Quercetin Inhibits Cell Survival and Metastatic Ability via the EMT-Mediated Pathway in Oral Squamous Cell Carcinoma
"> Figure 1
<p>Quercetin reduced cell viability and arrested the G2/M phase cell cycle in oral squamous cell carcinoma (OSCC) cells. (<b>A</b>) Cell viability was investigated by an MTT assay. Oral squamous cell carcinoma cell lines (OSC20, SAS, and HN22 cells) were treated with quercetin (10, 20, 40, 80, and 160 μM). (<b>B</b>) Quercetin was shown to induce cell cycle arrest in OSC20, SAS, and HN22 cells. Data are the means ± SEM. * <span class="html-italic">p</span> < 0.05 and ** <span class="html-italic">p</span> < 0.01 vs. corresponding control (quercetin 0 μM).</p> "> Figure 2
<p>Cell migration ability assessed by a wound-healing assay. (<b>A</b>) Changes in the wound area were observed after 24 h. In the quercetin-treated cells, the wound area was less closed. This indicates a decrease in migration capacity. (<b>B</b>) The wound area was calculated and presented as a graph. Data are the means ± SEM. * <span class="html-italic">p</span> < 0.05 and ** <span class="html-italic">p</span> < 0.01 vs. the corresponding control (quercetin 0 μM).</p> "> Figure 3
<p>Quercetin is shown to induce regulation of epithelial-mesenchymal transition (EMT) and matrix metalloproteinase (MMP). (<b>A</b>) Western blotting was conducted to examine the changes in the EMT inducers. The results showed that the epithelial markers (E-cadherin and claudin-1) were upregulated, and the mesenchymal markers (fibronectin, vimentin, and alpha-smooth muscle actin (α-SMA)) were downregulated upon treatment with quercetin. (<b>B</b>) Quantitation of A. The band intensities of each target protein were measured using an image analyzer and presented as relative ratio. (<b>C</b>) Gelatin zymography shows the MMP-2 and MMP-9 activities in oral cancer cell lines (OSC20, SAS, and HN22 cells) upon quercetin treatment. Data are the means ± SEM. * <span class="html-italic">p</span> < 0.05, and ** <span class="html-italic">p</span> < 0.01 vs. the corresponding control (quercetin 0 μM).</p> "> Figure 4
<p>EMT-activating transcription factors were detected using Western blot and immunofluorescence. (<b>A</b>) The Western blot showed that quercetin downregulated EMT transcription factors at the protein level. (<b>B</b>) Quantitation of A. The band intensities of each target protein were measured using an image analyzer and presented as relative ratio. (<b>C</b>) Representative fluorescence microscopy images of Twist in OSCC cell lines. (<b>D</b>) Representative fluorescence microscopy images of Slug in OSCC cell lines. Scale bars: 20 μM. Data are the means ± SEM. * <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 vs. the corresponding control (quercetin 0 μM).</p> "> Figure 5
<p>Quercetin was not toxic in normal keratocyte and transforming growth factor β1 (TGF-β1) stimulated EMT in HaCaT cells. (<b>A</b>) To examine the effect of quercetin on cell viability, an MTT assay was conducted on HaCaT and nHOK cells. Data are means ± SEM. * <span class="html-italic">p</span> < 0.05 vs. the corresponding control (quercetin 0 μM). (<b>B</b>) Expression of EMT-related markers such as in TGF-β1-treated HaCaT cells were analyzed by Western blotting. (<b>C</b>–<b>H</b>) Quantitation of B. The band intensities of each target protein were measured using an image analyzer and presented as relative ratio. Data are means ± SEM. * <span class="html-italic">p</span> < 0.05 and *** <span class="html-italic">p</span> < 0.005 vs. corresponding control (TGF-β1 0 ng/mL).</p> "> Figure 6
<p>Quercetin inhibited TGF-β1-induced EMT. (<b>A</b>) Experimental setup. (<b>B</b>) Western blot results showed that quercetin also regulated TGF-β1-induced EMT markers. (<b>C</b>) Treatment of TGF-β1 also changed the morphology of HaCaT. Further, quercetin induced morphological recovery. Scale bars: 50 μM. (<b>D</b>–<b>I</b>) Quantification of hRPTECs viability by MTT assay. (<b>J</b>,<b>K</b>) A wound-healing assay was conducted to evaluate the TGF-β1-induced EMT migration ability; quercetin attenuated EMT-induced migration in HaCaT cells. (<b>L</b>) Quercetin inhibited the invasion capacity of EMT-induced HaCaT cells. Data are the means ± SEM. * <span class="html-italic">p</span> < 0.05, and *** <span class="html-italic">p</span> < 0.001 vs. the corresponding control (without TGF-β1 and quercetin); <sup>#</sup> <span class="html-italic">p</span> < 0.05 vs. the corresponding control (with TGF-β1 and without quercetin).</p> ">
Abstract
:1. Introduction
2. Results
2.1. Quercetin Reduced Cell Viability and Arrested G2 Phase Cell Cycle in OSCC Cells
2.2. Quercetin Suppressed the Migration Potential of OSCC Cells
2.3. Quercetin Regulated EMT and MMPs in OSCC Cells
2.4. Quercetin Regulated EMT-Activating Transcription Factors in OSCC Cells
2.5. Transforming Growth Factor β1 (TGF-β1) Induced EMT in Human Keratinocyte HaCaT Cells
2.6. Quercetin Attenuated TGF-β1-induced EMT in HaCaT Cells
3. Discussion
4. Materials and Methods
4.1. Reagents and Antibodies
4.2. Cell Culture and Treatment
4.3. MTT Assay
4.4. Cell Cycle Analysis
4.5. Wound-Healing Assay
4.6. Western Blot Analysis
4.7. Gelatin Zymography
4.8. Immunofluorescence Staining
4.9. Invasion Assay
4.10. Statistical Analyses
Author Contributions
Funding
Conflicts of Interest
References
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Sample Availability: Samples are available from the corresponding authors. |
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Kim, S.R.; Lee, E.Y.; Kim, D.J.; Kim, H.J.; Park, H.R. Quercetin Inhibits Cell Survival and Metastatic Ability via the EMT-Mediated Pathway in Oral Squamous Cell Carcinoma. Molecules 2020, 25, 757. https://doi.org/10.3390/molecules25030757
Kim SR, Lee EY, Kim DJ, Kim HJ, Park HR. Quercetin Inhibits Cell Survival and Metastatic Ability via the EMT-Mediated Pathway in Oral Squamous Cell Carcinoma. Molecules. 2020; 25(3):757. https://doi.org/10.3390/molecules25030757
Chicago/Turabian StyleKim, So Ra, Eun Young Lee, Da Jeong Kim, Hye Jung Kim, and Hae Ryoun Park. 2020. "Quercetin Inhibits Cell Survival and Metastatic Ability via the EMT-Mediated Pathway in Oral Squamous Cell Carcinoma" Molecules 25, no. 3: 757. https://doi.org/10.3390/molecules25030757