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Apoptosis

, Volume 22, Issue 11, pp 1473–1484 | Cite as

Piperlongumine induces G2/M phase arrest and apoptosis in cholangiocarcinoma cells through the ROS-JNK-ERK signaling pathway

Original Paper

Abstract

Cholangiocarcinoma (CCA) is an aggressive, metastatic bile duct cancer. CCA is difficult to diagnose, and responds poorly to current radio- and chemo-therapy. Piperlongumine (PL) is a naturally-occurring small molecule selectively toxic to cancer cells by targeting reactive oxygen species (ROS). In this study, we demonstrated the potential anticancer activity of PL in CCA. PL markedly induced death in CCA cell lines in a dose- and time-dependent manner through the activation of caspase-3 and PARP. PL also stimulated ROS accumulation in CCA. Co-exposure of PL with the ROS scavenger N-acetyl-l-cysteine or GSH completely blocked PL-induced apoptosis in CCA cell lines. Increased p21 via the p53-independent pathway in PL-treated CCA cells led to G2/M phase arrest and cell apoptosis. In addition, the study showed that PL trigger CCA cell lines death through JNK-ERK activation. Furthermore, the different antioxidant capacity of CCA cell lines also indicates the susceptibility of the cells to PL treatment. Our findings reveal that PL exhibits anti-tumor activity and has potential to be used as a chemotherapeutic agent against CCA.

Keywords

Cholangiocarcinoma Piperlongumine Anti-tumor activity Reactive oxygen species Cell apoptosis 

Abbreviations

pAKT

A three letter name (not an abbreviation); “p” stands for the phosphorylated (active) form

BCL-2

B cell lymphoma gene 2

BAX

BCL-2-associated protein X

BAD

BCL-2-associated death promoter

pERK

Phosphorylated (active) extracellular-signal-regulated kinase

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

HRP

Horseradish peroxidase

pJNK

Phosphorylated (active) C-Jun NH2-terminal kinase

PARP

Poly (ADP-ribose) polymerase

p21

21 kDa cyclin-dependent kinase inhibitor

p53

53 kDa tumor suppressor protein

rpm

Revolutions per minute

Notes

Acknowledgements

The authors thank (a) Professor Banchob Sripa of the Department of Pathology, Faculty of Medicine, Khon Kaen University for providing the cholangiocarcinoma cells; (b) the Biochemistry Laboratory, the Center for Scientific and Technological Equipment, Suranaree University of Technology for providing the research facilities; (c) Professor Jong-Lyel Roh of Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea for providing some of the piperlongumine; and, (d) Mr. Bryan Roderick Hamman for assistance with the English-language presentation of the manuscript.

Funding

This work was supported by grants from Suranaree University of Technology (Grant Number: SUT1-102-57-24-23) and the Young Research Grants, Thailand Research Fund (MRG6080055) to CS. HC was supported by Grants (NRF-2015K1A4A3046807 and 2008-0062286) from the National Research Foundation of Korea and a Grant (2017-307) from the Asan Institute for Life Sciences, Seoul, Korea.

Author contributions

ST carried out all the experiments, performed data analysis, and prepared the manuscript draft. WS was provided advice on the cytotoxicity tests and data analysis. KJL supported and provided advice on the western blot analysis. HC was a grant holder, conceived the research, provided guidance on data analysis, revised and approved the manuscript. CT was a grant holder, designed the experiment, provided advice on the cell cycle analysis, ROS measurement, apoptosis assay, gene expression, and data analysis, revised and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

10495_2017_1422_MOESM1_ESM.tif (4.6 mb)
Supplement Fig1. Anit-proliferative activity of PL on KKU-100 and NIH3T3. (a) KKU-100 and NIH3T3 were grown in 96-well plates for 24 h then treated with various concentration of PL range from 0-100 µM for 24 and 48 h. Cell viability was measured using SRB assays. Percentages of cell viability relative to control (without PL) are shown as mean ± SD from three independent experiments. *P < 0.05. (b) Western blot analysis of PL treated with NIH3T3. NIH3T3 was treated with PL at 10 µM for 0, 3, 6, 12, and 24 h. Activation of caspase-3, and PARP were examined using western blot analysis. β-actin was used as an equal loading control for normalization. (TIF 4716 KB)

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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  1. 1.Biochemistry-Electrochemistry Research Unit and School of Chemistry, Institute of ScienceSuranaree University of TechnologyNakhon RatchasimaThailand
  2. 2.Center of Excellence on Advanced Functional MaterialsSuranaree University of TechnologyNakhon RatchasimaThailand
  3. 3.Department of Convergence Medicine, Asan Institute for Life SciencesUniversity of Ulsan College of Medicine, Asan Medical CenterSeoulSouth Korea
  4. 4.Department of Physiology, Asan-Minnesota Institute for Innovating Transplantation, Bio-Medical Institute of TechnologyUniversity of Ulsan College of Medicine, Asan Medical CenterSeoulSouth Korea

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