Activation of c-Jun N-terminal kinase is essential for mitochondrial membrane potential change and apoptosis induced by doxycycline in melanoma cells

作者：Shieh Jiunn Min; Huang Tur Fu; Hung Chi Feng; Chou Kuan Hsien; Tsai Yih Jeng; Wu Wen Bin^*

来源：British Journal of Pharmacology, 2010, 160(5): 1171-1184.

DOI：10.1111/j.1476-5381.2010.00746.x

• apoptosis
• ASK
• caspase
• doxycycline
• free radical
• JNK
• melanoma
• mitochondria
• ROS
• tetracycline

Background and purpose:
Tetracyclines were recently found to induce tumour cell death, but the early processes involved in this cytotoxic effect remain unclear.
Experimental approach:
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 (Delta Psi(m)) was analyzed by fluorescence microscopy and flow cytometry.
Key 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 Delta Psi(m) change during apoptosis. The JNK inhibitor/small interference RNA inhibited doxycycline-induced JNK activation, Delta Psi(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 and implications:
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.

• 出版日期2010-7