Nadifloxacin downregulates the production of matrix metalloproteinases in the human epidermal keratinocyte cell line HaCaT

Dear Editor, Nadifloxacin is a potent new quinolone antibiotic, approved for topical use in acne vulgaris patients in Japan. Matrix metalloproteinases (MMP) are enzymes effective in degrading the extracellular matrix (ECM), such as interstitial collagens and basement membranes and are abundantly found in acne lesions. These enzymes are thought to work in promoting inflammation and tissue remodeling, the exacerbating factors in scar formation. We investigated the effect of nadifloxacin on the production of MMP produced in HaCaT keratinocytes. HaCaT keratinocytes were a generous gift from Dr Kuroki (Showa University) with the permission of Dr Fusenig (Institute Fur Zellund Tumourbiologie, Deutsches Kresforschungszentrum, Heidelberg, Germany). The cells were seeded into 6-well plates and cultured with or without nadifloxacin, incubated overnight, and then stimulated with tumor necrosis factor (TNF)-a (10 ng ⁄ mL). The supernatants were collected after 48 h and subjected to enzyme-linked immunosorbent assay (ELISA). MMP-1, -9 and -13 ELISA Kits were from GE Healthcare Japan (Tokyo, Japan). The optical density of each well was determined using a microplate reader (Model 550; Bio-Rad, Richmond, CA, USA) set to 450 nm. Total RNA samples were isolated using ISOGEN (Nippon Gene, Tokyo, Japan). Reverse transcription was performed using the Superscript III RT Kit (Invitrogen Japan, Tokyo, Japan). The primers and probes for the MMP (TaqMan gene expression assays; MMP-9, Hs00957562 m1; MMP13, Hs00233992 m1; MMP-1, Hs00233958 m1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Applied Biosystems (Norwalk, CT, USA). Real-time polymerase chain reaction (RT–PCR) and the analysis were carried out with an ABI PRISM 7000 sequence detector (Applied Biosystems) using the TaqMan RT–PCR Master Mix Reagents Kit (Applied Biosystems). All results are expressed as the mean ± standard deviation. Statistical comparisons were made by Mann–Whitney’s U-test. The results were considered significantly different at P < 0.05. HaCaT keratinocytes produced MMP-1, -9 and -13, which were induced by the addition of TNF-a. The level of MMP-13 was the highest among those of the MMP (Fig. 1a). The induction of MMP-9 by TNF-a was less when the cells were 100% confluent compared