Activation of c-Jun N-terminal kinase is essential for mitochondrial membrane potential change and apoptosis induced by doxycycline in melanoma cells.
OBJECTIVE
Tetracyclines were recently found to induce tumour cell death, but the early processes involved in this cytotoxic effect remain unclear.


METHODS
Viability of human and mouse melanoma cells was determined by MTT assay and flow cytometry. Kinase/protein/caspase activation was measured by Western blotting and mitochondrial membrane potential (DeltaPsi(m)) was analyzed by fluorescence microscopy and flow cytometry.


RESULTS
Human and mouse melanoma cells were treated with doxycycline or minocycline but only doxycycline was cytotoxic. This cell death (apoptosis) in A2058 cells involved activation of caspase-3, -7 and -9 and contributed to inhibition, by doxycycline, of matrix metalloproteinase (MMP) activity and migration of these cells. Doxycycline induced intra-cellular reactive oxygen species (ROS) production, apoptosis signal-regulated kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activation at an early stage of treatment and induced mitochondrial cytochrome c release into cytosol and DeltaPsi(m) change during apoptosis. The JNK inhibitor/small interference RNA inhibited doxycycline-induced JNK activation, DeltaPsi(m) change and apoptosis, but did not affect ASK1 activation, suggesting a role of ASK1 for JNK activation in melanoma cell apoptosis. Two ROS scavengers reduced doxycycline-induced JNK and caspase activation, and apoptosis. Taken together, the results suggest the involvement of a ROS-ASK1-JNK pathway in doxycycline-induced melanoma cell apoptosis.


CONCLUSIONS
We have shown a promising cytotoxic effect of doxycycline on melanoma cells, have identified ROS and ASK1 as the possible initiators and have demonstrated that JNK activation is necessary for doxycycline-induced melanoma cell apoptosis.
Shieh
Jiunn-Min
JM
Department of Internal Medicine, Chi-Mei Medical Center, Tainan, Taiwan.
Huang
Tur-Fu
TF
Hung
Chi-Feng
CF
Chou
Kuan-Hsien
KH
Tsai
Yih-Jeng
YJ
Wu
Wen-Bin
WB
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D004318:Doxycycline / Q000008:administration & dosage / Q000494:pharmacology*; D004354:Drug Screening Assays, Antitumor; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000502:physiology; D048848:MAP Kinase Kinase Kinase 5 / Q000096:biosynthesis; D008545:Melanoma / Q000188:drug therapy / Q000503:physiopathology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008911:Minocycline / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects*; D048051:p38 Mitogen-Activated Protein Kinases / Q000096:biosynthesis
D016166:Free Radical Scavengers; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D045304:Cytochromes c; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; D008911:Minocycline; D004318:Doxycycline