Anne C.E. Moor

Signaling pathways in cell death and survival after photodynamic therapy

Photodynamic therapy (PDT) is a cytotoxic treatment, which can induce cells to initiate a rescue response, or to undergo cell death, either apoptosis or necrosis. The many signaling pathways involved in these processes are the topic of this review. The subcellular localization of the photosensitizer has been shown to be a key factor in the outcome of PDT. Mitochondrial localized photosensitizers are able to induce apoptosis very rapidly. Lysosomal localized photosensitizers can elicit either a necrotic or an apoptotic response. In the plasma membrane, a target for various photosensitizers, rescue responses, apoptosis and necrosis is initiated. Several protein phosphorylation cascades are involved in the regulation of the response to PDT. Finally, a number of stress-induced proteins play a role in the rescue response after PDT. Notably, the induction of apoptosis by PDT might not be crucial for an optimal outcome. Recent studies indicate that abrogation of the apoptotic pathway does alter the clonogenic survival of the cells after PDT. Further studies, both in vitro and especially in vivo could lead to more efficient combination therapies in which signaling pathways, involved in cell death or rescue, are either up- or downregulated before PDT.

Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library.

The first step in developing a targeted cancer therapeutic is generating a ligand that binds to a receptor which is either tumor specific or sufficiently overexpressed in tumors to provide targeting specificity. For this work, we generated human monoclonal antibodies to the EGF receptor (EGFR), an antigen overexpressed on many solid tumors. Single chain Fv (scFv) antibody fragments were directly selected by panning a phage display library on tumor cells (A431) overexpressing EGFR or Chinese hamster ovary cells (CHO/EGFR cells) transfected with the EGFR gene and recovering endocytosed phage from within the cell. Three unique scFvs were isolated, two from selections on A431 cells and two from selections on CHO/EGFR cells. All three scFv bound native receptor as expressed on a panel of tumor cells and did not bind EGFR negative cells. Phage antibodies and multivalent immunoliposomes constructed from scFv were endocytosed by EGFR expressing cells as shown by confocal microscopy. Native scFv primarily stained the cell surface, with less staining intracellularly. The results demonstrate how phage antibodies binding native cell surface receptors can be directly selected on overexpressing cell lines or transfected cells. Use of a transfected cell line allows selection of antibodies to native receptors without the need for protein expression and purification, significantly speeding the generation of targeting antibodies to genomic sequences. Depending upon the format used, the antibodies can be used to deliver molecules to the cell surface or intracellularly.

Epidermal growth factor receptor-targeted photosensitizer selectively inhibits EGFR signaling and induces targeted phototoxicity in ovarian cancer cells

Targeted photosensitizer delivery to EGFR-expressing cells was achieved in the present study using a high purity, targeted photoimmunoconjugate (PIC). When the PDT agent, benzoporphyrin derivative monoacid ring A (BPD) was coupled to an EGFR-targeting antibody (cetuximab), we observed altered cellular localization and selective phototoxicity of EGFR-positive cells, but no phototoxicity of EGFR-negative cells. Cetuximab in the PIC formulation blocked EGF-induced activation of the EGFR and downstream signaling pathways. Our results suggest that photoimmunotargeting is a useful dual strategy for the selective destruction of cancer cells and also exerts the receptor-blocking biological function of the antibody.

Chapter 3 Mechanism of action—molecular effects

Abstract Cells exposed to photodynamic therapy (PDT) respond with activation of molecular pathways that are involved in the regulation of gene expression, cell cycle and cell death. This chapter gives a review of the molecular responses that have been documented after PDT, including effects on cell fate decision, stress responses, and intra- and intercellular signaling. The subcellular localization of the photosensitizer is an important determinant for specific molecular response patterns and for the ultimate PDT effect. Divergent and sometimes, contradictory results of consequences of PDT at the molecular level are likely due to the variability of PDT regimens with respect to photosensitizers, PDT dose, and other variables of specific experimental model. We are beginning to understand a few of the molecular effects of PDT, and this learning process will form the basis for research that aims at developing improved PDT regimens. PDT may also be usful as a tool to analyze certain cellular-molecular pathways.