Blocking autophagy enhances meloxicam lethality to hepatocellular carcinoma by promotion of endoplasmic reticulum stress.
OBJECTIVE
Meloxicam, a selective cyclooxygenase-2 (COX-2) inhibitor, has been demonstrated to exert anti-tumour effects against various malignancies. However, up to now, mechanisms involved in meloxicam anti-hepatocellular carcinoma effects have remained unclear.


METHODS
Cell viability and apoptosis were assessed by CCK-8 and flow cytometry. Endoplasmic reticulum (ER) stress and autophagy-associated molecules were analysed by western blotting and immunofluorescence assay. GRP78 and Atg5 knock-down by siRNA or chemical inhibition was used to investigate cytotoxic effects of meloxicam treatment on HCC cells.


RESULTS
We found that meloxicam led to apoptosis and autophagy in HepG2 and Bel-7402 cells via a mechanism that involved ER stress. Up-regulation of GRP78 signalling pathway from meloxicam-induced ER stress was critical for activation of autophagy. Furthermore, autophagy activation attenuated ER stress-related cell death. Blocking autophagy by 3-methyladenine (3-MA) or Atg5 siRNA knock-down enhanced meloxicam lethality for HCC by activation of ER stress-related apoptosis. In addition, GRP78 seemed to lead to autophagic activation via the AMPK-mTOR signalling pathway. Blocking AMPK with a chemical inhibitor inhibited autophagy suggesting that meloxicam-regulated autophagy requires activation of AMPK.


CONCLUSIONS
Our results revealed that both ER stress and autophagy were involved in cell death evoked by meloxicam in HCC cells. This inhibition of autophagy to enhance meloxicam lethality, suggests a novel therapeutic strategy against HCC.
Department of General Surgery, Qianfoshan Hospital, Shandong University, Jinan, Shandong, 250014, China.
Jingtao
J
Zhong
Department of Hepatobiliary Surgery, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, 530021, China.
Xiaofeng
X
Dong
Department of General Surgery, Qianfoshan Hospital, Shandong University, Jinan, Shandong, 250014, China.
Peng
P
Xiu
Department of General Surgery, Qianfoshan Hospital, Shandong University, Jinan, Shandong, 250014, China.
Fuhai
F
Wang
Laboratory of Medicrovascular Medicine and Medical Research Center, Qianfoshan Hospital, Shandong University, Jinan, Shandong, 250014, China.
Ju
J
Liu
Department of General Surgery, Qianfoshan Hospital, Shandong University, Jinan, Shandong, 250014, China.
Honglong
H
Wei
Department of General Surgery, Qianfoshan Hospital, Shandong University, Jinan, Shandong, 250014, China.
Zongzhen
Z
Xu
Department of General Surgery, Qianfoshan Hospital, Shandong University, Jinan, Shandong, 250014, China.
Feng
F
Liu
Department of General Surgery, Qianfoshan Hospital, Shandong University, Jinan, Shandong, 250014, China.
Tao
T
Li
Department of General Surgery, Qianfoshan Hospital, Shandong University, Jinan, Shandong, 250014, China.
Jie
J
Li
D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D000071187:Autophagy-Related Protein 5; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy*; D000077239:Meloxicam; D008869:Microtubule-Associated Proteins / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering; D013843:Thiazines / Q000494:pharmacology*; D013844:Thiazoles / Q000494:pharmacology*
C502778:ATG5 protein, human; D000970:Antineoplastic Agents; D000071187:Autophagy-Related Protein 5; D052246:Cyclooxygenase 2 Inhibitors; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D013843:Thiazines; D013844:Thiazoles; C025946:3-methyladenine; D055372:AMP-Activated Protein Kinases; D000225:Adenine; D000077239:Meloxicam