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healing assay. Results (Figure 2A) showed an inhibition of migratory ability both at 6 and 24 h in AgNPs-EPSaer treated cells. AgNPs-EPSanaer treatment did not affect significantly the migratory capability of SKBR3 cells. Cell migration represent a complex process that involves the expression of a number of growth factors, cytokines and matrix metalloproteinases (MMPs) [32, 33]. Because of the significant effects induced by AgNPs-EPSaer on cell migration, we evaluated the effects on MMP-2 and MMP- 9 enzymatic activities and protein expression in SKBR3 cells using gelatin zymography and western blot. Data revealed a significant decrease of enzymatic activities in AgNPs-EPSaer treated cells with the maximum effect for pro MMP-9 that is almost disappeared. AgNPs-EPSanaer (Figure 2B, 2C and 2D) treatment instead did not affect significantly the activity levels of MMPs, accordingly to the scratch assay results. Morphological assessment of SKBR-3 cells treated with both AgNPs-EPS The morphology of SKBR3 cells treated with either AgNPs-EPSaer and AgNPs-EPSanaer were significantly changed in comparison to the untreated control cells: treated cells showed apoptotic-like symptoms such as cell shrinkage and detachment from the adjacent cells especially in the case of AgNPs-EPSaer treatment. Moreover, irregularity in shape, cytoplasmic blebbing, intracellular vacuoles, cellular debris and nuclear chromatin condensation were clearly observed (Figure 3A). No morphological changes were observed for free-metal EPS (data not shown). AO/EB staining technique was used to verify whether the AgNPs-EPS-cell death is due to apoptotic induction or unspecific necrosis. As expected (Figure 3B), the control cells emitted only green fluorescence due to the AO staining. On the contrary, the AgNPs-EPS treated cells exhibited orange fluorescence (sensitive to pH lowering) due to the loss in membrane integrity. In particular, in AgNPs-EPSanaer treated cells, only membrane blebbing and condensed chromatin were observed, while in AgNPs- EPSaer treated cells, loss of membrane integrity, condensed chromatin and nuclei stained with EB, were observed. Effect of AgNPs-EPS treatment on intracellular oxygen species Elevation of intracellular ROS levels is known to induce oxidative stress and cell death [34, 35]. Several studied reported that AgNP cytotoxicity is primarily Figure 2: AgNPs-EPSaer inhibit wound healing and MMP9/MMP2 activity in SKBR3 cells. (A) Inhibitory effect on cell migration assessed by wound healing assay and quantified by measuring the scratched area. (B) Zymographic analysis of MMP9 and MMP2 activity in SKBR3 cells. The gelatinolytic bands correspond to the pro MMP9 and pro MMP2. (C) Densitometric analyses of proMMP2 and MMP9 secretion. Statistical significance was assessed by the Student’s t-test: *p < 0.001. The data in the graphs are expressed as mean of the band-intensity ± SD. (D) Western blot validation of MMP9 and MMP2 protein expression in conditioned media. www.impactjournals.com/oncotarget 9688 OncotargetPDF Image | Anticancer activity of biogenerated silver nanoparticles
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