Immortalized Keratinocyte Cell Line Research Articles

TNIP1 Regulates Cutibacterium acnes-Induced Innate Immune Functions in Epidermal Keratinocytes.

Human skin cells recognize the presence of the skin microbiome through pathogen recognition receptors. Epidermal keratinocytes are known to activate toll-like receptors (TLRs) 2 and 4 in response to the commensal Cutibacterium acnes (C. acnes, formerly known as Propionibacterium acnes) bacterium and subsequently to induce innate immune and inflammatory events. These events may lead to the appearance of macroscopic inflammatory acne lesions in puberty: comedos, papules and, pustules. Healthy skin does not exhibit inflammation or skin lesions, even in the continuous presence of the same microbes. As the molecular mechanism for this duality is still unclear, we aimed to identify factors and mechanisms that control the innate immune response to C. acnes in keratinocytes using a human immortalized keratinocyte cell line, HPV-KER, normal human keratinocytes (NHEK) and an organotypic skin model (OSM). TNIP1, a negative regulator of the NF-κB signaling pathway, was found to be expressed in HPV-KER cells, and its expression was rapidly induced in response to C. acnes treatment, which was confirmed in NHEK cells and OSMs. Expression changes were not dependent on the C. acnes strain. However, we found that the extent of expression was dependent on C. acnes dose. Bacterial-induced changes in TNIP1 expression were regulated by signaling pathways involving NF-κB, p38, MAPKK and JNK. Experimental modification of TNIP1 levels affected constitutive and C. acnes-induced NF-κB promoter activities and subsequent inflammatory cytokine and chemokine mRNA and protein levels. These results suggest an important role for this negative regulator in the control of bacterially induced TLR signaling pathways in keratinocytes. We showed that all-trans retinoic acid (ATRA) induced elevated TNIP1 expression in HPV-KER cells and also in OSMs, where TNIP1 levels increased throughout the epidermis. ATRA also reduced constitutive and bacterium-induced levels of TNFα, CCL5 and TLR2, while simultaneously increasing CXCL8 and TLR4 expression. Based on these findings, we propose that ATRA may exhibit dual effects in acne therapy by both affecting the expression of the negative regulator TNIP1 and attenuating TLR2-induced inflammation. Overall, TNIP1, as a possible regulator of C. acnes-induced innate immune and inflammatory events in keratinocytes, may play important roles in the maintenance of epidermal homeostasis.

Synergistic antiproliferative and differentiating effect of 2,4-monofurfurylidene-tetra-O-methylsorbitol and 4,6-dimethyl-2-(3,4,5-trimethoxyphenylamino)pyrimidine on primary and immortalized keratinocytes

Psoriasis is one of the most common chronic autoinflammatory skin disease, associated with hyperproliferation and abnormal differentiation of keratinocytes, inflammation, and angiogenesis. The available treatments for psoriasis are not curative and may have numerous side effects, and topical administration is preferred over systemic therapy due to the reduced systemic burden of the drug. Thus, novel and more efficacious formulations of anti-inflammatory and/or differentiating compounds for topical application could be very useful for the disease management and for improving the quality of life of the patients. Here we evaluated the potential as anti-psoriatic of an equimolar mixture of two compounds, 2,4-Monofurfurylidene-tetra-O-methylsorbitol (Compound A) and 4,6-dimethyl-2-(3,4,5-trimethoxyphenylamino)pyrimidine (Compound B), that, used individually, are known to possess immunomodulating properties (Compound A) and keratolitic and anti-inflammatory activity (Compound B). Human immortalized keratinocyte cell line (HaCaT cells) and primary human keratinocyte cells from adult donor (HEKa) were used as in vitro experimental models. We show that the mix A + B exhibits antiproliferative activity and induces terminal differentiation more efficiently than compounds A and B used individually. We confirm that the compound B is the active ingredient of the mixture and the mainly responsible for anti-psoriatic activity, but the mix A + B is more effective and possesses lower cytotoxicity than the compound B alone. This could be ascribable to the association with compound A, that is known to possess, in addition to the immunomodulating ability, antioxidant and antiradical action. Our results indicate that mix A + B could be a suitable candidate for a new cosmeceutical formulation for topical treatment of psoriasis.

Efficacy of essential oils from plants cultivated in the Amazonian Biome against gastrointestinal nematodes in sheep.

The excessive use of anthelmintics to control nematodes has resulted in anthelminthic resistance. Essential oils (EOs) are a rich source of bioactive molecules that can be assessed for their ability to control resistant parasite populations. The aims of this study were to screen EOs from 10 plant species in vitro for anthelmintic activity against Haemonchus contortus, evaluate the cytotoxicity of those EOs in a human immortalized keratinocyte cell line (HaCaT), and test the most promising EO candidate in vivo in Santa Inês sheep. The efficacy was investigated in vitro using an egg hatch test (EHT) and a larval development test (LDT). EO cytotoxicity was evaluated with the sulforhodamine-B assay. In the in vivo experiment, 28 Santa Inês sheep naturally infected were distributed into groups: G1-Mentha arvensis (EO5), 200mgkg-1; G2-menthol, 160mgkg-1; G3-negative control; and G4-positive control (monepantel). EO5, from M. arvensis (86.7% menthol), had the lowest LC50 and LC90 values in the EHT (0.10, 0.27mgmL-1, respectively), good performance in the LDT (0.015, 0.072mgmL-1, respectively), and the lowest cytotoxicity (190.9µgmL-1) in HaCaT cells. In the in vivo test, a single dose of the EO5 (200mgkg-1 BW) had an efficacy of approximately 50% on days 1, 14, and 21; however, values were not significantly from day 0. Conversely, pure menthol at a dose of 160mgkg-1 BW showed no in vivo efficacy. This can be attributed to key factors related to bioavailability and pharmacology of terpenes in the host organism, as well as to the fact that menthol is mainly excreted as glucuronides in urine. Thus, further studies should be conducted with formulation systems that deliver bioactives directly to the abomasum, focusing on terpenes, whose excretion route is mainly via faeces.

1162 BMAL1 and CLOCK proteins in regulating the sensitivity of human keratinocytes to UVB-induced apoptosis

A diverse array of biological processes is under circadian controls. It has been reported that UV-induced apoptosis and DNA damage responses in mouse skin are time-of-day dependent and regulated by core clock proteins, including BMAL1, CLOCK, PERs, and CRYs. The aim of this study is to investigate the circadian controls of UV responses in human keratinocytes, using the siRNA-mediated loss-of-function approach. We found that low-dose (5 mJ/cm2) of UVB triggered an oscillation of BMAL1 and CLOCK mRNA expression in the immortal HaCat human keratinocyte cell line, as well as primary human keratinocytes. In HaCat cells, depletion of BMAL1 or CLOCK reduced the percentage of UVB-induced apoptotic cells from 43% (in controls) to 29% (p < 0.001) and 31% (P < 0.05), respectively. Moreover, knockdown of either clock gene strongly blocked UVB-induced expression of tumor necrosis factor α (TNFα) and phosphorylation of NF-κB. These results suggest that BMAL1-CLOCK proteins may promote cell death by regulating the TNFα-NF-κB mediated extrinsic apoptotic pathway in UVB-irradiated HaCat keratinocytes. Given the critical impact of UVB on photo-aging and photo-carcinogenesis, mechanistic elucidation and understanding of circadian controls on UVB effects in human skin will be beneficial for prevention and treatment of skin-related diseases including cancers.

Metformin Promotes HaCaT Cell Apoptosis through Generation of Reactive Oxygen Species via Raf-1-ERK1/2-Nrf2 Inactivation.

Although metformin (MET) may be useful for the treatment of psoriasis, the mechanisms underlying its method of action have yet to be fully elucidated. Here, the relationship between MET function and reactive oxygen species (ROS) levels and the underlying mechanism were explored in human immortalized keratinocyte cell line (HaCaT). HaCaT cells were incubated with MET at 0, 10, 20, 40, and 60mM for 24h. The cell viability was evaluated by the CCK-8 assay. The cell apoptosis rate and intracellular ROS levels were examined using flow cytometry. The protein expression and the phosphorylation levels of nuclear factor erythroid-derived 2 related factor 2 (Nrf2), Raf-1, and ERK1/2 were assessed by Western blot. The specific ROS scavenger N-acetyl-cysteine (NAC) and the specific Nrf2 agonist Oltipraz (OPZ) were used to analyze the effect of MET. MET decreased HaCaT cell proliferation and induced HaCaT cell apoptosis in a dose-dependent manner. MET was found to elevate intracellular ROS levels in a dose-dependent manner, while pretreatment with NAC attenuated these effects. MET inhibits the protein expression and the phosphorylation levels of Nrf2. The combination of OPZ and MET can significantly increase the cell viability, decrease the rate of apoptosis, and attenuate the intracellular ROS levels relative to MET alone. MET inhibits the protein expression and the phosphorylation levels of Raf-1 and ERK1/2. MET was found to attenuate Raf-1-ERK1/2 signaling in HaCaT cells to suppress the expression and phosphorylation levels of Nrf2, which contributed to the intracellular generation of ROS and the pro-apoptotic effects of MET.

Multifunctional Tannic Acid/Silver Nanoparticle-Based Mucoadhesive Hydrogel for Improved Local Treatment of HSV Infection: In Vitro and In Vivo Studies.

Mucoadhesive gelling systems with tannic acid modified silver nanoparticles were developed for effective treatment of herpes virus infections. To increase nanoparticle residence time after local application, semi solid formulations designed from generally regarded as safe (GRAS) excipients were investigated for their rheological and mechanical properties followed with ex vivo mucoadhesive behavior to the porcine vaginal mucosa. Particular effort was made to evaluate the activity of nanoparticle-based hydrogels toward herpes simplex virus (HSV) type 1 and 2 infection in vitro in immortal human keratinocyte cell line and in vivo using murine model of HSV-2 genital infection. The effect of infectivity was determined by real time quantitative polymerase chain reaction, plaque assay, inactivation, attachment, penetration and cell-to-cell assessments. All analyzed nanoparticle-based hydrogels exhibited pseudoplastic and thixotropic properties. Viscosity and mechanical measurements of hydrogels were found to correlate with the mucoadhesive properties. The results confirmed the ability of nanoparticle-based hydrogels to affect viral attachment, impede penetration and cell-to-cell transmission, although profound differences in the activity evoked by tested preparations toward HSV-1 and HSV-2 were noted. In addition, these findings demonstrated the in vivo potential of tannic acid modified silver nanoparticle-based hydrogels for vaginal treatment of HSV-2 genital infection.

Synthesis, Characterization, and Evaluation of Triptolide Cell-Penetrating Peptide Derivative for Transdermal Delivery of Triptolide.

Triptolide (TP) has been used as one of the most common systemic treatments for various diseases since the 1960s. However, TP displays diverse side effects on various organs, which limits its clinical application. To overcome this issue, numerous C-14-hydroxyl group derivatives of TP have been synthesized. In this research, the C-14-hydroxyl group of TP is modified by a cell-penetrating peptide polyarginine (R7). The derivative TP-disulfide-CR7 (TP-S-S-CR7) containing a disulfide linkage between TP and R7 possesses less toxicity at various concentrations on the immortal human keratinocyte (HaCaT) cell line by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay compared with free TP. Treating HaCaT cells with TP (100 nM) could increase intracellular ROS (reactive oxygen species) and decrease the activity of SOD (superoxide dismutase). Meanwhile, treating HaCaT cells with equimolar concentration of TP-S-S-CR7 did not cause both of the above TP-induced alterations. In addition, TP-S-S-CR7 did not show significant dermal toxicity on guinea pigs and could efficiently overcome the barrier of corneum and then reach epidermis and dermis within 2 h of transdermal administration. In addition, there was a relatively lower concentration of TP in blood indicating less toxicity on organs. Such results suggest that topical therapy using polyarginine is possible by the transdermal delivery of TP.

Supportive properties of basement membrane layer of human amniotic membrane enable development of tissue engineering applications.

Human amniotic membrane (HAM) has been widely used as a natural scaffold in tissue engineering due to many of its unique biological properties such as providing growth factors, cytokines and tissue inhibitors of metalloproteinases. This study aimed at finding the most suitable and supportive layer of HAM as a delivery system for autologous or allogeneic cell transplantation. Three different layers of HAM were examined including basement membrane, epithelial and stromal layers. In order to prepare the basement membrane, de-epithelialization was performed using 0.5M NaOH and its efficiency was investigated by histological stainings, DNA quantification, biomechanical testing and electron microscopy. Adipose-derived stromal cells (ASCs) and a human immortalized keratinocyte cell line(HaCaT) were seeded on the three different layers of HAM and cultured for 3weeks. The potential of the three different layers of HAM to support the attachment and viability of cells were then monitored by histology, electron microscopy and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, mechanical strengths of the basement membrane were assessed before and after cell culture. The results indicated that the integrity of extra cellular matrix (ECM)components was preserved after de-epithelialization and resulted in producing an intact basement amniotic membrane (BAM). Moreover, all three layers of HAM could support the attachment and proliferation of cells with no visible cytotoxic effects. However, the growth and viability of both cell types on the BAM were significantly higher than the other two layers. We conclude that growth stimulating effectors of BAM and its increased mechanical strength after culturing of ASCs, besides lack of immunogenicity make it an ideal model for delivering allogeneic cells and tissue engineering applications.

Myelin Oligodendrocyte Glycoprotein-Independent Rubella Infection of Keratinocytes and Resistance of First-Trimester Trophoblast Cells to Rubella Virus In Vitro.

Rubella virus (RuV), which belongs to the family Togaviridae and genus Rubivirus, causes systemic infection in children and young adults and congenital rubella syndrome in developing fetuses if the infection occurs during pregnancy. The mechanisms of fetal infection by RuV are not completely understood. Myelin oligodendrocyte glycoprotein (MOG) is reported to be a cellular receptor for RuV; however, it is mainly expressed in the central nervous system. Therefore, it is thought that other receptors are also responsible for virus entry into susceptible cells. In this study, we found that first-trimester trophoblast cells were resistant to RuV. In addition, we showed that HaCaT cells (an immortalized keratinocyte cell line) that did not express MOG on their surface were infected with RuV. This finding is one of the first demonstrations of MOG-independent RuV infection of susceptible host cells and suggests that it is important to continue searching for alternative RuV receptors. In addition, this study reports the resistance of first-trimester trophoblast cells to RuV and suggests that utilizing an epithelial–mesenchymal transition approach to study the mechanisms of transplacental vertical RuV infection.

Cytotoxicity and antitumoral activity by apoptosis induction of AC13: A brominated curcumin analogue

Background: Natural compounds with therapeutic potential have been explored as antitumoral agents, as curcumin (CUR), a substance which has activity against various tumor types and a tool used to improve the action of these compounds is the production of analogs. Objective: In this study, we investigated the antitumoral activity of AC13, a CUR analog. Materials and Methods: Cytotoxicity of AC13 and CUR for different cancer cell lines was analyzed by MTT assay after 24, and 48 h of exposure and caspases 3 and 7 enzymatic activity in CasKi and human spontaneously transformed immortal keratinocyte cell line cells was analyzed after 24 h of incubation with AC13 or CUR at 50 μM. Results: It was observed significant viability loss only for CasKi cells after incubation with AC13. Hence, it was made a more detailed screening of the cytotoxicity for these cells and nontumoral cells incubated with AC13 or CUR, showing concentration-dependent decrease of cell viability. Posteriorly, AC13 induces increase in the caspases activity in both cell lines, being that for tumor cells this increase was greater than that unleashed by CUR. Conclusion: Therefore, AC13 triggers cell death by apoptosis in CasKi and shows greater effect than CUR for these tumor cells, suggesting to be a promising compound for the treatment of cancer and should be studied more thoroughly. Abbreviations used: CUR: Curcumin; TLC: Thin-layer chromatography; DMSO: Dimethyl sulfoxide; Caski: Human cervical carcinoma cell line; HeLa: Human cervical carcinoma cell line; MDA-MB-231: Human breast adenocarcinoma cell line; 786-O: Human renal cell adenocarcinoma cell line; HaCaT: Human spontaneously transformed immortal keratinocyte cell line; DMEM: Dulbecco Modified Eagle Medium; DOXO: Doxorubicin; CLO: Bisphosphonamidate clodronate; U2-OS: Human osteosarcoma cell line; FLLL32: Cell-permeable analog of curcumin; BDMC-A: Analog of curcumin.

Immortalized N/TERT keratinocytes as an alternative cell source in 3D human epidermal models

The strong societal urge to reduce the use of experimental animals, and the biological differences between rodent and human skin, have led to the development of alternative models for healthy and diseased human skin. However, the limited availability of primary keratinocytes to generate such models hampers large-scale implementation of skin models in biomedical, toxicological, and pharmaceutical research. Immortalized cell lines may overcome these issues, however, few immortalized human keratinocyte cell lines are available and most do not form a fully stratified epithelium. In this study we compared two immortalized keratinocyte cell lines (N/TERT1, N/TERT2G) to human primary keratinocytes based on epidermal differentiation, response to inflammatory mediators, and the development of normal and inflammatory human epidermal equivalents (HEEs). Stratum corneum permeability, epidermal morphology, and expression of epidermal differentiation and host defence genes and proteins in N/TERT-HEE cultures was similar to that of primary human keratinocytes. We successfully generated N/TERT-HEEs with psoriasis or atopic dermatitis features and validated these models for drug-screening purposes. We conclude that the N/TERT keratinocyte cell lines are useful substitutes for primary human keratinocytes thereby providing a biologically relevant, unlimited cell source for in vitro studies on epidermal biology, inflammatory skin disease pathogenesis and therapeutics.

Acitretin modulates HaCaT cells proliferation through STAT1- and STAT3-dependent signaling

Acitretin has been a valuable option for the treatment of psoriasis, however, the molecular events of acitretin leading to the normalization of keratinocytes differentiation on psoriasis patients have not been fully explored. To investigate whether there were certain relationship between keratinocytes proliferation and JAK/STAT signaling pathways in psoriasis, and how acitretin modulated the signaling pathways. HaCaT cells, an in vitro immortal human keratinocyte cell line, was chosen as a in vitro model of psoriasis. The small interfering RNA targeting STAT1 (siRNA-STAT1) and STAT3 (siRNA-STAT3) were subsequently transfected into the HaCaT cells which were treated with or without acitretin. We found that HaCaT cells proliferation and the expression of STAT1 or STAT3 were inhibited by acitretin, siRNA-STAT1 and siRNA-STAT3. Our experimental data shows that acitretin might inhibit HaCaT cells proliferation in psoriasis by decreasing the expression of STAT- and STAT3-dependent mechanism.

Gene expression analysis combined with functional genomics approach identifies ITIH5 as tumor suppressor gene in cervical carcinogenesis.

Progression from human papillomavirus-induced premalignant cervical intraepithelial neoplasia (CIN) to cervical cancer (CC) is driven by genetic and epigenetic events. Our microarray-based expression study has previously shown that inter-α-trypsin-inhibitor heavy chain 5 (ITIH5) mRNA levels in CCs were significantly lower than in high-grade precursor lesions (CIN3s). Therefore, we aimed to analyze in depth ITIH5 expression during cervical carcinogenesis in biopsy material and cell culture. Moreover, functional analyses were performed by ectopic expression of ITIH5 in different cell lines. We were able to confirm the validity of our microarray differential expression data by qPCR, demonstrating a clear ITIH5 downregulation in CC as compared with CIN2/3 or normal cervix. ITIH5 protein loss, evaluated by immunohistochemistry, was evident in 81% of CCs, whereas ITIH5 showed weak to moderate cytoplasmic staining in 91% of CIN2/3 cases. In addition, ITIH5 was strongly reduced or absent in seven CC cell lines and in three immortalized keratinocyte cell lines. Moreover, ITIH5 mRNA loss was associated with ITIH5 promoter methylation. ITIH5 expression could be restored in CC cell lines by pharmacological induction of DNA demethylation and histone acetylation. Functionally, ITIH5 overexpression significantly suppressed proliferation of SW756 cells and further resulted in a significant reduction of colony formation and cell migration in both CaSki and SW756 tumor models, but had no effect on invasion. Remarkably, ITIH5 overexpression did not influence the phenotype of HeLa cells. Taken together, ITIH5 gene silencing is a frequent event during disease progression, thereby providing evidence for a tumor suppressive role in cervical carcinogenesis.

Biocompatibility and characterization of polyglycerol-based thermoresponsive nanogels designed as novel drug-delivery systems and their intracellular localization in keratinocytes

Novel nanogels that possess the capacity to change their physico-chemical properties in response to external stimuli are promising drug-delivery candidates for the treatment of severe skin diseases. As thermoresponsive nanogels (tNGs) are capable of enhancing penetration through biological barriers such as the stratum corneum and are taken up by keratinocytes of human skin, potential adverse consequences of their exposure must be elucidated. In this study, tNGs were synthesized from dendritic polyglycerol (dPG) and two thermoresponsive polymers. tNG_dPG_tPG are the combination of dPG with poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)) and tNG_dPG_pNIPAM the one with poly(N-isopropylacrylamide) (pNIPAM). Both thermoresponsive nanogels are able to incorporate high amounts of dexamethasone and tacrolimus, drugs used in the treatment of severe skin diseases. Cellular uptake, intracellular localization and the toxicological properties of the tNGs were comprehensively characterized in primary normal human keratinocytes (NHK) and in spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (HaCaT). Laser scanning confocal microscopy revealed fluorescently labeled tNGs entered into the cells and localized predominantly within lysosomal compartments. MTT assay, comet assay and carboxy-H2DCFDA assay, demonstrated neither cytotoxic or genotoxic effects, nor any induction of reactive oxygen species of the tNGs in keratinocytes. In addition, both tNGs were devoid of eye irritation potential as shown by bovine corneal opacity and permeability (BCOP) test and red blood cell (RBC) hemolysis assay. Therefore, our study provides evidence that tNGs are locally well tolerated and underlines their potential for cutaneous drug delivery.

Calcipotriol inhibits proliferation of human keratinocytes by downregulating STAT1 and STAT3 signaling

Psoriasis is an autoimmune disease, which is characterized by aberrantly high levels of inflammation, but the underlying pathogenic mechanisms are still not fully understood. Signal transducer and activator of transcription...

Using Organotypic Epithelial Tissue Culture to Study the Human Papillomavirus Life Cycle.

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which there is continued expression of the HPV E6 and E7 oncogenes, often as a consequence of integration of HPV DNA into the host genome. Such cancers represent "dead ends" for the virus as integration disrupts the viral genome and because the cancers are defective in normal epithelial differentiation, which is required for production of progeny papillomavirus. In order to study the full viral life cycle, from the establishment to maintenance to productive stages, our lab makes use of the organotypic epithelial tissue culture system. This system allows us to mimic the three-dimensional structure of epithelia whose differentiation is tightly linked to the completion of the HPV viral life cycle. In this chapter we describe how various aspects of the HPV life cycle are monitored in raft cultures making use of an immortalized keratinocyte cell line. © 2016 by John Wiley & Sons, Inc.

Synthesis, structure, bioactivity and biocompatibility of melt-derived P2O5‐CaO‐B2O3‐K2O‐MoO3 glasses

New glasses following the P2O5‐CaO‐B2O3‐K2O‐MoO3 composition were prepared using the classical melt quenching method in order to investigate and correlate their structural peculiarities with the composition and, moreover, with the bioactivity and biocompatibility behavior. The structure and surface morphology of samples were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Two complementary techniques were used to determine the elemental composition inside samples and on the outermost layer of their surface, namely energy dispersive X-ray (EDX) and X-ray photoelectron spectroscopy (XPS), respectively. For assessing in vitro the bioactivity of the synthesized glasses, the ability to form hydroxyapatite crystallites on their surface upon immersion in simulated body fluid (SBF) was checked using SEM and EDX analysis. The in vitro cytotoxic effect of samples was evaluated using a mitochondrial activity assay.The results reveal that the increase of MoO3 content in the P2O5‐CaO‐B2O‐K2O composition generates a continuously disordering of the local structure and the increase of Mo6+/Mo5+ ratio values on samples surface. No bioactivity was observed for this glass system after 15days of immersion in SBF. The in vitro toxicity studies show that samples containing MoO3 up to 7mol% have a good biocompatibility with healthy human immortalized keratinocyte (HaCaT) cell line.

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.

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.

Studies on Biosurfactant producing bacteria from Jeppiaar salt pan with special reference to anticancer activity

Biosurfactants are surface-active compounds produced by microorganisms. These molecules reduce surface tension between aqueous solutions and hydrocarbon mixtures. In this study, we collected water samples from Jeppiaar salt pan in Chennai, Tamilnadu, India. Ten different microorganisms were isolated from salt pan water samples. Screening of biosurfactants producing potential strain was done using Hemolytic activity, Oil displacement test, Drop Collapse assay. Four isolates were positive for biosuractant production among that one isolate was found as potential biosurfactant producer. Both biochemical and 16S rRNA sequencing methods one of the potential strain was identified as Bacillus cereus JS06. The biosurfactant was extracted from cell-free broth at 72-h grown cells by step-by-step purification of acid precipitation method. The dried surfactant was used for further characterization by Fourier transmission Infrared spectroscopy with attenuated total reflectance (FTIR-ATR) and anticancer studies. On FTIR-ATR analysis characteristic absorbance bands were observed for the extracted biosurfactant. The crude biosurfactant was screened for its anticancer activity against human laryngeal carcinoma (HEP-2) cell line and transformed aneuploid immortal keratinocyte (HeCAT) cell line. The results obtained showed that biosurfactants have better interfacial-activity and anticancer property, thus being more attractive to be applied in pharmaceuticals and medical field.

Alginate–hyaluronan composite hydrogels accelerate wound healing process

In this paper we propose polysaccharide hydrogels combining alginate (ALG) and hyaluronan (HA) as biofunctional platform for dermal wound repair. Hydrogels produced by internal gelation were homogeneous and easy to handle. Rheological evaluation of gelation kinetics of ALG/HA mixtures at different ratios allowed understanding the HA effect on ALG cross-linking process. Disk-shaped hydrogels, at different ALG/HA ratio, were characterized for morphology, homogeneity and mechanical properties. Results suggest that, although the presence of HA does significantly slow down gelation kinetics, the concentration of cross-links reached at the end of gelation is scarcely affected. The in vitro activity of ALG/HA dressings was tested on adipose derived multipotent adult stem cells (Ad-MSC) and an immortalized keratinocyte cell line (HaCaT). Hydrogels did not interfere with cell viability in both cells lines, but significantly promoted gap closure in a scratch assay at early (1 day) and late (5 days) stages as compared to hydrogels made of ALG alone (p<0.01 and 0.001 for Ad-MSC and HaCaT, respectively). In vivo wound healing studies, conducted on a rat model of excised wound indicated that after 5 days ALG/HA hydrogels significantly promoted wound closure as compared to ALG ones (p<0.001). Overall results demonstrate that the integration of HA in a physically cross-linked ALG hydrogel can be a versatile strategy to promote wound healing that can be easily translated in a clinical setting.