Down-Regulation of Claudin-2 Expression by Cyanidin-3-Glucoside Enhances Sensitivity to Anticancer Drugs in the Spheroid of Human Lung Adenocarcinoma A549 Cells
Abstract
:1. Introduction
2. Results
2.1. Effects of C3G, P3G, and Cyanidin on the Protein Level of CLDN2 in A549 Cells
2.2. Effect of Anthocyanins on the Cell Localization of CLDN2
2.3. Effect of Anthocyanins on the mRNA Level of CLDN2
2.4. Effect of C3G on Intracellular Signaling Pathways
2.5. Effect of C3G on the Protein Stability and Transcriptional Activity of CLDN2
2.6. Effects of Endocytosis, Lysosome and Protein Phosphatase Inhibitors on the C3G-Induced CLDN2 Reduction
2.7. Effect of p38 Mitogen-Activated Protein Kinase (MAPK) Inhibitor on the C3G-Induced CLDN2 Reduction
2.8. Effect of p38 MAPK Inhibitor on the Stability of CLDN2 Protein
2.9. Effect of C3G on Transepithelial Electrical Resistance (TER) and Paracellular Permeability
2.10. Effect of C3G on the DXR-Induced Cytotoxicity in the Spheroid Model
2.11. Effects of Anthocyanins on Apoptotic and Necrotic Cell Death Induced by DXR
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cell Culture
4.3. Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting
4.4. RNA Isolation and Quantitative Real-Time PCR
4.5. Immunofluorescence Measurement
4.6. Luciferase Reporter Assay
4.7. TER and Paracellular Permeability
4.8. D Spheroid Model
4.9. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
CAN | Cantharidin |
CHL | Chloroquine |
CLDN | Claudin |
Ct | Threshold cycle |
C3G | Cyanidin-3-glucoside |
DMEM | Dulbecco’s Modified Eagle’s medium |
DXR | Doxorubicin |
ERK | Extracellular signal-regulated kinase |
FasL | Fas ligand |
LY | Lucifer yellow |
MAPK | Mitogen-activated protein kinase |
MβCD | Methyl-β-cyclodextrin |
MDC | Monodansylcadaverine |
MEK | Mitogen-activated protein kinase |
PCR | Polymerase chain reaction |
PDK1 | Phosphoinositide-dependent kinase 1 |
PI3K | Phosphoinositide 3-kinase |
P3G | Pelargonidin-3-glucoside |
p-Ser | Phosphoserine |
p-Thr | Phosphothreonine |
SDS-PAGE | SDS-polyacrylamide gel electrophoresis |
siRNA | Small interfering RNA |
Stat3 | Signal transducer and activator of transcription 3 |
TER | Transepithelial electrical resistance |
TJ | Tight junction |
WST-1 | 4-[3-[4-Iodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] |
ZO-1 | Zonula occludens-1 |
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Eguchi, H.; Matsunaga, H.; Onuma, S.; Yoshino, Y.; Matsunaga, T.; Ikari, A. Down-Regulation of Claudin-2 Expression by Cyanidin-3-Glucoside Enhances Sensitivity to Anticancer Drugs in the Spheroid of Human Lung Adenocarcinoma A549 Cells. Int. J. Mol. Sci. 2021, 22, 499. https://doi.org/10.3390/ijms22020499
Eguchi H, Matsunaga H, Onuma S, Yoshino Y, Matsunaga T, Ikari A. Down-Regulation of Claudin-2 Expression by Cyanidin-3-Glucoside Enhances Sensitivity to Anticancer Drugs in the Spheroid of Human Lung Adenocarcinoma A549 Cells. International Journal of Molecular Sciences. 2021; 22(2):499. https://doi.org/10.3390/ijms22020499
Chicago/Turabian StyleEguchi, Hiroaki, Haruka Matsunaga, Saki Onuma, Yuta Yoshino, Toshiyuki Matsunaga, and Akira Ikari. 2021. "Down-Regulation of Claudin-2 Expression by Cyanidin-3-Glucoside Enhances Sensitivity to Anticancer Drugs in the Spheroid of Human Lung Adenocarcinoma A549 Cells" International Journal of Molecular Sciences 22, no. 2: 499. https://doi.org/10.3390/ijms22020499