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the result of oxidative stress [36, 37], which in turn induces cell death through several supposed molecular mechanisms such as activation of intrinsic pathway of apoptosis, autophagy or both [38–41]. We investigated the effect of AgNPs-EPS on intracellular levels of ROS by dichlorodihydrofluorescein diacetate (DCFH-DA) and Dihydroethidium (DHE) assays, two generic indicators of cellular ROS levels. As shown in Figure 4, after 24 h of exposure AgNPs-EPSaer induced a remarkable ROS increase in a concentration-dependent manner. Compared to the control cells, a maximum of 2.25 and 1.75 fold increases in ROS generation was observed with 10 μg/ml concentration of AgNPs-EPSaer treatment, in DCFH-DA and DHE assay respectively, suggesting an important role of ROS in cell death induction. AgNPs-EPSanaer treatment did not affect significantly intracellular levels of ROS in comparison to the control cells. Detection of Apoptosis and autophagy We examined whether AgNPs-EPS-induced cell death occurs via apoptosis, autophagy, or both. Flow cytometry analysis (Figure 5A) revealed that only the 11.57% of AgNPs-EPSaer treated cells underwent to apoptosis compared to the 5.50% in untreated cells and doxorubicin treated cells (27.81%), while no apoptosis was recorded for AgNPs-EPSanaer treated cells (5.58%) and for free-metal EPS treated cells (6.06%). Accordingly, no DNA fragmentation was evident in SKBR3 cells after 24 h of Ag- NPs-EPS exposure (Figure 5B), suggesting that apoptosis was not prominent in AgNPs-EPSaer -induced cell death, at least in the first 24 h of treatment. Therefore, three different staining specific for lysosomal/vacuolar activity include Lysotracker® (a fluorescent acidotropic probe used for labeling acidic organelles in live cells such as lysosomes/ vacuoles), MonoDansylCadaverine (MDC) (a weak base able to label autophagosomes and autolysosomes) and Acridine Orange (AO) (a fluorescent basic dye that accumulates in acidic compartments, such as lysosomes and vacuole) were used to investigate autophagy. AgNPs-EPSaer treated cells (Figure 5C) showed autophagolysosomes in the cytoplasm detected by fluorescence microscopy as bright punctate dots [42, 43]. Moreover the expression of autophagic markers such as AKT, p-AKT, ATG5, ATG7, HSP90, P62, LC3I/II protein and beclin I, were analyzed by Western blot (Figure 5D). As expected, we observed a concomitant down regulation of AKT, p-AKT, p62 and HSP90 expression and an up-regulation of ATG5, ATG7, LC3-II and beclin-1 in AgNPs-EPSaer treated cells, indicating a prominent mechanism of autophagic cell death. Effect of AgNPs-EPS on SKBR3 proteome Differential proteomic analysis (2D-DIGE) was performed to detect protein modulations induced by both AgNPs-EPS on SKBR-3 cells, after 24 h of treatments Figure 3: Morphological changes in SKBR3 cells treated with AgNPs-EPS. (A) Inverted phase-contrast micrographs Magnification 200X. (B) Fluorescence microscope micrographs after AO/EB double staining. Magnification 680X. Viable cells (blank arrow), membranes blebbing (yellow arrow), loss of membrane integrity, condensed chromatin and nuclei stained with EB (red arrows). Each experiment was performed in triplicate (n = 3) and generated similar morphological features. www.impactjournals.com/oncotarget 9689 OncotargetPDF Image | Anticancer activity of biogenerated silver nanoparticles
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