299 ALOE-EMODIN INHIBITS PROLIFERATION OF ADULT HUMAN KERATINOCYTES The differences in apoptosis of control versus treated keratinocytes were analyzed by paired t-test. The value of p 0.05 was considered significant. RESULTS AE DECREASES PROLIFERATION OF THE KERATINOCYTES CULTIVATED IN VITRO We first assessed the influence of AE on keratinocyte proliferation (Figure 1A). The proliferation of control cultures containing only DMSO was 9194 ± 3101 counts per Figure 1. Aloe-emodin (AE) downregulates keratinocyte proliferation. Keratinocytes were incubated with- out (0) or with AE at indicated concentrations for 72 h. The incorporation of [3H]thymidine was determined and results are presented as counts per minute (cpm). (A) Each value is a mean of triplicate cultures of one sample. (B) The observed antiproliferative effect of AE was not followed by an increase in lactate dehydroge- nase release. Keratinocytes from 10 different donors are labeled 1–10 (Figures 1A and 1B). ∗p 0.05.

JOURNAL OF COSMETIC SCIENCE 300 minute (cpm), in cultures treated with 1.25 μM AE 7647 ± 2 423 cpm, in cultures treated with 2.5 μM AE 4680 ± 1535 cpm, and in those treated with 5 μM AE 1815 ± 566 cpm (mean ± SEM). The results of ANOVA showed that AE significantly inhibited keratinocyte proliferation (Figure 1A) in a dose dependent manner ( p = 0.019), with the maximal inhibitory effect achieved at 5 μM AE ( p = 0.021 compared to control). Al- though AE was able to signifi cantly inhibit keratinocyte proliferation at 5 μM concen- tration, considerable differences in keratinocyte response were observed among the cultures obtained from different individuals (Figure 1A). The observed individual differences were confi rmed by comparing the IC50 values between different donors (Table I), which revealed that keratinocytes from some donors display markedly differ- ent sensitivity to the antiproliferative action of AE (e.g., compare donors No. 8 and 10). The observed antiproliferative effect of AE was not accompanied by a signifi cant in- crease in LDH release during a 72 h incubation period (Figure 1B) (ANOVA p = 0.66), as well as at earlier time points (6, 24, and 48 h data not shown), indicating that the inhibitory effect of the drug on keratinocyte proliferation was probably not due to ne- crotic cell death. THE INFLUENCE OF AE ON KERATINOCYTE APOPTOSIS In order to explore the mechanisms underlying the observed inhibition of keratinocyte proliferation, we assessed whether AE induces apoptotic cell death (Figure 2). A signifi - cant increase in the percentage of apoptotic (TB−/FDA−) keratinocytes could be observed 6 h upon addition of 5 μM AE (16.7 ± 1.3, mean ± SEM) compared to the untreated cultures (10.4 ± 0.1, mean ± SEM, p = 0.001) (Figure 2). The number of necrotic cells in keratinocyte cultures did not signifi cantly change upon treatment with AE (Figure 2), thus further supporting results of the LDH release assay (data not shown). DISCUSSION In this study, we demonstrated antiproliferative effect of AE on the primary human kera- tinocytes from 10 different donors cultivated in vitro (Figure 1A). The antiproliferative effect of AE was previously reported in various human cell cultures at 20–80 μM concen- tration (6). It was a consequence of induction of p21, p53 and subsequent G1 cell cycle arrest (7), or increased number of cells within S phase (8). In some experiments, increased proportion of cells cycling at a higher ploidy level ( G2/M) was observed (8). The latest may explain increased DNA synthesis in the rat hepatocytes reported by Wolfl e et al. in this particular experimental setup (4). In line with previously presented data (6), our Table I The Half Maximal Inhibitory Conc entrations of Aloe-emodin for Proliferation of Keratinocyte Cultures from 10 Different Donors Sample 1 2 3 4 5 6 7 8 9 10 Mean SEM IC50 (μM) 2.96 3.91 3.02 3.46 2.86 2.14 1.79 0.96 3.22 5.40 2.97 0.38 IC50 ( ppm) 1.10 1.45 1.12 1.28 1.06 0.79 0.66 0.36 1.19 2.00 1.10 0.14