L-Cystine-Containing Hair-Growth Formulation Supports Protection, Viability, and Proliferation of Keratinocytes.

PURPOSE: Clinical studies have confirmed that the hair-growth-promoting effect of approved oral drug combinations is beneficial for the treatment of diffuse telogen effluvium, which is characterized by the excessive loss of telogen club hairs. Since data elucidating the mode of action of such combinations are limited, our study focused on the identification of cellular processes potentially supporting the treatment of hair loss. MATERIALS AND METHODS: A minimal growth culture system (MGM) was used to mimic in vitro the reduced activity of human hair follicular keratinocytes (HHFKs). The effect of four core compounds (L-cystine, thiamine, calcium D-pantothenate, and folic acid) of a marketed oral combination (Panto[vi]gar®), which are approved for the treatment of diffuse hair loss, was examined by comparing HHFKs cultured either with or without the compounds. After determining their impact on metabolic activity and proliferation, we conducted a comparative whole-genome gene expression study with subsequent functional grouping of differentially expressed genes to identify cellular processes influenced by the tested compounds. RESULTS: The four core compounds of an oral hair-growth formulation enhanced proliferation and metabolic activity of HHFKs compared to HHFKs cultivated in MGM only. Functional grouping of differentially expressed genes confirmed the regulation of cell cycle-/proliferation-associated genes (cdk1, HJURP) and revealed regulation of cell death- and oxidative stress-associated gene groups. A supportive effect of the compounds on cell viability was demonstrated by lower sensitivity to solar-simulated UV-radiation and increased protection against oxidative stress. We established a central role for L-cystine, as changes in the expression of the anti-oxidative gene hmox1 were L-cystine-dependent. However, to reach a maximal stimulating effect on proliferation, the combination of all four compounds was necessary. CONCLUSION: The tested compound combination had positive effects on metabolic activity, cell viability, and proliferation of keratinocytes. Furthermore, this study suggested that L-cystine primarily contributes to the observed protection against endogenous oxidative stress.

Cystine-thiamin-containing hair-growth formulation modulates the response to UV radiation in an in vitro model for growth-limiting conditions of human keratinocytes.

BACKGROUND: Ultraviolet radiation (UVR) is known to be harmful to normal human epidermal keratinocytes (NHEKs) of the epidermal skin layer, as well as to hair-follicle-associated keratinocytes. An oral formulation containing l-cystine, thiamin, calcium d-pantothenate, medicinal yeast, keratin and p-aminobenzoic acid (Panto[vi]gar®) has demonstrated clinical efficacy for the treatment of diffuse telogen effluvium; however, its mode of action at the cellular level, and in particular whether protective mechanisms are involved, has yet to be elucidated. OBJECTIVES: To assess the capacity of ingredients of this oral formulation, both separately and in combination, to modulate the effects of UVR in growth-limited NHEKs in vitro. METHODS: NHEKs were incubated in keratinocyte basal medium, keratinocyte basal medium lacking cystine, thiamin, calcium d-pantothenate, folic acid and biotine (minimal growth medium [MGM]) or MGM plus test compound. Test compounds comprised the following four ingredients related to the oral formulation: l-cystine, thiamin, calcium d-pantothenate and folic acid (a proposed metabolite of p-aminobenzoic acid), and a combination of these (Panto[vi]gar®-in vitro correlate; P-IC). The effect of different doses of these compounds on the metabolic activity and proliferation of NHEKs was tested, as well as their influence on the impact of UV light on NHEKs assessed by monitoring metabolic activity, cell number and apoptosis induction. RESULTS: Compared with basal medium, MGM reduced the proliferation of NHEKs in a time-dependent manner. Reduced proliferation is a characteristic of the multifactorial and complex phenotype associated with diffuse hair loss. l-cystine (50 µM) increased metabolic activity and proliferation 3-fold versus MGM (p < 0.05). Thiamin also had a significant effect (p < 0.05) on proliferation and metabolic activity of NHEKs, but calcium d-pantothenate and folic acid did not when tested individually in this in vitro model. In the presence of P-IC, metabolic activity increased 4-fold and proliferation 3-fold compared with MGM alone (p < 0.05 for both). Following UV irradiation, cells in MGM showed a 72% reduction in metabolic activity, while P-IC-treated cells showed only a 12-18% reduction. The observed prevention of the UV-induced reduction in metabolic activity was not simply due to filtering UVR by the P-IC components, as P-IC-mediated reduction of this effect persisted even when P-IC was washed out during UV irradiation. CONCLUSION: This study demonstrated that l-cystine and thiamin are essential for proliferation of epidermal keratinocytes and suggests a novel, UV-protective potential of formulations combining l-cystine and thiamin in growth-limited inter-follicular NHEKs in vitro.

Expression of hurpin, a serine proteinase inhibitor, in normal and pathological skin: overexpression and redistribution in psoriasis and cutaneous carcinomas.

Hurpin was identified by differential display analysis studying UV-repressible genes in the keratinocyte cell line HaCaT. We have previously reported that hurpin mRNA is overexpressed in psoriatic skin compared to non-lesional or normal skin; hurpin inhibits cathepsin L and that, after overexpression in keratinocytes, hurpin decreases UV-induced apoptosis. To further study the expression of hurpin, we have isolated monoclonal antibodies against hurpin and analyzed its expression in normal and diseased skin by immunohistochemistry (IHC). In the epidermis of normal skin, we found hurpin to be mainly expressed in the stratum basale. In contrast, we found an enhanced expression of hurpin in the stratum spinosum and stratum granulosum in the majority of diseased skin samples. Within the dermis of normal and diseased skin, hurpin was detected in sebaceous and sweat glands, hair follicles, and endothelial cells of blood vessels. Hurpin was localized in the cytoplasm in normal and diseased skin. Additionally to IHC, we analyzed hurpin expression in selected skin diseases by semiquantitative reverse-transcription polymerase chain reaction. We found overexpression of hurpin mRNA in psoriasis, squamous cell carcinoma (SCC), and actinic keratosis. In contrast, expression of hurpin in melanoma and basal cell carcinoma was comparable to that in normal skin. Overall, the strongest overexpression was observed in SCC and psoriasis. Individual differences in hurpin expression between patients were observed. The increased expression and redistribution of hurpin in diseased skin suggests its possible involvement in inflammatory processes or the regulation of endogenous or pathogen-derived proteinase activity. Additional studies will elucidate the physiological role of hurpin.

Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet-induced apoptosis.

Hurpin (headpin/PI13/serpinB13) is an intracellular, differentially spliced member of the serpin superfamily that has been linked to differentiation and apoptosis of human keratinocytes. It is transiently downregulated by UV light and overexpressed in psoriatic skin lesions. Although it has all of the features of an inhibitory serpin, a productive interaction between hurpin and a proteinase has not yet been reported. Here we demonstrate that hurpin is a potent and selective inhibitor of the archetypal lysosomal cysteine proteinase cathepsin L (catL). Recombinant hurpin inhibits human catL with a stoichiometry of inhibition (SI) of 1.7 and a rate constant k(assoc) of (4.6 +/- 0.14) x 10(5) M(-1) s(-1). It inefficiently inhibits catV and does not inhibit papain, catB, or catK. To investigate the inhibitory mechanism, we determined the P1-P1' bond in the reactive center loop cleaved by catL ((356)Thr-(357)Ser) and expressed variants in which the proximal hinge, P1 residue, or differentially spliced CD loop was mutated. The results of assays using these proteins suggest that inhibition of catL by hurpin occurs via the conventional serpin inhibitory mechanism and that the CD loop plays no role in the process. Finally, it was found that the majority of hurpin is cytosolic and that its overexpression in human keratinocytes confers resistance to UV-induced apoptosis. Given that lysosomal disruption, release of catL, and catL-mediated caspase activation are known to occur in response to cellular stress, we propose that a physiological role of hurpin is to protect epithelial cells from ectopic catL.

Molecular basis of basal cell carcinoma: analysis of differential gene expression by differential display PCR and expression array.

Basal cell carcinoma (BCC) is the most common tumor in the Caucasian population. Although BCC rarely metastasize and cause death, they are problematic due to their destructive growth and the frequent localization on the face. Until now the knowledge of genes differentially expressed in BCC has been incomplete. To elucidate the complex alterations in BCC-associated gene expression, we took advantage of 2 techniques: the differential display RT-PCR (DD-PCR) and the differential hybridization of cDNA arrays. Using DD-PCR, we showed differential expression of genes known from other biological contexts (e.g., rac, ubiquitin hydrolase), which could now be associated with BCC. In addition, we detected unknown genes possibly contributing to the carcinogenesis of BCC. Of the 588 genes screened by differential hybridization of the Atlas human cDNA array, differences in the expression levels of BCC were observed for 10 genes. These data were obtained with RNA probes pooled from several BCC of different donors and were subsequently confirmed by semiquantitative RT-PCR for Janus protein tyrosine kinase 3 (Jak3), microsomal glutathione S-transferase 1 (GST 12), teratocarcinoma-derived growth factor cripto, glutaredoxin and the monocyte chemoattractant protein 1 (MCP-1) in 10 individual BCC specimens, 2 squamous cell carcinoma (SCC), the cell line HaCaT and cultured normal human keratinocytes (NHK) in comparison to normal skin. These genes are candidates from gene families with known association to tumors, but they have not been reported in the carcinogenesis of BCC yet. In summary, both approaches allow the detection of differentially expressed genes possibly involved in the carcinogenesis of BCC.