Kaempherol and Luteolin Decrease Claudin-2 Expression Mediated by Inhibition of STAT3 in Lung Adenocarcinoma A549 Cells
Abstract
:1. Introduction
2. Experimental Section
2.1. Materials
2.2. Cell Culture and Transfection
2.3. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting
2.4. Measurement of O2− Scavenging Activity
2.5. RNA Isolation and Polymerase Chain Reaction (PCR)
2.6. Luciferase Reporter Assay
2.7. Chromatin Immunoprecipitation (ChIP) Assay
2.8. Cell Proliferation
2.9. Statistics
3. Results
3.1. Effects of Flavonoids on Claudin-2 Expression in A549 Cells
3.2. Effects of Antioxidant Capacity on Claudin-2 Expression
3.3. Effects of Flavonoids on mRNA Levels of Junctional Protein and Intracellular Signaling Pathway
3.4. Effects of Flavonoids on the Promoter Activity of Claudin-2
3.5. Inhibition of Association between STAT3 and Claudin-2 Promoter by Flavonoids
3.6. Effects of Three Flavonoids and Ectopic Claudin-2 Expression on Cell Proliferation
4. Discussion
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
cdx2 | caudal-related homeobox 2 |
ChIP | chromatin immunoprecipitation |
COX | cyclooxygenase |
DMEM | Dulbecco’s Modified Eagle’s medium |
DMSO | Dimethyl sulfoxide |
ERK | extracellular signal-regulated kinase |
FCS | fetal calf serum |
NAC | N-acetyl cysteine |
MDCK | Madin Darby canine kidney |
MEK | mitogen-activated protein kinase kinase |
NF-κB | nuclear factor-κB |
PCR | polymerase chain reaction |
PI3K | phosphoinositide 3-kinase |
SDS-PAGE | SDS-polyacrylamide gel electrophoresis |
STAT | signal transducers and activators of transcription |
TJs | tight junctions |
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Name | Direction | Sequence |
---|---|---|
Claudin-1 | Forward | 5′-ATGAGGATGGCTGTCATTGG-3′ |
Claudin-1 | Reverse | 5′-ATTGACTGGGGTCATAGGGT-3′ |
Claudin-2 | Forward | 5′-ATTGTGACAGCAGTTGGCTT-3′ |
Claudin-2 | Reverse | 5′-CTATAGATGTCACACTGGGTGATG-3′ |
Occludin | Forward | 5′-TTTGTGGGACAAGGAACACA-3′ |
Occludin | Reverse | 5′-TCATTCACTTTGCCATTGGA-3′ |
E-cadherin | Forward | 5′-ACCCCCTGTTGGTGTCTTT-3′ |
E-cadherin | Reverse | 5′-TTCGGGCTTGTTGTCATTCT-3′ |
β-actin | Forward | 5′-CCTGAGGCACTCTTCCAGCCTT-3′ |
β-actin | Reverse | 5′-TGCGGATGTCCACGTCACACTTC-3′ |
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Sonoki, H.; Tanimae, A.; Endo, S.; Matsunaga, T.; Furuta, T.; Ichihara, K.; Ikari, A. Kaempherol and Luteolin Decrease Claudin-2 Expression Mediated by Inhibition of STAT3 in Lung Adenocarcinoma A549 Cells. Nutrients 2017, 9, 597. https://doi.org/10.3390/nu9060597
Sonoki H, Tanimae A, Endo S, Matsunaga T, Furuta T, Ichihara K, Ikari A. Kaempherol and Luteolin Decrease Claudin-2 Expression Mediated by Inhibition of STAT3 in Lung Adenocarcinoma A549 Cells. Nutrients. 2017; 9(6):597. https://doi.org/10.3390/nu9060597
Chicago/Turabian StyleSonoki, Hiroyuki, Asami Tanimae, Satoshi Endo, Toshiyuki Matsunaga, Takumi Furuta, Kenji Ichihara, and Akira Ikari. 2017. "Kaempherol and Luteolin Decrease Claudin-2 Expression Mediated by Inhibition of STAT3 in Lung Adenocarcinoma A549 Cells" Nutrients 9, no. 6: 597. https://doi.org/10.3390/nu9060597