5-Aminolevulinic Acid–based Photochemical Internalization of the Immunotoxin MOC31-gelonin Generates Synergistic Cytotoxic Effects In Vitro¶

Abstract

Photochemical internalization (PCI) is a novel method for the endosomal or lysosomal release of membrane-impermeable molecules into the cytosol of target cells. This novel technology is based on the photodynamically induced rupture of endocytic vesicles preloaded with molecules of therapeutic interest. PCI of the ribosome-inactivating plant toxin gelonin and the immunotoxin monoclonal antibody 31 (MOC31) gelonin has been performed previously by the use of the endocytic vesicle-localizing photosensitizers TPPS2a and AIPcS2a and light, demonstrating synergistic toxicity against the more than 20 different cell lines tested, most of them of neoplastic origin. In this study we demonstrate that 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) is also capable of inducing PCI of MOC31-gelonin in the human colon adenocarcinoma cell line WiDr. The cells were incubated with 1 mM 5-ALA for up to 8 h in serum-free medium and from 24 to 96 h in serum-containing medium. Fluorescence microscopical studies indicate a partial plasma membrane localization of PpIX when 5-ALA was applied under serum-free conditions. This plasma membrane localization was not seen when 5-ALA was given in the presence of serum. There was a granular component of the PpIX localization in addition to a diffuse cytoplasmic localization. The granular component resembled the localization of the fluorescent dye conjugate Alexa-gelonin and the lysosomal localizing dye acridine orange. Our present results provide evidence for an endocytic vesicle-associated fraction of PpIX after 5-ALA incubation of the WiDr cells. We demonstrate that PCI, by combining 5-ALA, MOC31-gelonin and light, induces a synergistic cytotoxic effect against the WiDr cells.

Photochemically stimulated drug delivery increases the cytotoxicity and specificity of EGF–saporin

“…PCI has been shown to increase the cytosolic delivery and subsequent therapeutic effect in vitro of many macromolecules such as proteins (Selbo et al 2000a, Dietze et al 2003, immunotoxins (Selbo et al 2000b, Selbo et al 2001a and DNA delivered by cationic polymers , Prasmickaite et al 2001, adenovirus , Engesaeter et al 2005, Engesaeter et al 2006a, Engesaeter et al 2006b) and adenoassociated virus . PCI in vitro has also been reported as an intracellular delivery system for peptides (Berg et al 1996, Berg et al 1999, PNAs (Shiraishi & Nielsen 2006, siRNA (Oliveira et al 2007) and some chemotherapeutics, such as bleomycin , doxorubicin , Lai et al 2007) and mitoxantrone (Adigbli et al 2007).…”

“…The susceptibility of TPPS 2a -PDT mediated EGFR damage seems to be cell line Photofrin (Wong et al 2003), 5-ALA induced protoporphyrin IX (PpIX) (Wong et al 2003), Hypericin (de Witte et al 1993) and RB (Zhuang et al 2003, Schieke et al 2004 as PSs may be caused by a direct oxidation of the receptor at light exposure time, as all of these PSs has been shown to localize to the plasma membrane (Thomas & Pardini 1992, Lin et al 2000, Selbo et al 2001a, Hsieh et al 2003b. In addition, the lipophilicity of these PSs indicate that they can freely diffuse through the plasma membrane, and thereby easily target the intracellular domain of EGFR upon light exposure.…”

Destination ‘Lysosome’: a target organelle for tumour cell killing?

“…A reactive oxygen species is then formed which oxidizes the lipids of endosomal and lysosomal membranes causing the cytosolic release of the cytotoxic drug along with lysosomal hydrolases. This technology has proved its efficacy in tumour-bearing mice treated with gelonin (Selbo et al, 2001). Photosensitizers have higher affinity for tumour cells than for normal cells, probably due to differences in lysosomal acidification, and this can be used in photodynamic therapy of cancer.…”

Enhancing the efficacy of cytotoxic agents for cancer therapy using photochemical internalisation

“…29 Gelonin has been covalently linked to MOC31 (an antibody recognising EGP-2) and has been used in several cancer models. 29,37,39 A PCI-based synergy was found between gelonin and two photosensitisers (TPPS 2a and AlPcS 2a ), with a greater effect seen with the targeted MOC31-gelonin in a small cell lung carcinoma cell line (NCI-H146). Lower cytotoxicity was seen with MOC31-gelonin alone, but were equally efficient on EGP-2 antigen negative cells.…”