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. 2019 May 15;12(2):72.
doi: 10.3390/ph12020072.

Colloidal Silver Induces Cytoskeleton Reorganization and E-Cadherin Recruitment at Cell-Cell Contacts in HaCaT Cells

Elena Montano  1 , Maria Vivo  2 , Andrea Maria Guarino  3 , Orsola di Martino  4 , Blanda Di Luccia  5 , Viola Calabrò  6 , Sergio Caserta  7 , Alessandra Pollice  8
Affiliations

Colloidal Silver Induces Cytoskeleton Reorganization and E-Cadherin Recruitment at Cell-Cell Contacts in HaCaT Cells

Elena Montano et al. Pharmaceuticals (Basel). .

Abstract

Up until the first half of the 20th century, silver found significant employment in medical applications, particularly in the healing of open wounds, thanks to its antibacterial and antifungal properties. Wound repair is a complex and dynamic biological process regulated by several pathways that cooperate to restore tissue integrity and homeostasis. To facilitate healing, injuries need to be promptly treated. Recently, the interest in alternatives to antibiotics has been raised given the widespread phenomenon of antibiotic resistance. Among these alternatives, the use of silver appears to be a valid option, so a resurgence in its use has been recently observed. In particular, in contrast to ionic silver, colloidal silver, a suspension of metallic silver particles, shows antibacterial activity displaying less or no toxicity. However, the human health risks associated with exposure to silver nanoparticles (NP) appear to be conflicted, and some studies have suggested that it could be toxic in different cellular contexts. These potentially harmful effects of silver NP depend on various parameters including NP size, which commonly range from 1 to 100 nm. In this study, we analyzed the effect of a colloidal silver preparation composed of very small and homogeneous nanoparticles of 0.62 nm size, smaller than those previously tested. We found no adverse effect on the cell proliferation of HaCaT cells, even at high NP concentration. Time-lapse microscopy and indirect immunofluorescence experiments demonstrated that this preparation of colloidal silver strongly increased cell migration, re-modeled the cytoskeleton, and caused recruitment of E-cadherin at cell-cell junctions of human cultured keratinocytes.

Keywords: E-cadherin; colloidal silver; keratinocytes; nanoparticles; skin; wound healing.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Effects of AgC on HaCaT cell viability. MTT assay of HaCaT cells incubated for 24 or 48 h with colloidal (grey bars) or ionic silver (dark bars) at 0.5 or 2 μg/mL. Data are expressed as absorbance at 570 nm and presented as mean ± SE of three independent experiments, each done in sestuplicate. Analysis of variance was performed by two-way Anova followed by the Bonferroni post-test. **** P < 0.0001 when compared with the control.
Figure 2
Figure 2
Effect of AgC on stress granules formation. HaCaT cells were seeded on a coverslip and treated (or not) with AgC at 0.5 or 2 μg/mL for 24 h. Cells were then fixed and analyzed by TRITC-conjugated phalloidin staining (red) or indirect IF with the anti-YB1 antibody (green). Nuclei were stained with DAPI. Scale bar, 7 μM. Images were acquired using a Nikon TE Eclipse 2000.
Figure 3
Figure 3
Effect of AgC on wound closure dynamics. (A) Representative phase contrast microscopy images of cells incubated (or not) with AgC at different time points showing the wound closure process over time. (B,C) The wound closure process was quantified by measuring the reduction of the wound area (A) over time, as described in the Materials and Methods Section. Evolution of the wound area A, normalized to the value A0 (A at time 0), is reported for the control (B) and AgC treated (C) cells by selecting random fields along the wound for each experiment. The linear range of each data series was fit in order to measure the wound closure velocity α. Values of α for the control and treated cells are indicated on each graph as the mean from three independent experiments analyzed in triplicate. Standard error of the mean was calculated to account for reproducibility, and the t-test was calculated to verify the statistical significance of the differences with respect to the control samples.
Figure 4
Figure 4
Effect of AgC on wound closure dynamics. Values of α (wound closure velocity) (A), τ duplication time) (B), and D (cell motility coefficient) (C) of the control and AgC treated cells are reported. Values of D were calculated according to the Fisher–Kolmogoroff equation from values of α and τ (see Materials and Methods). Data are expressed as the mean of at least three independent experiments. SEM is reported as error bars, statistical significance was assessed by the paired two-tailed t-test (* P = 0.04; ** P = 0.007).
Figure 5
Figure 5
E-cadherin localization in HaCaT cells upon AgC treatment. Cells were allowed to adhere onto coverslips for 24 h and then treated with 0.5 μg/mL AgC for 8 h. Cells were then fixed and subjected to IF with an anti-E-cadherin antibody followed by TRITC-conjugated phalloidin to visualize the actin cytoskeleton. Representative images of E-cadherin subcellular localization and phalloidin are shown. Merged images also show DAPI staining to visualize the nuclei. Images were taken with a Zeiss confocal laser-scanning microscope Axio Observer (scale bar, 15 μM). A ×40 objective was used and image analysis was performed using ImageJ.
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