Probucol suppresses human glioma cell proliferation in vitro via ROS production and LKB1-AMPK activation.
OBJECTIVE
Probucol, an anti-hyperlipidemic drug, has been reported to exert antitumor activities at various stages of tumor initiation, promotion and progression. In this study we examined whether the drug affected glioma cell growth in vitro and the underlying mechanisms.


METHODS
Human glioma U87 and glioblastoma SF295 cell lines were used. Cell proliferation was accessed using the cell proliferation assay and BrdU incorporation. The phosphorylation of AMPK, liver kinase B1 (LKB1) and p27(Kip1) was detected by Western blot. The activity of 26S proteasome was assessed with an in situ fluorescent substrate. siRNAs were used to suppress the expression of the relevant signaling proteins.


RESULTS
Treatment of U87 glioma cells with probucol (10-100 μmol/L) suppressed the cell proliferation in dose- and time dependent manners. Meanwhile, probucol markedly increased the ROS production, phosphorylation of AMPK at Thr172 and LKB1 at Ser428 in the cells. Furthermore, probucol significantly decreased 26S proteasome activity and increased p27(Kip1) protein level in the cells in an AMPK-dependent manner. Probucol-induced suppression of U87 cell proliferation could be reversed by pretreatment with tempol (a superoxide dismutase mimetic), MG132 (proteasome inhibitor) or compound C (AMPK inhibitor), or by gene silencing of LKB1, AMPK or p27(Kip1). Similar results were observed in probucol-treated SF295 cells.


CONCLUSIONS
Probucol suppresses human glioma cell proliferation in vitro via ROS production and LKB1-AMPK activation, which reduces 26S proteasome-dependent degradation of p27(Kip1).
Tumor Center, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Yong-sheng
YS
Jiang
Department of Neurology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Jing-an
JA
Lei
Department of Neurology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Fang
F
Feng
Department of Neurology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Qi-ming
QM
Liang
Department of Neurology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Fu-rong
FR
Wang
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005910:Glioma / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects*; D010766:Phosphorylation; D011341:Probucol / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
D000970:Antineoplastic Agents; D000975:Antioxidants; C436291:CDKN1B protein, human; D061988:Proteasome Inhibitors; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D011341:Probucol