No abstract

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose–response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

The role of Bax in the release of cytochrome c from mitochondria and the induction of apoptosis has been demonstrated in many systems. Using immunocytochemical staining, we observed that photodynamic therapy (PDT) with the photosensitiser Pc 4 induced Bax translocation from the cytosol to mitochondria, and the release of cytochrome c from mitochondria as early signalling for the intrinsic pathway of apoptosis in human breast cancer MCF-7c3 cells. To test the role of Bax in apoptosis, MCF-7c3 cells were treated with Bax antisense oligonucleotides, which resulted in as much as a 50% inhibition of PDT-induced apoptosis. In the second approach, Bax-negative human prostate cancer DU-145 cells were studied. Following PDT, the hallmarks of apoptosis, including the release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, caspase activation, and chromatin condensation and fragmentation, were completely blocked in these cells. Restoration of Bax expression in DU-145 cells restored apoptosis, indicating that the resistance of DU-145 cells to PDT-induced apoptosis is due to the lack of Bax rather than to another defect in the apoptotic machinery. However, despite the inhibition of apoptosis, the Bax-negative DU-145 cells were as photosensitive as Bax-replete MCF7c3 cells, as determined by clonogenic assay. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to Bax activation.

Photodynamic therapy (PDT) is an efficient inducer of apoptosis in many types of cells, except in cells deficient in one or more of the factors that mediate apoptosis. Recent reports have identified autophagy as a potential alternative cell death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145 cells) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v cells) and in apoptosis-competent cells (MCF-7c3 cells that stably overexpress human pro-caspase-3 and Chinese hamster ovary CHO 5A100 cells). Further, each of the cell lines was also studied with and without stably overexpressed Bcl-2. Autophagy was identified by electron microscopic observation of the presence of double-membrane-delineated autophagosomal vesicles in the cytosol and by immunoblot observation of the Pc 4-PDT dose- and time-dependent increase in the level of LC3-II, a component of the autophagosomal membrane. Autophagy was observed in all of the cell lines studied, whether or not they were capable of typical apoptosis and whether or not they overexpressed Bcl-2. The presence of stably overexpressed Bcl-2 in the cells protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO 5A100 cells). In contrast, Bcl-2 overexpression did not protect against the development of autophagy in any of the cell lines or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145 cells). Furthermore, 3-methyladenine and wortmannin, inhibitors of autophagy, provided greater protection against loss of viability to apoptosis-deficient than to apoptosis-competent cells. The results show that autophagy occurs during cell death following PDT in human cancer cells competent or not for normal apoptosis. Only the apoptosis-competent cells are protected by Bcl-2 against cell death.

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 upregulation 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‐2Δ33‐54 and Bcl‐2Δ37‐63 (each of which can be photodamaged) protected cells from apoptosis caused by Pc 4‐PDT. In contrast, Bcl‐2Δ210‐239, 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‐2Δ33‐54 or Bcl‐2Δ37‐63 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.

Abstract10-N-nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipindeficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.

No 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.