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and cells [1–3]. Nanotechnology offers a wealth of tools to treat cancer by passing biological barriers to deliver therapeutic agents directly [4]. The unique physicochemical characteristics of metal NPs, such as high surface-to-volume ratio, broad optical properties, ease of synthesis and surface functionalization offer new opportunities for cancer therapeutics. The production of silver nanoparticles (AgNPs) has become the object of intense research and several methods have been developed to synthesize noble metal NPs, including physical and chemical ones [5–7]. The biological synthesis of AgNPs have received increasing attention due to the growing need of developing eco-friendly (i. e. “green”) technologies in material synthesis [6,8–12]. Thus, different microorganisms, either bacteria or fungi, have been used as potential cell-factories for both intra and extra cellular production of AgNPs [9, 13]. The mechanism of biological formation of metal-NPs is mainly due to the capability of biopolimers and, in particular, microbial exopolysaccharides (EPS) to act as metal reducers and/ or stabilizers [14–16]. The Klebsiella oxytoca DSM 29614 EPS is composed of a branched heptasaccharide repeating unit (1 galactose, 4 rhamnoses, 2 glucuronic acids), with metal binding properties during the fermentative biosynthetic process [17, 18]. K. oxytoca strains are ubiquitous bacteria mainly studied as opportunistic pathogens which are responsible for nosocomial infections [19]. However, many K. oxytoca strains are also known to produce exopolysaccharides of environmental and pharmaceutical interest [17, 20]. Furthermore, the production of other metal nanoparticles embedded in this EPS has already been reported [21–25]. In this study, we tested the anticancer effect of biogenerated AgNPs-EPS on different cancer cell lines, and then investigated its molecular mechanism of action in the SKBR3 breast cancer cell line. In particular, we found that AgNPs-EPSaer caused: i) a significant decrease of cell viability and motility, ii) an impairment of MMP-2 and MMP-9 activity, and iii) a promotion of ROS generation, which, in turn, induced cell death through apoptosis and autophagy. These evidences were confirmed by a differential proteomic analysis, in which proteomic changes are consistent with the activation of important pathways including endoplasmic reticulum stress, oxidative stress and mitochondrial disfunction triggering cell death trough apoptosis and/ or autophagy activation. Finally, TEM micrographs and the determination of total silver in subcellular fractions reinforce the idea that Ag+ released from AgNPs-EPSaer firstly in mitochondria and then in nuclei determines cell damage and death. To the best of our knowledge, this is the first study reporting the mechanism of action of biosynthesized AgNPs through an integrated proteomic approach. RESULTS Cytotoxic effects of AgNPs-EPS The cytotoxic effect of AgNPs biogenerated by Klebsiella oxytoca DSM 29614 under aerobic (AgNPs- EPSaer) and anaerobic conditions (AgNPs-EPSanaer) was investigated after 24 h of treatment on two human breast cancer cell lines (SKBR3 and 8701-BC) and three human colon cancer cell lines (HT-29, HCT 116 and Caco-2) by using the MTT assay. The results, expressed as IC50 values, calculated from the dose-survival curves, are reported in Table 1. The AgNPs-EPSaer is more active than AgNPs- EPSanaer, with SKBR3 and 8701-BC cell lines being more sensitive to AgNPs-EPSaer treatment in comparison to HT-29, HCT 116 and Caco-2 colon cancer cell lines. In particular, SKBR3 cells proliferation was significantly inhibited by AgNPs-EPSaer with an IC50 value of 5 μg/ml, while an IC50 value of 8 μg/ml was found for 8701-BC cell line. These values were found well within the clinically acceptable concentration of 100 μg/ml [26], suggesting a potential anticancer effect of both biogenerated AgNPs- EPS. Since AgNPs-EPSaer contains more total silver than AgNPs-EPSanaer [24] and the amount of Ag+ released from AgNPs-EPSaer is significantly higher than for AgNPs- EPSanaer [25], we believe that the biological activity of AgNPs-EPS is Ag-dependent. None toxic effect was observed for metal-free EPS. Since AgNPs-EPSaer had the highest cytotoxic activity towards breast cancer SKBR3 cells, the selectivity index (SI) was investigated by using a non tumoral mammary epithelial cell line (HB2), as previously reported [27]. Cytotoxic assays were performed for 24h and 48h, and doxorubicin was used as a positive control (Table 2). The SI were quite similar between AgNPs-EPSaer and doxorubicin, a commonly used chemotherapeutic drug [28]. Moreover, this investigation revealed high selectivity for SKBR3 cells [29], with higher SI value for higher treatment time, being SI 3.4 and 5.0 after 24 and 48 h, respectively. Based on these results, SKBR3 cells were further used to study the mechanisms of cytotoxicity associated with the AgNPs-EPS treatments. Colony formation assay In order to assess SKBR3 cell viability in terms of reproductive capacity after treatments with the two kinds of AgNPs-EPS, a colony formation assay was performed [30]. To this aim, cells were seeded in appropriate dilutions and treated with two different concentrations (50 and 5 μg/ml) for 1 and 24 h, maintained under normal culture conditions and analysed two weeks later for the formation of colonies. The results (Figure 1) showed that AgNPs- EPSaer treatment inhibited significantly the colony-forming ability of SKBR3 cells in a dose- and time-dependent manner and, interestingly, resulted effective already www.impactjournals.com/oncotarget 9686 OncotargetPDF Image | Anticancer activity of biogenerated silver nanoparticles
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