The activation of c-Jun NH₂-terminal kinase is required for dihydroartemisinin-induced autophagy in pancreatic cancer cells.
BACKGROUND
c-Jun NH₂-terminal kinases (JNKs) are strongly activated by a stressful cellular environment, such as chemotherapy and oxidative stress. Autophagy is a protein-degradation system in which double-membrane vacuoles called autophagosomes are formed. The autophagy-related gene Beclin 1 plays a key role in this process. We previously found that autophagy was induced by dihydroartemisinin (DHA) in pancreatic cancer cells. However, little is known about the complex relationship between ROS, JNK activation, autophagy induction, and Beclin 1 expression.


METHODS
Cell viability and CCK-8 assays were carried out to determine the cell proliferation; small interfering RNAs (siRNAs) were used to knockdown c-Jun NH₂-terminal kinases (JNK1/2) genes; western blot was performed to detect the protein expression of LC3, JNK, Beclin 1, caspase 3 and β-actin; production of intracellular ROS was analyzed using FACS flow cytometry; autophagy induction was confirmed by electron microscopy.


RESULTS
In the present study, we explored the role of DHA and Beclin 1 expression in autophagy. DHA-treated cells showed autophagy characteristics, and DHA also activated the JNK pathway and up-regulated the expression of Beclin 1. Conversely, blocking JNK signaling inhibited Beclin 1 up-regulation. JNK activation was found to primarily depend on reactive oxygen species (ROS) resulting from the DHA treatment. Moreover, JNK pathway inhibition and Beclin 1 silencing prevented the induction of DHA-induced autophagy.


CONCLUSIONS
These results suggest that the induction of autophagy by DHA is required for JNK-mediated Beclin 1 expression.
Guang
G
Jia
Rui
R
Kong
Zhi-Bin
ZB
Ma
Bing
B
Han
Yong-Wei
YW
Wang
Shang-Ha
SH
Pan
Ying-Hua
YH
Li
Department of Pancreatic and Biliary Surgery; Key Laboratory of Hepatosplenic Surgery, the First Affiliated Hospital of Harbin Medical University, Harbin, China. wuliang_227@163.com.
Bei
B
Sun
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D037621:Artemisinins / Q000494:pharmacology*; D001343:Autophagy; D000071186:Beclin-1; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D004789:Enzyme Activation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D010190:Pancreatic Neoplasms; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D037621:Artemisinins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D017382:Reactive Oxygen Species; C039060:artenimol; D048031:JNK Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; D053148:Caspase 3