Photodynamic therapy-induced death of MCF-7 human breast cancer cells: a role for caspase-3 in the late steps of apoptosis but not for the critical lethal event.
Photodynamic therapy (PDT) causes mitochondrial damage and induces apoptosis through release of cytochrome c and activation of caspase-3. To test whether caspase 3 is the sole executioner of apoptosis and its role in overall cell lethality, we compared the response of MCF-7c3 cells that express a stably transfected CASP-3 gene to that of parental MCF-7:SW8 cells transfected with vector alone (MCF-7v). Following photosensitization with the phthalocyanine Pc 4 and red light, cytochrome c was released from the mitochondria to equivalent extents in the two cell lines. However, the appearance of apoptotic indicators, such as active caspase-3 (DEVDase), cleavage of poly(ADP-ribose) polymerase, and oligonucleosomal DNA fragmentation, was observed only in MCF-7c3 cells during the first 6 h after photosensitization. Although production of 50-kb DNA fragments and chromatin condensation were found in PDT-treated MCF-7v cells by 20-24 h posttreatment, the rate and extent of apoptosis were much less than in MCF-7c3 cells. MCF-7c3 cells were more sensitive to photosensitization than were MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to photodynamic killing when evaluated by a clonogenic assay. These results show (a) the importance of assessing overall cell death by clonogenic assay; (b) that the critical lethal event is independent of caspase-3, perhaps at or near the release of cytochrome c from mitochondria; and (c) that the caspase-3-mediated events appear to be irrelevant in determining overall killing of cells.
Xue
L Y
LY
Department of Radiation Oncology, and CWRU/Ireland Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Chiu
S M
SM
Oleinick
N L
NL
D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D048429:Cell Size; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D007211:Indoles / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D003574:Cytochrome c Group; D007211:Indoles; D017319:Photosensitizing Agents; C107448:phthalocyanine Pc 4; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases