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Hit compounds from in silico screening for inhibitors of the EGFR dimerization process were evaluated for their anti-proliferative (CCD-1106 keratinocytes) and anti-oxidant (TBA assay) activity and their effect on EGFR dimerization (BS(3) chemical crosslinking assay). 7-Benzyl-8-{N'-[1-(3-ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]hydrazino}-1,3-dimethylxanthine 2a (127 μM) leads to 37% inhibition of p-EGFR dimerization in the CCD-1106 cell line and also inhibits phosphorylation of proteins in the MAPK/ERK pathway, ERK 1/2 and p-38. Based on this initial data, 2a was selected for further study and was evaluated for its anti-proliferative activity in a range of keratinocyte (CCD-1106, HaCaT and NHEK) and monocyte (ThP1 and U937) cell lines. Xanthine 2a is pro-apoptotic in HaCaT keratinocytes, as shown by electron microscopy, caspase 3/7, and annexin V-FITC/PI flow cytometric assays. It is significantly less cytotoxic than the established antipsoriatic agent dithranol 14, as determined by MTT and LDH release assays, and thus has potential as a lead compound for the treatment of psoriasis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Established treatments for psoriasis are generally based on antiproliferative, anti-inflammatory, or differentiation-modifying activity, or a combination of these effects. New agents for the treatment of psoriasis could be identified by high-throughput screening (HTS) of large compound libraries using keratinocyte proliferation models. Although several new proliferation assays have been developed, the radioactive [(3)H]-thymidine incorporation assay is still considered to be the gold standard for the evaluation of keratinocyte proliferation in vitro. In this study, we compare a number of simple, and reliable, colorimetric (MTT, NRU, SRB, and CVS), and fluorimetric (CAM and AB) methods with the [(3)H]-thymidine incorporation assay for the measurement of keratinocyte proliferation in the exponential growth phase in 96-well formats. The concentrations that induced 50% growth inhibition (GI(50)) were determined by each assay for the established antipsoriatics, dithranol, and methotrexate. Strong correlations were observed between the percentage growth inhibitions determined by the radioactive and the colorimetric assays, with no significant differences (P > 0.05) between their GI(50) values. The colorimetric assays are thus suitable alternatives to the radioactive assay for quantifying keratinocyte growth inhibition. We have also validated the use of the HaCaT cell line as a representative of the hyperproliferative psoriatic epidermis, in the preclinical screening of experimental anti-psoriatic agents.

Objectives Dithranol, one of the most successful topical agents for the treatment of psoriasis, has been shown to exert its therapeutic effect by inducing keratinocyte apoptosis. To gain further insights into dithranol-induced apoptotic events in vitro, a detailed investigation of its time- and dose-dependent effects has been performed through the evaluation of selected apoptotic markers, using a human keratinocyte cell line ( HaCaT) as a model. Methods The time- and dose-dependent effects of dithranol on a human keratinocyte cell line ( HaCaT) were investigated through the evaluation of a series of apoptotic markers; morphological changes (electron microscopy), phosphatidylserine externalisation (flow cytometry), and caspase-3/7 activation. Key findings The dithranol-induced apoptotic cascade was found to follow a well-defined dose and time-course, with the concentration and the period of exposure to the drug acting as the two major factors influencing the events and nature of cell death. The earliest apoptotic event detected was caspase activation (after 6 h), followed by the occurrence of phosphatidylserine externalisation (after 9 h) and subsequently the morphological characteristics associated with early and late stage apoptosis/necrosis (after 12 h). Conclusions This study has elucidated the dose- and time-response effects of dithranol-induced apoptosis in human keratinocytes in vitro. [ABSTRACT FROM AUTHOR]

Abstract: Gossypol 1, gossypolone 2, and a series of bis 3 and half Schiff’s bases 4 of gossypol were synthesised and tested for anti-proliferative and anti-oxidant activity. (−)-Gossypol (−)-1 was the most potent inhibitor of the proliferation of the HPV-16 keratinocyte cell line (using an MTT viability assay) with a GI50 of 4.8μM. The bis Schiff’s base of (−)-gossypol with l-tyrosine ethyl ester (−)-3b was the most potent inhibitor of iron/ascorbate dependent lipid peroxidation (using the thiobarbituric acid test), with an IC50 of 11.7μM, with (−)-gossypol being the next most potent of the series, with an IC50 of 13.1μM. The results from these initial assays suggest that gossypol, as either a racemic mixture rac-1, or the individual atropisomers (−)-1 or (+)-1, has potential for the treatment of psoriasis. [Copyright &y& Elsevier]