells (ADSCs) on wound healing mechanisms for human tympanic membrane-derived keratinocytes (hTM) and immortalized human keratinocytes (HaCaT). ADSC conditioned media (CMADSC) were assessed for paracrine activity on keratinocyte proliferation and migration, with hypoxic conditions for ADSC culture used to generate contrasting effects on cytokine gene expression. Keratinocytes cultured in CMADSC showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CMADSC. Assessment of ADSC gene expression on a cytokine array showed a range of wound healing cytokines expressed and under stringent hypoxic and serum-free conditions was upregulated (VEGF A, MMP9, Tissue Factor, PAI-1) or downregulated (CXCL5, CCL7, TNF-α). Several of these may contribute to the activity of conditioned media on the keratinocytes with potential applications in TM perforation repair. VEGFA protein was confirmed by immunoassay to be increased in conditioned media. Together with gene regulation associated with hypoxia in ADSCs, this study has provided several strong leads for a stem cell-derived approach to TM wound healing.

  19. Toxic effects of several types of antifouling paints in human and rat hepatic or epidermal cells.

    PubMed

    de Sousa, G; Delescluse, C; Pralavorio, M; Perichaud, M; Avon, M; Lafaurie, M; Rahmani, R

    1998-08-01

    Fouling is the successive development of marine organisms on immersed surfaces, a process which has heavy negative economic impacts. Several antifouling technologies, generally based on the leaching of biocides from painted surfaces, have been developed, but these biocides are toxic to the environment. Hence, we compared the toxicity of several currently used paint lixiviats in rat hepatocytes, human HepG2 and HaCaT cells. Acute toxicity was assessed by the Neutral Red and MTT assays. Chronic effect was tested using induction of the 7-ethoxyresorufin-O-deethylase (EROD) activity as a marker. Large variations were observed among the various cell types or the antifouling formulations, both in terms of IC50 values (from approximately 0.5 to approximately 10%, v/v) and EROD induction (from approximately 1 to 10-fold over control). These differences appear to be related to variable biocide (copper compounds, organotins, etc...) concentrations in the different paint formulations, or to the specific metabolic capabilities of the cell system used.

  20. Influence of cyclodextrins on the proliferation of HaCaT keratinocytes in vitro.

    PubMed

    Hipler, U C; Schönfelder, U; Hipler, C; Elsner, P

    2007-10-01

    Safety and efficacy of pharmaceutical agents can be greatly improved by encapsulation within, or covalent attachment to, a biomaterial carrier. Drug delivery systems must deliver the necessary amount of drug to the targeted site for a necessary period of time, both efficiently and precisely. Various kinds of high-performance biomaterials are being constantly developed for this purpose. Cyclodextrins are potential candidates for such a role, because of their ability to alter physical, chemical, and biological properties of guest molecules through the formation of inclusion complexes. The alpha-, beta-, and gamma-cyclodextrins are widely used natural cyclodextrins, consisting of six, seven, and eight D-glucopyranose residues, respectively, linked by -1,4 glycosidic bonds into a macro cycle. Each cyclodextrin has its own ability to form inclusion complexes with specific guests, an ability, which depends on a proper fit of the guest molecule into the hydrophobic cyclodextrin cavity. The most common pharmaceutical application of cyclodextrins is to enhance the solubility, stability, and bioavailability of drug molecules. Such kinds of ligand-receptor complexes can be used for different applications, e.g., for a transdermal therapeutic system (TTS) or in biofunctional textiles. The aim of this study was the investigation of the influence of the different cyclodextrins on the cell proliferation using HaCaT keratinocytes as an in vitro test system. Moreover, the study was performed to find harmless and nontoxic cyclodextrin concentrations for dermal applications. By means of different independent in vitro tests could be confirmed that alpha-, beta-, and gamma-cyclodextrins in concentrations up to 0.1% (w/v) do not show any antiproliferative influence on HaCaT keratinocytes. Sometimes even proliferative effects could be found. However, all used cyclodextrins (besides gamma-cyclodextrin and its derivatives) in concentrations of 0.5 and 1% (w/v), respectively, exert a

  1. Extracts from Calendula officinalis offer in vitro protection against H2 O2 induced oxidative stress cell killing of human skin cells.

    PubMed

    Alnuqaydan, Abdullah M; Lenehan, Claire E; Hughes, Rachel R; Sanderson, Barbara J

    2015-01-01

    The in vitro safety and antioxidant potential of Calendula officinalis flower head extracts was investigated. The effect of different concentrations (0.125, 0.5, 1.0, 2.0 and 5.0% (v/v)) of Calendula extracts on human skin cells HaCaT in vitro was explored. Doses of 1.0% (v/v) (0.88 mg dry weight/mL) or less showed no toxicity. Cells were also exposed to the Calendula extracts for either 4, 24 or 48 h before being exposed to an oxidative insult (hydrogen peroxide H2 O2 ) for 1 h. Using the MTT cytotoxicity assay, it was observed that two independent extracts of C. officinalis gave time-dependent and concentration-dependent H2 O2 protection against induced oxidative stress in vitro using human skin cells. Pre-incubation with the Calendula extracts for 24 and 48 h increased survival relative to the population without extract by 20% and 40% respectively following oxidative challenge. The antioxidant potential of the Calendula extracts was confirmed using a complimentary chemical technique, the DPPH(●) assay. Calendula extracts exhibited free radical scavenging abilities. This study demonstrates that Calendula flower extracts contain bioactive and free radical scavenging compounds that significantly protect against oxidative stress in a human skin cell culture model. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Cells of human breast milk.

    PubMed

    Witkowska-Zimny, Malgorzata; Kaminska-El-Hassan, Ewa

    2017-01-01

    Human milk is a complex fluid that has developed to satisfy the nutritional requirements of infants. In addition to proteins, lipids, carbohydrates and other biologically active components, breast milk contains a diverse microbiome that is presumed to colonize the infant gastrointestinal tract and a heterogeneous population of cells with unclear physiological roles and health implications. Noteworthy cellular components of breast milk include progenitor/stem cells. This review summarizes the current state of knowledge of breast milk cells, including leukocytes, epithelial cells, stem cells and potentially probiotic bacteria.

  3. IgG and IgA with Potential Microbial-Binding Activity Are Expressed by Normal Human Skin Epidermal Cells

    PubMed Central

    Jiang, Dongyang; Ge, Jing; Liao, Qinyuan; Ma, Junfan; Liu, Yang; Huang, Jing; Wang, Chong; Xu, Weiyan; Zheng, Jie; Shao, Wenwei; Lee, Gregory; Qiu, Xiaoyan

    2015-01-01

    The innate immune system of the skin is thought to depend largely on a multi-layered mechanical barrier supplemented by epidermis-derived antimicrobial peptides. To date, there are no reports of antimicrobial antibody secretion by the epidermis. In this study, we report the expression of functional immunoglobulin G (IgG) and immunoglobulin A (IgA), previously thought to be only produced by B cells, in normal human epidermal cells and the human keratinocyte line HaCaT. While B cells express a fully diverse Ig, epidermal cell-expressed IgG or IgA showed one or two conservative VHDJH rearrangements in each individual. These unique VDJ rearrangements in epidermal cells were found neither in the B cell-derived Ig VDJ databases published by others nor in our positive controls. IgG and IgA from epidermal cells of the same individual had different VDJ rearrangement patterns. IgG was found primarily in prickle cells, and IgA was mainly detected in basal cells. Both epidermal cell-derived IgG and IgA showed potential antibody activity by binding pathogens like Staphylococcus aureus, the most common pathogenic skin bacteria, but the microbial-binding profile was different. Our data indicates that normal human epidermal cells spontaneously express IgG and IgA, and we speculate that these Igs participate in skin innate immunity. PMID:25625513

  4. Subcellular Raman Microspectroscopy Imaging of Nucleic Acids and Tryptophan for Distinction of Normal Human Skin Cells and Tumorigenic Keratinocytes.

    PubMed

    Piredda, Paola; Berning, Manuel; Boukamp, Petra; Volkmer, Andreas

    2015-07-07

    At present, tumor diagnostic imaging is commonly based on hematoxylin and eosin or immunohistochemical staining of biopsies, which requires tissue excision, fixation, and staining with exogenous marker molecules. Here, we report on label-free tumor imaging using confocal spontaneous Raman scattering microspectroscopy, which exploits the intrinsic vibrational contrast of endogenous biomolecular species. We present a chemically specific and quantitative approach to monitoring normal human skin cells (keratinocytes and fibroblasts) as well as the human HaCaT in vitro skin carcinogenesis model and the tumor-derived MET in vivo skin cancer progression model. Mapping the amplitudes of two spectrally well isolated Raman bands at 752 and 785 cm(-1) allowed for direct visualization of the distributions representative of tryptophan-rich proteins and nucleic acids, respectively, with subcellular spatial resolution. Using these Raman markers, it was feasible to discriminate between normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) and to confine all tumorigenic cells from both the NHEK and NHDF. First evidence for the successful application of the proposed intracellular nucleic acid and tryptophan Raman signatures for skin cancer diagnosis was further demonstrated in an organotypic cutaneous squamous cell carcinomas model, allowing for the identification of tumor cells and their surrounding stroma in the tissue context.

  5. Identification of Key Proteins in Human Epithelial Cells Responding to Bystander Signals From Irradiated Trout Skin

    PubMed Central

    Smith, Richard; Wang, Jiaxi; Seymour, Colin; Mothersill, Carmel; Howe, Orla

    2015-01-01

    Radiation-induced bystander signaling has been found to occur in live rainbow trout fish (Oncorhynchus mykiss). This article reports identification of key proteomic changes in a bystander reporter cell line (HaCaT) grown in low-dose irradiated tissue-conditioned media (ITCM) from rainbow trout fish. In vitro explant cultures were generated from the skin of fish previously exposed to low doses (0.1 and 0.5 Gy) of X-ray radiation in vivo. The ITCM was harvested from all donor explant cultures and placed on recipient HaCaT cells to observe any change in protein expression caused by the bystander signals. Proteomic methods using 2-dimensional (2D) gel electrophoresis and mass spectroscopy were employed to screen for novel proteins expressed. The proteomic changes measured in HaCaT cells receiving the ITCM revealed that exposure to 0.5 Gy induced an upregulation of annexin A2 and cingulin and a downregulation of Rho-GDI2, F-actin-capping protein subunit beta, microtubule-associated protein RP/EB family member, and 14-3-3 proteins. The 0.1 Gy dose also induced a downregulation of Rho-GDI2, hMMS19, F-actin-capping protein subunit beta, and microtubule-associated protein RP/EB family member proteins. The proteins reported may influence apoptotic signaling, as the results were suggestive of an induction of cell communication, repair mechanisms, and dysregulation of growth signals. PMID:26673684

  6. Perspectives on human stem cell research.

    PubMed

    Jung, Kyu Won

    2009-09-01

    Human stem cell research draws not only scientists' but the public's attention. Human stem cell research is considered to be able to identify the mechanism of human development and change the paradigm of medical practices. However, there are heated ethical and legal debates about human stem cell research. The core issue is that of human dignity and human life. Some prefer human adult stem cell research or iPS cell research, others hES cell research. We do not need to exclude any type of stem cell research because each has its own merits and issues, and they can facilitate the scientific revolution when working together.

  7. Multiple biological effects of inhibitors of arachidonic acid metabolism on human keratinocytes.

    PubMed

    Pacherník, Jirí; Hampl, Ales; Soucek, Karel; Kovaríková, Martina; Andrysík, Zdenek; Hofmanová, Jirina; Kozubík, Alois

    2002-02-01

    Various compounds that inhibit processing of arachidonic acid (AA) are being intensively tested for their antitumour activity. However, the mechanisms responsible for such activity remain rather elusive. To approach this issue, we examined the effects of several structurally different inhibitors of AA metabolism in the human keratinocyte HaCaT cell line. Several parameters were determined in HaCaT cells exposed to increasing concentrations of the inhibitors for 24 and/or 48 h. These included (1) oxidoreductase activity, total protein mass and cell cycle distribution to assess cell proliferation, (2) degradation of PARP protein to assess apoptosis, and (3) cell morphology, distribution of F-actin and expression of cytokeratins and E-cadherin to evaluate changes in differentiation status. While eicosatetraynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), esculetin and MK-886 reduced proliferation of HaCaT cells, the cyclooxygenase inhibitors indomethacin and piroxicam had no such effects. Esculetin and NDGA arrested cells in S phase, and ETYA and MK-886 delayed cell progression through G(1) phase. Higher concentrations of NDGA, MK886 and/or ETYA caused cleavage of PARP. No changes in the expression of cytokeratins and E-cadherin were observed upon treatment with any of the inhibitors. However, esculetin induced redistribution of F-actin accompanied by increased cell adhesion and size. Our findings indicate that, in addition to their ability to inhibit cell proliferation and to induce apoptosis, lipoxygenase inhibitors and/or ETYA may also elicit other important physiological responses in HaCaT keratinocytes.

  8. CD44v6 expression in human skin keratinocytes as a possible mechanism for carcinogenesis associated with chronic arsenic exposure

    PubMed Central

    Huang, S.; Guo, S.; Guo, F.; Yang, Q.; Xiao, X.; Murata, M.; Ohnishi, S.; Kawanishi, S.; Ma, N.

    2013-01-01

    Inorganic arsenic is a well-known human skin carcinogen. Chronic arsenic exposure results in various types of human skin lesions, including squamous cell carcinoma (SCC). To investigate whether mutant stem cells participate in arsenic-associated carcinogenesis, we repeatedly exposed the human spontaneously immortalized skin keratinocytes (HaCaT) cell line to an environmentally relevant level of arsenic (0.05 ppm) in vitrofor 18 weeks. Following sodium arsenite administration, cell cycle, colony-forming efficiency (CFE), cell tumorigenicity, and expression of CD44v6, NF-κB and p53, were analyzed at different time points (0, 5, 10, 15, 20, 25 and 30 passages). We found that a chronic exposure of HaCaT cells to a low level of arsenic induced a cancer stem-like phenotype. Furthermore, arsenictreated HaCaT cells also became tumorigenic in nude mice, their growth cycle was predominantly in G2/M and S phases. Relative to nontreated cells, they exhibited a higher growth rate and a significant increase in CFE. Western blot analysis found that arsenic was capable of increasing cell proliferation and sprouting of cancer stem-like phenotype. Additionally, immunohistochemical analysis demonstrated that CD44v6 expression was upregulated in HaCaT cells exposed to a low level of arsenic during early stages of induction. The expression of CD44v6 in arsenic-treated cells was positively correlated with their cloning efficiency in soft agar (r=0.949, P=0.01). Likewise, the expressions of activating transcription factor NF-κB and p53 genes in the arsenic-treated HaCaT cells were significantly higher than that in non-treated cells. Higher expressions of CD44v6, NF-κB and p53 were also observed in tumor tissues isolated from Balb/c nude mice. The present results suggest that CD44v6 may be a biomarker of arsenicinduced neoplastic transformation in human skin cells, and that arsenic promotes malignant transformation in human skin lesions through a NF-κB signaling pathway

  9. Extremely low frequency electromagnetic field enhances human keratinocyte cell growth and decreases proinflammatory chemokine production.

    PubMed

    Vianale, G; Reale, M; Amerio, P; Stefanachi, M; Di Luzio, S; Muraro, R

    2008-06-01

    Proliferation and differentiation of keratinocytes are central processes in tissue regeneration after injury. Chemokines, produced by a wide range of cell types including keratinocytes, play a regulatory role in inflammatory skin diseases. Several studies have shown that an electromagnetic field (EMF) can influence both inflammatory processes and repair mechanisms including wound healing on different tissue models. To elucidate the effect of extremely low frequency EMF (ELF-EMF) on keratinocyte proliferation and production of chemokines [RANTES, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha and interleukin (IL)-8] in order to evaluate a potential therapeutic use of magnetic fields. The human keratinocyte cell line HaCaT was exposed at 1 mT, 50 Hz for different lengths of time and compared with unexposed control cells. Cell growth and viability were evaluated at different exposure times by cell count and trypan blue exclusion. Chemokine production and expression were analysed by enzyme-linked immunosorbent assay (ELISA) and by real-time polymerase chain reaction. Total NF-kappaB p65 was quantified by ELISA. Significantly increased growth rates were observed after 48 h of EMF exposure as compared with control cells, while no difference in cell viabilities were detected. Gene expression and release of RANTES, MCP-1, MIP-1 alpha and IL-8 were significantly reduced after 72 h of exposure. NF-kappaB levels became almost undetectable after only 1 h of EMF exposure, and were inversely correlated with cell density. Our results show that ELF-EMF modulates chemokine production and keratinocyte growth through inhibition of the NF-kappaB signalling pathway and thus may inhibit inflammatory processes. ELF-EMF could represent an additional therapeutic approach in the treatment of skin injury.

  10. Diffusion inside living human cells

    NASA Astrophysics Data System (ADS)

    Leijnse, N.; Jeon, J.-H.; Loft, S.; Metzler, R.; Oddershede, L. B.

    2012-04-01

    Naturally occurring lipid granules diffuse in the cytoplasm and can be used as tracers to map out the viscoelastic landscape inside living cells. Using optical trapping and single particle tracking we found that lipid granules exhibit anomalous diffusion inside human umbilical vein endothelial cells. For these cells the exact diffusional pattern of a particular granule depends on the physiological state of the cell and on the localization of the granule within the cytoplasm. Granules located close to the actin rich periphery of the cell move less than those located towards to the center of the cell or within the nucleus. Also, granules in cells which are stressed by intense laser illumination or which have attached to a surface for a long period of time move in a more restricted fashion than those within healthy cells. For granules diffusing in healthy cells, in regions away from the cell periphery, occurrences of weak ergodicity breaking are observed, similar to the recent observations inside living fission yeast cells [1].

  11. Niacin restriction upregulates NADPH oxidase and ROS in human keratinocytes

    PubMed Central

    Benavente, Claudia A.; Jacobson, Elaine L.

    2008-01-01

    NAD+ is a substrate for many enzymes, including poly(ADP-ribose) polymerases and sirtuins, which are involved in fundamental cellular processes including DNA repair, stress responses, signaling, transcription, apoptosis, metabolism, differentiation, chromatin structure, and life span. Because these molecular processes are important early in cancer development, we developed a model to identify critical NAD-dependent pathways potentially important in early skin carcinogenesis. Removal of niacin from the cell culture medium allowed control of intracellular NAD. Unlike many non-immortalized human cells, HaCaT keratinocytes, which are immortalized and have a mutant p53 and aberrant NF-kB activity, become severely NAD depleted but divide indefinitely under these conditions. Niacin deficient HaCaTs develop a decreased growth rate due to an increase in apoptotic cells and an arrest in the G2/M phase of the cell cycle. Long- term survival mechanisms in niacin deficient HaCats involve accumulation of reactive oxygen species and increased DNA damage. These alterations result, at least in part, from increased expression and activity of NADPH oxidase, whose downstream effects can be reversed by nicotinamide or NADPH oxidase inhibitors. Our data support the hypothesis that glutamine is a likely alternative energy source during niacin deficiency and we suggest a model for NADPH generation important in ROS production. PMID:17997992

  12. Oxidative stress mediated Ca(2+) release manifests endoplasmic reticulum stress leading to unfolded protein response in UV-B irradiated human skin cells.

    PubMed

    Farrukh, Mufti R; Nissar, Ul A; Afnan, Quadri; Rafiq, Rather A; Sharma, Love; Amin, Shajrul; Kaiser, Peerzada; Sharma, Parduman R; Tasduq, Sheikh A

    2014-07-01

    Exposure of skin to ultraviolet (UV) radiation, an environmental stressor induces number of adverse biological effects (photodamage), including cancer. The damage induced by UV-irradiation in skin cells is initiated by the photochemical generation of reactive oxygen species (ROS) and induction of endoplasmic reticulum (ER) stress and consequent activation of unfolded protein response (UPR). To decipher cellular and molecular events responsible for UV-B mediated ER stress and UPR activation in skin cells. The study was performed on human skin fibroblast (Hs68) and keratinocyte (HaCaT) cells exposed to UV-B radiations in lab conditions. Different parameters of UVB induced cellular and molecular changes were analyzed using Western-blotting, microscopic studies and flow cytometry. Our results depicted that UV-B induces an immediate ROS generation that resulted in emptying of ER Ca(2+) stores inducing ER stress and activation of PERK-peIF2α-CHOP pathway. Quenching ROS generation by anti-oxidants prevented Ca(2+) release and subsequent induction of ER stress and UPR activation. UV-B irradiation induced PERK dependent G2/M phase cell cycle arrest in Hs68 and G1/S phase cell cycle arrest in HaCaT. Also our study reflects that UV-B exposure leads to loss of mitochondrial membrane potential, activation of apoptotic cascade as evident by AnnexinV/PI staining, decreased expression of Bcl-2 and increased cleavage of PARP-1 protein. UV-B induced Ca(2+) deficit within ER lumen was mediated by immediate ROS generation. Insufficient Ca(2+) concentration within ER lumen developed ER stress leading to UPR activation. These changes were reversed by use of anti-oxidants which quench ROS. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  13. Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells.

    PubMed

    Boonkaew, Benjawan; Kempf, Margit; Kimble, Roy; Cuttle, Leila

    2014-12-01

    A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver(®) and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver(®) showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells.

  14. Nickel compounds induce apoptosis in human bronchial epithelial Beas-2B cells by activation of c-Myc through ERK pathway

    SciTech Connect

    Li Qin; Suen, T.-C.; Sun Hong; Arita, Adriana; Costa, Max

    2009-03-01

    Nickel compounds are carcinogenic to humans and have been shown to alter epigenetic homeostasis. The c-Myc protein controls 15% of human genes and it has been shown that fluctuations of c-Myc protein alter global epigenetic marks. Therefore, the regulation of c-Myc by nickel ions in immortalized but not tumorigenic human bronchial epithelial Beas-2B cells was examined in this study. It was found that c-Myc protein expression was increased by nickel ions in non-tumorigenic Beas-2B and human keratinocyte HaCaT cells. The results also indicated that nickel ions induced apoptosis in Beas-2B cells. Knockout of c-Myc and its restoration in a rat cell system confirmed the essential role of c-Myc in nickel ion-induced apoptosis. Further studies in Beas-2B cells showed that nickel ion increased the c-Myc mRNA level and c-Myc promoter activity, but did not increase c-Myc mRNA and protein stability. Moreover, nickel ion upregulated c-Myc in Beas-2B cells through the MEK/ERK pathway. Collectively, the results demonstrate that c-Myc induction by nickel ions occurs via an ERK-dependent pathway and plays a crucial role in nickel-induced apoptosis in Beas-2B cells.

  15. CD44v6 expression in human skin keratinocytes as a possible mechanism for carcinogenesis associated with chronic arsenic exposure.

    PubMed

    Huang, S; Guo, S; Guo, F; Yang, Q; Xiao, X; Murata, M; Ohnishi, S; Kawanishi, S; Ma, N

    2013-01-14

    Inorganic arsenic is a well-known human skin carcinogen. Chronic arsenic exposure results in various types of human skin lesions, including squamous cell carcinoma (SCC). To investigate whether mutant stem cells participate in arsenic-associated carcinogenesis, we repeatedly exposed the HaCaT cells line to an environmentally relevant level of arsenic (0.05 ppm) in vitro for 18 weeks. Following sodium arsenic arsenite administration, cell cycle, colony-forming efficiency (CFE), cell tumorigenicity, and expression of CD44v6, NF-κB and p53, were analyzed at different time points (0, 5, 10, 15, 20, 25 and 30 passages). We found that a chronic exposure of HaCaT cells to a low level of arsenic induced a cancer stem- like phenotype. Furthermore, arsenic-treated HaCaT cells also became tumorigenic in nude mice, their growth cycle was predominantly in G2/M and S phases. Relative to nontreated cells, they exhibited a higher growth rate and a significant increase in CFE. Western blot analysis found that arsenic was capable of increasing cell proliferation and sprouting of cancer stem-like phenotype. Additionally, immunohistochemical analysis demonstrated that CD44v6 expression was up-regulated in HaCaT cells exposed to a low level of arsenic during early stages of induction. The expression of CD44v6 in arsenic-treated cells was positively correlated with their cloning efficiency in soft agar (r=0.949, P=0.01). Likewise, the expressions of activating transcription factor NF-κB and p53 genes in the arsenic-treated HaCaT cells were significantly higher than that in non-treated cells. Higher expressions of CD44v6, NF-κB and p53 were also observed in tumor tissues isolated from Balb/c nude mice. The present results suggest that CD44v6 may be a biomarker of arsenic-induced neoplastic transformation in human skin cells, and that arsenic promotes malignant transformation in human skin lesions through a NF-κB signaling pathway-stimulated expression of CD44v6.

  16. The additive effects of minoxidil and retinol on human hair growth in vitro.

    PubMed

    Yoo, Hyeon Gyeong; Chang, In-Young; Pyo, Hyun Keol; Kang, Yong Jung; Lee, Seung Ho; Kwon, Oh Sang; Cho, Kwang Hyun; Eun, Hee Chul; Kim, Kyu Han

    2007-01-01

    Minoxidil enhances hair growth by prolonging the anagen phase and induces new hair growth in androgenetic alopecia (AGA), whereas retinol significantly improves scalp skin condition and promotes hair growth. We investigated the combined effects of minoxidil and retinol on human hair growth in vitro and on cultured human dermal papilla cells (DPCs) and epidermal keratinocytes (HaCaT). The combination of minoxidil and retinol additively promoted hair growth in hair follicle organ cultures. In addition, minoxidil plus retinol more effectively elevated phosphorylated Erk, phosphorylated Akt levels, and the Bcl-2/Bax ratio than minoxidil alone in DPCs and HaCaT. We found that the significant hair shaft elongation demonstrated after minoxidil plus retinol treatment would depend on the dual kinetics associated with the activations of Erk- and Akt-dependent pathways and the prevention of apoptosis by increasing the Bcl-2/Bax ratio.

  17. The impact of extracellular syntaxin4 on HaCaT keratinocyte behavior

    SciTech Connect

    Kadono, Nanako; Miyazaki, Takafumi; Okugawa, Yoji; Nakajima, Kiichiro; Hirai, Yohei

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer A subpopulation of syntaxin4 localizes extracellularly in the keratinocytes. Black-Right-Pointing-Pointer Epimorphin and syntaxin4 confer the resistance to the oxidative stress. Black-Right-Pointing-Pointer Epimorphin suppresses and syntaxin4 accelerates the CCE formation. Black-Right-Pointing-Pointer The antagonistic peptide to syntaxin4 blocks the syntaxin4-dependent CCE formation. -- Abstract: Syntaxin4 belongs to t-SNARE protein family and functions as a vesicular fusion mediator in the plasma membrane in a wide variety of cell types. This protein resembles another family member, epimorphin, a subpopulation of which has been shown to be secreted extracellularly in order to exert signaling functions. Here, we demonstrate the secretion of syntaxin4 via a non-classical pathway and its extracellular functions by using the functionally normal keratinocyte HaCaT. Extracellularly presented syntaxin4 appeared to elicit many cell responses similar to epimorphin with an important exception: it clearly facilitated keratinocyte cornification. The circularized peptide ST4n1 was synthesized from the putative functional core of syntaxin4 (a.a. 103-108), which is equivalent to the previously generated antagonist of epimorphin, and neutralized this contradictory effect. Intriguingly, an epimorphin mutant (EP4M) in which the functional core was replaced by that of syntaxin4 behaved like epimorphin, which was again antagonized by ST4n1. Electrophoresis-based analyses demonstrated the distinct structure of syntaxin4 compared to epimorphin or EP4M. These results revealed, for the first time, the extracellular role of syntaxin4 and shed light on the division of the extracellular effects exerted by epimorphin and syntaxin4 on keratinocyte cornification.

  18. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

    During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

  19. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

    During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

  20. Photosensitized methyl paraben induces apoptosis via caspase dependent pathway under ambient UVB exposure in human skin cells.

    PubMed

    Dubey, Divya; Chopra, Deepti; Singh, Jyoti; Srivastav, Ajeet K; Kumari, Smita; Verma, Ankit; Ray, Ratan Singh

    2017-10-01

    Methyl paraben (MP), is a widely used preservative in pharmaceutical, food and cosmetic products. Its molecular mechanism under ambient ultraviolet radiation is not well understood. We investigated photosensitizing mechanism of MP under ambient UVB (0.6 mW/cm(2)) intensity. MP showed dose dependent decrease in cell viability of human keratinocyte cell line (HaCaT) by MTT and NRU assays. Study showed 40% reduction in antimicrobial activity of UVB irradiated MP through E. coli culture. Photosensitized MP (25 μg/ml) significantly enhanced lipid peroxidation, intracellular ROS generation and disrupted mitochondrial membrane integrity. MP induced loss of lysosomal membrane integrity and endoplasmic reticulum (ER) mediated stress evident from Ca(+2) release. Phototoxicity of MP showed nuclear fragmentation, phosphatidylserine translocation, 30% tail DNA and micronuclei formation. Study showed mitochondria mediated apoptosis via upregulation of Bax, Apaf-1, Cytochrome C and Caspase-3. Upregulation of Caspase-12 (2 folds) specifically showed role of ER in apoptosis. Specific caspase inhibitor, Z-VAD-FMK showed involvement of caspase cascade pathway in apoptosis. Results indicate that photosensitive MP leads to oxidative stress mediated DNA damage and apoptosis through mitochondria and ER. MP causes deleterious effects and its long term exposure to human skin may promote skin diseases. Therefore, MP should be replaced by other photosafe preservatives for humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Triggering apoptotic death of human epidermal keratinocytes by malic Acid: involvement of endoplasmic reticulum stress- and mitochondria-dependent signaling pathways.

    PubMed

    Hsiao, Yu-Ping; Lai, Wan-Wen; Wu, Shi-Bei; Tsai, Chung-Hung; Tang, Sheau-Chung; Chung, Jing-Gung; Yang, Jen-Hung

    2015-01-09

    Malic acid (MA) has been commonly used in cosmetic products, but the safety reports in skin are sparse. To investigate the biological effects of MA in human skin keratinocytes, we investigated the potential cytotoxicity and apoptotic effects of MA in human keratinocyte cell lines (HaCaT). The data showed that MA induced apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells and normal human epidermal keratinocytes (NHEKs). Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX) but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR) was significantly decreased whereas extracellular acidification rate (ECAR) was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways.

  2. Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

    PubMed Central

    Hsiao, Yu-Ping; Lai, Wan-Wen; Wu, Shi-Bei; Tsai, Chung-Hung; Tang, Sheau-Chung; Chung, Jing-Gung; Yang, Jen-Hung

    2015-01-01

    Malic acid (MA) has been commonly used in cosmetic products, but the safety reports in skin are sparse. To investigate the biological effects of MA in human skin keratinocytes, we investigated the potential cytotoxicity and apoptotic effects of MA in human keratinocyte cell lines (HaCaT). The data showed that MA induced apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells and normal human epidermal keratinocytes (NHEKs). Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX) but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR) was significantly decreased whereas extracellular acidification rate (ECAR) was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways. PMID:25584429

  3. Human fetal mesenchymal stem cells.

    PubMed

    O'Donoghue, Keelin; Chan, Jerry

    2006-09-01

    Stem cells have been isolated at all stages of development from the early developing embryo to the post-reproductive adult organism. However, the fetal environment is unique as it is the only time in ontogeny that there is migration of stem cells in large numbers into different organ compartments. While fetal neural and haemopoietic stem cells (HSC) have been well characterised, only recently have mesenchymal stem cells from the human fetus been isolated and evaluated. Our group have characterised in human fetal blood, liver and bone marrow a population of non-haemopoietic, non-endothelial cells with an immunophenotype similar to adult bone marrow-derived mesenchymal stem cells (MSC). These cells, human fetal mesenchymal stem cells (hfMSC), are true multipotent stem cells with greater self-renewal and differentiation capacity than their adult counterparts. They circulate in first trimester fetal blood and have been found to traffic into the maternal circulation, engrafting in bone marrow, where they remain microchimeric for decades after pregnancy. Though fetal microchimerism has been implicated in the pathogenesis of autoimmune disease, the biological role of hfMSC microchimerism is unknown. Potential downstream applications of hfMSC include their use as a target cell for non-invasive pre-natal diagnosis from maternal blood, and for fetal cellular and gene therapy. Using hfMSC in fetal therapy offers the theoretical advantages of avoidance of immune rejection, increased engraftment, and treatment before disease pathology sets in. Aside from allogeneic hfMSC in utero transplantation, the use of autologous hfMSC has been brought a step forward with the development of early blood sampling techniques, efficient viral transduction and clonal expansion. Work is ongoing to determine hfMSC fate post-transplantation in murine models of genetic disease. In this review we will examine what is known about hfMSC biology, as well as discussing areas for future research. The

  4. The role of plant metabolism in the mutagenic and cytotoxic effects of four organophosphorus insecticides in Salmonella typhimurium and in human cell lines.

    PubMed

    Cortés-Eslava, Josefina; Gómez-Arroyo, Sandra; Arenas-Huertero, Francisco; Flores-Maya, Saúl; Díaz-Hernández, Martha E; Calderón-Segura, María Elena; Valencia-Quintana, Rafael; Espinosa-Aguirre, Jesús Javier; Villalobos-Pietrini, Rafael

    2013-08-01

    This study used a cell/microbe co-incubation assay to evaluate the effect of four organophosphorus insecticides (parathion-methyl, azinphos-methyl, omethoate, and methamidophos) metabolized by coriander (Coriandrum sativum). The reverse mutation of Salmonella typhimurium strains TA98 and TA100 was used as an indicator of genetic damage. Treatments with these insecticides inhibited peroxidase activity in plant cells by between 17% (omethoate) and 98% (azinphos-methyl) and decreased plant protein content by between 36% (omethoate) and 99.6% (azinphos-methyl). Azinphos-methyl was the most toxic when applied directly. In the Ames test, treatments applied directly to strain TA100 killed the bacteria; however, the presence of plant metabolism detoxified the system and permitted the growth of bacteria. In strain TA98, plant metabolites of insecticides were mutagenic. This result suggests that the tested pesticides produce mutations through frameshifting. The same pesticides were applied to human skin (HaCaT) and lung (NL-20) cell lines to evaluate their effects on cell viability. Pesticides applied directly were more cytotoxic than the combination of pesticide plus coriander metabolic fraction. Omethoate and methamidophos did not affect the viability of HaCaT cells, but azinphos-methyl and parathion-methyl at 100 and 1000μgmL(-1) significantly decreased viability (p<0.05). The NL-20 cell line was remarkably sensitive to the direct application of insecticides. All of the treatment conditions caused decreases in NL-20 cell viability (e.g., viability decreased to 12.0% after parathion-methyl treatment, to 14.7% after azinphos-methyl treatment, and to 6.9% after omethoate treatment). Similar to the Ames test, all of the insecticides showed decreased toxicity in human cells when they were cultured in the presence of plant metabolism. In conclusion, when the studied organophosphorus insecticides were plant-metabolized, they induced mutations in the bacterial strain TA98. In

  5. Trehalose, sucrose and raffinose are novel activators of autophagy in human keratinocytes through an mTOR-independent pathway

    PubMed Central

    Chen, Xu; Li, Min; Li, Li; Xu, Song; Huang, Dan; Ju, Mei; Huang, Ju; Chen, Kun; Gu, Heng

    2016-01-01

    Trehalose is a natural disaccharide that is found in a diverse range of organisms but not in mammals. Autophagy is a process which mediates the sequestration, lysosomal delivery and degradation of proteins and organelles. Studies have shown that trehalose exerts beneficial effects through inducing autophagy in mammalian cells. However, whether trehalose or other saccharides can activate autophagy in keratinocytes is unknown. Here, we found that trehalose treatment increased the LC3-I to LC3-II conversion, acridine orange-stained vacuoles and GFP-LC3B (LC3B protein tagged with green fluorescent protein) puncta in the HaCaT human keratinocyte cell line, indicating autophagy induction. Trehalose-induced autophagy was also observed in primary keratinocytes and the A431 epidermal cancer cell line. mTOR signalling was not affected by trehalose treatment, suggesting that trehalose induced autophagy through an mTOR-independent pathway. mTOR-independent autophagy induction was also observed in HaCaT and HeLa cells treated with sucrose or raffinose but not in glucose, maltose or sorbitol treated HaCaT cells, indicating that autophagy induction was not a general property of saccharides. Finally, although trehalose treatment had an inhibitory effect on cell proliferation, it had a cytoprotective effect on cells exposed to UVB radiation. Our study provides new insight into the saccharide-mediated regulation of autophagy in keratinocytes. PMID:27328819

  6. Nuclear expression of Rac1 in cervical premalignant lesions and cervical cancer cells

    PubMed Central

    2012-01-01

    Background Abnormal expression of Rho-GTPases has been reported in several human cancers. However, the expression of these proteins in cervical cancer has been poorly investigated. In this study we analyzed the expression of the GTPases Rac1, RhoA, Cdc42, and the Rho-GEFs, Tiam1 and beta-Pix, in cervical pre-malignant lesions and cervical cancer cell lines. Methods Protein expression was analyzed by immunochemistry on 102 cervical paraffin-embedded biopsies: 20 without Squamous Intraepithelial Lesions (SIL), 51 Low- grade SIL, and 31 High-grade SIL; and in cervical cancer cell lines C33A and SiHa, and non-tumorigenic HaCat cells. Nuclear localization of Rac1 in HaCat, C33A and SiHa cells was assessed by cellular fractionation and Western blotting, in the presence or not of a chemical Rac1 inhibitor (NSC23766). Results Immunoreacivity for Rac1, RhoA, Tiam1 and beta-Pix was stronger in L-SIL and H-SIL, compared to samples without SIL, and it was significantly associated with the histological diagnosis. Nuclear expression of Rac1 was observed in 52.9% L-SIL and 48.4% H-SIL, but not in samples without SIL. Rac1 was found in the nucleus of C33A and SiHa cells but not in HaCat cells. Chemical inhibition of Rac1 resulted in reduced cell proliferation in HaCat, C33A and SiHa cells. Conclusion Rac1 is expressed in the nucleus of epithelial cells in SILs and cervical cancer cell lines, and chemical inhibition of Rac1 reduces cellular proliferation. Further studies are needed to better understand the role of Rho-GTPases in cervical cancer progression. PMID:22443139

  7. Human stem cell ethics: beyond the embryo.

    PubMed

    Sugarman, Jeremy

    2008-06-05

    Human embryonic stem cell research has elicited powerful debates about the morality of destroying human embryos. However, there are important ethical issues related to stem cell research that are unrelated to embryo destruction. These include particular issues involving different types of cells used, the procurement of such cells, in vivo use of stem cells, intellectual property, and conflicts of interest.

  8. Effects of Soft Denture Liners on L929 Fibroblasts, HaCaT Keratinocytes, and RAW 264.7 Macrophages

    PubMed Central

    Chaves, Carolina de Andrade Lima; de Souza Costa, Carlos Alberto; Vergani, Carlos Eduardo; Chaves de Souza, Pedro Paulo; Machado, Ana Lucia

    2014-01-01

    The effects of six soft liners (Ufi Gel P (UG), Sofreliner S (SR), Durabase Soft (D), Trusoft (T), Coe Comfort (CC), and Softone (ST)) on L929, HaCat, and RAW 264.7 cells were investigated. Eluates (24 and 48 h) from the materials were applied on the cells and the viability, type of cell death, and morphology were evaluated. Cells were also seeded on the specimens' surfaces (direct contact) and incubated (24 or 48 h), and viability was analyzed. Controls were cells in culture medium without eluates or specimens. For cell viability, no significant differences were found among materials or between extraction periods, and the liners were noncytotoxic or slightly cytotoxic. Morphology of RAW 264.7 cells was altered by the 24 h eluates from CC and D and the 48 h eluates from SR, CC, and D. The 24 and 48 h eluates from all materials (except T) increased the percentages of L929 necrotic cells. For direct contact tests, the lowest cytotoxicity was observed for UG and SR. Although eluates did not reduce viability, morphology alterations and increase in necrosis were seen. Moreover, in the direct contact, effects on viability were more pronounced, particularly for D, T, CC and ST. Thus, the use of UG and SR might reduce the risk of adverse effects. PMID:25295276

  9. Differentiated human stem cells resemble fetal, not adult, β cells.

    PubMed

    Hrvatin, Sinisa; O'Donnell, Charles W; Deng, Francis; Millman, Jeffrey R; Pagliuca, Felicia Walton; DiIorio, Philip; Rezania, Alireza; Gifford, David K; Melton, Douglas A

    2014-02-25

    Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional β cells.

  10. [Effect of plant hormones on the components of secretory pathway in human normal and tumor cells].

    PubMed

    Vil'danova, M S; Savitskaia, M A; Onishchenko, G E; Smirnova, E A

    2014-01-01

    Plant hormones play a key role in plant growth and differentiation. Many hormones are known as potential antitumor agents, yet others appear to affect the secretory activity and are produced by mammalian cells as pro-inflammatory cytokines. The goal of this research was to study the effect of abscisic and gibberellic acids on the secretory system of human cultured epidermoid carcinoma cells A431 and keratinocytes HaCat. Immunocytochemical and morphometric analysis demonstrated that subtoxic concentration of plant hormones induced the broadening of the ER network and increased the size of Golgi complex. Electron microscopy studies confirmed the hypertrophic changes of the Golgi apparatus, specifically, the swelling of cisternae in the trans-compartment of dictyosomes after exposure to abscisic acid, and swelling of cis- and trans-compartment of dictyosomes after exposure to abscisic acid, and swelling of cis- and trans-compartments of dictyosomes after exposure to gibberellic acid. Using of Click-iT technique allowed to detect the elevation of the total protein synthesis only in A431 cells exposed to abscisic acid. Cumulative data suggests that, under these conditions, the hypertrophy of Golgi apparatus may reflect the enhanced secretory activity of cells. In other experiments, the hypertrophy of Golgi is not related to increased protein synthesis and therefore may suggest the stress-related changes of ER and Golgi apparatus. Our results demonstrate that morphologically similar reaction of cellular organelles, such as hypertrophy of Golgi apparatus, is the result of different functional activities, and that molecular mechanisms underlying the changes induced in cells need further investigations.

  11. Embryonic stem cell patents and human dignity.

    PubMed

    Resnik, David B

    2007-09-01

    This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells.

  12. Embryonic Stem Cell Patents and Human Dignity

    PubMed Central

    Resnik, David B.

    2009-01-01

    This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells. PMID:17922198

  13. Stimulating effects of Bacillus subtilis natto-fermented Radix astragali on hyaluronic acid production in human skin cells.

    PubMed

    Hsu, Mei-Fang; Chiang, Been-Huang

    2009-09-25

    Radix astragali, a well-known Chinese herb, which has been traditionally used for skincare, and microbial fermentation is one of the conventional methods for processing Chinese herbs. This research studied the effects of non-fermented (HQNB) and fermented preparations (HQB) of Radix astragali on hyaluronic acid (HA) production in primary human skin cells. HQB and HQNB were prepared and added to the cultures of primary human skin cells. Hyaluronic acid content was determined using ELISA. Real-time RT-PCR was used to evaluate hyaluronan synthase gene expression. The bioactive compounds were analyzed by HPLC. The growth-stimulating effect of HQNB on both of keratinocytes and fibroblasts were significantly higher than that of HQB. Conversely, HQB, but not HQNB significantly stimulated HA production in both cultured primary human epidermal keratinocytes and human dermal fibroblasts in dose-dependent manners. In addition, HQB markedly and dose-dependently increased the expression of hyaluronan synthase 3 and hyaluronan synthase 2 mRNA in HaCaT cells and human fibroblasts, respectively. Therefore, HQB might be a promising candidate for preventing the age-dependent loss of HA content in aged human skin, and its effect on the enhancement of HA synthesis in skin cells is highly related to its effect on the expression of hyaluronan synthase genes. The three major active isoflavonoids in Radix astragali were identified as ononin, calycosin, and formononetin. After fermentation, all of these three compounds in HQB were significantly reduced. However, HQB still had significantly higher enhancement effect on the production of HA than HQNB. It appeared that isoflavonoid aglycones or other metabolites, converted from their primary isoflavones during fermentation, might be responsible for the skincare functions found in this study. This study demonstrated the low toxicity and the stimulating effects of HQB on HA synthesis, and suggests that HQB may play a promising role in anti

  14. Effect of Culture Supernatant Derived from Trichophyton Rubrum Grown in the Nail Medium on the Innate Immunity-related Molecules of HaCaT

    PubMed Central

    Huang, Xin-Zhu; Liang, Pan-Pan; Ma, Han; Yi, Jin-Ling; Yin, Song-Chao; Chen, Zhi-Rui; Li, Mei-Rong; Lai, Wei; Chen, Jian

    2015-01-01

    Background: Trichophyton rubrum is superficial fungi characteristically confined to dead keratinized tissues. These observations suggest that the soluble components released by the fungus could influence the host immune response in a cell in contact-free manner. Therefore, this research aimed to analyze whether the culture supernatant derived from T. rubrum grown in the nail medium could elicit the immune response of keratinocyte effectively. Methods: The culture supernatants of two strains (T1a, TXHB) were compared for the β-glucan concentrations and their capacity to impact the innate immunity of keratinocytes. The β-glucan concentrations in the supernatants were determined with the fungal G-test kit and protein concentrations with bicinchoninic acid protein quantitative method, then HaCaT was stimulated with different concentrations of culture supernatants by adopting morphological method to select a suitable dosage. Expressions of host defense genes were assessed by quantitative polymerase chain reaction after the HaCaT was stimulated with the culture supernatants. Data were analyzed with one-way analysis of variance, followed by the least significant difference test. Results: The T. rubrum strains (T1a and TXHB) released β-glucan of 87.530 ± 37.581 pg/ml and 15.747 ± 6.453 pg/ml, respectively into the media. The messenger RNA (mRNA) expressions of toll-like receptor-2 (TLR2), TLR4, and CARD9 were moderately up-regulated in HaCaT within 6-h applications of both supernatants. HaCaT cells were more responsive to T1a than TXHB. The slight increase of dendritic cells-specific intercellular adhesion molecule 3-grabbing nonintegrin expression was faster and stronger, induced by T1a supernatant than TXHB. The moderate decreases of RNase 7, the slight up-regulations of Dectin-1 and interleukin-8 at the mRNA level were detected only in response to T1a rather than TXHB. After a long-time contact, all the elevated defense genes decreased after 24 h. Conclusion: The

  15. The vitamin D receptor inhibits the respiratory chain, contributing to the metabolic switch that is essential for cancer cell proliferation.

    PubMed

    Consiglio, Marco; Destefanis, Michele; Morena, Deborah; Foglizzo, Valentina; Forneris, Mattia; Pescarmona, Gianpiero; Silvagno, Francesca

    2014-01-01

    We recently described the mitochondrial localization and import of the vitamin D receptor (VDR) in actively proliferating HaCaT cells for the first time, but its role in the organelle remains unknown. Many metabolic intermediates that support cell growth are provided by the mitochondria; consequently, the identification of proteins that regulate mitochondrial metabolic pathways is of great interest, and we sought to understand whether VDR may modulate these pathways. We genetically silenced VDR in HaCaT cells and studied the effects on cell growth, mitochondrial metabolism and biosynthetic pathways. VDR knockdown resulted in robust growth inhibition, with accumulation in the G0G1 phase of the cell cycle and decreased accumulation in the M phase. The effects of VDR silencing on proliferation were confirmed in several human cancer cell lines. Decreased VDR expression was consistently observed in two different models of cell differentiation. The impairment of silenced HaCaT cell growth was accompanied by sharp increases in the mitochondrial membrane potential, which sensitized the cells to oxidative stress. We found that transcription of the subunits II and IV of cytochrome c oxidase was significantly increased upon VDR silencing. Accordingly, treatment of HaCaT cells with vitamin D downregulated both subunits, suggesting that VDR may inhibit the respiratory chain and redirect TCA intermediates toward biosynthesis, thus contributing to the metabolic switch that is typical of cancer cells. In order to explore this hypothesis, we examined various acetyl-CoA-dependent biosynthetic pathways, such as the mevalonate pathway (measured as cholesterol biosynthesis and prenylation of small GTPases), and histone acetylation levels; all of these pathways were inhibited by VDR silencing. These data provide evidence of the role of VDR as a gatekeeper of mitochondrial respiratory chain activity and a facilitator of the diversion of acetyl-CoA from the energy-producing TCA cycle

  16. The Vitamin D Receptor Inhibits the Respiratory Chain, Contributing to the Metabolic Switch that Is Essential for Cancer Cell Proliferation

    PubMed Central

    Morena, Deborah; Foglizzo, Valentina; Forneris, Mattia; Pescarmona, Gianpiero; Silvagno, Francesca

    2014-01-01

    We recently described the mitochondrial localization and import of the vitamin D receptor (VDR) in actively proliferating HaCaT cells for the first time, but its role in the organelle remains unknown. Many metabolic intermediates that support cell growth are provided by the mitochondria; consequently, the identification of proteins that regulate mitochondrial metabolic pathways is of great interest, and we sought to understand whether VDR may modulate these pathways. We genetically silenced VDR in HaCaT cells and studied the effects on cell growth, mitochondrial metabolism and biosynthetic pathways. VDR knockdown resulted in robust growth inhibition, with accumulation in the G0G1 phase of the cell cycle and decreased accumulation in the M phase. The effects of VDR silencing on proliferation were confirmed in several human cancer cell lines. Decreased VDR expression was consistently observed in two different models of cell differentiation. The impairment of silenced HaCaT cell growth was accompanied by sharp increases in the mitochondrial membrane potential, which sensitized the cells to oxidative stress. We found that transcription of the subunits II and IV of cytochrome c oxidase was significantly increased upon VDR silencing. Accordingly, treatment of HaCaT cells with vitamin D downregulated both subunits, suggesting that VDR may inhibit the respiratory chain and redirect TCA intermediates toward biosynthesis, thus contributing to the metabolic switch that is typical of cancer cells. In order to explore this hypothesis, we examined various acetyl-CoA-dependent biosynthetic pathways, such as the mevalonate pathway (measured as cholesterol biosynthesis and prenylation of small GTPases), and histone acetylation levels; all of these pathways were inhibited by VDR silencing. These data provide evidence of the role of VDR as a gatekeeper of mitochondrial respiratory chain activity and a facilitator of the diversion of acetyl-CoA from the energy-producing TCA cycle

  17. Discrimination of skin sensitizers from non-sensitizers by interleukin-1α and interleukin-6 production on cultured human keratinocytes.

    PubMed

    Jung, Daun; Che, Jeong-Hwan; Lim, Kyung-Min; Chun, Young-Jin; Heo, Yong; Seok, Seung Hyeok

    2016-09-01

    In vitro testing methods for classifying sensitizers could be valuable alternatives to in vivo sensitization testing using animal models, such as the murine local lymph node assay (LLNA) and the guinea pig maximization test (GMT), but there remains a need for in vitro methods that are more accurate and simpler to distinguish skin sensitizers from non-sensitizers. Thus, the aim of our study was to establish an in vitro assay as a screening tool for detecting skin sensitizers using the human keratinocyte cell line, HaCaT. HaCaT cells were exposed to 16 relevant skin sensitizers and 6 skin non-sensitizers. The highest dose used was the dose causing 75% cell viability (CV75) that we determined by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The levels of extracellular production of interleukin-1α (IL-1α) and IL-6 were measured. The sensitivity of IL-1α was 63%, specificity was 83% and accuracy was 68%. In the case of IL-6, sensitivity: 69%, specificity: 83% and accuracy: 73%. Thus, this study suggests that measuring extracellular production of pro-inflammatory cytokines IL-1α and IL-6 by human HaCaT cells may potentially classify skin sensitizers from non-sensitizers. Copyright © 2015 John Wiley & Sons, Ltd.

  18. Neoplastic transformation of human cells in vitro.

    PubMed

    Rhim, J S

    1993-01-01

    Efforts to investigate the progression of events that lead normal human cells in culture to become neoplastic in response to carcinogenic agents have been aided by the development of the suitable in vitro model systems. For initial human cell transformation studies, a flat, nontumorigenic clonal line, originally derived from a human osteosarcoma (HOS), was used. When treated with chemical carcinogens such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and 3-methyl-cholanthrene (3MC), the HOS cells underwent morphological alterations and acquired tumorigenic properties. These cell lines were very useful inasmuch as a non-ras cellular transforming gene, met, and an activated H-ras oncogene have been isolated from MNNG-transformed and 3MC-transformed HOS lines, respectively, by DNA transfection procedure. Alteration of p53 gene in chemically transformed HOS cell lines has recently been shown. Although carcinogens cause human cancer, normal human cells in culture have proven difficult to achieve. Neoplastic transformation of human cells in culture has recently been achieved by a stepwise fashion-immortalization and conversion of the immortalized cells to tumorigenic cells. One of the critical initial events in the progression of normal human cells to tumor cells is the escape from cellular senescence. With few exceptions, normal human cells require immortalization to provide a practical system for transformation studies. Thus, the role of carcinogenic agents in the development of human cancers is now being defined using a variety of human cells. The neoplastic transformation in human cell cultures is reviewed. In doing so, this author attempts to put into perspective the history of human cell transformation by carcinogenic agents, and to discuss the current state of the art in transformation of human cells in culture; thus providing insight into the molecular and cellular mechanisms involved in the conversion of normal cells to a neoplastic state of growth.

  19. Nicotinamide downregulates gene expression of interleukin-6, interleukin-10, monocyte chemoattractant protein-1, and tumour necrosis factor-α gene expression in HaCaT keratinocytes after ultraviolet B irradiation.

    PubMed

    Monfrecola, G; Gaudiello, F; Cirillo, T; Fabbrocini, G; Balato, A; Lembo, S

    2013-03-01

    Ultraviolet (UV) radiation has profound effects on human skin, causing sunburn, inflammation, cellular-tissue injury, cell death, and skin cancer. Most of these effects are mediated by a number of cytokines produced by keratinocytes. In this study we investigated whether nicotinamide (NCT), the amide form of vitamin B3, might have a protective function in reducing the expression of interleukin (IL)-1β, IL-6, IL-8, IL-10, monocyte chemoattractant protein (MCP)-1 and tumour necrosis factor (TNF)-α in UV-irradiated keratinocytes. HaCaT cells were treated with UVB in the presence or absence of NCT, and cytokine mRNA levels were examined by quantitative real-time PCR. NCT significantly downregulated IL-6, IL-10, MCP-1 and TNF-α mRNA expression, whereas it did not exert any significant effect on IL-1β or IL-8 expression. Because of its ability to decrease these cytokine mediators after UV exposure, NCT is a possible therapy to improve or prevent conditions induced or aggravated by UV light.

  20. Human satellite cells: identification on human muscle fibres

    PubMed Central

    Boldrin, Luisa; Morgan, Jennifer E

    2012-01-01

    Satellite cells, normally quiescent underneath the myofibre basal lamina, are skeletal muscle stem cells responsible for postnatal muscle growth, repair and regeneration. Since their scarcity and small size have limited study on transverse muscle sections, techniques to isolate individual myofibres, bearing their attendant satellite cells, were developed. Studies on mouse myofibres have generated much information on satellite cells, but the limited availability and small size of human muscle biopsies have hampered equivalent studies of satellite cells on human myofibres. Here, we identified satellite cells on fragments of human and mouse myofibres, using a method applicable to small muscle biopsies. PMID:22333991

  1. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

    PubMed Central

    Ramos-Jerz, Maria del R.; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M.

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

  2. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    PubMed

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

  3. Fat cell turnover in humans.

    PubMed

    Arner, Peter; Spalding, Kirsty L

    2010-05-21

    Obesity is a condition where excess body fat accumulates to such an extent that one's health may be affected. Owing to the cardiovascular and metabolic disorders associated with obesity, and the epidemic of obesity facing most countries today, life expectancy in the developed world may start to decrease for the first time in recent history. Other conditions, such as anorexia nervosa and cachexia, are characterised by subnormal levels of adipose tissue and as with obesity lead to morbidity and mortality. Given the significant personal and economic costs of these conditions and their increasing prevalence in society, understanding the factors that determine the fat mass is therefore of prime interest and may lead to effective treatments and/or interventions for these disorders. Fat mass can be regulated in two ways. The lipid filling of pre-existing fat cells could be altered and the number of fat cells could be changed by the generation of new fat cells or the dying of old ones (i.e. adipocyte turnover). This review summarizes what is known about fat cell turnover in humans and the potential clinical implications. 2010 Elsevier Inc. All rights reserved.

  4. Neoplastic transformation of human cells

    NASA Technical Reports Server (NTRS)

    Goth-Goldstein, Regine

    1995-01-01

    The goal of this project was to gain a better understanding of the cellular mechanisms of cancer induction by ionizing radiation as a risk assessment for workers subjected to high LET irradiation such as that found in space. The following ions were used for irradiation: Iron, Argon, Neon, and Lanthanum. Two tests were performed: growth in low serum and growth in agar were used as indicators of cell transformation. The specific aims of this project were to: (1) compare the effectiveness of various ions on degree of transformation of a single dose of the same RBE; (2) determine if successive irradiations with the same ion (Ge 600 MeV/u) increases the degree of transformation; (3) test if clones with the greatest degree of transformation produce tumors in nude mice; and (4) construct a cell hybrid of a transformed and control (non-transformed) clone. The cells used for this work are human mammary epithelial cells with an extended lifespan and selected for growth in MEM + 10% serum.

  5. TELOMERE AND TELOMERASE MODULATION BY BERGAMOT POLYPHENOLIC FRACTION IN EXPERIMENTAL PHOTOAGEING IN HUMAN KERATINOCYTES.

    PubMed

    Nisticò, S; Ehrlich, J; Gliozzi, M; Maiuolo, J; Del Duca, E; Muscoli, C; Mollace, V

    2015-01-01

    Photoageing represents the addition of extrinsic chronic ultraviolet radiation-induced damage on intrinsic ageing and accounts for most age-associated changes in skin appearance. In this study, we evaluated the effect of 38% BPF, a highly concentrated extract of the bergamot fruit (Citrus bergamia) on UVB-induced photoageing by examining inflammatory cytokine expression, telomere length/telomerase alterations and cellular viability in human immortalized HaCaT keratinocytes. Our results suggest that 38% BPF protects HaCaT cells against UVB-induced oxidative stress and markers of photoageing in a dose-dependent manner and could be a useful supplement in skin care products. Together with antioxidant properties, BPF, a highly concentrated extract of the bergamot fruit, appears to modulate basic cellular signal transduction pathways leading to anti-proliferative, anti-aging and immune modulating responses.

  6. Montivipera bornmuelleri venom selectively exhibits high cytotoxic effects on keratinocytes cancer cell lines.

    PubMed

    Sawan, Saly; Yaacoub, Tania; Hraoui-Bloquet, Souad; Sadek, Riyad; Hleihel, Walid; Fajloun, Ziad; Karam, Marc

    2017-04-04

    The Viperidae family venom is a rich source of bioactive compounds such as many proteases, which cause tissue necrosis and affect mostly the vascular system. However, the venom exhibits therapeutic potentials and has contributed to the development of some medical drugs. Specifically, the Montivipera bornmuelleri venom has shown to exhibit antibacterial, pro-inflammatory and antifungal activities. This work evaluates the cytotoxic effect of the M. bornmuelleri venom on human-derived keratinocytes including the non-tumorigenic HaCaT, the benign A5 and the low-grade malignant II4 cells. The toxicity of different venom concentrations (0.9, 1.87, 3.75, 7.5, 15, 30 and 60μg/mL) and their effect on the viability of the cells lines were assessed using the Lactate Dehydrogenase (LDH) activity and the Trypan blue tests after 24h of incubation. The venom was able to reduce the viability of all cell lines in a dose dependent manner with the HaCat cells being the least affected. For example, the 60μg/mL dose induced a more significant decrease the viability of A5 (44%) and II4 (21.33%) keratinocytes as compared to HaCaT cells (70.63%). Also, this venom showed a higher cytotoxic activity on the A5 (52.45%) and II4 (98.67%) cells as compared to HaCaT cells (30.14%) with an IC50 estimated at 10μg/mL on II4 and at 60μg/mL on benign A5. Those differential cytotoxic effects of the M. bornmuelleri venom pave the road for more advanced studies which might unravel the potential anticancer effects of this venom. Copyright © 2017 Elsevier GmbH. All rights reserved.

  7. The human airway epithelial basal cell transcriptome.

    PubMed

    Hackett, Neil R; Shaykhiev, Renat; Walters, Matthew S; Wang, Rui; Zwick, Rachel K; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G

    2011-05-04

    The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium.

  8. The Human Airway Epithelial Basal Cell Transcriptome

    PubMed Central

    Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

    2011-01-01

    Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem

  9. Silver nanoparticles protect human keratinocytes against UVB radiation-induced DNA damage and apoptosis: potential for prevention of skin carcinogenesis.

    PubMed

    Arora, Sumit; Tyagi, Nikhil; Bhardwaj, Arun; Rusu, Lilia; Palanki, Rohan; Vig, Komal; Singh, Shree R; Singh, Ajay P; Palanki, Srinivas; Miller, Michael E; Carter, James E; Singh, Seema

    2015-07-01

    Ultraviolet (UV)-B radiation from the sun is an established etiological cause of skin cancer, which afflicts more than a million lives each year in the United States alone. Here, we tested the chemopreventive efficacy of silver-nanoparticles (AgNPs) against UVB-irradiation-induced DNA damage and apoptosis in human immortalized keratinocytes (HaCaT). AgNPs were synthesized by reduction-chemistry and characterized for their physicochemical properties. AgNPs were well tolerated by HaCaT cells and their pretreatment protected them from UVB-irradiation-induced apoptosis along with significant reduction in cyclobutane-pyrimidine-dimer formation. Moreover, AgNPs pre-treatment led to G1-phase cell-cycle arrest in UVB-irradiated HaCaT cells. AgNPs were efficiently internalized in UVB-irradiated cells and localized into cytoplasmic and nuclear compartments. Furthermore, we observed an altered expression of various genes involved in cell-cycle, apoptosis and nucleotide-excision repair in HaCaT cells treated with AgNPs prior to UVB-irradiation. Together, these findings provide support for potential utility of AgNPs as novel chemopreventive agents against UVB-irradiation-induced skin carcinogenesis. Excessive exposure to the sun is known to increase the risk of skin cancer due to DNA damage. In this work, the authors tested the use of silver nanoparticles as protective agents against ultraviolet radiation. The positive results may open a door for the use of silver nanoparticle as novel agents in the future. Published by Elsevier Inc.

  10. Association of Toxicity of Sorafenib and Sunitinib for Human Keratinocytes with Inhibition of Signal Transduction and Activator of Transcription 3 (STAT3)

    PubMed Central

    Yamamoto, Kazuhiro; Mizumoto, Atsushi; Nishimura, Kohji; Uda, Atsushi; Mukai, Akira; Yamashita, Kazuhiko; Kume, Manabu; Makimoto, Hiroo; Bito, Toshinori; Nishigori, Chikako; Nakagawa, Tsutomu; Hirano, Takeshi; Hirai, Midori

    2014-01-01

    Hand–foot skin reaction is a most common multi-kinase inhibitor-related adverse event. This study aimed to examine whether the toxicity of sorafenib and sunitinib for human keratinocytes was associated with inhibiting signal transduction and activator of transcription 3 (STAT3). We studied whether STAT3 activity affects sorafenib- and sunitinib-induced cell growth inhibition in HaCaT cells by WST-8 assay. Stattic enhanced the cell-growth inhibitory and apoptotic effects of sorafenib and sunitinib. HaCaT cells transfected with constitutively-active STAT3 (STAT3C) were resistant to the sorafenib- and sunitinib-induced cell growth inhibition. STAT3 activity decreased after short-term treatment with sorafenib and sunitinib in a dose-dependent manner and recovered after long-term treatment with sorafenib and sunitinib at low doses. Moreover, the expression of survivin and bcl-2 decreased after treatment with sorafenib and sunitinib was concomitant with variations in STAT3 activity. Sorafenib-induced STAT3 inhibition was mediated by regulation via MAPK pathways in HaCaT cells, while sunitinib-induced STAT3 inhibition was not. Thus, STAT3 activation mediating apoptosis suppressors may be a key factor in sorafenib and sunitinib-induced keratinocyte cytotoxicity. PMID:25013907

  11. Human immunodeficiency virus can productively infect cultured human glial cells.

    PubMed

    Cheng-Mayer, C; Rutka, J T; Rosenblum, M L; McHugh, T; Stites, D P; Levy, J A

    1987-05-01

    Six isolates of the human immunodeficiency virus (HIV) showed differences in their ability to productively infect glioma-derived cell lines and early-passage human brain cell cultures. Susceptibility to HIV infection correlated well with the expression of the astrocyte marker glial fibrillary acidic protein. The CD4 molecule was expressed on some, but not all, of the brain-derived cells; however, no correlation was observed between CD4 protein expression and susceptibility to virus infection. The results show that HIV can productively infect human brain cells, particularly those of glial origin, and suggest that these cell types in the brain can harbor the virus.

  12. Cell motion predicts human epidermal stemness

    PubMed Central

    Toki, Fujio; Tate, Sota; Imai, Matome; Matsushita, Natsuki; Shiraishi, Ken; Sayama, Koji; Toki, Hiroshi; Higashiyama, Shigeki

    2015-01-01

    Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation. PMID:25897083

  13. Human regulatory B cells control the TFH cell response.

    PubMed

    Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

    2017-07-01

    Follicular helper T (TFH) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of TFH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on TFH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate TFH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing TFH cell maturation. In cocultures they differentiated B cells into CD138(+) plasma and IgD(-)CD27(+) memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented TFH cell development. Added to TFH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3(+)CXCR5(+)PD-1(+) follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on TFH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control TFH cell maturation, expand follicular regulatory T cells, and inhibit the TFH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the TFH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  14. Satellite cells in human skeletal muscle plasticity.

    PubMed

    Snijders, Tim; Nederveen, Joshua P; McKay, Bryon R; Joanisse, Sophie; Verdijk, Lex B; van Loon, Luc J C; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

  15. Skin metabolism of aminophenols: Human keratinocytes as a suitable in vitro model to qualitatively predict the dermal transformation of 4-amino-2-hydroxytoluene in vivo

    SciTech Connect

    Goebel, C. Hewitt, N.J.; Kunze, G.; Wenker, M.; Hein, D.W.; Beck, H.; Skare, J.

    2009-02-15

    4-Amino-2-hydroxytolune (AHT) is an aromatic amine ingredient in oxidative hair colouring products. As skin contact occurs during hair dyeing, characterisation of dermal metabolism is important for the safety assessment of this chemical class. We have compared the metabolism of AHT in the human keratinocyte cell line HaCaT with that observed ex-vivo in human skin and in vivo (topical application versus oral (p.o.) and intravenous (i.v.) route). Three major metabolites of AHT were excreted, i.e. N-acetyl-AHT, AHT-sulfate and AHT-glucuronide. When 12.5 mg/kg AHT was applied topically, the relative amounts of each metabolite were altered such that N-acetyl-AHT product was the major metabolite (66% of the dose in comparison with 37% and 32% of the same applied dose after i.v. and p.o. administration, respectively). N-acetylated products were the only metabolites detected in HaCaT cells and ex-vivo whole human skin discs for AHT and p-aminophenol (PAP), an aromatic amine known to undergo N-acetylation in vivo. Since N-acetyltransferase 1 (NAT1) is the responsible enzyme, kinetics of AHT was further compared to the standard NAT1 substrate p-aminobenzoic acid (PABA) in the HaCaT model revealing similar values for K{sub m} and V{sub max}. In conclusion NAT1 dependent dermal N-acetylation of AHT represents a 'first-pass' metabolism effect in the skin prior to entering the systemic circulation. Since the HaCaT cell model represents a suitable in vitro assay for addressing the qualitative contribution of the skin to the metabolism of topically-applied aromatic amines it may contribute to a reduction in animal testing.

  16. Skin metabolism of aminophenols: human keratinocytes as a suitable in vitro model to qualitatively predict the dermal transformation of 4-amino-2-hydroxytoluene in vivo.

    PubMed

    Goebel, C; Hewitt, N J; Kunze, G; Wenker, M; Hein, D W; Beck, H; Skare, J

    2009-02-15

    4-Amino-2-hydroxytolune (AHT) is an aromatic amine ingredient in oxidative hair colouring products. As skin contact occurs during hair dyeing, characterisation of dermal metabolism is important for the safety assessment of this chemical class. We have compared the metabolism of AHT in the human keratinocyte cell line HaCaT with that observed ex-vivo in human skin and in vivo (topical application versus oral (p.o.) and intravenous (i.v.) route). Three major metabolites of AHT were excreted, i.e. N-acetyl-AHT, AHT-sulfate and AHT-glucuronide. When 12.5 mg/kg AHT was applied topically, the relative amounts of each metabolite were altered such that N-acetyl-AHT product was the major metabolite (66% of the dose in comparison with 37% and 32% of the same applied dose after i.v. and p.o. administration, respectively). N-acetylated products were the only metabolites detected in HaCaT cells and ex-vivo whole human skin discs for AHT and p-aminophenol (PAP), an aromatic amine known to undergo N-acetylation in vivo. Since N-acetyltransferase 1 (NAT1) is the responsible enzyme, kinetics of AHT was further compared to the standard NAT1 substrate p-aminobenzoic acid (PABA) in the HaCaT model revealing similar values for K(m) and V(max). In conclusion NAT1 dependent dermal N-acetylation of AHT represents a 'first-pass' metabolism effect in the skin prior to entering the systemic circulation. Since the HaCaT cell model represents a suitable in vitro assay for addressing the qualitative contribution of the skin to the metabolism of topically-applied aromatic amines it may contribute to a reduction in animal testing.

  17. Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device

    PubMed Central

    Jung, Moon Hee; Jung, Sang-Myung; Shin, Hwa Sung

    2016-01-01

    Artificial skin or skin equivalents have been used for clinical purpose to skin graft and as substitutes for animal experiments. The culture of cell lines such as HaCaT has the potential to produce large amounts of artificial skin at a low cost. However, there is a limit to keratinization due to the restriction of differentiation in HaCaT. In this study, a culture device that mimics the in vivo keratinization mechanism, co-stimulated by air-exposure and mechanical stimulation, was developed to construct skin equivalents. The device can reconstruct the epidermal morphology, including the cornified layer, similar to its formation in vivo. Under the condition, epidermis was differentiated in the spinous and granular layers. Formation of the stratum corneum is consistent with the mRNA and protein expressions of differentiation markers. The device is the first of its kind to combine air-exposure with mechanical stress to co-stimulate keratinization, which can facilitate the economically viable production of HaCaT-based artificial skin substitutes. PMID:27670754

  18. Lonicera caerulea and Vaccinium myrtillus fruit polyphenols protect HaCaT keratinocytes against UVB-induced phototoxic stress and DNA damage.

    PubMed

    Svobodová, Alena; Zdarilová, Adéla; Vostálová, Jitka

    2009-12-01

    Sunlight is a very potent environmental factor in skin pathogenesis and can induce skin cancer. UVB irradiation is known to cause oxidative stress, inflammation and especially DNA damage. Topical application of agents with UV absorbing, antioxidant and anti-inflammatory activities is a successful strategy in the protection of the skin against UV-caused damage. To examine the ability of the phenolic fraction of Lonicera caerulea and Vaccinum myrtillus fruits to moderate UVB-induced damage. HaCaT keratinocytes, a well-established in vitro system for investigations on UV radiation induced cell damage, were used to assess the effects of pre- and post-treatment with L. caerulea (LCE) and V. myrtillus (VME) phenolic fractions (5-50 mg/l) on keratinocyte damage induced by a solar simulator (295-315 nm). In this study, a model of UVB-induced damage to HaCaT was established. LCE and VME efficiently reduced the extent of DNA breakage (especially at concentrations of 25 and 10 mg/l) together with caspase-3 and -9 activity and DNA laddering induced by UVB (100 or 200 mJ/cm(2)). LCE and VME significantly decreased RONS generation and partially diminished IL-6 expression. LCE pre-treatment also prevented keratinocytes proliferation. The results suggest that the phenolic fraction of L. caerulea and V. myrtillus fruits suppress UVB-caused injury to keratinocytes. These results now need to be demonstrated in vivo.

  19. Interferon Production by Human Cells In Vitro

    PubMed Central

    Spina, Celsa A.; Chang, R. Shihman; Mishra, L.; Golden, H. Dean

    1972-01-01

    The relative capacity of several types of human cells and tissue to produce interferon was studied. Types of cells and tissue included were fibroblasts from embryos, foreskins, and biopsied skins; amnion cells; peripheral leukocytes; established lymphoid cell lines; established heteroploid cell lines; and chorioamniotic membrane. When Newcastle disease virus was used as the inducer, fibroblasts and amnion cells produced more interferon per 106 cells than leukocytes, lymphoid cells, and heteroploid cells. Only minor variations in interferon-producing capacity were observed among fibroblasts from 36 persons. Culture passage level, cell concentration, and inducer were factors that significantly affected interferon production. PMID:4344957

  20. The interaction between Staphylococcus aureus SdrD and desmoglein 1 is important for adhesion to host cells

    PubMed Central

    Askarian, Fatemeh; Ajayi, Clement; Hanssen, Anne-Merethe; van Sorge, Nina M.; Pettersen, Ingvild; Diep, Dzung B.; Sollid, Johanna U. E.; Johannessen, Mona

    2016-01-01

    Staphylococcus aureus is known as a frequent colonizer of the skin and mucosa. Among bacterial factors involved in colonization are adhesins such as the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Serine aspartate repeat containing protein D (SdrD) is involved in adhesion to human squamous cells isolated from the nose. Here, we identify Desmoglein 1 (Dsg1) as a novel interaction partner for SdrD. Genetic deletion of sdrD in S. aureus NCTC8325-4 through allelic replacement resulted in decreased bacterial adherence to Dsg1- expressing HaCaT cells in vitro. Complementary gain-of-function was demonstrated by heterologous expression of SdrD in Lactococcus lactis, which increased adherence to HaCaT cells. Also ectopic expression of Dsg1 in HEK293 cells resulted in increased adherence of S. aureus NCTC8325-4 in vitro. Increased adherence of NCTC8325-4, compared to NCTC8325-4ΔsdrD, to the recombinant immobilized Dsg1 demonstrated direct interaction between SdrD and Dsg1. Specificity of SdrD interaction with Dsg1 was further verified using flow cytometry and confirmed binding of recombinant SdrD to HaCaT cells expressing Dsg1 on their surface. These data demonstrate that Dsg1 is a host ligand for SdrD. PMID:26924733

  1. Endothelial cells derived from human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  2. Human embryonic stem cells: prospects for development.

    PubMed

    Pera, Martin F; Trounson, Alan O

    2004-11-01

    It is widely anticipated that human embryonic stem (ES) cells will serve as an experimental model for studying early development in our species, and, conversely, that studies of development in model systems, the mouse in particular, will inform our efforts to manipulate human stem cells in vitro. A comparison of primate and mouse ES cells suggests that a common underlying blueprint for the pluripotent state has undergone significant species-specific modification. As we discuss here, technical advances in the propagation and manipulation of human ES cells have improved our understanding of their growth and differentiation, providing the potential to investigate early human development and to develop new clinical therapies.

  3. Modulating prime molecular expressions and in vitro wound healing rate in keratinocyte (HaCaT) population under characteristic honey dilutions.

    PubMed

    Chaudhary, Amrita; Bag, Swarnendu; Mandal, Mousumi; Krishna Karri, Sri Phani; Barui, Ananya; Rajput, Monika; Banerjee, Provas; Sheet, Debdoot; Chatterjee, Jyotirmoy

    2015-05-26

    In traditional medicines honey is known for healing efficacy and vividly used as "Anupan" in Ayurvedic medicines appreciating roles in dilutions. Validating efficacy of physico-chemically characterized honey in dilutions, studies on in vitro wound healing and attainment of cellular confluence epithelial cells including expressions of cardinal genes is crucial. To evaluate effects of characterized honey in varied dilutions on cellular viability, in vitro wound healing and modulation of prime epithelial gene expressions. Six Indian honey-samples from different sources were physico-chemically characterized and optimal one was explored in dilutions (v/v%) through in vitro studies on human epithelial (HaCaT) cells for viability, wound healing and expressions of genes p63, E-cadherin, β-catenin, GnT-III and GnT-V. Studied honey samples (i.e. A-F) depicted range of pH (2-4), water (12.48-23.95), electrical conductivity (2.57-14.34), carbohydrate (68.73-98.65), protein (.316-5.36) and antioxidant potential. Though sample A and F showed physico-chemical proximity, but overall bio-impact of the earlier was better, thus studied in 8-.1% (v/v) dilution range. Four dilutions (.01, .04, .1, .25 v/v%) augmented cellular viability but in vitro wound healing was fastest (p<.05) under .1%. Such efficacy was further documented for p63 up-regulation by immunocytochemistry and mRNA studies. The E-cadherin and β-catenin mRNA-expressions were also up-regulated and their proteins were predominantly cytoplasmic. E-cadherin up-regulation was corroborative with down-regulation and up-regulation of GnT-III and GnT-V respectively. Present study illustrated efficacy of particular honey dilution (.1%) with characteristic free radical scavenging activity in facilitating cell proliferation and attainment of confluence towards faster wound healing and modulation of cardinal epithelial genes (viz. p63, E-cadherin, β-catenin, Gnt-III and V). Copyright © 2015 Elsevier Ireland Ltd. All rights

  4. A hyperosmotic stimulus elevates intracellular calcium and inhibits proliferation of a human keratinocyte cell line.

    PubMed

    Dascalu, A; Matithyou, A; Oron, Y; Korenstein, R

    2000-10-01

    Occlusion has previously been used to treat psoriatic plaques and was shown to improve the condition. We investigated the consequences of applying a mechanical stress, in vitro, on the HaCaT keratinocyte cell line. A mechanical load applied to cells can be mimicked by a hyperosmotic stimulus. Exposure of HaCaT keratinocytes to different hyperosmotic solutions (final osmolarity in the range 350-600 mOsm, produced by sucrose addition) resulted in an inhibition of cell proliferation after 96 h of treatment. As keratinocyte maturation is regulated by calcium levels, we measured hyperosmotic-stimulus-induced changes of intracellular calcium ([Ca2+]i) by single-cell image analysis employing FURA-2/AM. The hyperosmotic stimulus produced a rapid transient 2.6-fold elevation of [Ca2+]i followed by a gradual decay to the basal level. The transients originated from extracellular as well as from intracellular calcium pools and did not respond to voltage-sensitive calcium channel blockers. The hyperosmotic stimulus was shown to increase the cellular expression of involucrin, a differentiation marker, following 72 h of incubation, as measured by flow cytometry. Treatment of cells with the [Ca2+]i chelator BAPTA/AM almost completely blocked the [Ca2+]i elevation, but did not alter cellular growth or the induction of differentiation observed after hyperosmotic stimulus. It is suggested that treatment of keratinocytes with hyperosmotic stimulus can induce short-time effects (calcium transients) as well as long-term cellular maturation.

  5. Evidence for Human Lung Stem Cells

    PubMed Central

    Kajstura, Jan; Rota, Marcello; Hall, Sean R.; Hosoda, Toru; D’Amario, Domenico; Sanada, Fumihiro; Zheng, Hanqiao; Ogórek, Barbara; Rondon-Clavo, Carlos; Ferreira-Martins, João; Matsuda, Alex; Arranto, Christian; Goichberg, Polina; Giordano, Giovanna; Haley, Kathleen J.; Bardelli, Silvana; Rayatzadeh, Hussein; Liu, Xiaoli; Quaini, Federico; Liao, Ronglih; Leri, Annarosa; Perrella, Mark A.; Loscalzo, Joseph; Anversa, Piero

    2011-01-01

    BACKGROUND Although progenitor cells have been described in distinct anatomical regions of the lung, description of resident stem cells has remained elusive. METHODS Surgical lung-tissue specimens were studied in situ to identify and characterize human lung stem cells. We defined their phenotype and functional properties in vitro and in vivo. RESULTS Human lungs contain undifferentiated human lung stem cells nested in niches in the distal airways. These cells are self-renewing, clonogenic, and multipotent in vitro. After injection into damaged mouse lung in vivo, human lung stem cells form human bronchioles, alveoli, and pulmonary vessels integrated structurally and functionally with the damaged organ. The formation of a chimeric lung was confirmed by detection of human transcripts for epithelial and vascular genes. In addition, the self-renewal and long-term proliferation of human lung stem cells was shown in serial-transplantation assays. CONCLUSIONS Human lungs contain identifiable stem cells. In animal models, these cells participate in tissue homeostasis and regeneration. They have the undemonstrated potential to promote tissue restoration in patients with lung disease. (Funded by the National Institutes of Health.) PMID:21561345

  6. Trophoblast lineage cells derived from human induced pluripotent stem cells

    SciTech Connect

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  7. Stereoselective suppressive effects of protopanaxadiol epimers on UV-B-induced reactive oxygen species and matrix metalloproteinase-2 in human dermal keratinocytes.

    PubMed

    Oh, Sun-Joo; Lee, Sihyeong; Kho, Ye Eun; Kim, Kyunghoon; Jin, Chang Duck; Lim, Chang-Jin

    2015-01-01

    This study aimed to assess the skin-related anti-photoaging activities of the 2 epimeric forms of protopanaxadiol (PPD), 20(S)-PPD and 20(R)-PPD, in cultured human keratinocytes (HaCaT cells). The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), as well as cell viability for HaCaT cells under UV-B irradiation. The activities for MMP-2 and -1 in conditioned medium were determined using gelatin zymography, and MMP-2 protein in the conditioned medium was detected using Western blot analysis. 20(S)-PPD, but not 20(R)-PPD, suppressed UV-B-induced ROS elevation. Neither of the epimers, at the concentrations used, exhibited cytotoxicity, irrespective of UV-B irradiation. 20(S)-PPD, but not 20(R)-PPD, exhibited an inhibitory effect on UV-B-induced MMP-2 activity and expression in HaCaT cells. In brief, only 20(S)-PPD, a major metabolic product of PPD-type ginsenosides, inhibits UV-B-induced ROS and MMP-2 elevation, implying its stereospecific anti-photoaging activity on the skin.

  8. Isolation and generation of human dendritic cells.

    PubMed

    Nair, Smita; Archer, Gerald E; Tedder, Thomas F

    2012-11-01

    Dendritic cells are highly specialized antigen-presenting cells (APC), which may be isolated or generated from human blood mononuclear cells. Although mature blood dendritic cells normally represent ∼0.2% of human blood mononuclear cells, their frequency can be greatly increased using the cell enrichment methods described in this unit. More highly purified dendritic cell preparations can be obtained from these populations by sorting of fluorescence-labeled cells. Alternatively, dendritic cells can be generated from monocytes by culture with the appropriate cytokines, as described here. In addition, a negative selection approach is provided that may be employed to generate cell preparations that have been depleted of dendritic cells to be used for comparison in functional studies.

  9. The Haemophilus ducreyi serum resistance antigen DsrA confers attachment to human keratinocytes.

    PubMed

    Cole, Leah E; Kawula, Thomas H; Toffer, Kristen L; Elkins, Christopher

    2002-11-01

    Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. H. ducreyi serum resistance protein A (DsrA) is a member of a family of multifunctional outer membrane proteins that are involved in resistance to killing by human serum complement. The members of this family include YadA of Yersinia species, the UspA proteins of Moraxella catarrhalis, and the Eib proteins of Escherichia coli. The role of YadA, UspA1, and UspA2H as eukaryotic cell adhesins and the function of UspA2 as a vitronectin binder led to our investigation of the cell adhesion and vitronectin binding properties of DsrA. We found that DsrA was a keratinocyte-specific adhesin as it was necessary and sufficient for attachment to HaCaT cells, a keratinocyte cell line, but was not required for attachment to HS27 cells, a fibroblast cell line. We also found that DsrA was specifically responsible for the ability of H. ducreyi to bind vitronectin. We then theorized that DsrA might use vitronectin as a bridge to bind to human cells, but this hypothesis proved to be untrue as eliminating HaCaT cell binding of vitronectin with a monoclonal antibody specific to integrin alpha(v)beta(5) did not affect the attachment of H. ducreyi to HaCaT cells. Finally, we wanted to examine the importance of keratinocyte adhesion in chancroid pathogenesis so we tested the wild-type and dsrA mutant strains of H. ducreyi in our swine models of chancroid pathogenesis. The dsrA mutant was less virulent than the wild type in both the normal and immune cell-depleted swine models of chancroid infection.

  10. Varying the morphology of silver nanoparticles results in differential toxicity against micro-organisms, HaCaT keratinocytes and affects skin deposition.

    PubMed

    Holmes, Amy M; Lim, Julian; Studier, Hauke; Roberts, Michael S

    2016-12-01

    The use of silver nanoparticles (Ag NPs) within the healthcare sector and consumer products is rapidly increasing. There are now a range of diverse-shaped Ag NPs that are commercially available and many of the products containing nanosilver are topically applied to human skin. Currently, there is limited data on the extent to which the antimicrobial efficacy and cytotoxicity of Ag NPs is related to their shape and how the shape of the Ag NPs affects their distribution in both intact and burn wounded human skin after topical application. In this study, we related the relative Ag NP cytotoxicity to potential skin pathogens and HaCaT keratinocytes in vitro with the shape of the Ag NPs. We employed multiphoton fluorescence lifetime imaging to map the distribution of the native and unlabeled Ag NPs after topical application to both intact and burn wounded human skin using the localized surface plasmon resonance signal of the Ag NPs. Truncated plate shaped Ag NPs led to the highest cytotoxicity against both bacteria (IC50 ranges from 31.25 to 125 μg/mL depending on the bacterial species) and HaCaT keratinocytes (IC50 78.65 μg/mL [95%CI 63.88, 96.83]) thus both with similar orders of magnitude. All Ag NPs were less cytotoxic than solutions of silver nitrate (IC50 of 7.85 μg/mL [95%CI 1.49, 14.69]). Plate-shaped Ag NPs displayed the highest substantivity within the superficial layers of the stratum corneum when topically applied to intact skin and the highest deposition into the wound bed when applied to burned ex vivo human skin relative to other Ag NP shapes.

  11. Photosensitized 2-amino-3-hydroxypyridine-induced mitochondrial apoptosis via Smac/DIABLO in human skin cells.

    PubMed

    Goyal, Shruti; Amar, Saroj Kumar; Dwivedi, Ashish; Mujtaba, Syed Faiz; Kushwaha, Hari Narayan; Chopra, Deepti; Pal, Manish Kumar; Singh, Dhirendra; Chaturvedi, Rajnish Kumar; Ray, Ratan Singh

    2016-04-15

    The popularity of hair dyes use has been increasing regularly throughout the world as per the demand of hair coloring fashion trends and other cosmetic products. 2-Amino-3-hydroxypyridine (A132) is widely used as a hair dye ingredient around the world. We are reporting first time the phototoxicity mechanism of A132 under ambient environmental UV-B radiation. It showed maximum absorption in UV-B region (317 nm) and forms a photoproduct within an hour exposure of UV-B irradiation. Photocytotoxicity of A132 in human keratinocytes (HaCaT) was measured by mitochondrial (MTT), lysosomal (NRU) and LDH assays which illustrated the significant reduction in cell viability. The role of reactive oxygen species (ROS) generation for A132 phototoxicity was established photo- chemically as well as intracellularly. Noteworthy, formation of tail DNA (comet assay), micronuclei and cyclobutane pyrimidine dimers (CPDs) (immunocytochemistry) formation confirmed the photogenotoxic potential of dye. Cell cycle study (sub-G1peak) and staining with EB/AO revealed the cell cycle arrest and apoptosis. Further, mitochondrial mediated apoptosis was corroborated by reduced MMP, release of cytochrome c and upregulation of caspase-3. Release of mitochondrial Smac/DIABLO in cytoplasm demonstrated the caspase dependent apoptotic cell death by photolabile A132 dye. In-addition increased Bax/Bcl2 ratio again proved the apoptosis. Thus, study suggests that A132 induces photogenotoxicity, phototoxicity and apoptotic cell death through the involvement of Smac/DIABLO in mitochondrial apoptosis via caspase dependent manner. Therefore, the long term use of A132 dye and sunlight exposure jointly increased the oxidative stress in skin which causes premature hair loss, damage to progenitor cells of hair follicles. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Agerarin, identified from Ageratum houstonianum, stimulates circadian CLOCK-mediated aquaporin-3 gene expression in HaCaT keratinocytes.

    PubMed

    Shin, Soon Young; Lee, Da Hyun; Gil, Ha-Na; Kim, Beom Soo; Choe, Jeong-Sook; Kim, Jung-Bong; Lee, Young Han; Lim, Yoongho

    2017-09-11

    The juice of Ageratum houstonianum is used in folk medicine as an external wound healing aid for skin injuries. However, the active component of A. houstonianum and its mode of action in skin wound healing has not been investigated. This study was conducted to investigate the effect of A. houstonianum ethanolnolic extract (AHE) on the expression of aquaporin-3 (AQP3), an integral membrane protein for water and glycerol transport in keratinocytes, and to identify the structure of the A. houstonianum bioactive compound. Here, we show that AHE increased AQP3 gene expression at the transcriptional level through the p38 MAPK pathway in HaCaT cells. Furthermore, AHE ameliorated suppression of AQP3 expression caused by ultraviolet B (UVB) irradiation. Agerarin (6,7-dimethoxy-2,2-dimethyl-2H-chromene) was identified as the bioactive compound responsible for the up-regulation of AQP3 expression by enhancing the expression of the transcription factor circadian locomotor output cycles kaput (CLOCK). In conclusion, agerarin is a bioactive compound in AHE responsible for CLOCK-mediated AQP3 expression in keratinocytes.

  13. Hepatic Differentiation from Human Ips Cells Using M15 Cells.

    PubMed

    Umeda, Kahoko; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    Here, we describe a procedure of human iPS cells differentiation into the definitive endoderm, further into albumin-expressing and albumin-secreting hepatocyte, using M15, a mesonephros- derived cell line. Approximately 90 % of human iPS cells differentiated into SOX17-positive definitive endoderm then approximately 50 % of cells became albumin-positive cells, and secreted ALB protein. This M15 feeder system for endoderm and hepatic differentiation is a simple and efficient method, and useful for elucidating molecular mechanisms for hepatic fate decision, and could represent an attractive approach for a surrogate cell source for pharmaceutical studies.

  14. Competition between human cells by entosis

    PubMed Central

    Sun, Qiang; Luo, Tianzhi; Ren, Yixin; Florey, Oliver; Shirasawa, Senji; Sasazuki, Takehiko; Robinson, Douglas N; Overholtzer, Michael

    2014-01-01

    Human carcinomas are comprised of complex mixtures of tumor cells that are known to compete indirectly for nutrients and growth factors. Whether tumor cells could also compete directly, for example by elimination of rivals, is not known. Here we show that human cells can directly compete by a mechanism of engulfment called entosis. By entosis, cells are engulfed, or cannibalized while alive, and subsequently undergo cell death. We find that the identity of engulfing (“winner”) and engulfed (“loser”) cells is dictated by mechanical deformability controlled by RhoA and actomyosin, where tumor cells with high deformability preferentially engulf and outcompete neighboring cells with low deformability in heterogeneous populations. We further find that activated Kras and Rac signaling impart winner status to cells by downregulating contractile myosin, allowing for the internalization of neighboring cells that eventually undergo cell death. Finally, we compute the energy landscape of cell-in-cell formation, demonstrating that a mechanical differential between winner and loser cells is required for entosis to proceed. These data define a mechanism of competition in mammalian cells that occurs in human tumors. PMID:25342560

  15. Activin A programs human TFH cell differentiation

    PubMed Central

    Locci, Michela; Wu, Jennifer; Arumemi, Fortuna; Mikulski, Zbigniew; Dahlberg, Carol; Miller, Andrew T.; Crotty, Shane

    2016-01-01

    SUMMARY Follicular helper T (TFH) cells are CD4+ T cells specialized in helping B cells and are associated both with protective antibody responses and autoimmune diseases. The promise of targeting TFH cells therapeutically has been limited by fragmentary understanding of extrinsic signals regulating human TFH cell differentiation. A screen of a human protein library identified activin A as new regulator of TFH cell differentiation. Activin A orchestrated expression of multiple TFH-associated genes, independently or in concert with additional signals. TFH programming by activin A was antagonized by the cytokine IL-2. Activin A’s capacity to drive TFH cell differentiation in vitro was conserved for non-human primates but not mice. Finally, activin A-induced TFH programming was dependent on SMAD2 and SMAD3 signaling and blocked by pharmacological inhibitors. PMID:27376469

  16. Human skin cells support thymus-independent T cell development

    PubMed Central

    Clark, Rachael A.; Yamanaka, Kei-ichi; Bai, Mei; Dowgiert, Rebecca; Kupper, Thomas S.

    2005-01-01

    Thymic tissue has previously been considered a requirement for the generation of a functional and diverse population of human T cells. We report that fibroblasts and keratinocytes from human skin arrayed on a synthetic 3-dimensional matrix support the development of functional human T cells from hematopoietic precursor cells in the absence of thymic tissue. Newly generated T cells contained T cell receptor excision circles, possessed a diverse T cell repertoire, and were functionally mature and tolerant to self MHC, indicating successful completion of positive and negative selection. Skin cell cultures expressed the AIRE, Foxn1, and Hoxa3 transcription factors and a panel of autoantigens. Skin and bone marrow biopsies can thus be used to generate de novo functional and diverse T cell populations for potential therapeutic use in immunosuppressed patients. PMID:16224538

  17. Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.

    PubMed

    Mangerich, Aswin; Debiak, Malgorzata; Birtel, Matthias; Ponath, Viviane; Balszuweit, Frank; Lex, Kirsten; Martello, Rita; Burckhardt-Boer, Waltraud; Strobelt, Romano; Siegert, Markus; Thiermann, Horst; Steinritz, Dirk; Schmidt, Annette; Bürkle, Alexander

    2016-02-26

    Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of

  18. Human embryonic stem cells derived by somatic cell nuclear transfer.

    PubMed

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L; Wolf, Don; Mitalipov, Shoukhrat

    2013-06-06

    Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

  19. Differentiation of Neural Lineage Cells from Human Pluripotent Stem Cells

    PubMed Central

    Schwartz, Philip H.; Brick, David J.; Stover, Alexander E.; Loring, Jeanne F.; Müller, Franz Josef

    2008-01-01

    Human pluripotent stem cells have the unique properties of being able to proliferate indefinitely in their undifferentiated state and to differentiate into any somatic cell type. These cells are thus posited to be extremely useful for furthering our understanding of both normal and abnormal human development, providing a human cell preparation that can be used to screen for new reagents or therapeutic agents, and generating large numbers of differentiated cells that can be used for transplantation purposes. Critical among the applications for the latter are diseases and injuries of the nervous system, medical approaches to which have been, to date, primarily palliative in nature. Differentiation of human pluripotent stem cells into cells of the neural lineage, therefore, has become a central focus of a number of laboratories. This has resulted in the description in the literature of several dozen methods for neural cell differentiation from human pluripotent stem cells. Among these are methods for the generation of such divergent neural cells as dopaminergic neurons, retinal neurons, ventral motoneurons, and oligodendroglial progenitors. In this review, we attempt to fully describe most of these methods, breaking them down into five basic subdivisions: 1) starting material, 2) induction of loss of pluripotency, 3) neural induction, 4) neural maintenance and expansion, and 5) neuronal/glial differentiation. We also show data supporting the concept that undifferentiated human pluripotent stem cells appear to have an innate neural differentiation potential. In addition, we evaluate data comparing and contrasting neural stem cells differentiated from human pluripotent stem cells with those derived directly from the human brain. PMID:18593611

  20. Cloning the human SUMO1 promoter.

    PubMed

    Nanos-Webb, Angela; Deyrieux, Adeline; Bian, Xue-lin; Rosas-Acosta, Germán; Wilson, Van G

    2010-03-01

    Regulation of the sumoylation system at the level of gene expression has not yet been explored. To begin to define transcriptional regulatory features, the promoter region for the SUMO1 gene was cloned from human genomic DNA and characterized. Initially, a 532 base pair fragment upstream of and including the predicted SUMO1 transcription start site (TSS) was cloned and shown to possess promoter activity. Subsequent deletion analysis showed that a smaller fragment containing 158 bp upstream of the TSS region exhibited basal promoter activity in both human and rodent cell lines. Within this basal promoter fragment, there were predicted binding sites for numerous transcription factors, including the nude mouse gene product, Whn (FoxN1). Electrophoretic mobility shift assays showed that Whn could bind to an ACGC motif adjacent to the TSR, and in transfection studies Whn stimulated a 3-fold increase in transcription from this cloned promoter in keratinocytes (HaCaT cells). Mutation of the ACGC motif abrogated both Whn binding and transcriptional activation, indicating that the Whn effect is likely due to direct interaction with this promoter element. Consistent with these observations on the cloned promoter region, Whn also modestly stimulated transcription from the endogenous, genomic SUMO1 promoter in HaCaT cells, consistent with Whn potentially playing a regulatory role for SUMO1 transcription in keratinocytes.

  1. Development of human mast cells in vitro.

    PubMed Central

    Furitsu, T; Saito, H; Dvorak, A M; Schwartz, L B; Irani, A M; Burdick, J F; Ishizaka, K; Ishizaka, T

    1989-01-01

    Nucleated cells of human umbilical cord blood were cocultured with mouse skin-derived 3T3 fibroblasts. After 7-8 weeks in culture, when the number of the other hematopoietic cells declined, metachromatic granule-containing mononuclear cells appeared in the cultures, and the number of the cells increased up to 12 weeks. After 11-14 weeks in culture, the metachromatic mononuclear cells comprised a substantial portion of the cultured cells. These cells contained 1.8-2 micrograms of histamine per 10(6) cells and bore receptors for IgE. All of the cells contained tryptase in their granules. Electron microscopic analysis showed that these cells were mature human mast cells, clearly different from the basophilic granulocytes or eosinophils that arise in a variety of circumstances in cord blood cell cultures. Most of the cultured mast cells expressed some granules with regular crystalline arrays and contained both tryptase and chymase, and thus resembled human skin mast cells. Images PMID:2532357

  2. Expression cloning of human B cell immunoglobulins.

    PubMed

    Wardemann, Hedda; Kofer, Juliane

    2013-01-01

    The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.

  3. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

    PubMed Central

    Varma, Sandeep R.; Sivaprakasam, Thiyagarajan O.; Mishra, Abheepsa; Kumar, L. M. Sharath; Prakash, N. S.; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  4. Cells immunoreactive for neuropeptide in human thymomas.

    PubMed Central

    Lauriola, L; Maggiano, N; Larocca, L M; Ranelletti, F O; Ricci, R; Piantelli, M; Capelli, A

    1990-01-01

    The presence of opioid peptides, bombesin, and substance P was investigated by immunohistochemistry in tissue sections from six human thymomas. The number of immunoreactive cells seemed to vary from one case to another. Ultrastructural investigation, showing the presence of desmosomes in labelled cells, allowed these cells to be classified as epithelial lineage cells. The occurrence of cells containing neuropeptide in thymomas suggest that peptide molecules could have modulated the behaviour of this tumour, given the reported influence of these molecules on immune functions and their growth promoting activity on several cell types, including mesenchymal and epithelial cells. Images PMID:1699978

  5. Reprogramming of human exocrine pancreas cells to beta cells.

    PubMed

    Staels, Willem; Heremans, Yves; Heimberg, Harry

    2015-12-01

    One of the key promises of regenerative medicine is providing a cure for diabetes. Cell-based therapies are proving their safety and efficiency, but donor beta cell shortages and immunological issues remain major hurdles. Reprogramming of human pancreatic exocrine cells towards beta cells would offer a major advantage by providing an abundant and autologous source of beta cells. Over the past decade our understanding of transdifferentiation processes greatly increased allowing us to design reprogramming protocols that fairly aim for clinical trials.

  6. Ex Vivo Expanded Human NK Cells Survive and Proliferate in Humanized Mice with Autologous Human Immune Cells.

    PubMed

    Vahedi, Fatemeh; Nham, Tina; Poznanski, Sophie M; Chew, Marianne V; Shenouda, Mira M; Lee, Dean; Ashkar, Ali A

    2017-09-21

    Adoptive immune cell therapy is emerging as a promising immunotherapy for cancer. Particularly, the adoptive transfer of NK cells has garnered attention due to their natural cytotoxicity against tumor cells and safety upon adoptive transfer to patients. Although strategies exist to efficiently generate large quantities of expanded NK cells ex vivo, it remains unknown whether these expanded NK cells can persist and/or proliferate in vivo in the absence of exogenous human cytokines. Here, we have examined the adoptive transfer of ex vivo expanded human cord blood-derived NK cells into humanized mice reconstituted with autologous human cord blood immune cells. We report that ex vivo expanded NK cells are able to survive and possibly proliferate in vivo in humanized mice without exogenous cytokine administration, but not in control mice that lack human immune cells. These findings demonstrate that the presence of autologous human immune cells supports the in vivo survival of ex vivo expanded human NK cells. These results support the application of ex vivo expanded NK cells in cancer immunotherapy and provide a translational humanized mouse model to test the lifespan, safety, and functionality of adoptively transferred cells in the presence of autologous human immune cells prior to clinical use.

  7. Influence of the stabilizers on the toxicity of metallic nanomaterials in aquatic organisms and human cell lines.

    PubMed

    Andreani, Tatiana; Nogueira, Verónica; Pinto, Vera V; Ferreira, Maria José; Rasteiro, Maria Graça; Silva, Amélia M; Pereira, Ruth; Pereira, Carlos M

    2017-12-31

    In this study, following a systematic approach, we used aquatic species (bacteria Vibrio fischeri and microalgae Raphidocelis subcapitata) and different human cell lines (Caco-2, HepG2, SV-80 and HaCaT) representing different tissues and exposure pathways, to investigate how two organic stabilizers (PVA and DMSO) used for NMs dispersion influence their physicochemical properties, the persistence of metals in suspension and the toxicity/ecotoxicity of two metallic NMs (nano-Ag and nano-Cu). Although the stabilizers are expected to contribute to improve the dispersion and stability of NMs, the results obtained clearly showed that no similar changes in toxicity and morphological properties of the nano-Ag can be expected after its stabilization with PVA. Thus, regarding human cell lines, the reduction in the average size of the PVA-nano-Ag was followed by a reduction or maintenance of its toxicity, but the opposite was observed for the aquatic species tested since an increase in the average size enhanced its toxicity. As far as nano-Cu is considered DMSO contributed for a better dispersion of this nanomaterial, however this was not translated in a similar toxicity/ecotoxicity modification. In summary, even for nano-Cu, for which few or no data exists regarding its toxicity after stabilization with organic compounds, it was confirmed with consistent data, that the toxicity of metallic NMs is a complex combination of average size, chemical composition, solubilization or persistence in suspension of the metallic forms, interaction with test medium components and sensitivity of test species and cell lines. The combination of all of these factors makes the toxicity of metallic NMs unpredictable and points for the need of an extensive evaluation of each new formulation. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Human CD4+ T Cell Response to Human Herpesvirus 6

    PubMed Central

    Nastke, Maria-D.; Becerra, Aniuska; Yin, Liusong; Dominguez-Amorocho, Omar; Gibson, Laura; Calvo-Calle, J. Mauricio

    2012-01-01

    Following primary infection, human herpesvirus 6 (HHV-6) establishes a persistent infection for life. HHV-6 reactivation has been associated with transplant rejection, delayed engraftment, encephalitis, muscular dystrophy, and drug-induced hypersensitivity syndrome. The poor understanding of the targets and outcome of the cellular immune response to HHV-6 makes it difficult to outline the role of HHV-6 in human disease. To fill in this gap, we characterized CD4 T cell responses to HHV-6 using peripheral blood mononuclear cell (PBMC) and T cell lines generated from healthy donors. CD4+ T cells responding to HHV-6 in peripheral blood were observed at frequencies below 0.1% of total T cells but could be expanded easily in vitro. Analysis of cytokines in supernatants of PBMC and T cell cultures challenged with HHV-6 preparations indicated that gamma interferon (IFN-γ) and interleukin-10 (IL-10) were appropriate markers of the HHV-6 cellular response. Eleven CD4+ T cell epitopes, all but one derived from abundant virion components, were identified. The response was highly cross-reactive between HHV-6A and HHV-6B variants. Seven of the CD4+ T cell epitopes do not share significant homologies with other known human pathogens, including the closely related human viruses human herpesvirus 7 (HHV-7) and human cytomegalovirus (HCMV). Major histocompatibility complex (MHC) tetramers generated with these epitopes were able to detect HHV-6-specific T cell populations. These findings provide a window into the immune response to HHV-6 and provide a basis for tracking HHV-6 cellular immune responses. PMID:22357271

  9. Cell proliferation in human coronary arteries.

    PubMed Central

    Gordon, D; Reidy, M A; Benditt, E P; Schwartz, S M

    1990-01-01

    Despite the lack of direct evidence for cell multiplication, proliferation of smooth muscle cells in human atherosclerotic lesions has been assumed to play a central role in ontogeny of the plaque. We used antibodies to cell cycle-related proteins on tissue sections of human arteries and coronary atherosclerotic plaques. Specific cell types were identified by immunochemical reagents for smooth muscle, monocyte-macrophages, and other blood cells. Low rates of smooth muscle cell proliferation were observed. Macrophages were also observed with rates of proliferation comparable to that of the smooth muscle. Additional replicating cells could not be defined as belonging to specific cell types with the reagents used in this study. These findings imply that smooth muscle replication in advanced plaques is indolent and raise the possibility of a role for proliferating leukocytes. Images PMID:1972277

  10. Th17 cells in human disease

    PubMed Central

    Tesmer, Laura A.; Lundy, Steven K.; Sarkar, Sujata; Fox, David A.

    2012-01-01

    Summary Our understanding of the role of T cells in human disease is undergoing revision as a result of the discovery of T-helper 17 (Th17) cells, a unique CD4+ T-cell subset characterized by production of interleukin-17 (IL-17). IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues. Recent data in humans and mice suggest that Th17 cells play an important role in the pathogenesis of a diverse group of immune-mediated diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and asthma. Initial reports also propose a role for Th17 cells in tumorigenesis and transplant rejection. Important differences, as well as many similarities, are emerging when the biology of Th17 cells in the mouse is compared with corresponding phenomena in humans. As our understanding of human Th17 biology grows, the mechanisms underlying many diseases are becoming more apparent, resulting in a new appreciation for both previously known and more recently discovered cytokines, chemokines, and feedback mechanisms. Given the strong association between excessive Th17 activity and human disease, new therapeutic approaches targeting Th17 cells are highly promising, but the potential safety of such treatments may be limited by the role of these cells in normal host defenses against infection. PMID:18613831

  11. Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells

    NASA Astrophysics Data System (ADS)

    Winter, J.; Wende, K.; Masur, K.; Iseni, S.; Dünnbier, M.; Hammer, M. U.; Tresp, H.; Weltmann, K.-D.; Reuter, S.

    2013-07-01

    In this study, the effect of feed gas humidity on the reactive component generation of an atmospheric-pressure argon plasma jet and its effect on human skin cells are investigated. Feed gas humidity is identified as one key parameter that strongly influences stability and reproducibility of plasma medical studies. The plasma jet is investigated by absorption spectroscopy in the ultraviolet and infrared spectral region for its ozone production depending on the humidity concentration in the feed gas. By optical emission spectroscopy the dependence of present excited plasma species such as hydroxyl radicals, molecular nitrogen, argon and atomic oxygen on the feed gas humidity is investigated. As an interface layer between the plasma jet effluent and the biological cell, a buffer solution is treated and the hydrogen peroxide (H2O2) production is studied with two independent colorimetric assays as a function of humidity admixture to the feed gas. Ultimately, the effect of varying feed gas humidity on the cell viability of indirect plasma treated adherent HaCAT cells is investigated. The highest viability is found for the driest feed gas condition. Furthermore, this work shows answers for the relevance of unwanted—or intended—feed gas humidity in plasma medical experiments and their comparatively large relevance with respect to ambient humidity. The findings will lead to more reproducible experiments in the field of plasma medicine.

  12. Human genome project and sickle cell disease.

    PubMed

    Norman, Brenda J; Miller, Sheila D

    2011-01-01

    Sickle cell disease is one of the most common genetic blood disorders in the United States that affects 1 in every 375 African Americans. Sickle cell disease is an inherited condition caused by abnormal hemoglobin in the red blood cells. The Human Genome Project has provided valuable insight and extensive research advances in the understanding of the human genome and sickle cell disease. Significant progress in genetic knowledge has led to an increase in the ability for researchers to map and sequence genes for diagnosis, treatment, and prevention of sickle cell disease and other chronic illnesses. This article explores some of the recent knowledge and advances about sickle cell disease and the Human Genome Project.

  13. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    NASA Technical Reports Server (NTRS)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  14. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    NASA Technical Reports Server (NTRS)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  15. Identification of human brain tumour initiating cells.

    PubMed

    Singh, Sheila K; Hawkins, Cynthia; Clarke, Ian D; Squire, Jeremy A; Bayani, Jane; Hide, Takuichiro; Henkelman, R Mark; Cusimano, Michael D; Dirks, Peter B

    2004-11-18

    The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Although the existence of CSCs in human leukaemia is established, little evidence exists for CSCs in solid tumours, except for breast cancer. Recently, we prospectively isolated a CD133+ cell subpopulation from human brain tumours that exhibited stem cell properties in vitro. However, the true measures of CSCs are their capacity for self renewal and exact recapitulation of the original tumour. Here we report the development of a xenograft assay that identified human brain tumour initiating cells that initiate tumours in vivo. Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains. Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 10(5) CD133- cells engrafted but did not cause a tumour. Thus, the identification of brain tumour initiating cells provides insights into human brain tumour pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumours, and establishes a previously unidentified cellular target for more effective cancer therapies.

  16. Immortalisation of human urothelial cells.

    PubMed

    Petzoldt, J L; Leigh, I M; Duffy, P G; Sexton, C; Masters, J R

    1995-01-01

    A cell line derived from the urothelium lining the ureter of a 12-year-old girl was immortalised using a temperature-sensitive SV40 large T-antigen gene construct, and designated UROtsa. Following immortalisation, UROtsa cells expressed SV40 large T-antigen, but did not acquire characteristics of neoplastic transformation, including growth in soft agar or the development of tumours in nude mice. Metaphase spreads had a normal chromosomal appearance and number. UROtsa cells remained permissive for cell growth at 39 degrees C, indicating that they did not retain temperature sensitivity. UROtsa provides an in vitro model of "normal" urothelium.

  17. The Human Natural Killer Cell Immune Synapse

    NASA Astrophysics Data System (ADS)

    Davis, Daniel M.; Chiu, Isaac; Fassett, Marlys; Cohen, George B.; Mandelboim, Ofer; Strominger, Jack L.

    1999-12-01

    Inhibitory killer Ig-like receptors (KIR) at the surface of natural killer (NK) cells induced clustering of HLA-C at the contacting surface of target cells. In this manner, inhibitory immune synapses were formed as human NK cells surveyed target cells. At target/NK cell synapses, HLA-C/KIR distributed into rings around central patches of intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, the opposite orientation to mature murine T cell-activating synapses. This organization of protein was stable for at least 20 min. Cells could support multiple synapses simultaneously, and clusters of HLA-C moved as NK cells crawled over target cells. Clustering required a divalent metal cation, explaining how metal chelators inhibit KIR function. Surprisingly, however, formation of inhibitory synapses was unaffected by ATP depletion and the cytoskeletal inhibitors, colchicine and cytochalsins B and D. Clearly, supramolecular organization within plasma membranes is critical for NK cell immunosurveillance.

  18. TRPV4 exhibits a functional role in cell-volume regulation.

    PubMed

    Becker, Daniel; Blase, Christopher; Bereiter-Hahn, Juergen; Jendrach, Marina

    2005-06-01

    Tight regulation of the cell volume is important for the maintenance of cellular homeostasis. In a hypotonic environment, cells swell owing to osmosis. With many vertebrate cells, swelling is followed by an active reduction of volume, a process called regulatory volume decrease (RVD). A possible participant in RVD is the non-selective cation channel TRPV4, a member of the TRP superfamily that has been shown to react to hypotonic stimuli with a conductance for Ca2+. As a model for cell-volume regulation, we used a human keratinocyte cell line (HaCaT) that produces TRPV4 endogenously. When HaCaT cells were exposed to a hypotonic solution (200 mOsm) maximal swelling was followed by RVD. During swelling and volume regulation, a strong Ca2+ influx was measured. Gd3+, an inhibitor of TRPV4, blocked RVD of HaCaT cells and the accompanying rise of cytosolic Ca2+. To define the role of TRPV4 in volume regulation, a TRPV4-EGFP fusion protein was produced in CHO cells. CHO cells are unable to undergo RVD under hypotonic conditions and do not produce TRPV4 endogenously. Fluorescence imaging revealed that recombinant TRPV4 was localized to the cell membrane. Production of TRPV4 enabled CHO cells to undergo typical RVD after hypo-osmolarity-induced cell swelling. RVD of TRPV4-transfected CHO cells was significantly reduced by Gd3+ treatment or in Ca2+-free solution. Taken together, these results show a direct participation of TRPV4 in RVD.

  19. Nucleofection of human embryonic stem cells.

    PubMed

    Siemen, Henrike; Nix, Michael; Endl, Elmar; Koch, Philipp; Itskovitz-Eldor, Joseph; Brüstle, Oliver

    2005-08-01

    Human embryonic stem (hES) cells provide an important tool for the study of human development, disease, and tissue regeneration. Technologies for efficient genetic modification are required to exploit hES cells fully for these applications. Here we present a customized protocol for the transfection of hES cells with the Nucleofector technology and compare its efficiency with conventional electroporation and lipofection. Cell survival and transfection efficiency were quantified using an enhanced green fluorescent protein (EGFP) reporter construct. Our optimized nucleofection parameters yielded survival rates >70%. Under these conditions, 66% of the surviving cells showed transgene expression 24 h after nucleofection. Transfected cells maintained expression of the pluripotency- associated markers Tra-1-60, Tra-1-81, and Oct4 and could be expanded to stably transgene-expressing clones. The low quantities of hES cells and DNA required for nucleofection could make this method an attractive tool for miniaturized high throughput screening (HTS) applications.

  20. Calcium signaling in human pluripotent stem cells.

    PubMed

    Apáti, Ágota; Berecz, Tünde; Sarkadi, Balázs

    2016-03-01

    Human pluripotent stem cells provide new tools for developmental and pharmacological studies as well as for regenerative medicine applications. Calcium homeostasis and ligand-dependent calcium signaling are key components of major cellular responses, including cell proliferation, differentiation or apoptosis. Interestingly, these phenomena have not been characterized in detail as yet in pluripotent human cell sates. Here we review the methods applicable for studying both short- and long-term calcium responses, focusing on the expression of fluorescent calcium indicator proteins and imaging methods as applied in pluripotent human stem cells. We discuss the potential regulatory pathways involving calcium responses in hPS cells and compare these to the implicated pathways in mouse PS cells. A recent development in the stem cell field is the recognition of so called "naïve" states, resembling the earliest potential forms of stem cells during development, as well as the "fuzzy" stem cells, which may be alternative forms of pluripotent cell types, therefore we also discuss the potential role of calcium homeostasis in these PS cell types.

  1. Satellite cells in human skeletal muscle plasticity

    PubMed Central

    Snijders, Tim; Nederveen, Joshua P.; McKay, Bryon R.; Joanisse, Sophie; Verdijk, Lex B.; van Loon, Luc J. C.; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models. PMID:26557092

  2. Human progenitor cells for bone engineering applications.

    PubMed

    de Peppo, G M; Thomsen, P; Karlsson, C; Strehl, R; Lindahl, A; Hyllner, J

    2013-06-01

    In this report, the authors review the human skeleton and the increasing burden of bone deficiencies, the limitations encountered with the current treatments and the opportunities provided by the emerging field of cell-based bone engineering. Special emphasis is placed on different sources of human progenitor cells, as well as their pros and cons in relation to their utilization for the large-scale construction of functional bone-engineered substitutes for clinical applications. It is concluded that, human pluripotent stem cells represent a valuable source for the derivation of progenitor cells, which combine the advantages of both embryonic and adult stem cells, and indeed display high potential for the construction of functional substitutes for bone replacement therapies.

  3. Cadmium increases human fetal germ cell apoptosis.

    PubMed

    Angenard, Gaëlle; Muczynski, Vincent; Coffigny, Hervé; Pairault, Catherine; Duquenne, Clotilde; Frydman, René; Habert, René; Rouiller-Fabre, Virginie; Livera, Gabriel

    2010-03-01

    Cadmium (Cd) is a common environmental pollutant and a major constituent of tobacco smoke. Adverse effects of this heavy metal on reproductive function have been identified in adults; however, no studies have examined its effects on human reproductive organs during development. Using our previously developed organ culture system, we investigated the effects of cadmium chloride on human gonads at the beginning of fetal life, a critical stage in the development of reproductive function. Human fetal gonads were recovered during the first trimester (711 weeks postconception) and cultured with or without Cd. We used different concentrations of Cd and compared results with those obtained with mouse fetal gonads at similar stages. Cd, at concentrations as low as 1 microM, significantly decreased the germ cell density in human fetal ovaries. This correlated with an increase in germ cell apoptosis, but there was no effect on proliferation. Similarly, in the human fetal testis, Cd (1 microM) reduced germ cell number without affecting testosterone secretion. In mouse fetal gonads, Cd increased only female germ cell apoptosis. This is the first experimental demonstration that Cd, at low concentrations, alters the survival of male and female germ cells in humans. Considering data demonstrating extensive human exposure, we believe that current environmental levels of Cd could be deleterious to early gametogenesis.

  4. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    SciTech Connect

    Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva; Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs; Apati, Agota

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  5. Cell senescence in human aging and disease.

    PubMed

    Fossel, Michael

    2002-04-01

    The most common causes of death and suffering, even in most underdeveloped nations, are age-related diseases. These diseases share fundamental and often unappreciated pathology at the cellular and genetic levels, through cell senescence. In cancer, enforcing cell senescence permits us to kill cancer cells without significantly harming normal cells. In other age-related diseases, cell senescence plays a direct role, and we may be able to prevent and reverse much of the pathology. While aging is attributed to "wear and tear," genetic studies show that these effects are avoidable (as is the case in germ cell lines) and occur only when cells down-regulate active (and sufficient) repair mechanisms, permitting degradation to occur. Aging occurs when cells permit accumulative damage by wear and tear, by altering their gene expression rather than vice versa. Using telomerase in laboratory settings, we can currently reset this pattern and its consequences both within cells and between cells. Doing so resets not only cell behavior but the pathological consequences within tissues comprising such cells. We can currently grow histologically young, reconstituted human skin using old human skin cells (keratinocytes and fibroblasts). Technically we could now test this approach in joints, vessels, the immune system, and other tissues. This model is consistent with all available laboratory data and known aging pathology. Within the next decade, we will be able to treat age-related diseases more effectively than ever before.

  6. Human Stem Cells for Craniomaxillofacial Reconstruction

    PubMed Central

    Kirkpatrick, William Niall Alexander; Cameron, Malcolm Gregor

    2014-01-01

    Human stem cell research represents an exceptional opportunity for regenerative medicine and the surgical reconstruction of the craniomaxillofacial complex. The correct architecture and function of the vastly diverse tissues of this important anatomical region are critical for life supportive processes, the delivery of senses, social interaction, and aesthetics. Craniomaxillofacial tissue loss is commonly associated with inflammatory responses of the surrounding tissue, significant scarring, disfigurement, and psychological sequelae as an inevitable consequence. The in vitro production of fully functional cells for skin, muscle, cartilage, bone, and neurovascular tissue formation from human stem cells, may one day provide novel materials for the reconstructive surgeon operating on patients with both hard and soft tissue deficit due to cancer, congenital disease, or trauma. However, the clinical translation of human stem cell technology, including the application of human pluripotent stem cells (hPSCs) in novel regenerative therapies, faces several hurdles that must be solved to permit safe and effective use in patients. The basic biology of hPSCs remains to be fully elucidated and concerns of tumorigenicity need to be addressed, prior to the development of cell transplantation treatments. Furthermore, functional comparison of in vitro generated tissue to their in vivo counterparts will be necessary for confirmation of maturity and suitability for application in reconstructive surgery. Here, we provide an overview of human stem cells in disease modeling, drug screening, and therapeutics, while also discussing the application of regenerative medicine for craniomaxillofacial tissue deficit and surgical reconstruction. PMID:24564584

  7. Mitochondrial and lipogenic effects of vitamin D on differentiating and proliferating human keratinocytes.

    PubMed

    Consiglio, Marco; Viano, Marta; Casarin, Stefania; Castagnoli, Carlotta; Pescarmona, Gianpiero; Silvagno, Francesca

    2015-10-01

    Even in cells that are resistant to the differentiating effects of vitamin D, the activated vitamin D receptor (VDR) can downregulate the mitochondrial respiratory chain and sustain cell growth through enhancing the activity of biosynthetic pathways. The aim of this study was to investigate whether vitamin D is effective also in modulating mitochondria and biosynthetic metabolism of differentiating cells. We compared the effect of vitamin D on two cellular models: the primary human keratinocytes, differentiating and sensitive to the genomic action of VDR, and the human keratinocyte cell line HaCaT, characterized by a rapid growth and resistance to vitamin D. We analysed the nuclear translocation and features of VDR, the effects of vitamin D on mitochondrial transcription and the consequences on lipid biosynthetic fate. We found that the negative modulation of respiratory chain is a general mechanism of action of vitamin D, but at high doses, the HaCaT cells became resistant to mitochondrial effects by upregulating the catabolic enzyme CYP24 hydroxylase. In differentiating keratinocytes, vitamin D treatment promoted intracellular lipid deposition, likewise the inhibitor of respiratory chain stigmatellin, whereas in proliferating HaCaT, this biosynthetic pathway was not inducible by the hormone. By linking the results on respiratory chain and lipid accumulation, we conclude that vitamin D, by suppressing respiratory chain transcription in all keratinocytes, is able to support both the proliferation and the specialized metabolism of differentiating cells. Through mitochondrial control, vitamin D can have an essential role in all the metabolic phenotypes occurring in healthy and diseased skin.

  8. Signaling hierarchy regulating human endothelial cell development

    USDA-ARS?s Scientific Manuscript database

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

  9. Interspecies chimeras for human stem cell research.

    PubMed

    Masaki, Hideki; Nakauchi, Hiromitsu

    2017-07-15

    Interspecies chimeric assays are a valuable tool for investigating the potential of human stem and progenitor cells, as well as their differentiated progeny. This Spotlight article discusses the different factors that affect interspecies chimera generation, such as evolutionary distance, developmental timing, and apoptosis of the transplanted cells, and suggests some possible strategies to address them. A refined approach to generating interspecies chimeras could contribute not only to a better understanding of cellular potential, but also to understanding the nature of xenogeneic barriers and mechanisms of heterochronicity, to modeling human development, and to the creation of human transplantable organs. © 2017. Published by The Company of Biologists Ltd.

  10. Retrospective birth dating of cells in humans.

    PubMed

    Spalding, Kirsty L; Bhardwaj, Ratan D; Buchholz, Bruce A; Druid, Henrik; Frisén, Jonas

    2005-07-15

    The generation of cells in the human body has been difficult to study, and our understanding of cell turnover is limited. Testing of nuclear weapons resulted in a dramatic global increase in the levels of the isotope 14C in the atmosphere, followed by an exponential decrease after 1963. We show that the level of 14C in genomic DNA closely parallels atmospheric levels and can be used to establish the time point when the DNA was synthesized and cells were born. We use this strategy to determine the age of cells in the cortex of the adult human brain and show that whereas nonneuronal cells are exchanged, occipital neurons are as old as the individual, supporting the view that postnatal neurogenesis does not take place in this region. Retrospective birth dating is a generally applicable strategy that can be used to measure cell turnover in man under physiological and pathological conditions.

  11. Human embryonic stem cells and lung regeneration.

    PubMed

    Varanou, A; Page, C P; Minger, S L

    2008-10-01

    Human embryonic stem cells are pluripotent cells derived from the inner cell mass of preimplantation stage embryos. Their unique potential to give rise to all differentiated cell types has generated great interest in stem cell research and the potential that it may have in developmental biology, medicine and pharmacology. The main focus of stem cell research has been on cell therapy for pathological conditions with no current methods of treatment, such as neurodegenerative diseases, cardiac pathology, retinal dysfunction and lung and liver disease. The overall aim is to develop methods of application either of pure cell populations or of whole tissue parts to the diseased organ under investigation. In the field of pulmonary research, studies using human embryonic stem cells have succeeded in generating enriched cultures of type II pneumocytes in vitro. On account of their potential of indefinite proliferation in vitro, embryonic stem cells could be a source of an unlimited supply of cells available for transplantation and for use in gene therapy. Uncovering the ability to generate such cell types will expand our understanding of biological processes to such a degree that disease understanding and management could change dramatically.

  12. Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis

    PubMed Central

    Feng, Dingqing; Yan, Keqin; Zhou, Ying; Liang, Haiyan; Liang, Jing; Zhao, Weidong; Dong, Zhongjun; Ling, Bin

    2016-01-01

    The human papillomavirus (HPV) oncoproteins E6 and E7 are risk factors that are primarily responsible for the initiation and progression of cervical cancer, and they play a key role in immortalization and transformation by reprogramming differentiating host epithelial cells. It is unclear how cervical epithelial cells transform into tumor-initiating cells (TICs). Here, we observed that the germ stem cell protein Piwil2 is expressed in pre-cancerous and malignant lesions of the cervix and cervical cancer cell lines with the exception of the non-HPV-infected C33a cell line. Knockdown of Piwil2 by shRNA led to a marked reduction in proliferation and colony formation, in vivo tumorigenicity, chemo-resistance, and the proportion of cancer stem-like cells. In contrast, Piwil2 overexpression induced malignant transformation of HaCaT cells and the acquisition of tumor-initiating capabilities. Gene-set enrichment analysis revealed embryonic stem cell (ESC) identity, malignant biological behavior, and specifically, activation targets of the cell reprogramming factors c-Myc, Klf4, Nanog, Oct4, and Sox2 in Piwil2-overexpressing HaCaT cells. We further confirmed that E6 and E7 reactivated Piwil2 and that E6 and E7 overexpression resulted in a similar gene-set enrichment pattern as Piwil2 overexpression in HaCaT cells. Moreover, Piwil2 overexpression or E6 and E7 activation induced H3K9 acetylation but reduced H3K9 trimethylation, which contributed to the epigenetic reprogramming and ESC signature maintenance, as predicted previously. Our study demonstrates that Piwil2, reactivated by the HPV oncoproteins E6 and E7, plays an essential role in the transformation of cervical epithelial cells to TICs via epigenetics-based cell reprogramming. PMID:27602489

  13. Noncovalent inhibitors of human 20S and 26S proteasome based on trypsin inhibitor SFTI-1.

    PubMed

    Dębowski, Dawid; Cichorek, Mirosława; Lubos, Marta; Wójcik, Sławomir; Łęgowska, Anna; Rolka, Krzysztof

    2016-09-01

    Sunflower trypsin inhibitor (SFTI-1) is recognized as an attractive scaffold to designed potent inhibitors of various proteases. We have recently found that its analogues inhibit noncovalently both human and yeast 20S proteasomes. Here, a set of novel and more potent in vitro inhibitors is presented. The inhibitory potency of the peptides was assessed with human 20S proteasome in the presence or absence of sodium dodecyl sulfate and with human 26 proteasome. Their antiproliferative action against tumor (human melanoma cells A375) and normal cells (46 BR.1N human fibroblasts and HaCaT keratinocytes) was determined. The selected fluoresceine-labeled inhibitors were able to internalize into A375 cells and were sometimes present as foci in the cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 685-696, 2016. © 2016 Wiley Periodicals, Inc.

  14. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2007-10-01

    Epithelial Stem Cells PRINCIPAL INVESTIGATOR: Peter D. Eirew CONTRACTING ORGANIZATION: British Columbia Cancer Agency...NUMBER Characterization of Human Mammary Epithelial Stem Cells 5b. GRANT NUMBER W81XWH-06-1-0702 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Abstract The mammary epithelium in normal adult female mice contains undifferentiated stem cells with extensive in vivo regenerative and self-renewal

  15. Human brain glial cells synthesize thrombospondin.

    PubMed Central

    Asch, A S; Leung, L L; Shapiro, J; Nachman, R L

    1986-01-01

    Thrombospondin, a 450-kDa multinodular glycoprotein with lectin-type activity, is found in human platelets, endothelial cells, fibroblasts, smooth muscle cells, monocytes, and granular pneumocytes. Thrombospondin interacts with heparin, fibrinogen, fibronectin, collagen, histidine-rich glycoprotein, and plasminogen. Recently, thrombospondin synthesis by smooth muscle cells has been reported to be augmented by platelet-derived growth factor. We present evidence that thrombospondin is present within and synthesized by astrocytic neuroglial cells. Heparin-Sepharose affinity chromatography of material derived from a human brain homogenate yielded a protein that, when reduced, had an apparent size of 180 kDa and comigrated with reduced platelet thrombospondin on NaDodSO4/PAGE. Immunoblot analysis with monospecific anti-thrombospondin confirmed the presence of immunoreactive thrombospondin. Indirect immunofluorescence of cultured human glial cells indicated the presence of thrombospondin. Metabolic labeling of glial cell cultures with [35S]methionine followed by immunoprecipitation with monospecific anti-thrombospondin revealed synthesis of a 180-kDa polypeptide that comigrated with platelet thrombospondin on NaDodSO4/PAGE. Cultured human glial cells were incubated for 48 hr in serum-free medium with purified platelet-derived growth factor at concentrations up to 50 ng/ml. Aliquots taken at intervals were analyzed by a quantitative double-antibody ELISA. The growth factor stimulated the release of thrombospondin into the culture medium by as much as 10-fold over control cultures. The presence of thrombospondin within glial cells of the central nervous system and the augmentation of its synthesis by platelet-derived growth factor suggest that thrombospondin may play an important role in regulating cell-cell and cell-matrix interactions during periods of cell division and growth. Images PMID:2939460

  16. Pancreastatin producing cell line from human pancreatic islet cell tumor.

    PubMed

    Funakoshi, A; Tateishi, K; Tsuru, M; Jimi, A; Wakasugi, H; Ikeda, Y; Kono, A

    1990-04-30

    It has been characterized that cell line QGP-1 derived from human non-functioning pancreatic islet cell tumor produces human pancreastatin. Exponentially growing cultures produced 5.7 fmol of pancreastatin/10(6) cells/hr. Human pancreastatin immunoreactivities in plasma and tumor after xenografting with QGP-1 into nude mouse were 92.7 fmol/ml and 160.2 pmol/g wet weight, respectively. Immunocytochemical study revealed both chromogranin A and pancreastatin immunoreactive cells in the tumor. Gel filtrations of culture medium and tumor extract identified heterogenous molecular forms of PST-LI which eluted as large and smaller molecular species. These results suggest that plasma pancreastatin levels may be useful as a tumor marker of endocrine tumor of the pancreas, and the pancreastatin producing cell line may be useful for studies of the mechanism of secretions and processing of chromogranin A and pancreastatin.

  17. Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions.

    PubMed

    Debiak, Malgorzata; Lex, Kirsten; Ponath, Viviane; Burckhardt-Boer, Waltraud; Thiermann, Horst; Steinritz, Dirk; Schmidt, Annette; Mangerich, Aswin; Bürkle, Alexander

    2016-02-26

    Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard-induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy. PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP-ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2-chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence-based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose- and time-dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES-induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the

  18. Myeloid derived suppressor cells in human diseases

    PubMed Central

    Greten, Tim F.; Manns, Michael P.; Korangy, Firouzeh

    2012-01-01

    Myeloid derived suppressor cells (MDSC) have been described as a heterogeneous cell population with potent immune suppressor function in mice. Limited data are available on MDSC in human diseases. Interpretation of these data is complicated by the fact that different markers have been used to analyze human MDSC subtypes in various clinical settings. Human MDSC are CD11b+, CD33+, HLA-DRneg/low and can be divided into granulocytic CD14− and monocytic CD14+ subtypes. Interleukin 4Rα, VEGFR, CD15 and CD66b have been suggested to be more specific markers for human MDSC, however these markers can only be found on some MDSC subsets. Until today the best marker for human MDSC remains their suppressor function, which can be either direct or indirect through the induction of regulatory T cells. Immune suppressor activity has been associated with high arginase 1 and iNOS activity as well as ROS production by MDSC. Not only in murine models, but even more importantly in patients with cancer, different drugs have been shown to either reverse the immune suppressor function of MDSC or directly target these cells. Systemic treatment with all-trans-retinoic acid has been shown to mature human MDSC and reverse their immune suppressor function. Alternatively, MDSC can be targeted by treatment with the multi-targeted receptor tyrosine kinase inhibitor sunitinib. In this review will provide a comprehensive summary of the recent literature on human MDSC. PMID:21237299

  19. Interaction of Staphylococci with Human B cells

    PubMed Central

    Nygaard, Tyler K.; Kobayashi, Scott D.; Freedman, Brett; Porter, Adeline R.; Voyich, Jovanka M.; Otto, Michael; Schneewind, Olaf; DeLeo, Frank R.

    2016-01-01

    Staphylococcus aureus is a leading cause of human infections worldwide. The pathogen produces numerous molecules that can interfere with recognition and binding by host innate immune cells, an initial step required for the ingestion and subsequent destruction of microbes by phagocytes. To better understand the interaction of this pathogen with human immune cells, we compared the association of S. aureus and S. epidermidis with leukocytes in human blood. We found that a significantly greater proportion of B cells associated with S. epidermidis relative to S. aureus. Complement components and complement receptors were important for the binding of B cells with S. epidermidis. Experiments using staphylococci inactivated by ultraviolet radiation and S. aureus isogenic deletion mutants indicated that S. aureus secretes molecules regulated by the SaeR/S two-component system that interfere with the ability of human B cells to bind this bacterium. We hypothesize that the relative inability of B cells to bind S. aureus contributes to the microbe’s success as a human pathogen. PMID:27711145

  20. Activation of human T cells in hypertension: Studies of Humanized Mice and Hypertensive Humans

    PubMed Central

    Itani, Hana A.; McMaster, William G.; Saleh, Mohamed A.; Nazarewicz, Rafal R.; Mikolajczyk, Tomasz P.; Kaszuba, Anna; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E.; Chen, Wei; Bonami, Rachel H.; Marshall, Andrew F.; Poffenberger, Greg; Weyand, Cornelia M.; Madhur, Meena S.; Moore, Daniel J.; Harrison, David G.; Guzik, Tomasz J.

    2016-01-01

    Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We employed a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 mm Hg vs. 116 mm Hg for sham treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta and kidney revealed increased infiltration of human leukocytes (CD45+) and T lymphocytes (CD3+ and CD4+) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3+/CD45RO+) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T cell proliferation. We also observed an increase in circulating IL-17A producing CD4+ T cells and both CD4+ and CD8+ T cells that produce IFN-γ in hypertensive compared to normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II. PMID:27217403

  1. Intrinsic radiation resistance in human chondrosarcoma cells

    SciTech Connect

    Moussavi-Harami, Farid; Mollano, Anthony; Martin, James A.; Ayoob, Andrew; Domann, Frederick E.; Gitelis, Steven; Buckwalter, Joseph A. . E-mail: joseph-buckwalter@uiowa.edu

    2006-07-28

    Human chondrosarcomas rarely respond to radiation treatment, limiting the options for eradication of these tumors. The basis of radiation resistance in chondrosarcomas remains obscure. In normal cells radiation induces DNA damage that leads to growth arrest or death. However, cells that lack cell cycle control mechanisms needed for these responses show intrinsic radiation resistance. In previous work, we identified immortalized human chondrosarcoma cell lines that lacked p16{sup ink4a}, one of the major tumor suppressor proteins that regulate the cell cycle. We hypothesized that the absence of p16{sup ink4a} contributes to the intrinsic radiation resistance of chondrosarcomas and that restoring p16{sup ink4a} expression would increase their radiation sensitivity. To test this we determined the effects of ectopic p16{sup ink4a} expression on chondrosarcoma cell resistance to low-dose {gamma}-irradiation (1-5 Gy). p16{sup ink4a} expression significantly increased radiation sensitivity in clonogenic assays. Apoptosis did not increase significantly with radiation and was unaffected by p16{sup ink4a} transduction of chondrosarcoma cells, indicating that mitotic catastrophe, rather than programmed cell death, was the predominant radiation effect. These results support the hypothesis that p16{sup ink4a} plays a role in the radiation resistance of chondrosarcoma cell lines and suggests that restoring p16 expression will improve the radiation sensitivity of human chondrosarcomas.

  2. Bone tissue engineering with human stem cells

    PubMed Central

    2010-01-01

    Treatment of extensive bone defects requires autologous bone grafting or implantation of bone substitute materials. An attractive alternative has been to engineer fully viable, biological bone grafts in vitro by culturing osteogenic cells within three-dimensional scaffolds, under conditions supporting bone formation. Such grafts could be used for implantation, but also as physiologically relevant models in basic and translational studies of bone development, disease and drug discovery. A source of human cells that can be derived in large numbers from a small initial harvest and predictably differentiated into bone forming cells is critically important for engineering human bone grafts. We discuss the characteristics and limitations of various types of human embryonic and adult stem cells, and their utility for bone tissue engineering. PMID:20637059

  3. CD161-expressing human T cells.

    PubMed

    Fergusson, Joannah R; Fleming, Vicki M; Klenerman, Paul

    2011-01-01

    Expression of the Natural Killer cell receptor CD161 has recently been identified on a subset of T cells, including both CD4+ T helper and CD8+ T cells. Expression of this molecule within the adult circulation is restricted to those T cells with a memory phenotype. However, the distinct properties of these T cell populations is yet to be fully determined, although expression of CD161 has been related to the secretion of interleukin-17, and therefore to a type 17 phenotype. Recent studies have aimed to determine both the origin of these cells and the significance of CD161 expression as either a marker of specific cell types or as an effector and regulator of lymphocyte function, and hence to characterize the role of these CD161+ cells within a variety of human diseases in which they have been implicated.

  4. Local anesthetics induce human renal cell apoptosis.

    PubMed

    Lee, H Thomas; Xu, Hua; Siegel, Cory D; Krichevsky, Igor E

    2003-01-01

    Renal cell apoptosis contributes significantly to the pathogenesis of acute renal failure. Local anesthetics induce apoptosis in neuronal and lymphocytic cell lines. We examined the effects of chronic (48 h) local anesthetic treatment (lidocaine, bupivacaine and tetracaine) on human proximal tubular (HK-2) cells. Apoptosis induction was assessed by detecting poly(ADP)-ribose polymerase fragmentation, caspase activation, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, DNA laddering and by cellular morphology. Cell death was quantified by measuring neutral red dye uptake and lactate dehydrogenase released into the cell culture medium. All 3 local anesthetics caused concentration-dependent cell death, induced HK-2 cell apoptosis and potentiated TNF-alpha induced apoptosis. Local anesthetics induced HK-2 cell apoptosis by activation of caspases 3, 6, 7, 8 and 9. ZVAD-fmk, a pan-caspase inhibitor, blocked the local anesthetic induced HK-2 cell apoptosis. Local anesthetics also inhibited the activities of anti-apoptotic kinases protein kinase B (Akt) and extracellular signal regulated mitrogen-activated protein kinase. Local anesthetic's pro-apoptotic effects are independent of sodium channel inhibition as tetrodotoxin, a selective voltage-gated sodium channel blocker, failed to mimic local anesthetic-mediated induction or potentiation of HK-2 cell apoptosis. We conclude that local anesthetics induce human renal cell apoptotic signaling by caspase activation and via inhibition of pro-survival signaling pathways.

  5. Blockade of Tumor Cell TGF-Betas: A Strategy to Reverse Antiestrogen Resistance in Human Breast Cancer

    DTIC Science & Technology

    2002-01-01

    junctions and the acquisition of spindle cell morphology. LY294002, an inhibitor of the pl 10 catalytic subunit of P13K, and a dominant- negative...kinase- phorylation of Smad2 in HaCaT cells was not altered by inactive version of Akt in which Lys179 in the catalytic dn Akt. domain has been mutated...that result in LY294002, a small molecule inhibitor of the p110 catalytic growth inhibition may be independent from those involved subunit of P13K

  6. Xenobiotic metabolism capacities of human skin in comparison with a 3D-epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: phase II enzymes.

    PubMed

    Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Ruwiedel, Karsten; Hübenthal, Ulrike; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Abel, Josef; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

    2012-05-01

    The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing.

  7. Genotoxicity of citrate-coated silver nanoparticles to human keratinocytes assessed by the comet assay and cytokinesis blocked micronucleus assay.

    PubMed

    Bastos, V; Duarte, I F; Santos, C; Oliveira, H

    2017-02-01

    Silver nanoparticles (AgNPs) are widely used in industrial, cosmetic, and biomedical products, and humans are frequently exposed to these products through the skin. It is widely recognized that the characteristics of AgNPs (e.g., size, coating) may influence their cytotoxic effects, but their correlation with DNA damage and mitotic disorders remains poorly explored. In this study, human keratinocytes (HaCaT cell line) were exposed to well-characterized 30 nm AgNPs coated with citrate, and their effects on viability, DNA fragmentation (assessed by the comet assay), and micronuclei (MNi) induction (assessed by the cytokinesis-block micronucleus cytome assays, CBMN) were investigated. The results showed that 10 and 40 μg/mL AgNPs decreased cell proliferation and viability, and induced a significant genetic damage. This was observed by an increase of DNA amount in comet tail, which linearly correlated with dose and time of exposure. Also, cytostaticity (increase of mononucleated cells) and MNi rates increased in treated cells. In contrast, no significant changes were observed in nucleoplasmatic bridges (NPBs) or nuclear buds (NBUDs), although NBUDs tended to increase in all conditions and periods. The cytostatic effects on HaCaT cells were also shown by the decrease of their nuclear division index. Thus, both comet and CBMN assays supported the observation that citrate-AgNPs induced genotoxic effects on HaCaT cells. Considering that AgNPs are present in a vast number of consumer products and also in multiple nanomedicine skin applications and formulations, more research is needed to determine the properties that confer less toxicity of AgNPs to different cell lines.

  8. Coriandrum sativum L. protects human keratinocytes from oxidative stress by regulating oxidative defense systems.

    PubMed

    Park, G; Kim, H G; Kim, Y O; Park, S H; Kim, S Y; Oh, M S

    2012-01-01

    Oxidative radicals are major environmental causes of human skin damage. Oxidative defense factors, including nuclear factor erythroid-derived 2-related factor 2 (Nrf2), are centrally involved in repairing skin cells or protecting them from oxidative damage. Coriandrum sativum L. (coriander; CS) is a commonly consumed food and a traditional phytomedicine in Asia and Europe. In this study, we examined the protective effects of a standardized CS leaf extract against oxidative stress in human HaCaT keratinocytes. CS significantly and dose-dependently protected cells against reduced cell viability caused by H2O2-induced damage, as assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Other assays demonstrated that CS protected HaCaT cells by increasing the levels of glutathione and activities of oxidative defense enzymes, such as superoxide dismutase and catalase. Moreover, it increased the expression of activated Nrf2, which plays a crucial role in protecting skin cells against oxidative stress. These results suggest that CS protects human keratinocytes from H2O2-induced oxidative stress through antioxidant effects. Copyright © 2012 S. Karger AG, Basel.

  9. Identification of cells initiating human melanomas

    PubMed Central

    Schatton, Tobias; Murphy, George F.; Frank, Natasha Y.; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M.; Weishaupt, Carsten; Fuhlbrigge, Robert C.; Kupper, Thomas S.; Sayegh, Mohamed H.; Frank, Markus H.

    2012-01-01

    Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies1,2 and solid cancers3–6. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5− bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ sub-populations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5− progeny, whereas ABCB5− tumour populations give rise, at lower rates, exclusively to ABCB5− cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy. PMID:18202660

  10. Identification of cells initiating human melanomas.

    PubMed

    Schatton, Tobias; Murphy, George F; Frank, Natasha Y; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M; Weishaupt, Carsten; Fuhlbrigge, Robert C; Kupper, Thomas S; Sayegh, Mohamed H; Frank, Markus H

    2008-01-17

    Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies and solid cancers. If such minority populations are associated with tumour progression in