Caffeic acid phenethyl ester inhibits proliferation of human keratinocytes and interferes with the EGF regulation of ornithine decarboxylase.

Caffeic acid phenethyl ester (CAPE) was evaluated for its potential in regulating keratinocyte proliferation. CAPE inhibited the proliferation of SV40 transformed keratinocytes (Z114) in a concentration- and time-dependent manner. Inhibition by CAPE was seen with 0.5 to 5.0 micrograms/ml at 48 h. Cell toxicity was observed at 10 micrograms/ml by changes in morphology and decreased viability. Pretreatment of Z114 cells with CAPE significantly prevented the full induction of ornithine decarboxylase (ODC) by epidermal growth factor (EGF) in a concentration- and time-dependent manner. Inhibition was observed with a concentration of CAPE as low as 1 microgram/ml, and complete inhibition of ODC induction by EGF occurred at 5 micrograms/ml. Northern analysis showed that treatment of cells with CAPE for 24 h suppressed EGF induction of ODC gene expression. Incubation of Z114 cells with CAPE for 24 h resulted in a concentration-dependent decrease in EGF binding and a 30% reduction in the EGF induced autophosphorylation of the EGF receptor. CAPE decreased both membranous and cytosolic PKC activity in a concentration- and time-dependent manner. Because significant inhibition of keratinocyte proliferation occurred at concentrations of CAPE that interfered with PKC activity and EGF signal transduction but did not cause overt toxicity, CAPE may prove useful for the treatment of hyperproliferative skin diseases.

A simple method to predict whether topical agents will interfere with phototherapy.

The objective of this study was to assess the potential interference of topical agents commonly used in psoriasis with concurrent phototherapy. Twenty-one commercially available topical agents were tested. To create solutions from the creams, lotions, and ointments, extractions were made using three different solvents (95 percent ethanol, hexanes, and 1,4-dioxane) and their absorbance from 260 to 400 nm was measured. The absorbance value of the solutions at 310 nm was used to rank the various agents in terms of potential interference with ultraviolet B (UVB) phototherapy. The absorbance at 360 nm was used to rank the agents for potential interference with psoralen/ultraviolet A (PUVA) therapy. Salicylic acid-containing preparations had substantial absorption in the UVB (280 to 320 nm) range. The tar-based products had impressive absorbance in both the UVA (320 to 400 nm) and UVB ranges. Calcipotriene (Dovonex) showed a maximal absorbance in the ultraviolet C (UVC; 200 to 280 nm) and UVB range. Tretinoin (Retin-A) had substantial absorbance in the UVA range. Anthralin (Drithocreme) revealed maximal absorbance within the UVC and UVB ranges. Topical steroid preparations and ammonium lactate (Lac-Hydrin) had low absorbance in both UVB and UVA ranges. In conclusion, salicylic acid-containing preparations, tar-based products, calcipotriene, anthralin, and most tretinoin preparations should be removed before and/or applied after phototherapy.

Regulation of the induction of ornithine decarboxylase in keratinocytes by retinoids.

Treatment of SV40-transformed keratinocytes (Z114) with epidermal growth factor (EGF) resulted in an increase in ornithine decarboxylase (ODC) activity and a dose-dependent increase in ODC mRNA levels. Pretreatment of keratinocytes with all-trans-retinoic retinoic acid inhibited the EGF induction of ODC activity. In both quiescent and EGF-stimulated cells, all-trans-retinoic acid inhibited ODC gene transcription and lowered ODC mRNA levels, whereas glyceraldehyde phosphate dehydrogenase expression remained unaffected. Treatment with all-trans-retinoic acid for 24 h resulted in a dose- and time-dependent decrease of up to 52% in EGF binding to EGF receptors and a 30-75% decrease in EGF-receptor quantity. In addition, when cells were treated with both UV radiation and all-trans-retinoic acid, their effects were additive in causing a decrease in EGF binding. Blocking of EGF receptors with a neutralizing antibody for EGF receptors inhibited the induction of ODC activity by EGF. The effects of several other retinoids, including Ro15-0778, etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cis-retinoic acid and acitretin, were also studied to determine their effects on EGF binding and ODC activity. Two of these other retinoids, 13-cis-retinoic acid and Ro13-7410, inhibited EGF binding the most (35-46%, P < 0.001); several others (etarotene, Ro40-8757 and etretinate) were less effective (7-16%), but significantly decreased EGF binding (P < 0.05), and two retinoids (Ro15-0778 and acitretin) showed no significant effect on EGF binding. In contrast, all of the retinoids tested inhibited the induction of ODC activity by EGF, although etretinate and Ro15-0778 were less effective. EGF signal transduction is important in ODC gene regulation, and retinoids are significant modulators of this pathway.

Phorbol 12-myristate 13-acetate inhibits epidermal growth factor signalling in human keratinocytes, leading to decreased ornithine decarboxylase activity.

Several studies have suggested that murine and human keratinocytes respond differently to phorbol 12-myristate 13-acetate (PMA). Using an in vitro assay, we found that in contrast to its effect on murine skin, PMA did not induce ornithine decarboxylase (ODC) activity in human skin biopsies. To explore the signalling induced by PMA and to determine whether an in vitro culture system could be used to predict biological activity of retinoids in human keratinocytes, we studied a simian virus 40 (SV40)-transformed human keratinocyte cell line. Epidermal growth factor (EGF) stimulates ODC activity and increases the steady-state level of ODC mRNA in a dose- and time-dependent manner in these cells [Prystowsky, Clevenger and Zheng (1993) Exp. Dermatol. 2, 125-132]. In this report, 10(-10) M-10(-7) M PMA induced ODC mRNA and enzyme synthesis at 7 h, but did not significantly induce ODC activity and inhibited the EGF induction of ODC activity. To explore the mechanism whereby PMA interfered with EGF signalling, the effect of PMA on EGF binding to its cell-surface receptor was studied; acute treatment with PMA (within 7 h) decreased EGF binding to 41-57% of the baseline level. In contrast, chronic treatment with PMA (24 h) increased EGF binding to 156% of the baseline level and was associated with an increase in quantity of EGF receptor protein. Protein kinase C (PKC) activation correlated with the acute decrease in EGF binding following PMA treatment. In summary, PMA induced ODC mRNA and ODC enzyme synthesis, while steady-state levels of immunoprecipitable ODC enzyme protein and ODC activity were not increased, demonstrating possible increased turnover of ODC enzyme protein. Additionally, PMA inhibited the induction of ODC by EGF through decreased EGF binding, possibly mediated by PKC activation. Finally treatment of the keratinocytes with retinoids including etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cisretinoic acid, and acitretin blocked the PMA induction of ODC mRNA, suggesting this in vitro model could be a valuable screening assay for predicting biological activity in humans.