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  • br Discussion br The unique chemical and physical


    4. Discussion
    The unique chemical and physical properties of CAP have received considerable attention for its potential biomedical applications. Emerging fields of application of CAP include wound healing, ster-ilization of infected tissue, inactivation of microorganisms, tooth bleaching, blood coagulation, skin regeneration, and cancer therapy [5,8,40]. On the other hand, MNPs with appropriate surface coatings are increasingly being used clinically for various biomedical applica-tions. Due to requirements of nontoxicity and biocompatibility, iron oxide-based MNPs are predominantly used, despite some attempts to 
    develop ‘more MNPs’ based on cobalt, nickel, gadolinium and other compounds [41]. Iron oxide-based MNPs can bind to an external magnetic field to the target site by attaching the targeting molecules, such as proteins or antibodies. In this paper, we combined CAP with iron oxide-based MNPs and applied them to lung cancer both in vivo and in vitro situations, and evaluate its underlying mechanism.
    In our study, the shapes of A549 cancer BMPO changed from fusiform to circular when they were treated by CAP (Fig. 3a and b). These cells were also difficult to be digested into a single-cell suspension, in-dicating the appearance of some similar characteristic with normal lung epithelial and recovery of cell-cell junction. In vitro and in vivo ex-periments have shown that CAP is effect against lung tumors (Fig. 3a and Fig. 9a). One of main mechanisms involved CAP on cell biological function is ROS [40,42,43]. Generally, ROS includes superoxide (O2-), nitrite (NO), atomic oxygen (O), ozone (O3), hydroxyl radical (•OH), singlet delta oxygen (SOD, O2(1 g)), peroxynitrite (ONOO-), and hy-drogen peroxide (H2O2). Hydrogen peroxide was characterized to show ROS level. In our goup previous study, we have determined the con-centration of hydrogen peroxide indicating time-dependent behavior [44]. The production of intracellular ROS in A549 cancer cells was detected via the DCFH-DA probe (Fig. 4). The intracellular ROS in-tensity substantially increased in CAP treated A549 cancer cells with dose-dependent behavior. The proliferation of A549 cancer cells was significantly repressed by CAP. Cell migration and invasion are also critical evidence for CAP suppressing A549 cancer cells (Fig. 6a). Transwell and wound healing assays demonstrated that CAP abrogated the migration and invasion ability of A549 cancer cells (Fig. 6b). The synergistic effect of CAP and iron oxide-based MNPs on A549 cancer cells were investigated and indicated a noticeably decreased number of
    Fig. 8. Iron oxide-based MNPs and CAP Inhibited expression of EGFR and its downstream signaling pathway activates: (a) expression of EGFR mRNA was greatly reduced by CAP in a dose-dependent manner, (b) western blot analysis showed downregulation of EGFR protein in CAP treated A549 cells, (c) synergistic inhibition of EGFR protein by CAP and iron oxide-based MNPs, (d) western blot showed that phosphorylation of pEKR1/2 and pAKT which were downstream kinase of EGFR were decreased in the CAP treated A549 cells, and (e) dual treatment of CAP and iron oxide-based MNPs decreased the expression of pERK and pAKT.
    colonies, which suggested that iron oxide-based MNPs enhanced the efficiency of CAP in restraining tumor growth (Fig. 5). Moreover, the high fluorescence intensity in the co-treated cells indicated that iron oxide-based MNPs could sensitize the cellular cytotoxicity of CAP. MNPs could be phagocytized by tumor cell and combined with lyso-some, under the action of enzymes, ferrous ions or ferric iron are re-leased into cytoplasm as shown by Prussian blue staining (Fig. 2e) and iron ion promote the conversion of hydrogen peroxide into ROS and enhance the killing effect of CAP. The effects of A549 cancer cells treated by CAP or iron oxide-based MNPs either individually or in combination on gene expression demonstrated E-cadherin was upre-gulated, whereas the mesenchymal marker vimentin was significantly suppressed (Fig. 7). Previous studies demonstrated that phorbol ester elicited EMT processes was regulated by histone H3 acetylation and NADPH oxidase (NOX) 2-derived ROS signaling [45], indicating CAP modulated EMT processes via production of ROS. On the other hand, plasma generated ROS either enter the near-surface or act as ligands to membrane surface receptors and thereby initiate various cellular re-sponses, including DNA damage, cell cycle arrest, and other redox-mediated stresses [42]. Cells experiencing DNA damage result in ROS levels to increase from mitochondrial sources, which initiates signaling cascades inducing gap junction signaling to adjacent cells and the re-lease of extracellular signaling compounds that the signal cell further away [46]. Therefore, there is the possibility that co-treatment with CAP and MNPs facilitates entry of MNPs into cells via gap junction.