Mechanisms of PKC-Mediated Enhancement of HIF-1α Activity and its Inhibition by Vitamin K2 in Hepatocellular Carcinoma Cells
Abstract
:1. Introduction
2. Results
2.1. PKC-δ—But not PKC-α or PKC-ε—Is Involved in the Activation of HIF-1α and Inhibition by VK2 Under Both Normoxic and Hypoxic Conditions
2.2. PKC-δ is Involved in the HIF-1α Protein Expression, and VK2 Suppresses the TPA-Induced HIF-1α Protein Expression
2.3. PKC-δ Regulates the TPA-Induced Recruitment of HIF-1α, and VK2 Abrogates the Induction of HIF-1α Recruitment by TPA in Huh7 Cells
2.4. PKC-δ Knockdown and VK2 Suppresses the Nuclear Translocation of HIF-1α Stimulated by TPA
3. Discussion
4. Materials and Methods
4.1. Cell Culture, Hypoxic Conditions and Reagents
4.2. Luciferase Reporter Gene Assay
4.3. Real-Time qRT-PCR, Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction
4.4. Western Blotting
4.5. Chromatin Immunoprecipitation (ChIP) Assay
4.6. Fluorescence Microscope Assay
4.7. SiRNA-Mediated Knockdown of PKC
4.8. Statistical Analyses
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
HIF-1α | Hypoxia-inducible factor-1 alpha |
PKC | Protein kinase C |
TPA | 12-O-tetradecanoylphorbor-13-acetate |
TF | Transcription factor |
VK2 | Vitamin K2 |
HRE | Hypoxia responsive element |
VEGF | Vascular endothelial growth factor |
EPO | Erythropoietin |
VHL | Von Hippel-Lindau |
NF-κB | Nuclear factor kappa B |
FBS | Fetal bovine serum |
DMEM | Dulbecco’s modified Eagle`s medium |
DMSO | Dimethyl sulfoxide |
RT-PCR | Reverse transcription polymerase chain reaction |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
ChIP assay | Chromatin immunoprecipitation assay |
HCC | Hepatocellular carcinoma |
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Xia, J.; Ozaki, I.; Matsuhashi, S.; Kuwashiro, T.; Takahashi, H.; Anzai, K.; Mizuta, T. Mechanisms of PKC-Mediated Enhancement of HIF-1α Activity and its Inhibition by Vitamin K2 in Hepatocellular Carcinoma Cells. Int. J. Mol. Sci. 2019, 20, 1022. https://doi.org/10.3390/ijms20051022
Xia J, Ozaki I, Matsuhashi S, Kuwashiro T, Takahashi H, Anzai K, Mizuta T. Mechanisms of PKC-Mediated Enhancement of HIF-1α Activity and its Inhibition by Vitamin K2 in Hepatocellular Carcinoma Cells. International Journal of Molecular Sciences. 2019; 20(5):1022. https://doi.org/10.3390/ijms20051022
Chicago/Turabian StyleXia, Jinghe, Iwata Ozaki, Sachiko Matsuhashi, Takuya Kuwashiro, Hirokazu Takahashi, Keizo Anzai, and Toshihiko Mizuta. 2019. "Mechanisms of PKC-Mediated Enhancement of HIF-1α Activity and its Inhibition by Vitamin K2 in Hepatocellular Carcinoma Cells" International Journal of Molecular Sciences 20, no. 5: 1022. https://doi.org/10.3390/ijms20051022