Photosensitizers for photodynamic therapy (PDT) are most commonly delivered to patients or experimental animals
via intravenous injection. After initial distribution throughout the body, there can be some preferential accumulation
within tumors or other abnormal tissue in comparison to the surrounding normal tissue. In contrast, the photosensitizer
precursor, 5-aminolevulinic acid (ALA) or one of its esters, is routinely administered topically, and more specifically, to
target skin lesions. Following metabolic conversion to protoporphyrin IX, the target area is photoilluminated, limiting
peripheral damage and targeting the effective agent to the desired region. However, not all skin lesions are responsive to
ALA-PDT. Topical administration of fully formed photosensitizers is less common but is receiving increased attention,
and some notable advances with selected approved and experimental photosensitizers have been published. Our team
has examined topical administration of the phthalocyanine photosensitizer Pc 4 to mammalian (human, mouse, pig) skin.
Pc 4 in a desired formulation and concentration was applied to the skin surface at a rate of 5-10 μL/cm<sup>2</sup> and kept under
occlusion. After various times, skin biopsies were examined by confocal microscopy, and fluorescence within regions of
interest was quantified. Early after application, images show the majority of the Pc 4 fluorescence within the stratum
corneum and upper epidermis. As a function of time and concentration, penetration of Pc 4 across the stratum corneum
and into the epidermis and dermis was observed. The data indicate that Pc 4 can be delivered to skin for photodynamic
activation and treatment of skin pathologies.
The phthalocyanine photosensitizer Pc 4 binds preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, including Bcl-2, are photodamaged and apoptosis is triggered. We recently prepared analogues of Pc 4 containing two axial ligands, one identical to the single ligand in Pc 4, and the other containing one or two hydroxyl groups on a dimethylsiloxy alkyl chain. Pc 181 is representative of this group of photosensitizers. In MCF-7 human breast cancer cells, the new analogues preferentially localized in lysosomes and were highly efficient in inducing apoptosis and overall cell death. The Bcl-2 family member Bid is required for
signaling to mitochondria for apoptosis in response to primary lysosomal photodamage. To further evaluate the role of Bid, we compared the effects of PDT with Pc 4 or Pc 181 in wild-type murine embryonic fibroblasts and those knocked out for Bid. We find that the two cell lines are equally sensitive to killing by Pc 4-PDT, but the Bid<sup>-/-</sup> cells are significantly more resistant to killing and apoptosis induction by Pc 181-PDT than are the Bid<sup>+/+</sup> cells. The data show that low levels of lysosomal photodamage are not alone lethal and that a specific defect in a factor required for apoptosis
can severely compromise cell response to PDT.
Pc 4, a photosensitizer first synthesized at Case Western Reserve University and now in clinical trial at University
Hospitals Case Medical Center, has been shown to bind preferentially and with high affinity to mitochondrial and
endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially the
anti-apoptotic protein Bcl-2, are photodamaged. Apoptosis, as indicated by activation of caspase-3 and cleavage of
poly(ADP-ribose) polymerase, is triggered by the initial photodamage. A series of analogues of Pc 4 has been
synthesized containing two axial ligands, one identical to the single ligand of Pc 4 and the other either the same as the Pc
4 ligand or bearing one or more hydroxyl groups. The hydroxyl-bearing axial ligands reduce the aggregation of the Pc in
polar environments and direct the Pc's to lysosomes. Photoirradiation of cells that have taken up these Pc's into their
lysosomes is 4-6 times more efficient at killing cells, as defined by loss of clonogenicity, than with Pc 4. Whereas PDT
with Pc 4 photodamages Bcl-2 and Bcl-xL over the same dose response range as for cell killing, PDT with Pc 181 or the
other lysosome-localizing Pc's causes much less photodamage to Bcl-2 relative to cell killing. Furthermore, in the case
of the lysosome-bound Pc's, little or no caspase-3-dependent apoptosis is observed.
In response to photodynamic therapy (PDT), many cells in culture or within experimental tumors are eliminated by apoptosis. PDT with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, causes prompt release of cytochrome c into the cytoplasm and activation of caspases-9 and -3, among other caspases, that are responsible for initiating cell degradation. Some cells appear resistant to apoptosis after PDT; however, if they have sustained sufficient damage, they will die by a necrotic process or through a different apoptotic pathway. In the case of PDT, the distinction between apoptosis and necrosis may be less important than the mechanism that triggers both processes, since critical lethal damage appears to occur during treatment and does not require the major steps in apoptosis to be expressed. We earlier showed, for example, that human breast cancer MCF-7 cells that lack caspase-3 are resistant to the induction of apoptosis by PDT, but are just as sensitive to the loss of clonogenicity as MCF-7 cells stably expressing transfected procaspase-3. Many photosensitizers that target mitochondria specifically attack the anti-apoptotic protein Bcl-2, generating a variety of crosslinked and cleaved photoproducts. Recent evidence suggests that the closely related protein Bcl-xL is also a target of Pc 4-PDT. Transient transfection of an expression vector encoding deletion mutants of Bcl-2 have identified the critical sensitive site in the protein that is required for photodamage. This region contains two alpha helices that form a secondary membrane anchorage site and are thought to be responsible for pore formation by Bcl-2. As specific protein targets are identified, we are becoming better able to model the critical events in PDT-induced cell death.
Early molecular damages have been studied in a series ofhuman tumor and rodent cell lines following photodynamic therapy (PDT) sensitized by the silicon phthalocyanine Pc 4. Pc 4 preferentially localizes in mitochondria, and upon photoirradiation, immediate photodamage to the anti-apoptotic oncoprotein Bcl-2 is observed. The loss ofthe native 26-kDa protein, as evidenced by western blot analysis, is accompanied by the generation of a 23 -kDa fragment from a small portion of the molecules as well as a variety ofhigher molecular weight protein bands indicative ofphotochemical crosslinking ofBcl-2 to itself, to (pro-apoptotic) homologs, or to other nearby proteins. The changes in Bcl-2 are apparent immediately upon exposure ofPc 4-loaded cells to activating red light, occur in the cold, and are not dependent upon caspase-3 or other proteases. Crosslinking is also observed for the intermediate filament protein vimentin. The results implicate Bcl-2 (and perhaps vimentin) as important molecular targets that lead to apoptosis in Pc 4-PDT-treated cells.