High sensitivity of human epidermal keratinocytes (HaCaT) to topoisomerase inhibitors

Abstract

In the panorama of the numerous established cell lines, the human keratinocyte line HaCaT has a very interesting feature, having a close similarity in functional competence to normal keratinocytes. This cell line has been used in many studies as a paradigm for epidermal cells and therefore we selected HaCaT as a cell model for investigating the activity of three antitopoisomerase drugs (Camptothecin, Doxorubicin, Ciprofloxacin) on in vitro cell growth. The effect was evaluated both by a 24-h cytotoxicity test and by a 7-day antiproliferation assay, in which the cell viability was assessed by an MTT (3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenil-2-H-tetrazolium bromide) test. DNA topoisomerase I was also partially purified from a nuclear extract of HaCaT cells, the level of topo I catalytic activity was measured by a pBR322 DNA relaxation assay and then the in vitro effect of antitopoisomerase drugs on the target enzyme was also assessed. The results indicated that the in vitro sensitivity of human epidermal HaCaT cells to antitopoisomerase drugs is comparable to that of many human tumour cell lines. HaCaT cells express a high level of topoisomerase I activity that is significantly inhibited by both Camptothecin and Doxorubicin and to a minor degree by Ciprofloxacin. A high correlation between the cell sensitivity to the antitopoisomerase I drug measured by the MTT test and the in vitro direct inhibition of HaCaT topoisomerase I was observed, suggesting that HaCaT cells can represent a very interesting model both for studying cellular pharmacokinetics of antineoplastic drugs on keratinocytes and for predicting possible secondary effects, exerted by these drugs on cutaneous cells, during treatment with chemotherapy.

Nadifloxacin, an antiacne quinolone antimicrobial, inhibits the production of proinflammatory cytokines by human peripheral blood mononuclear cells and normal human keratinocytes

“…To our knowledge, it is the first report to show the inhibition of keratinocyte cytokine production by fluoroquinolones. Ciprofloxacin has been shown to possess a cytotoxic activity against keratinocytes [35], however, potential cytotoxicity of NDFX to keratinocytes is unlikely to be responsible for the inhibition of cytokine production, because no morphological changes of the cells were noted after the addition of NDFX by phase-contrast microscopic observations.…”

Mitochondrial superoxide mediates doxorubicin-induced keratinocyte apoptosis through oxidative modification of ERK and Bcl-2 ubiquitination

“…Human keratinocytes were tested in this study since they are the primary target for DOX-induced toxicities. HaCaT keratinocytes, although exhibited alterations in their life-span (immortalizion), were used as they express typical epidermal phenotype [25-27], of which making them common in vitro models to aid the mechanistic studies of skin and hair follicle [28,29]. …”

Nadifloxacin downmodulates antigen-presenting functions of epidermal Langerhans cells and keratinocytes

“…Norfloxacin also suppresses the production of IFN-g by peripheral lymphocytes [21]. Ciprofloxacin markedly abolish the function of keratinocyte cell line [22]. Since NDFX inhibited the IFN-g-promoted expression of MHC class II molecules, it is possible that it blocks at least a certain step of signaling induced by IFN-g.…”