title: Association between the photodynamic loss of Bcl-2 and the sensitivity to apoptosis caused by phthalocyanine photodynamic therapy.
abstract: We have reported that photodynamic therapy (PDT) using the photosensitizer phthalocyanine (Pc) 4 and red light damages the antiapoptotic protein Bcl-2. Recently, using transient transfection of Bcl-2 deletion mutants, we identified the membrane anchorage domains of Bcl-2 as necessary to form the photosensitive target. However, it is not clear how Bcl-2 photodamage sensitizes cells to Pc 4-PDT-induced apoptosis, whether overall cell killing is also sensitized or how up-regulation of Bcl-2 in tumors might make them more or less responsive to Pc 4-PDT. In this study we report on MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) overexpressing wild-type Bcl-2 or certain deletion mutants in either a transient or a stable mode. By flow cytometric analysis of transiently transfected cells, we found that wild-type Bcl-2, Bcl-2delta33-54 and Bcl-2delta37-63 (each of which can be photodamaged) protected cells from apoptosis caused by Pc 4-PDT. In contrast, Bcl-2delta210-239, which lacks the C-terminal transmembrane domain and cannot be photodamaged, afforded no protection. We then evaluated the PDT sensitivity of transfected cell lines stably overexpressing high levels of wild-type Bcl-2 or one of the Bcl-2 mutants. Overexpression of wild-type Bcl-2, Bcl-2delta33-54 or Bcl-2delta37-63 resulted in relative resistance of cells to Pc 4-PDT, as assessed by morphological apoptosis or loss of clonogenicity. Furthermore, overexpression of Bcl-2 also inhibited the activation-associated conformational change of the proapoptotic protein Bax, and higher doses of Pc 4 and light were required to activate Bax in cells expressing high levels of Bcl-2. Many advanced cancer cells have elevated amounts of Bcl-2. Our results show that increasing the dose of Pc 4-PDT can overcome the resistance afforded by either Bcl-2 or the two mutants. PDT regimens that photodamage Bcl-2 lead to activation of Bax, induction of apoptosis and elimination of the otherwise resistant tumor cells.
authors.affiliation: Department of Radiation Oncology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4942, USA.
authors.forename: Jitsuo
authors.initials: J
authors.lastname: Usuda
authors.forename: Kashif
authors.initials: K
authors.lastname: Azizuddin
authors.forename: Song-mao
authors.initials: SM
authors.lastname: Chiu
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Oleinick
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000737:chemistry; D005260:Female; D049452:Green Fluorescent Proteins; D006801:Humans; D007211:Indoles / Q000494:pharmacology* / Q000627:therapeutic use; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D008928:Mitochondria / Q000737:chemistry; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D009685:Nuclear Envelope / Q000737:chemistry; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology* / Q000627:therapeutic use; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014410:Tumor Stem Cell Assay; D015854:Up-Regulation; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein
chemical_list: C494772:BAK1 protein, human; C494826:BAX protein, human; D007211:Indoles; D008164:Luminescent Proteins; D008565:Membrane Proteins; D017319:Photosensitizing Agents; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C107448:phthalocyanine Pc 4; D049452:Green Fluorescent Proteins