Cytotoxicity of Moscatilin to H23 Cells To investigate the inhibitory effect of moscatilin on cancer migration and invasion, prerequisite information regarding its cytotoxicity is crucial. 15]. In present study, we investigated that the administration of moscatilin attenuates migration and invasion in lung cancer cells. Its negative regulator is associated with the ability of compound to suppress endogenous ROS generation and consequently inhibits FAK and Akt activation-mediating cancer motility and invasiveness. Our finding reveals the novel mechanism of moscatilin on the regulation of cancer migration and invasion, which could be an advantage in development of this compound for cancer therapy. Open in a separate window Figure 1 Chemical structure of moscatilin. 2. Material and Methods 2.1. Cells and Reagents Human lung adenocarcinoma H23 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in RPMI-1640 medium containing 10% fetal bovine serum, 2?mM L-glutamine, 100?IU/mL penicillin, and 100?as previously described [16]. Moscatilin Tenofovir hydrate was dissolved in DMSO and deionized water for the indicated working concentrations. The amount of DMSO in the final solution was less than 0.1% which showed no cytotoxic in H23 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst33342, propidium iodide (PI), phalloidin tetramethylrhodamine B isothiocyanate, ribonuclease A, bovine serum albumin (BSA), and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical, Inc. (St. Louis, MO, USA). Annexin V Apoptosis Detection Kit was obtained from BD Biosciences (Woburn, MA, USA). Antibodies for phosphorylated Akt (S473), Akt, phosphorylated FAK (Y397), FAK, phosphorylated ERK (Thr202/Tyr204), ERK, Cdc42, test at a significance level of < 0.05 using SPSS version 19.0. 3. Results 3.1. Cytotoxicity of Moscatilin to H23 Cells To investigate the inhibitory effect of moscatilin on cancer migration and invasion, prerequisite information regarding its cytotoxicity is crucial. Human lung H23 cells were treated with various concentrations of moscatilin (0C5?= 4). *< 0.05 versus nontreated control cells. 3.2. Effect of Moscatilin on H23 Cells Migration The negative regulatory role of moscatilin on lung cancer migration was investigated by wound healing and Boyden chamber assays. Figures 3(a) and 3(c) show that treatment of the cells with nontoxic doses of moscatilin (0-1?= 4). *< 0.05 versus nontreated control cells. (b) Confluent monolayer of H23 cells was wounded using a 1?mm width tip and incubated with moscatilin (1?< 0.05 versus nontreated control cells. (c) After indicated treatment, migrating cells in the denuded zone were photographed. (d) H23 cell migration was examined by transwell assay for 24?h. Data were plotted as an average number of cells in each field and represented the means SD Tenofovir hydrate (= 4). *< 0.05 versus nontreated control cells. (e) Cells were treated with moscatilin (1?= 4). *< 0.05 versus nontreated control cells. (f) Migratory cells at the basolateral side of membrane were stained with Hoechst33342 for 30?min and visualized under fluorescence microscopy. 3.3. Effect of Moscatilin on H23 Cells Invasion and Filopodia Formation To further investigate the effect of moscatilin in lung cancer cell invasion, H23 cells were treated with nontoxic concentrations of moscatilin (0-1?= 4). *< 0.05 versus nontreated control cells. (c) Invading cells were stained with Hoechst33342 and visualized under fluorescence microscopy. (d) Effect of moscatilin on filopodia formation and cell Tenofovir hydrate morphology. After being treated with nontoxic dose of moscatilin for 24?h, cells were stained with either phalloidin or sulforhodamine B and visualized under fluorescence microscope. Filopodia was indicated by arrow. Since filopodia has been shown to play an essential role in cell motility and invasion by protrusion at the edge of motile cells for attachment and gliding [5], we further clarified whether the antimigrative and antiinvasive effects of moscatilin were related to the presence of filopodia. H23 cells were treated with nontoxic concentrations of moscatilin (0-1?= 4). *< 0.05 versus nontreated control cells of each time point. (b) After the indicated treatment for 3?h, cells were incubated with hydroxyphenyl fluorescein (HPF) probe. Hydroxyl radical level was detected using fluorescence microplate reader. *< 0.05 versus nontreated control cells. (c) Hydrogen peroxide level was examined using amplex red probe. *< 0.05 versus nontreated control cells. (d) Superoxide anion level SOX18 was detected by dihydroethidium (DHE) probe. *< 0.05 versus nontreated control cells. (e) Cells were pretreated with 50?= 4). *< 0.05 versus nontreated control cells. # < 0.05 versus ferrous sulfate treated cells. (f) Confluent Tenofovir hydrate monolayer of H23 cells was wounded using a 1?mm width tip and treated with moscatilin (1?< 0.05 versus nontreated control cells. # < 0.05 versus ferrous sulfate treated cells. Parallel study was conducted to investigate the relevance of antioxidant effect of moscatilin on cancer migration, and cells were preincubated with OH? generator in the presence or absence of moscatilin treatment. Wound healing assay shows that ferrous.