Mechanisms of cytotoxicity in human ovarian carcinoma cells exposed to free Mce^sub 6^ or HPMA copolymer-Mce^sub 6^ conjugates[para]

Photochemistry and Photobiology, Jun 2003 by Tijerina, Monica, Kope[cbreve]kova, Pavla, Kope[cbreve]ek, Jindrich

ABSTRACT

It is essential to understand cellular responses on photodynamic therapy (PDT) to design delivery systems that maximize cytotoxic effects coupled with minimal induction of side effects or protective mechanisms (or both). Here, we investigated mechanisms of toxicity in human ovarian carcinoma A2780 cells treated with structurally diverse N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer (P)-mesochlorin e^sub 6^ monoethylenediamine (Mce^sub 6^) conjugates that possessed differential subcellular accumulation or covalent attachments of photosensitizers (or both). Apoptosis and necrosis were observed after photoactivation, with increased apoptotic responses observed in cells exposed to conjugates possessing Mce^sub 6^ linkage via a lysosomally degradable tetrapeptide spacer (HPMA copolymer-Mce^sub 6^ conjugates containing Mce^sub 6^ bound via glycylphenylalanylleucylglycine [GFLG] linker [P-GFLG-Mce^sub 6^], HPMA copolymer-Mce^sub 6^ conjugates containing Mce^sub 6^ bound via a GFLG spacer and containing nuclear localization sequence, PKKKRKV^sub 132^K(FITC)C [NLS(fluorescein-5-isothiocyanate [FITC])] bound via a thioether linkage [P-NLS(FITC)-GFLG-Mce^sub 6^]). Furthermore, the induction of necrosis was more pronounced in cells exposed to conjugates containing both a nuclear localization sequence (NLS) and Mce^sub 6^ bound by a degradable linker (P-NLS(FITC)-GFLG-Mce^sub 6^). Caspase-independent mechanisms of cell death were identified in cells treated with nuclear-targeted conjugates possessing Mce^sub 6^ attached using a nondegradable tether (HPMA copolymer-Mce^sub 6^ conjugates containing Mce^sub 6^ bound via a GG spacer and containing NLS(FITC) bound via a thioether linkage [P-NLS(FITC)-GG-Mce^sub 6^]), whereas low levels of apoptosis and necrosis were detected in cells exposed to photoactivated nontargeted HPMA copolymer-Mce^sub 6^ conjugates containing Mce^sub 6^ coupled through a nondegradable spacer (HPMA copolymer-Mce^sub 6^ conjugates containing Mce^sub 6^ bound via GG linker [P-GG-Mce^sub 6^]). Variations in gene expression were observed in cells on PDT. Specifically, HSP70 expression was solely detected in cells treated with P-GFLG-Mce^sub 6^, whereas the loss of detection of several genes were observed in cells treated with P-NLS(FITC)-GFLG-Mce^sub 6^. Variations in cellular responses on PDT using different HPMA copolymer-Mce^sub 6^ conjugates will prove useful in the design of optimal HPMA copolymer PDT delivery systems.

INTRODUCTION

Photodynamic therapy (PDT) is a minimally invasive procedure that has great potential for the treatment of malignant disease (1). On the administration of photosensitizers, light is used to photoactivate sensitizers at the site of neoplastic growth, enabling physicians to direct photodamage, reducing nonspecific damage to normal tissue.

On cellular damage induced by chemotherapy, subcellular organelles sense stress-initiating biochemical signals inducing recovery mechanisms. For oxidative damage the endoplasmic reticulum and cytosol can upregulate expression of glucose-regulated proteins (GRP) and heat shock proteins (HSP), respectively (2). These proteins aid in the recovery of stress by acting as chaperones assisting in the refolding of damaged proteins or by acting as proteases degrading damaged proteins to restore homeostasis (3). Alterations in GRP78, GRP94 and HSP70 gene expression on PDT have been reported (4-6). Conjugation of electrophilic toxicants to glutathione (GSH)-producing water-soluble adducts targeted for excretion provides cells an alternative mechanism of detoxification (7). Depletion of GSH by buthionine sulfoximine sensitized several cell lines to PDT (8,9). Repair mechanisms facilitate the recovery of cells from cellular damage induced by cytotoxic agents and may result in resistance, a major obstacle in successful chemotherapy.

PDT can induce apoptosis or necrosis depending on cell genotype, PDT dose, physical properties and subcellular localization of the sensitizer (10). Morphologically, apoptosis entails rapid chromatin condensation, budding of cells and formation of apoptotic bodies, which are phagocytosed and digested by neighboring cells (11,12). Biochemically, it involves complex signaling machinery influenced by a variety of genes and proteins (13,14). Key proteins that monitor repair and damage within cells are p53 and bcl-2. Cell cycle regulation, DNA repair and apoptosis are all mediated by the protein product of the p53 tumor suppressor gene (15,16), whereas bcl-2, a mitochondrial membrane protein, functions as an antagonist to proapoptotic signals (17,18). Two principal apoptosis-inducing pathways are the Fas signaling and mitochondrion-elicited death pathways involving the activation of caspases, cysteine proteases triggered in response to proapoptotic signals (19). Caspase-independent mechanisms of cell death have been documented; however, the cellular components have not been elucidated to date (20-23). Necrosis, a form of uncontrolled cell death generally accompanied by inflammation, entails irregular clumping of chromatin, cell swelling and subsequent disintegration. Mutations in genes regulating apoptosis are common in malignancies. Although mutations do not necessarily play a role in the sensitivity of cells to death induced by chemotherapeutic agents, they may alter mechanisms of cell death from apoptosis to necrosis and vice versa (10,24).