Betulinic acid induces apoptosis in skin cancer cells and differentiation in normal human keratinocytes.

Betulinic acid (BA), a pentacyclic triterpene of plant origin, induces cell death in melanoma cells and other malignant cells of neuroectodermal origin. Little is known about additional biological effects in normal target cells. We show, in this study, that BA induces differentiation as well as cell death in normal human keratinocytes (NHK). Cytotoxicity profiles of BA are compared among normal human keratinocytes, HaCaT cells, IGR1 melanoma cells and normal melanocytes. As expected, BA is toxic to all cell types, normal and malignant, but varies in its cytotoxic potency and in the extent of induction of apoptotic vs. necrotic cell death in the four different skin cell types. Apoptosis is proved by annexin V and Apo2.7 binding and by DNA fragmentation. Induction of differentiation-associated antigens in keratinocytes--filaggrin and involucrin--is demonstrated, together with specific morphological changes in treated cell cultures. BA, apart from its cytotoxic activities in cellular systems, is capable of inducing differentiation of NHK into corneocytes without immediately provoking apoptotic cell death.

Further investigations on the role of ascorbic acid in stratum corneum lipid models after UV exposure.

This study is the continuation of our research into vitamin C and its possible effects on human skin after topical administration. The effects of ascorbic acid, iron ions and UV irradiation on stratum corneum lipid models were investigated. The lipid models used were: a simple system (linolenic acid dispersion), a complex system (liposomes consisting of dipalmitoylphosphatidylcholine, cholesterol and linolenic acid) and complex systems with additionally incorporated ceramides (types III and IV). The lipid peroxidation was quantified by the thiobarbituric acid assay. A human adult low-calcium high-temperature (HaCaT) keratinocytes cell culture was used as a second in-vitro model. The amount of intracellular peroxides was determined by measuring the fluorescence intensity using the dihydrorhodamine 123 assay. Electron paramagnetic resonance spectroscopy was used to study the influence of ascorbic acid and iron ions on the signal intensity of 5-doxylstearic acid during UV exposure. Ascorbic acid showed prooxidative properties in the thiobarbituric acid assay whereas cell protection was measured in the HaCaT keratinocytes experiments. Electron paramagnetic resonance investigations revealed different extents of free radical production generated by iron ions, ascorbic acid and UV irradiation. In evaluating the results from this study new aspects of the mechanism of lipid damage caused by these three factors were suggested, transcending the simple redox behaviour of ascorbic acid.

Microemulsions as colloidal vehicle systems for dermal drug delivery. Part V: Microemulsions without and with glycolipid as penetration enhancer.

The aim of this study was to investigate the dermal administration of a highly hydrophilic model drug, diphenhydramine (DPH), in colloidal systems with an aqueous colloidal phase in the presence of a glycolipid (GL) as a penetration modifier. Dermal penetration of DPH, GL, and isopropylpalmitate (IPP) from ME systems without GL and with GL as well as from a hydrogel used as standard formulation were estimated in vitro using human skin. The penetration of the drug, the oil (IPP), and the GL was measured with highly sensitive HPLC, HPLC-MS, and GC-MS assays, respectively. It could be shown that penetration modifier GL is penetrating very fast, and to a high extent into and through the human skin. In contrast, the penetration of IPP used as oily phase in the ME is limited. When incorporated in the ME systems GL and DPH was accumulated in the viable epidermis and in the dermis. Using ME containing a penetration modifier such as GL, a slight additional enhancing effect could be observed, particularly concerning the penetration of DPH into the acceptor fluid when a highly hydrophilic drug such as DPH was applied.

Functional characterization of sodium- and chloride-dependent taurine transport in human keratinocytes.

In the skin, taurine acts as an important osmolyte required for keratinocyte hydration. It has antioxidant effects, protects cells from UV-induced stress and has effects on cell proliferation, inflammation and collagenogenesis. This study was performed to find and characterize functionally a taurine transport system in keratinocytes and to establish a cell culture model for skin taurine transport studies. Uptake of [(3)H]taurine was studied both in the human adult low calcium high temperature (HaCaT) cell line and in human native epidermal keratinocytes. Uptake of [(3)H]taurine in HaCaT cells was strictly dependent on extracellular sodium and chloride. The taurine uptake rate was saturable and indicated participation of a single transport system with kinetic parameters of Kt = 5.1 +/- 0.2 microm and Vmax = 320.5 +/- 2.8 pmol/10 min per mg of protein. Uptake was strongly inhibited by beta-amino acids (taurine, beta-alanine, hypotaurine, beta-guanidinopropionic acid), whereas alpha- and gamma-amino acids had little or no effect. Taurine uptake in normal keratinocytes was very similar to that in HaCaT cells with respect to substrate specificity and affinity. We conclude that keratinocytes express the Na(+) and Cl(-) dependent, high-affinity taurine transporter. This system accepts beta- and certain gamma-amino acids as substrates.

Carrier-mediated transport of clonidine in human keratinocytes.

The alpha-2 agonist clonidine is frequently used in transdermal therapeutic systems for antihypertensive therapy. This study was performed to characterize transport of clonidine into human keratinocytes. The uptake of [3H]clonidine was measured into monolayers of the human cell line HaCaT and normal human epidermal keratinocytes in primary culture. The uptake of clonidine was linear for up to 1min, independent of Na(+), but pH-dependent. Uptake was carrier-mediated with an affinity constant (K(t)) of 0.30mM and a maximal velocity (V(max)) of 15.7nmol/min per mg of protein. Diphenhydramine, guanabenz, procainamide, tryptamine, quinine, and quinidine, but not choline markedly inhibited clonidine uptake. We conclude that clonidine is transported into keratinocytes in a pH-dependent manner by a saturable uptake system different from the keratinocyte choline transporter. The substrate specificity of the system corresponds to that of the recently characterized system for tertiary amines. After diffusion of the drugs through the stratum corneum, this transport system might contribute to the passage of clonidine and diphenhydramine across the living epidermis after dermal administration.

Examinations of the antioxidative properties of the topically administered drug bufexamac reveal new insights into its mechanism of action.

The effect of bufexamac on UV-irradiation-induced lipid peroxidation was investigated. Linolenic acid was used as a model lipid. Bufexamac was shown to be capable of reducing the amount of lipid peroxidation. The quantification was carried out by the thiobarbituric acid assay. Irradiation experiments were also performed using HaCaT keratinocytes as a model system. The oxidative changes were quantified by DNA synthesis measurements and cell viability determinations. Bufexamac was found to act antioxidatively again. To investigate free radical involvement, electron paramagnetic resonance studies were carried out. The influence of bufexamac on the concentration of hydroxyl radicals generated by the Fenton system was examined using the spin trapping technique. Moreover, the hydroxamic acid's ability to react with stable radicals was checked. The quantification assay of 2,2-diphenyl-1-picrylhydrazyl hydrate showed no concentration changes of the stable radical caused by bufexamac. In the Fenton assay antioxidative effects were measured after the addition of the drug. The qualitative changes after irradiating bufexamac were studied at a molecular level by electrospray mass spectrometry. Multiple-stage mass spectrometry experiments enabled the establishment of fragmentation schemes. Phenolic degradation products were detected. The results suggest a new interpretation of the controversially debated mechanism of action of bufexamac and indicate possible reasons for its eczema provoking potential.

Transport of biotin in human keratinocytes.

Biotin is an essential micronutrient for normal cellular function, growth, and development. Biotin deficiency leads to pathologic, dermatologic, and neurocutaneous manifestations in skin and its appendages. Previous studies described the presence of specific biotin transport systems in the epithelia of the intestine, liver, kidney, and placenta, and in blood mononuclear cells. The aim of this study was to examine biotin transport into human keratinocytes. Uptake of [3H]biotin was measured both in the HaCaT cell line and in native keratinocytes in primary culture. Uptake of [3H]biotin (6 nM) in HaCaT cells was linear for up to 5 min of incubation. In the presence of an Na+ gradient total biotin uptake was 4- to 5-fold higher than in the absence of sodium ions. Biotin uptake was not altered by H+ and Cl- gradients. This transport system exhibited a Michaelis-Menten constant for biotin of 22.7+/-1.0 microM and a maximal velocity of 163.6+/-3.5 pmol per 5 min per mg protein. [3H]Biotin uptake (6 nM) was strongly inhibited by lipoic acid (oxidized form, Ki=4.6 microM; reduced form, Ki=11.4 microM), pantothenic acid (Ki=1.2 microM), and desthiobiotin (Ki=15.2 microM), but not by biocytin or biotin methyl ester. Measured at [3H]biotin concentrations of 0.1-10 nM we obtained kinetic evidence for the presence of a second transport component that is saturable at very low biotin concentrations (Kt=2.6+/-0.1 nM). Unlabeled lipoic acid and pantothenic acid (20 nM) did not inhibit the [3H]biotin uptake (1 nM). We conclude that human keratinocytes express the Na+-dependent multivitamin transporter with preference for pantothenate and a very high affinity transport component with specificity for biotin.

Relevance of reactive oxygen species in the induction of 8-oxo-2'-deoxyguanosine in HaCaT keratinocytes.

There is growing evidence that solar radiation-induced oxidative DNA damage may play an important role in carcinogenesis of the skin. One substantial modification in this context is the oxidation of the guanine base to 8-oxo-2'-deoxyguanosine. Using HaCaT keratinocytes, measurement of the 8-oxo-2'-deoxyguanosine content in this study was performed by flow cytometry on whole cells. Hydrogen peroxide and hydroxyl radicals seem not to be involved in the process of this DNA alteration. However, our results demonstrate that ultraviolet A can cause DNA damage at guanine sites primarily via photosensitized reactions. Although singlet oxygen can also lead to 8-oxo-2'-deoxyguanosine, the major mechanism seems to be based on formation of the guanylcation radical through excited riboflavin and can therefore proceed without the involvement of reactive oxygen species.

Functional characterization of a high-affinity choline transport system in human keratinocytes.

This study was performed to characterize the mechanism of choline transport into human keratinocytes. Uptake of [3H]choline was measured both in the HaCaT cell line and in native keratinocytes. Uptake in HaCaT cells was linear with time at least up to 10 min. There was little dependence of choline transport on sodium. Choline uptake was slightly stimulated by extracellular H+ with the pH optimum being 7.5. The uptake rate was saturable and indicated participation of a single transport system (Kt = 14.8 +/- 1.0 micro M, Vmax = 1.0 +/- 0.01 nmol per 10 min per mg protein). The choline uptake into HaCaT cells was inhibited by unlabeled choline, hemicholinium-3, and acetylcholine. The prototypical organic cation tetraethylammonium showed very little affinity for the choline uptake system in these cells. Several cationic drugs such as diphenhydramine, clonidine, and atropine also interacted with the transport system. Choline uptake in normal keratinocytes was very similar to that in HaCaT cells with respect to substrate specificity and affinity. We conclude that keratinocytes express a Na+ independent, high-affinity choline transport system. This system accepts many pharmacologically important organic cations as substrates. It is similar or identical to the choline carrier described in intestinal epithelial cells and in endothelial cells of the blood-brain barrier. The choline carrier seems to have relevance not only for the uptake of cationic drugs into the keratinocytes but also for the biosynthesis of skin lipids.

Evidence for a role of nitric oxide in the mediation of antiproliferative UVA effects in keratinocytes.

Using cultured human keratinocytes, the present study investigates the role of nitric oxide (NO) in the mediation of the antiproliferative effects of ultraviolet light A (UVA). UVA treatment of cells (3-21 J cm (-2)) caused a time- and dose-dependent increase in nitrite formation in a micromolar range. This effect was accompanied by a decrease in DNA synthesis by 53.5%. Moreover, UVA treatment slightly reduced cell viability by 23.8%. Preincubation of keratinocytes with the NO scavenger 4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO, 10-100 microM) or the NO synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA, 30-300 microM) significantly diminished the UVA-induced increase in nitrite. PTIO as well as l-NMMA partially protected keratinocytes from UVA-induced antiproliferative effects and increased DNA synthesis by 67 or 49% of the control. The co-application of UVA irradiation (10 J cm (-2)) and the essential cofactor of NO synthases tetrahydrobiopetrin (BH4, 500 microM) led to an overadditive increase in the release of nitrite as well as to a decrease in DNA synthesis. These results imply that NO is involved in the antiproliferative UVA effects in keratinocytes.

Is the photoprotective effect of vitamin E based on its antioxidative capacity?

Vitamin E (alpha-tocopherol) is widely used in the prevention of ultraviolet (UV)-induced skin damages. Mostly, the photoprotective effect of vitamin E is attributed to its radical scavenger capacity. We discuss here other possible explanations for its preventive potential.