Abstract
Hepatocellular carcinoma (HCC) is one of the most fatal malignancies with high mortality worldwide. Here the underlying antitumor mechanism of gallotannin was elucidated in HCC cells. Gallotannin suppressed viability and colony formation, increased subG1 portion and also induced senescence via upregulation of p21, G0/G1 arrest and higher SA-β-gal activity in HepG2 and SK-Hep1 cells. However, pan-caspase inhibitor Z-VAD-FMK reversed the ability of gallotannin to activate caspase 3 at 48 h after treatment in two HCC cells. Of note, gallotannin also induced autophagic features by increasing LC3 punctae, LC3B-II conversion, autophagic vacuoles and decreasing the expression of Beclin1 in two HCC cells. Furthermore, autophagy flux assay using GFP–mRFP–LC3 plasmid revealed increased yellowish color and late autophagy inhibitor CQ or NH4Cl enhanced cytotoxicity, LC3B-II conversion, and LC3 punctae in gallotannin-treated HepG2 and SK-Hep1 cells compared to early autophagy inhibitor 3-MA or wortmannin. Interestingly, gallotannin attenuated the expression of SIRT1 and mTOR and activated phosphorylation of AMPK in two HCC cells. Furthermore, AMPK activator AICAR significantly enhanced SA-β-gal activity and antiproliferation induced by gallotannin, while AMPK inhibitor compound C did not in two HCC cells. Consistently, LC3B-II conversion by gallotannin was not shown in AMPKα1 −/− MEF cells compared to WT AMPK +/+ MEF cells. Consistently, gallotannin reduced in vivo growth of HepG2 cells implanted in NCr nude mice along with decreased expression of PCNA and SIRT1 and increased AMPKα1 and TUNEL. Overall, these findings highlight evidence that regulation of SIRT1/AMPK is critically involved in gallotannin-induced senescence and impaired autophagy leading to cell death in HCC cells.
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16 July 2018
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Abbreviations
- p62/SQSTM1:
-
p62 sequestosome 1
- Gal:
-
Gallotannin
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- HCC:
-
Human hepatocellular carcinoma
- HepG2:
-
Human epithelial hepatocellular carcinoma
- SK-Hep1:
-
Human epithelial hepatic adenocarcinoma
- MEF:
-
Mouse embryo fibroblast
- CQ:
-
Chloroquine
- NH4Cl:
-
Ammoniun chloride
- AICAR:
-
5-Aminoimidazole-4-carboxamide ribonucleotide
- Com. C:
-
Compound C
- LC3:
-
Microtubule-associated protein 1 light chain 3
- LC3-I:
-
Soluble unlipidated form of LC3
- LC3-II:
-
LC3–phospholipid conjugate
- AMPK:
-
AMP-activated protein kinase
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- FITC:
-
Fluorescein isothiocyanate
- GFP/RFP:
-
Green fluorescent protein/red fluorescent protein
- SIRT1:
-
NAD-dependent deacetylase sirtuin-1
- Caspase:
-
Cysteine aspartyl-specific protease
- 3-MA:
-
3-Methyladenine
- SA-β-gal:
-
Senescence-associated β-galactosidase
- TEM:
-
Transmission electron microscopy
- TUNEL:
-
Terminal deoxynucleotidyl transferase dUTP nick end labeling
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Acknowledgements
This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (2014R1A2A10052872).
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HY Kwon designed this project, performed most of the experiments, and drafted the manuscript; JH Kim contributed to animal study; SK. Srivastava and BL Kim advised us of the experiment design and helped proofreading; SH Kim supervised all experiments and wrote the manuscript. All authors read and approved the final manuscript.
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204_2017_2021_MOESM1_ESM.jpg
Supplementary Figure 1. Gallotannin induced weak apoptosis in HCC cells in a time and concentration dependent fashion. a Cell cycle analysis of gallotannin treated HepG2 and SK-Hep1 cells. Cells were treated with gallotannin indicated concentrations and for different times. Then the cell cycle analysis was performed using FACs Calibur, and the cell cycle was analyzed using CellQuest Software. b SubG1 portion was increased dependent on time and concentration in gallotannin treated HepG2 and SK-Hep1 cells. The results of cell cycle analysis were represented as relative percentage ± SD. SubG1 portion of each treatment was represented (means ± SD of three independent experiments, **p < 0.01 and *** p < 0.001 vs. control by One-Way ANOVA). (JPEG 3334 kb)
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Kwon, H.Y., Kim, JH., Kim, B. et al. Regulation of SIRT1/AMPK axis is critically involved in gallotannin-induced senescence and impaired autophagy leading to cell death in hepatocellular carcinoma cells. Arch Toxicol 92, 241–257 (2018). https://doi.org/10.1007/s00204-017-2021-y
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DOI: https://doi.org/10.1007/s00204-017-2021-y