Cell Proliferation Of Human Keratinocytes Research Articles

Homogeneous preparation of water-soluble products from chitin under alkaline conditions and their cell proliferation in vitro

The purpose of this study was to prepare water-soluble products by homogeneous depolymerization of chitin with H2O2 under alkaline conditions and investigate their potential application in wound healing. For the first time, water-soluble products were successfully prepared using a chitin-NaOH/urea solution; the products were chitosans with molecular weights (Mw) of 3.48–33.5 kDa and degrees of deacetylation (DD) > 0.5. Their Mw, DD and yield were affected by the reaction temperature, reaction time, concentration of H2O2 and chitin DD. The deacetylation and depolymerization of chitin were achieved simultaneously. The depolymerization of chitin was caused by hydrogen abstraction of HO, whereas the deacetylation resulted from the cleavage of amide bonds by HO− and HO2−, although the latter played a more important role. All water-soluble chitosans markedly promoted the proliferation of human skin fibroblast (HSF) cells, but they inhibited the proliferation of human keratinocyte cells. For the proliferation of HSF, a low concentration of chitosans was important. In addition, water-soluble chitosans with an Mw of 3.48–16.4 kDa markedly stimulated the expression of growth factors such as PDGF and TGF-β by macrophages. Water-soluble chitosans could be used as a potential active component in wound dressings.

Controlled dual release of dihydrotestosterone and flutamide from polycaprolactone electrospun scaffolds accelerate burn wound healing.

Wound healing is a complex process involving multiple independent and overlapping sequential physiological mechanisms. In addition to cutaneous injury, a severe burn stimulates physiological derangements that induce a systemic hypermetabolic response resulting in impaired wound healing. Topical application of the anti‐androgen drug, flutamide accelerates cutaneous wound healing, whereas paradoxically systemic dihydrotestosterone (DHT) improves burn wound healing. We developed and characterized a PCL scaffold that is capable of controlled release of androgen (DHT) and anti‐androgen (F) individually or together. This study aims to investigate whether local modification of androgen actions has an impact on burn injury wound healing. In a full‐thickness burn wound healing, mouse model, DHT/F‐scaffold showed a significantly faster wound healing compared with F‐scaffold or DHT‐scaffold. Histology analysis confirmed that DHT/F‐scaffold exhibited higher re‐epithelization, cell proliferation, angiogenesis, and collagen deposition. Dual release of DHT and F from PCL scaffolds promoted cell proliferation of human keratinocytes and alters the keratinocyte cell cycle. Lastly, no adverse effects on androgen‐dependent organs, spleen and liver were observed. In conclusion, we demonstrated DHT plus F load PCL scaffolds accelerated burn wound healing when loading alone did not. These findings point to a complex role of androgens in burn wound healing and open novel therapeutic avenues for treating severe burn patients.

Physicochemical characteristics of thermo-responsive gelatin membranes containing carboxymethyl chitosan and poly(N-isopropylacrylamide-co-acrylic acid)

Gelatin has many superior biological properties and can absorb a large volume of water to form hydrogels, making it a promising material for skin wound care. In this study, gel membranes were prepared based on gelatin and added with different amounts of the synthesized carboxymethyl chitosan (CMCS) and poly(N-isopropylacrylamide-co-acrylic acid) (P(NI-co-AA)) copolymer for providing antibacterial and thermo-responsive properties, respectively. The synthesized CMCS had a degree of carboxymethyl substitution at 0.77 and the P(NI-co-AA) had an AA content of 5.7 mol %. Both CMCS and P(NI-co-AA) had carboxylate groups that could further provide ionic-crosslinking sites for the membranes. Though the P(NI-co-AA) copolymer had a higher phase transition temperature than poly(N-isopropylacrylamide) (PNIPAAm) owing to its hydrophilic carboxylate groups, the prepared gelatin/CMCS/P(NI-co-AA) membranes had similar phase transition temperatures to the PNIPAAm at around 30.5 − 32.4 °C because of ionic crosslinking. Moreover, the equilibrium swelling ratio and water vapor transmission rate of the gel membranes were in the range of 769 − 1226% and 1678 − 2496 g/m2- day, respectively. These membranes could promote cell proliferation of human keratinocytes (HaCaT) and mouse fibroblasts (L-929), indicating they were non-toxic to the cells. They also had antimicrobial properties against S. aureus and E. coli, where the antibacterial efficacy increased with increasing the CMCS content. These hydrogel membranes had good biocompatibility, suitable swelling ratio and water vapor transmission rate, antibacterial and thermo-responsive properties. Therefore, they have potential to be applied as functional wound dressing material.

Phloroglucinol derivatives from Hypericum species induce in vitro proliferation of cells involved in the wound healing process

The genus Hypericum (Hypericaceae) is a recognized source of therapeutic agents, being some species widely used due to their wound healing properties. In a previous study, south Brazilian species H. caprifoliatum, H. carinatum, H. connatum, H. myrianthum and H. polyanthemum demonstrated potential to induce proliferation of keratinocytes. In the present study, the effect of phloroglucinol derivatives isolated from Hypericum on cell proliferation of human keratinocytes, fibroblasts and stem cells was investigated. The best results, determined by the MTT assay, were achieved with cariphenone B at concentrations of 0.01 and 0.1 µM (122.3% and 114%, respectively) on HaCaT cells. Uliginosin B was able to induce the proliferation of mesenchymal stem cells (129% at 10 µM) and MRC5 fibroblasts (152.5% at 5 µM). These findings confirm the capacity of phloroglucinol derivatives to induce the in vitro cellular proliferation and reinforce the importance of Hypericum species as potential sources of wound healing compounds.

Abstract 5777: Translation of HPV mediated immortalization to cancer precision medicine

Abstract The E6/E7 oncogenes of the high-risk HPVs are both necessary and sufficient to immortalize HFKs and their presence and expression is required for the continued proliferation of HPV-positive cervical cancer cells. We and others have shown previously that hTERT induced by E6 and cytoskeleton alteration by E7 are critical. Both E6 and feeder cells activate telomerase, while both E7 and Rock inhibitor (Y-27632) disrupt the actin cytoskeleton and inactivate Rho. Feeders and Y-27632 to induce unlimited cell proliferation of human keratinocytes. Unexpectedly, we observed that feeders and Y-27632 could be used to establish both normal and tumor cell cultures from non-keratinocyte tissues. This culture has been termed as “conditional reprogramming”, since CR cultures stop proliferating or terminally differentiate after its removal, depending on culture conditions. The Combination of CR and Organoids ( (Matrigel, air-liquid interface (ALI)) cultures represents next generation human cancer models and functional diagnostics for cancer precision medicine as described in August 2015 in the NCI precision medicine initiative and three nature review articles (Nat Rev Cancer. 2015 Dec; Nat Rev Genet. 2015 Jul; Nat Rev Clin Oncol. 2014 Nov.). The technique is relatively simple and has been reproduced in more than 50 laboratories (including an original and independent article in Science from Massachusetts General Hospital) (Science. 2014, 346(6216):1480-6.). Importantly, the CR technology can generate 2x10<6cells in a week from small biopsies, and can generate cultures from cryopreserved tissue and small biopsies. We therefore initiated studies to examine whether CR cultures reflected the biology and genotype of the original tumor and whether cultures might be used to predict clinical responses. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment. Thus, the CR method significantly advances applications for regenerative medicine. Citation Format: Aleksandra Dakic, Nancy Palechor-Ceron, Ewa Krawczyk, Hang Yuan, Frank Suprynowicz, Seema Agarwal, Richard Schlegel, Vera Simic, Praathibha Sripadhan, Chen Chen, Jie Lu, Tung-Wei Hou, Sujata Choudhury, Xuefeng Liu. Translation of HPV mediated immortalization to cancer precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5777. doi:10.1158/1538-7445.AM2017-5777

Promotion of Hair Growth by Traditionally Used Delphinium Staphisagria Seeds through Inducti on of Angiogenesis.

How the seeds of Delphinium staphisagria promote hair growth in humans is yet to be discovered. This lack of information leads us to the investigation of hair promoting effects of seeds of Delphinium staphisagria in-vitro. Extract prepared from the Seed of Delphinium staphisagria (ESDS) - traditionally used for hair loss treatment - was selected and tested for the cytotoxic and angiogenic potential in endothelial cells (HUVECs) and human keratinocytes (HaCaT) cells. The effects of extract was determined by using in-vitro colorimetric MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] cytotoxicity assay. To identify the compounds that induce angiogenesis, we applied matrigel capillary assay in-vitro using HUVECs. Vinegar and water extracts of D. staphisagria seeds significantly promoted the proliferation of human keratinocyte cells by 137, 139, 143, 149 and 147 % at the concentration of 100, 120, 200, 250 and 300 μg/mL compared with vehicle -treated control, respectively at 24 h. HUVECs viability remained the same with the control group at the concentration 1, 10, 20 and 40 μg/mL after 24 h. Results demonstrated that ESDS did not cause toxicity in human keratinocytes and endothelial cells, while inducing the angiogenic activity in-vitro. D. staphisagria seeds promote hair growth without overt cytotoxicity and through inducing angiogenesis. Based on the information from the traditional uses and our experimental results, D. staphisagria's seeds appear to be a good candidate for the promotion of hair growth.

Safety aspects of non-ionic surfactant vesicles: a toxicity study related to the physicochemical characteristics of non-ionic surfactants.

Two different toxicity models were used to assess the relationship between the physicochemical properties of non-ionic surfactant vesicles (NSVs), and the safety of these vesicles for topical drug administration. The vesicles used in this study consisted of polyoxyethylene alkyl ethers (CnEOm) in which the number of C atoms (n) varied between 12 and 18 and the number of oxyethylene units (m) between 3 and 7. The physicochemical properties of the vesicles are described in terms of hydrophilic-lipophilic balance (HLB) values, and critical micelle concentrations (CMC), and the rigidity of the bilayers as determined by the gel-liquid transition temperatures and the cholesterol content of the bilayers. The first toxicity model, comprising the measurement of the ciliary beat frequency, is a tool to assess the safety of intranasally applied formulations. Studies using this ciliotoxicity model revealed that by increasing the length of the alkyl chain of the surfactant, a decrease in toxicity was observed. The opposite correlation was found if the length of the polyoxyethylene headgroup was increased. Furthermore, it was observed that gel-state vesicles produce less of an effect on the ciliary beat frequency than liquid state vesicles. The second toxicity model, comprising the determination of cell proliferation of human keratinocytes, is a method to assess skin irritancy. In contrast to the ciliotoxicity model the length of the polyoxyethylene headgroup and of the alkyl chains did not seem to have an effect on the safety of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

PI‐3K/Akt signal pathway plays a crucial role in arsenite‐induced cell proliferation of human keratinocytes through induction of cyclin D1

Exposure of arsenite can induce hyperproliferation of skin cells, which is believed to play important roles in arsenite-induced carcinogenesis by affecting both promotion and progression stages. However, the signal pathways and target genes activated by arsenite exposure responsible for the proliferation remain to be defined. In the present study, we found that: (1) exposure of human keratinocytic HaCat cells to arsenite caused an increase in cell proliferation, which was significantly inhibited by pretreatment of wortmannin, a specific chemical inhibitor of PI-3K/Akt signal pathway; (2) arsenite exposure was also able to activate PI-3K/Akt signal pathway, which thereby induced the elevation of cyclin D1 expression level in both HaCat cells and human primary keratinocytes based on that inhibition of PI-3K/Akt pathway by either pretreatment of wortmannin or the transfection of their dominant mutants, significantly inhibited cyclin D1 expression upon arsenite exposure; (3) PI-3K/Akt pathway is implicated in arsenite-induced proliferation of HaCat cells through the induction of cyclin D1 because either knockdown of cyclin D1 by its siRNA or inhibition of PI-3K/Akt signal pathway by their dominant mutants markedly impaired the proliferation of HaCat cells induced by arsenite exposure. Taken together, we provide the direct evidence that PI-3K/Akt pathway plays a role in the regulation of cell proliferation through the induction of cyclin D1 in human keratinocytes upon arsenite treatment. Given the importance of aberrant cell proliferation in cell transformation, we propose that the activation of PI-3K/Akt pathway and cyclin D1 induction may be the important mediators of human skin carcinogenic effect of arsenite.

Insulin-like growth factor-II regulates the 12-lipoxygenase gene expression and promotes cell proliferation in human keratinocytes via the extracellular regulatory kinase and phosphatidylinositol 3-kinase pathways

To study the relationship between insulin-like growth factor-II (IGF-II) and 12-lipoxygenase (12-LOX) that are upregulated in psoriasis, we monitored 12-lipoxygenase expression in the insulin-like growth factor-II treated human keratinocytes and explored the signaling pathways of 12-lipoxygenase expression. Insulin-like growth factor-II induced 12-lipoxygenase mRNA and protein levels in human keratinocytes through two major signal transduction pathways, namely, the extracellular signaling-regulated kinase (ERK)-mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. The IGF-II-induced upregulation of 12-lipoxygenase was attenuated by pretreating the cells with selective inhibitors or by overexpressing dominant-negative MEK. In addition, treatment of HaCaT cells with the 12-lipoxygenase metabolite 12 (S)-hydroxyeicosatetraenoic acid (12(S)-HETE) directly stimulated DNA synthesis and mitogenesis, and injection of insulin-like growth factor-II into the skin of hairless mice induced epidermal hyperplasia. These results suggest that insulin-like growth factor-II is involved in the pathogenesis of psoriasis as a paracrine inducer of 12-lipoxygenase.

Kiwi fruit (Actinidia chinensis L.) polysaccharides exert stimulating effects on cell proliferation via enhanced growth factor receptors, energy production, and collagen synthesis of human keratinocytes, fibroblasts, and skin equivalents.

Within physiological engineering exogenous carbohydrates were recently confirmed as pharmacologically active compounds. To investigate potential dermatological activity purified polysaccharides from kiwi fruits (Actinidia chinensis L., Actinidiaceae) were characterized concerning monomer composition, linkage types and molecular weights and were tested under in vitro conditions for regulating activities on cell physiology of human keratinocytes, fibroblasts, and skin equivalents. Ten micrograms per milliliter of raw polysaccharide, neutral type-II-arabinogalactans, and acidic arabinorhamnogalacturonans of kiwi fruits stimulated cell proliferation of human keratinocytes (NHK, HaCaT) up to 30% significantly while mitochondrial activity was stimulated for nearly 25% in regard to control cells. Fibroblasts were not as sensitive as keratinocytes but >130 microg/ml kiwi fruit polysaccharides increased proliferation and ATP-synthesis significantly, too. Proliferation-stimulating activity was dependent on terminal 1-alpha-l-arabinose residues since enzymatic release of these sugar moieties caused significantly decreased proliferation of HaCaT and fibroblasts of about 10% in regard to untreated cells. In three dimensional skin equivalents, it was shown that the polysaccharides led to a doubled collagen synthesis of fibroblasts compared to the normally strongly reduced biosynthetic activity.

Characterization of Receptors of Insect Cytokine, Growth-blocking Peptide, in Human Keratinocyte and Insect Sf9 Cells

Insect cytokine, growth-blocking peptide (GBP), enhances cell proliferation of human keratinocyte cells with a potency almost equivalent to that of human epidermal growth factor (EGF). GBP consists of 25 amino acid residues containing a core region that shows a striking similarity to the C-terminal beta-loop domain of EGF and disordered N and C termini. The present study demonstrates that, although GBP lacks the N-terminal half-portion of EGF molecule, at least five amino acids of the disordered N-terminal six-amino acid region are indispensable for affecting the cell growth activity of GBP. Upon stimulating mitogenesis in keratinocyte cells, GBP directly binds and activates their EGF receptors. GBP also effects proliferative activity on insect Sf9 cells through the binding and activation of the specific receptor, which consists of a heterodimeric complex: a binding subunit (60 kDa) and a tyrosine phosphorylation subunit (58 kDa). These results indicate that GBP enhances cell proliferation of human keratinocyte and insect Sf9 cells through the activation of EGF and GBP receptors, respectively.

The vitamin D 3 analogue, calcipotriol, induces sphingomyelin hydrolysis in human keratinocytes

The possible role of sphingomyelin cycle for the regulation of cell proliferation was investigated in human keratinocytes. The time-dependent breakdown of sphingomyelin was observed in the immortalized human keratinocyte cell line HaCaT as well as in primary human keratinocytes thereby providing evidence that the sphingomyelin cycle might be of importance in the epidermis. Peak levels of 20–30% sphingomyelin hydrolysis were measured 3 h after treatment of the cells with 1α,25-dihydroxyvitamin D 3 or with the vitamin D 3 analogue, calcipotriol. The decrease of sphingomyelin upon addition of vitamin D 3 or calcipotriol was accompanied by an approximately 70% increase of ceramide in the cells. The effects of vitamin D 3 and calcipotriol on sphingomyelin breakdown were paralleled by their antiproliferative potency. Furthermore, the cell-permeable ceramide, N-acetylsphingosine, and natural ceramide inhibited cell proliferation of human keratinocytes. The results presented suggest that induction of the sphingomyelin cycle represents one mechanism mediating the therapeutic effect of calcipotriol in treatment of psoriasis.