Evaluation of a Range of Anti-Proliferative Assays for the Preclinical Screening of Anti-Psoriatic Drugs: A Comparison of Colorimetric and Fluorimetric Assays with the Thymidine Incorporation Assay

Abstract

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.

A novel control of human keratin expression: cannabinoid receptor 1-mediated signaling down-regulates the expression of keratins K6 and K16 in human keratinocytesin vitroandin situ

“…This transformed human KC line is well-appreciated to constitutively express K6 and to be hyperproliferative (just like human wounded and psoriatic KC) (Balato et al, 2012; Farkas et al, 2001; George et al, 2010; Kim et al, 2011; Ronpirin & Tencomnao, 2012; Ryle et al, 1989; Saelee, Thongrakard & Tencomnao, 2011). K6 is expressed in hyper-proliferative cells (Weiss, Eichner & Sun, 1984) and both K6 expression and basal layer epidermal KC proliferation are increased in psoriasis lesions (Donetti et al, 2012; Griffiths & Barker, 2007; Korver et al, 2006; Litvinov et al, 2011; Mommers et al, 2000).…”

“…Since HaCaT KCs share some other characteristics with psoriatic KCs and are often employed as surrogate “psoriatic” KCs (Balato et al, 2012; Farkas et al, 2001; George et al, 2010; Kim et al, 2011; Ronpirin & Tencomnao, 2012; Saelee, Thongrakard & Tencomnao, 2011), we also tested whether and how ACEA modulated K6 expression in these cells in vitro . In order to delineate whether any such effects on keratin expression resulted only indirectly from a possible down-regulation of KC proliferation by CB1 stimulation (Toth et al, 2011), double-labeling and quantitative immunohistomorphometry for both K6 and Ki-67 was performed.…”

Dithranol downregulates expression of Id1 mRNA in human keratinocytes in vitro

“…This cell line, originally documented by Boukamp et al (1988), is believed to be an appropriate cellular model of psoriasis based on previous reports (Farkas et al, 2001;Tencomnao et al, 2009;George et al, 2010;Saelee et al, 2011;Ronpirin and Tencomnao, 2012). Hamajima et al (2010) demonstrated the role of Id1 in the hyperproliferation of keratinocytes, possibly via the nuclear factor-kappa B signaling pathway.…”

Peer Review #2 of "A novel control of human keratin expression: cannabinoid receptor 1-mediated signaling down-regulates the expression of keratins K6 and K16 in human keratinocytes in vitro and in situ (v0.1)"

“…HaCaT cells are a highly proliferating human KC line known to overexpress K6 (Ryle et al 1989). Since HaCaT KCs share some other characteristics with psoriatic KCs and are often employed as surrogate "psoriatic" KCs (Balato et al 2012;Farkas et al 2001;George et al 2010;Ronpirin & Tencomnao 2012;Saelee, Thongrakard & Tencomnao 2011), we also tested whether and how ACEA modulated K6 expression in these cells in vitro.…”