Characterization of apoptosis induced by photodynamic treatment with hypericin in A431 human epidermoid carcinoma cells.
Hypericin is a naturally occurring metabolite extracted from Hypericum plants and is regarded as a promising photosensitizing agent for applications in the frame of photodynamic treatment (PDT). This treatment procedure is based on the light-induced formation of reactive oxygen species and subsequent destruction of target cells. We used an in vitro model system consisting of human epidermoid carcinoma cells (A431) and hypericin as a photosensitizer to study the time- and dose-dependent characteristics of hypericin-PDT-based induction of cytotoxicity and apoptotic cell death. The induction of apoptosis by hypericin-PDT was found to follow a strict dose-dependent manner with a transition to necrotic cell death at higher doses. Apoptosis was analyzed by characteristical biochemical and morphological markers (activation of caspases, nuclear fragmentation and membrane blebbing). Time-course analysis of an almost homogenous apoptotic population of cells (at 1.44 J/cm2) showed a rapid increase in nuclear fragmentation and activation of caspases reaching a maximum at 5 hr after irradiation. Using specific caspase substrates, significant activation of caspase-2, -3, -6, and -9 was found. Mitochondrial involvement during hypericin-PDT-induced apoptosis could be proven by a rapid reduction of the mitochondrial membrane potential; interestingly, the level of intracellular adenosine-5'-triphosphate (ATP) remains at control level for up to 6 hr post irradiation suggesting upregulation of glycolysis as a compensating mechanism of energy supply. Our data contribute to a deeper understanding of the processes involved in apoptotic cell death following photodynamic treatment with hypericin.
Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
Juergen
J
Berlanda
Tobias
T
Kiesslich
Christian Benno
CB
Oberdanner
Franz Josef
FJ
Obermair
Barbara
B
Krammer
Kristjan
K
Plaetzer
D000255:Adenosine Triphosphate / Q000032:analysis; D000873:Anthracenes; D017209:Apoptosis / Q000187:drug effects*; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000473:pathology / Q000503:physiopathology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D010569:Perylene / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000378:metabolism / Q000494:pharmacology*
D000873:Anthracenes; D017319:Photosensitizing Agents; D010569:Perylene; C004965:hypericin; D000255:Adenosine Triphosphate; D020169:Caspases