Inhibition of autophagosome-lysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint.
Modulation of autophagy has been increasingly regarded as a promising cancer therapeutic approach. In this study, we screened several ginsenosides extracted from Panax ginseng and identified ginsenoside Ro (Ro) as a novel autophagy inhibitor. Ro blocked the autophagosome-lysosome fusion process by raising lysosomal pH and attenuating lysosomal cathepsin activity, resulting in the accumulation of the autophagosome marker MAP1LC3B/LC3B and SQSTM1/p62 (sequestosome 1) in various esophageal cancer cell lines. More detailed studies demonstrated that Ro activated ESR2 (estrogen receptor 2), which led to the activation of NCF1/p47(PHOX) (neutrophil cytosolic factor 1), a subunit of NADPH oxidase, and subsequent reactive oxygen species (ROS) production. Treatment with siRNAs or inhibitors of the ESR2-NCF1-ROS axis, such as N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), apocynin (ACN), Tiron, and Fulvestrant apparently decreased Ro-induced LC3B-II, GFP-LC3B puncta, and SQSTM1, indicating that ROS instigates autophagic flux inhibition triggered by Ro. More importantly, suppression of autophagy by Ro sensitized 5-fluorouracil (5-Fu)-induced cell death in chemoresistant esophageal cancer cells. 5-Fu induced prosurvival autophagy, and by inhibiting such autophagy, siRNAs against BECN1/beclin 1, ATG5, ATG7, and LC3B enhanced 5-Fu-induced autophagy-associated and apoptosis-independent cell death. We observed that Ro potentiates 5-Fu cytotoxicity via delaying CHEK1 (checkpoint kinase 1) degradation and downregulating DNA replication process, resulting in the delayed DNA repair and the accumulation of DNA damage. In summary, these data suggest that Ro is a novel autophagy inhibitor and could function as a potent anticancer agent in combination therapy to overcome chemoresistance.
a Department of Pharmacy , School of Medicine, Innovation Platform for Natural Small Molecule Drugs, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, Shenzhen University , Shenzhen , China.|b Guangzhou Jinan Biomedicine Research and Development Center, College of Life Science and Technology, Jinan University , Guangzhou , China.
Kai
K
Zheng
c The First Affiliated Hospital of Kunming Medical University , Kunming , China.
Yan
Y
Li
a Department of Pharmacy , School of Medicine, Innovation Platform for Natural Small Molecule Drugs, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, Shenzhen University , Shenzhen , China.
Shaoxiang
S
Wang
b Guangzhou Jinan Biomedicine Research and Development Center, College of Life Science and Technology, Jinan University , Guangzhou , China.
Xiao
X
Wang
a Department of Pharmacy , School of Medicine, Innovation Platform for Natural Small Molecule Drugs, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, Shenzhen University , Shenzhen , China.
Chenghui
C
Liao
a Department of Pharmacy , School of Medicine, Innovation Platform for Natural Small Molecule Drugs, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, Shenzhen University , Shenzhen , China.
Xiaopeng
X
Hu
a Department of Pharmacy , School of Medicine, Innovation Platform for Natural Small Molecule Drugs, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, Shenzhen University , Shenzhen , China.
Long
L
Fan
a Department of Pharmacy , School of Medicine, Innovation Platform for Natural Small Molecule Drugs, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, Shenzhen University , Shenzhen , China.
Qiangrong
Q
Kang
c The First Affiliated Hospital of Kunming Medical University , Kunming , China.
Yong
Y
Zeng
a Department of Pharmacy , School of Medicine, Innovation Platform for Natural Small Molecule Drugs, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, Shenzhen University , Shenzhen , China.
Xuli
X
Wu
a Department of Pharmacy , School of Medicine, Innovation Platform for Natural Small Molecule Drugs, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, Shenzhen University , Shenzhen , China.
Haiqiang
H
Wu
a Department of Pharmacy , School of Medicine, Innovation Platform for Natural Small Molecule Drugs, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, Shenzhen University , Shenzhen , China.
Jian
J
Zhang
b Guangzhou Jinan Biomedicine Research and Development Center, College of Life Science and Technology, Jinan University , Guangzhou , China.
Yifei
Y
Wang
a Department of Pharmacy , School of Medicine, Innovation Platform for Natural Small Molecule Drugs, Shenzhen Key Laboratory of Novel Natural Health Care Products, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, Shenzhen University , Shenzhen , China.
Zhendan
Z
He
D000818:Animals; D017209:Apoptosis; D000071182:Autophagosomes / Q000378:metabolism*; D001343:Autophagy; D002453:Cell Cycle; D002470:Cell Survival; D000071877:Checkpoint Kinase 1 / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004249:DNA Damage; D004938:Esophageal Neoplasms / Q000378:metabolism*; D047629:Estrogen Receptor beta / Q000378:metabolism; D005472:Fluorouracil / Q000494:pharmacology; D036145:Ginsenosides / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D019255:NADPH Oxidases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D014709:Vero Cells
C000600034:ESR2 protein, human; D047629:Estrogen Receptor beta; D036145:Ginsenosides; D017382:Reactive Oxygen Species; D049452:Green Fluorescent Proteins; C064378:ginsenoside Ro; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; D005472:Fluorouracil
DNA damage checkpoint; NADPH oxidases; ROS; autophagic flux; chemoresistance; esophageal cancer; estrogen receptor; ginsenoside Ro