Early release of mitochondrial cytochrome c and expression of mitochondrial epitope 7A6 with a porphyrin-derived photosensitizer: Bcl-2 and Bcl-x(L) overexpression do not prevent early mitochondrial events but still depress caspase activity

Certain nonmetallic porphyrins have potent antitumor activity upon visible light irradiation. Treatment of HeLa cells with nanomolar amounts of the ph otochemo therapeutic agent verteporfin and red light mobilized caspases 2, 3, 6, 7, 8, and 9, caused degradation of specific caspase substrates, and r esulted in morphologic changes consistent with apoptosis. Caspase processin g was detectable by 1 hour after light irradiation. The mitochondrial 7A6 e pitope, recognized by monoclonal antibody APO2.7, became accessible, and cy tochrome c was detectable within the cytosolic fraction of cells treated wi th verteporfin im mediately after light irradiation. The general caspase in hibitor benzyloxycarboyl-Val-Ala-Asp-fluoromethylketone did not prevent 7A6 expression produced by photosensitization at peptide concentrations which completely prevented caspase activation and cleavage of caspase-specific su bstrates. Enforced overexpression of Bcl-2 or Bcl-x(L) prevented cytochrome c release and 7A6 expression produced by ultraviolet B light treatment, bu t did not prevent cytochrome c release or 7A6 expression elicited by vertep orfin photosensitization. Bcl-2 or Bcl-x(L) overexpression delayed morpholo gic changes, depressed caspase activation, and limited substrate degradatio n, but did not protect against loss of viability after verteporfin photosen sitization. This observation indicates that cells overexpressing Bcl-2 or B cl-x(L) exhibit resistance to caspase activation even after the appearance of cytochrome c in the cytosol. Porphyrin photosensitizers are effective ch emotherapeutic agents that elicit:primary proapoptotic mitochondrial events even in the setting of heightened Bcl-2 or Bcl-x(L) expression.