Amorphous Silica Particles Relevant in Food Industry Influence Cellular Growth and Associated Signaling Pathways in Human Gastric Carcinoma Cells
"> Figure 1
<p>Influence of the suspension medium and the fetal bovine serum (FBS) amount on size distribution of 12 nm SiO<sub>2</sub> NPs analyzed by nanoparticle tracking analysis after 0 h and 24 h of incubation. Depicted are exemplary particle size distribution profiles (mean ± SEM of five measurements) of 1 mg/mL particle stock suspensions in (<b>A</b>) double-distilled water (FBS-free) or (<b>B</b>) 9% FBS-containing RPMI 1640 cell culture medium. Please mind the varying ordinate scaling; (<b>C</b>) Represented are D10, D50 and D90 values of the particle stock suspensions (1 mg/mL) suspended in either double-distilled water or RPMI 1640 cell culture medium, as well as, the medium incubation suspensions with area concentrations of 31.3 and 93.8 µg/cm<sup>2</sup>. The final FBS amount varied. The D values indicate percentage undersize distribution, for example D10 indicates 10% particles are smaller than the D10 value. This gives indication of the distribution of particle sizes and their corresponding merged particle diameter in [nm] (<span class="html-italic">n</span> ≥ 2).</p> "> Figure 2
<p>Influence of 12 nm SiO<sub>2</sub> NPs on cell proliferation and cell death of GXF251L cells monitored microscopically after an incubation period of 4 h, 24 h and 72 h. Staurosporine (800 nM) was applied as a positive control (+Cntrl) resulting in apoptotic and necrotic cell death. (<b>A</b>) Viable (Via), early apoptotic (EA) and late apoptotic (LA), as well as, necrotic cells (N) are plotted in relation to the total cell count; (<b>B</b>) The depicted bars represent the total cell counts and the respective amount of viable cells. Total cell count was quantified in relation to the negative control (−Cntrl). Cell viability was quantified in relation to the viability of the negative control, as well as, the total cell count of the negative control (T/C [%]). The solid line represents the negative control set to 100%; (<b>C</b>) Represented are microscopic bright field (bf) images of the negative and positive control and the incubation sample with 31.3 µg/cm<sup>2</sup> of 12 nm SiO<sub>2</sub> NPs after an incubation time of 24 h. Furthermore, the software analysis after detection in the Hoechst-channel is shown distinguishing between viable cells (green), early (blue) and late (violet) apoptotic cells, as well as, necrotic cells (red). Scale bars are equivalent to 50 µm. Statistical analysis was performed by One-way ANOVA followed by Fisher’s LSD test. Significances indicated as * refer to a comparison to the respective negative control (* ≡ <span class="html-italic">p</span> ≤ 0.05; ** ≡ <span class="html-italic">p</span> ≤ 0.01 and *** ≡ <span class="html-italic">p</span> ≤ 0.001; <span class="html-italic">n</span> ≥ 3). The same letters indicate a significant difference of these data (in (A): refers to necrotic cells only) with a statistical level of at least <span class="html-italic">p</span> ≤ 0.05.</p> "> Figure 3
<p>Influence of 200 nm SiO<sub>2</sub> particles on cell proliferation and cell death of GXF251L cells monitored microscopically after an incubation period of 4 h, 24 h and 72 h. Staurosporine (800 nM) was applied as a positive control (+Cntrl) resulting in apoptotic and necrotic cell death. (<b>A</b>) Viable (Via), early apoptotic (EA) and late apoptotic (LA), as well as, necrotic cells (N) are plotted in relation to the total cell count; (<b>B</b>) The depicted bars represent the total cell counts and the respective amount of viable cells. Total cell count was quantified in relation to the negative control (−Cntrl). Cell viability was quantified in relation to the viability of the negative control, as well as, the total cell count of the negative control (T/C [%]). The solid line represents the negative control set to 100%. Statistical analysis was performed by One-way ANOVA followed by Fisher’s LSD test. Significances indicated as * refer to a comparison to the respective negative control (* ≡ <span class="html-italic">p</span> ≤ 0.05; ** ≡ <span class="html-italic">p</span> ≤ 0.01 and *** ≡ <span class="html-italic">p</span> ≤ 0.001; <span class="html-italic">n</span> ≥ 3). The same letters indicate a significant difference of these data (in (<b>A</b>): refers to necrotic cells only) with a statistical level of at least <span class="html-italic">p</span> ≤ 0.05.</p> "> Figure 4
<p>Analysis of the cytotoxic effects of 12 nm SiO<sub>2</sub> NPs on GXF251L cells. (<b>A</b>) Influences on the mitochondrial activity determined by WST-1 assay and (<b>B</b>) on the membrane integrity determined by LDH leakage assay after 45 min and 24 h of incubation; (<b>C</b>) Growth effects after 24 h, 48 h and 72 h of incubation determined by SRB assay. For WST-1 and SRB assay all effects were quantified in relation to the negative assay control (T/C [%]). For the LDH leakage assay effects were quantified in relation to the positive assay control (T/C [%]). The solid lines represent the respective control set to 100%. Triton X-100 (1% <span class="html-italic">v</span>/<span class="html-italic">v</span>) was used as positive control. Statistical analysis was performed by One-way ANOVA followed by Fisher’s LSD test. Significances indicated as * refer to a comparison to the respective negative control (* ≡ <span class="html-italic">p</span> ≤ 0.05; ** ≡ <span class="html-italic">p</span> ≤ 0.01 and *** ≡ <span class="html-italic">p</span> ≤ 0.001; <span class="html-italic">n</span> ≥ 3).</p> "> Figure 5
<p>Intracellular levels and localization of Ki-67. Representative images of GXF251L cells treated with 12 nm SiO<sub>2</sub> NPs for 24 h after immunofluorescence staining of Ki-67 (<b>red</b>), α-Tubulin (<b>green</b>) and Lamin B (<b>blue</b>). White arrows indicate enhanced presence of Ki-67 in the nucleus. Scale bars are equivalent to 5 µm.</p> "> Figure 6
<p>(<b>A</b>) Representative appearance of Ki-67 and Lamin B in GXF251L cells treated with 12 nm SiO<sub>2</sub> NPs for 24 h after the application of the co-localization tool on the images. White fields represent the co-localization of Ki-67 (red) and Lamin B (blue). α-Tubulin is presented in green. Scale bars are equivalent to 5 µm; (<b>B</b>) Quantification of the red fluorescence associated with Ki-67 nuclear staining. [%]). The solid line represents the negative control set to 100%. Statistical analysis was performed by Student’s t-test. Significances indicated as * refer to a comparison to the samples treated with SiO<sub>2</sub> NP concentration of 31.3 µg/cm<sup>2</sup>. (** ≡ <span class="html-italic">p</span> ≤ 0.01 and *** ≡ <span class="html-italic">p</span> ≤ 0.001; 31.3 µg/cm<sup>2</sup> <span class="html-italic">n</span> = 66 nuclei; 93.8 µg/cm<sup>2</sup> <span class="html-italic">n</span> = 72 nuclei; EGF <span class="html-italic">n</span> = 67 nuclei).</p> "> Figure 7
<p>Endogenous epidermal growth factor (EGF) receptor levels and their activation status (pEGFR) in GXF251L cells after 45 min and 24 h of incubation with 12 nm SiO<sub>2</sub> NPs. EGF was applied as a positive control for phosphorylation of EGFR. (<b>A</b>) The relative amount was quantified as arbitrary light units in relation to the negative control (T/C [%]). The solid line represents the negative control set to 100%; (<b>B</b>) Representative images of a Western blot experiment. α-Tubulin was used as loading control and detected on the same membrane. Statistical analysis was obtained by one-way ANOVA followed by Fisher’s LSD test. Significances indicated as * refer to a comparison to the respective negative control (* ≡ <span class="html-italic">p</span> ≤ 0.05; ** ≡ <span class="html-italic">p</span> ≤ 0.01 and *** ≡ <span class="html-italic">p</span> ≤ 0.001; <span class="html-italic">n</span> ≥ 3). The same letters indicate a significant difference of these data with a statistical level of at least <span class="html-italic">p</span> ≤ 0.05.</p> "> Figure 8
<p>Endogenous ERK1/2 protein levels and their activation status (pERK1/2) in GXF251L cells after 45 min and 24 h of incubation with 12 nm SiO<sub>2</sub> NPs. EGF was applied as a positive control for phosphorylation of ERK1/2. (<b>A</b>) The relative amounts were quantified as arbitrary light units in relation to the negative control (T/C [%]). The solid line represents the negative control set to 100%; (<b>B</b>) Representative images of a Western blot experiment. α-Tubulin was used as loading control and detected on the same membrane. Statistical analysis was performed by One-way ANOVA followed by Fisher’s LSD test. Significances indicated as * refer to a comparison to the respective negative control (* ≡ <span class="html-italic">p</span> ≤ 0.05; ** ≡ <span class="html-italic">p</span> ≤ 0.01 and *** ≡ <span class="html-italic">p</span> ≤ 0.001; <span class="html-italic">n</span> ≥ 3). The same letters indicate a significant difference of these data with a statistical level of at least <span class="html-italic">p</span> ≤ 0.05.</p> "> Figure 9
<p>Investigation of the molecular mechanisms influencing the EGF receptor at the cellular level by analysis of its relative mRNA transcription rates and its localization in GXF251L cells. (<b>A</b>) Relative gene transcription rates of the EGFR after incubation with 12 nm SiO<sub>2</sub> NPs for 2 h, 6 h, 16 h and 24 h. The depicted bars represent the relative transcription rates in relation to the negative control (solid line) after normalization to β-actin and GAPDH expression (T/C [%]) (<span class="html-italic">n</span> ≥ 3); (<b>B</b>) 3D reconstruction of the appearance of the EGFR localization (red) and tubulin cytoskeleton (green) after immunocytochemical analysis. Represented are the results of the negative control, the incubation with 31.3 µg/cm<sup>2</sup> and with 93.8 µg/cm<sup>2</sup> of 12 nm SiO<sub>2</sub> NPs after 45 min of incubation and EGF stimulation as positive control. Scale bar distances are expressed in 10 µm.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Particle Characterization
2.2. Influence of SiO2 NPs on Cell Proliferation and Cell Death
2.3. Influence of SiO2 NPs on Mitochondrial Activity, Membrane Integrity, and Cell Growth
2.4. Influence of SiO2 NPs on the Proliferation Marker Ki-67
2.5. Influence of SiO2 NPs on the Epidermal Growth Factor Receptor Expression
2.6. Influence of SiO2 NP on the Extracellular Signal-Regulated Kinases 1/2
2.7. Evaluation of Molecular Mechanisms Sustaining the EGFR Increase Triggered by SiO2 NPs
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Particles and Incubation Conditions
4.3. Nanoparticle Tracking Analysis
4.4. ζ-Potential
4.5. Microscopy-Based Determination of Cell Counts, Apoptosis and Necrosis
4.6. Cytotoxicity
4.6.1. Water Soluble Tetrazolium (WST-1) Assay
4.6.2. Lactate Dehydrogenase (LDH) Assay
4.6.3. Sulforhodamine B (SRB) Assay
4.7. Western Blot Analysis
4.8. Immunocytochemistry
4.9. RNA Extraction and qRT-PCR
4.10. Statistics
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
JNK1/2 | c-Jun NH2-terminal kinases1/2 |
DMEM | Dulbecco’s Modified Eagle Medium |
DSMZ | German Collection of Microorganisms and Cell Cultures |
EGFR | epidermal growth factor receptor |
ERK1/2 | extracellular signal-regulated kinases 1/2 |
FBS | fetal bovine serum |
Fisher’s LSD | Fisher’s Least Significant Differences |
NTA | nanoparticle tracking analysis |
GIT | gastrointestinal tract |
LDH | lactate dehydrogenase |
MAPK | mitogen-activated protein kinases |
PI | propidium iodide |
PSD | particle size distribution |
qRT-PCR | quantitative real-time polymerase chain reaction |
RPMI 1640 | Roswell Park Memorial Institute Medium 1640 |
SiO2 NPs | silica nanoparticles |
SRB | sulforhodamine B |
SOP | standard operating procedure |
T/C | test over control |
WST-1 | water soluble tetrazolium |
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12 nm SiO2 NP Suspensions | Mean Diameter ± SD [nm] | ζ-Potential [mV] | ||||
---|---|---|---|---|---|---|
Designation | Particle concentration | Suspension media (final FBS-content) | 0 h | 24 h | 0 h | 24 h |
stock suspension | 1 mg/mL | RPMI 1640 (9% FBS) | 336 ± 7 | 301 ± 58 | −11.5 ± 0.3 | −11.3 ± 0.4 |
1 mg/mL | dd water (FBS-free) | 224 ± 17 | 220 ± 27 | −33.1 ± 6.6 | −25.0 ± 5.0 | |
incubation suspension | 31.3 µg/cm2 (100 µg/mL) | RPMI 1640 (0.9% FBS) | 333 ± 26 | 317 ± 20 | −11.0 ± 0.0 | −12.0 ± 0.4 |
RPMI 1640 (2.7% FBS) | 323 ± 29 | 261 ± 98 | −9.9 ± 0.9 | −10.3 ± 0.0 | ||
RPMI 1640 (9% FBS) | - | - | −9.5 ± 0.3 | −9.3 ± 0.6 | ||
93.8 µg/cm2 (300 µg/mL) | RPMI 1640 (2.7% FBS) | 315 ± 1 | 314 ± 25 | −11.8 ± 0.2 | −11.3 ± 0.1 | |
RPMI 1640 (9% FBS) | - | - | −10.5 ± 0.1 | −10.0 ± 0.5 |
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Wittig, A.; Gehrke, H.; Del Favero, G.; Fritz, E.-M.; Al-Rawi, M.; Diabaté, S.; Weiss, C.; Sami, H.; Ogris, M.; Marko, D. Amorphous Silica Particles Relevant in Food Industry Influence Cellular Growth and Associated Signaling Pathways in Human Gastric Carcinoma Cells. Nanomaterials 2017, 7, 18. https://doi.org/10.3390/nano7010018
Wittig A, Gehrke H, Del Favero G, Fritz E-M, Al-Rawi M, Diabaté S, Weiss C, Sami H, Ogris M, Marko D. Amorphous Silica Particles Relevant in Food Industry Influence Cellular Growth and Associated Signaling Pathways in Human Gastric Carcinoma Cells. Nanomaterials. 2017; 7(1):18. https://doi.org/10.3390/nano7010018
Chicago/Turabian StyleWittig, Anja, Helge Gehrke, Giorgia Del Favero, Eva-Maria Fritz, Marco Al-Rawi, Silvia Diabaté, Carsten Weiss, Haider Sami, Manfred Ogris, and Doris Marko. 2017. "Amorphous Silica Particles Relevant in Food Industry Influence Cellular Growth and Associated Signaling Pathways in Human Gastric Carcinoma Cells" Nanomaterials 7, no. 1: 18. https://doi.org/10.3390/nano7010018