Novel CHOP activator LGH00168 induces necroptosis in A549 human lung cancer cells via ROS-mediated ER stress and NF-κB inhibition.
OBJECTIVE
C/EBP homologous protein (CHOP) is a transcription factor that is activated at multiple levels during ER stress and plays an important role in ER stress-induced apoptosis. In this study we identified a novel CHOP activator, and further investigated its potential to be a therapeutic agent for human lung cancer.


METHODS
HEK293-CHOP-luc reporter cells were used in high-throughput screening (HTS) to identify CHOP activators. The cytotoxicity against cancer cells in vitro was measured with MTT assay. The anticancer effects were further examined in A549 human non-small cell lung cancer xenograft mice. The mechanisms underlying CHOP activation were analyzed using luciferase assays, and the anticancer mechanisms were elucidated in A549 cells.


RESULTS
From chemical libraries of 50 000 compounds, LGH00168 was identified as a CHOP activator, which showed cytotoxic activities against a panel of 9 cancer cell lines with an average IC50value of 3.26 μmol/L. Moreover, administration of LGH00168 significantly suppressed tumor growth in A549 xenograft bearing mice. LGH00168 activated CHOP promoter via AARE1 and AP1 elements, increased DR5 expression, decreased Bcl-2 expression, and inhibited the NF-κB pathway. Treatment of A549 cells with LGH00168 (10 μmol/L) did not induce apoptosis, but lead to RIP1-dependent necroptosis, accompanied by cell swelling, plasma membrane rupture, lysosomal membrane permeabilization, MMP collapse and caspase 8 inhibition. Furthermore, LGH00168 (10 and 20 μmol/L) dose-dependently induced mito-ROS production in A549 cells, which was reversed by the ROS scavenger N-acetyl-L-cysteine (NAC, 10 mmol/L). Moreover, NAC significantly diminished LGH00168-induced CHOP activation, NF-κB inhibition and necroptosis in A549 cells.


CONCLUSIONS
LGH00168 is a CHOP activator that inhibits A549 cell growth in vitro and lung tumor growth in vivo.
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Yi-Ming
YM
Ma
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Yan-Min
YM
Peng
Department of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
Qiong-Hua
QH
Zhu
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
An-Hui
AH
Gao
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100871, China.
Bo
B
Chao
Department of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
Qiao-Jun
QJ
He
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Jia
J
Li
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
You-Hong
YH
Hu
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100871, China.
Yu-Bo
YB
Zhou
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor / Q000187:drug effects; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D009336:Necrosis; D011719:Pyrazines / Q000494:pharmacology / Q000627:therapeutic use*; D011743:Pyrimidines / Q000494:pharmacology / Q000627:therapeutic use*; D017382:Reactive Oxygen Species / Q000378:metabolism*
D000970:Antineoplastic Agents; C000613885:LGH00168; D016328:NF-kappa B; D011719:Pyrazines; D011743:Pyrimidines; D017382:Reactive Oxygen Species