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 als o cause apoptosis. In the present study the cascade of signalling events in volved in PDT-induced apoptosis was investigated using Rose Bengal (RB) as the photosensitizer, and human epidermal carcinoma A431 cells as the cell m odel. We show that a 36-kDa kinase detected by an in-gel kinase assay is ma rkedly activated during PDT-triggered apoptosis. Immunoblot analysis reveal ed 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. Th e specific caspase inhibitors (acetyl-Asp-Glu-Val-Asp-aldehyde and acetyl-T yr-Val-Ala-Asp-chloromethylketone) block the PDT-induced caspase-3 activati on 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 r adical scavenger, mannitol, demonstrating that singlet oxygen is an immedia te early-apoptotic signal generated by PDT. In addition, PDT can induce a t wo-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 activatio n, and that the late-stage JNK activation is regulated by the caspase-media ted cleavage/activation of PAK2. Collectively, a model for the PDT-triggere d apoptotic signalling cascade with RB is proposed, which involves singlet oxygen, JNK, caspase-3 and PAK2, sequentially.