Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11–18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11–18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11–18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.

Ozenoxacin is a topical quinolone showing potent antimicrobial activities against Gram-negative and Gram-positive bacteria and is widely used for the treatment of inflammatory acne. However, the anti-inflammatory activities of ozenoxacin have not been examined so far. In the present study, we investigated the in vitro and in vivo anti-inflammatory effects of ozenoxacin. The production of interleukin (IL)-6 and IL-8 by human epidermal keratinocytes stimulated by heat-killed Cutibacterium acnes was significantly inhibited by ozenoxacin at concentrations from 1 to 30 μg ml −1. Likewise, the production of IL-6, IL-8, and tumor necrosis factor alpha by stimulated THP-1 cells, a human monocyte cell line, was inhibited by ozenoxacin at concentrations from 1 to 30 μg ml −1. The production of IL-1β by THP-1 was also inhibited by ozenoxacin at the concentration of 30 μg ml −1. Phosphorylation of the mitogen-activated protein kinases and degradation of IκB-α, an inhibitory factor of NF-κB in keratinocytes and THP-1 cells, was increased by stimulation with heat-killed C. acnes. Of these activated intracellular pathways, the p38 phosphorylation pathway was remarkably reduced by ozenoxacin in both keratinocytes and THP-1 cells. In addition, the application of 2% ozenoxacin suppressed the increase in the ear thickness of rats induced by an intracutaneous injection of heat-killed C. acnes. These findings suggest that ozenoxacin possesses an antiinflammatory activity, which may contribute to its therapeutic effects on inflammatory acne.

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When selecting external medicines for the treatment of skin diseases, it is thought to be very important to consider diŠerences in characteristics of their bases, because the bases may in‰uence the clinical e‹cacy of the medicines. In this study, we investigated whether the diŠerences in characteristics of three kinds of bases, white petrolatum, macrogol ointment, and aqueous gel aŠect wound healing. In vitro moisture permeability tests demonstrated that these bases have diŠerent characteristics in coatability and water retentivity, with the rank order of the intensity of coatability as white petrolatum＞macrogol ointment＞aqueous gel, and that of water retentivity as macrogol ointment＞white petrolatum＞ aqueous gel. Similar rank order of these bases was observed for transepidermal water loss and stratum corneum water content in the dry skin on the abdomen of guinea pigs induced by topical application of acetone/ether mixture, followed by water. In addition, we found that treatment with macrogol ointment, but not white petrolatum or aqueous gel, sig-niˆcantly accelerated wound healing in rat skin, and that the contents of basicˆbroblast growth factor and epidermal growth factor in the skin treated with macrogol ointment were signiˆcantly higher compared with non-treated skin. In conclusion, these results imply an important role of the bases of external medicines in the treatment of skin diseases.