Photodynamic therapy with the phthalocyanine photosensitizer Pc 4: the case experience with preclinical mechanistic and early clinical-translational studies

Abstract

Photodynamic therapy (PDT) is emerging as a promising non-invasive treatment for cancers. PDT involves either local or systemic administration of a photosensitizing drug, which preferentially localizes within the tumor, followed by illumination of the involved organ with light, usually from a laser source. Here, we provide a selective overview of our experience with PDT at Case Western Reserve University, specifically with the silicon phthalocyanine photosensitizer Pc 4. We first review our in vitro studies evaluating the mechanism of cell killing by Pc 4-PDT. Then we briefly describe our clinical experience in a Phase I trial of Pc 4-PDT and our preliminary translational studies evaluating the mechanisms behind tumor responses. Preclinical work identified (a) cardiolipin and the anti-apoptotic proteins Bcl-2 and Bcl-xL as targets of Pc 4-PDT, (b) the intrinsic pathway of apoptosis, with the key participation of caspase-3, as a central response of many human cancer cells to Pc 4-PDT, (c) signaling pathways that could modify apoptosis, and (d) a formulation by which Pc 4 could be applied topically to human skin and penetrate at least through the basal layer of the epidermis. Clinical-translational studies enabled us to develop an immunohistochemical assay for caspase-3 activation, using biopsies from patients treated with topical Pc 4 in a Phase I PDT trial for cutaneous T-cell lymphoma. Results suggest that this assay may be used as an early biomarker of clinical response.

Figure 1

The structure of (a) the porphyrin ring (b) the phthalocyanine ring, and (c) Pc 4.

Figure 2. Main mechanism of induction of apoptosis by Pc 4-PDT in vitro

Primary photodynamic damage to mitochondria causes opening of the permeabilitiy transition pore complex, releasing cytochrome c into the cytosol. There, cytochrome c combines with other cytoplasmic proteins to form the apoptosome, resulting in the activation of caspases-9 and -3, cleavage of various substrates, such as the inhibitor (ICAD) of the caspase-activated DNase (CAD), fragmentation of DNA, and condensation of chromatin to generate morphologically apoptotic cells.

Figure 3. Mitochondrial targets of Pc 4-PDT

Pc 4 may bind to both the outer and inner mitochondrial membranes. Colocalization of Pc 4 and NAO probably occurs within proximity of cardiolipin and key components of the mitochondrial inner membrane. Oxidation of cardiolipin may result in release of cytochrome c through opening of the permeability transition pore complex formed by the voltage-dependent anion channel (VDAC) and the adenine-nucleotide translocator (ANT). Photodamage to Bcl-2 may be an independent event or secondary to lipid oxidation.

Figure 4. Immunohistochemical identification of cells with caspase-3 protein

Biopsy sections are stained blue-purple with hematoxylin and brown for caspase-3. Four sections are shown: a) untreated non-responder, b) treated non-responder, c) untreated responder, and d) treated responder.

Figure 4. Immunohistochemical identification of cells with caspase-3 protein

Biopsy sections are stained blue-purple with hematoxylin and brown for caspase-3. Four sections are shown: a) untreated non-responder, b) treated non-responder, c) untreated responder, and d) treated responder.

Figure 4. Immunohistochemical identification of cells with caspase-3 protein

Biopsy sections are stained blue-purple with hematoxylin and brown for caspase-3. Four sections are shown: a) untreated non-responder, b) treated non-responder, c) untreated responder, and d) treated responder.

Figure 4. Immunohistochemical identification of cells with caspase-3 protein

Biopsy sections are stained blue-purple with hematoxylin and brown for caspase-3. Four sections are shown: a) untreated non-responder, b) treated non-responder, c) untreated responder, and d) treated responder.