Apoptotic signalling cascade in photosensitized human epidermal carcinoma A431 cells: involvement of singlet oxygen, c-Jun N-terminal kinase, caspase-3 and p21-activated kinase 2.
Photodynamic treatment (PDT) elicits diverse cellular responses and can also cause apoptosis. In the present study the cascade of signalling events involved in PDT-induced apoptosis was investigated using Rose Bengal (RB) as the photosensitizer, and human epidermal carcinoma A431 cells as the cell model. We show that a 36-kDa kinase detected by an in-gel kinase assay is markedly activated during PDT-triggered apoptosis. Immunoblot analysis revealed that this 36-kDa kinase represents the C-terminal catalytic fragment of p21-activated kinase (PAK)2. Generation of this active fragment of PAK2 is mediated by the caspase family of proteases, which are activated by PDT. The specific caspase inhibitors (acetyl-Asp-Glu-Val-Asp-aldehyde and acetyl-Tyr-Val-Ala-Asp-chloromethylketone) block the PDT-induced caspase-3 activation and subsequent PAK2 cleavage/activation, indicating a major role for the caspase family proteases in PDT-induced apoptosis. Both PDT-induced caspase-3 activation and PAK2 cleavage/activation can be inhibited by the singlet oxygen scavengers, L-histidine and alpha-tocopherol, but not the hydroxyl radical scavenger, mannitol, demonstrating that singlet oxygen is an immediate early-apoptotic signal generated by PDT. In addition, PDT can induce a two-stage activation of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) in A431 cells; the early-stage JNK activation is singlet oxygen-dependent, whereas the late-stage JNK activation is mediated by the singlet oxygen-triggered caspase activation. Experiments using anti-sense oligonucleotides against JNK1 and PAK2 further show that during PDT-induced apoptosis the early-stage JNK activation is required for caspase activation, and that the late-stage JNK activation is regulated by the caspase-mediated cleavage/activation of PAK2. Collectively, a model for the PDT-triggered apoptotic signalling cascade with RB is proposed, which involves singlet oxygen, JNK, caspase-3 and PAK2, sequentially.
Chan
W H
WH
Department of Cell and Molecular Biology, Institute of Basic Medicine, Medical College of Chang Gung University, Tao-Yuan, Taiwan, Republic of China.
Yu
J S
JS
Yang
S D
SD
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D020134:Catalytic Domain; D004789:Enzyme Activation / Q000187:drug effects; D016166:Free Radical Scavengers / Q000494:pharmacology; D006639:Histidine / Q000494:pharmacology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D008353:Mannitol / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008970:Molecular Weight; D016376:Oligonucleotides, Antisense / Q000235:genetics; D010100:Oxygen / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012395:Rose Bengal / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D026082:Singlet Oxygen; D014407:Tumor Cells, Cultured; D014810:Vitamin E / Q000494:pharmacology; D054462:p21-Activated Kinases
D061945:Caspase Inhibitors; D016166:Free Radical Scavengers; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments; D017319:Photosensitizing Agents; D014810:Vitamin E; D026082:Singlet Oxygen; D012395:Rose Bengal; D008353:Mannitol; D006639:Histidine; C515907:PAK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D010100:Oxygen