Photodynamic inhibition of enzymatic detachment of human cancer cells from a substratum Anatoly Uzdensky a,b, * , Asta Juzeniene a , Li-Wei Ma a , Johan Moan a a Institute for Cancer Research, 0310 Montebello, Oslo, Norway b Rostov State University, 344090, Rostov-on-Don, Russia Received 20 May 2003; received in revised form 16 October 2003; accepted 17 October 2003 Abstract Photodynamic therapy (PDT) is currently used for cancer treatment. It is shown that sublethal PDT of human WiDr adenocarcinoma cells and D54Mg glioblastoma cells with 5-aminolevulinic acid (ALA), disulfonated tetraphenylporphyrine (TPPS 2a ), or MitoTracker Red (MTR) inhibits their trypsin-induced detachment from a plastic substratum. TPPS 2a was bound selectively to the plasma membrane, whereas MTR was found in mitochondria. Both granular and diffuse fluorescence of ALA-derived protoporphyrin IX (PpIX) was observed in the perinuclear cytoplasm but not in the plasma membrane of WiDr cells stained for 2 h with 1 mM ALA. In D54Mg cells, PpIX fluorescence was observed not only in the cytoplasm but also in the plasma membrane. Fluorescence measurements showed a progressive accumulation of PpIX in the WiDr cells during incubation with ALA and a PpIX efflux into the medium after 1 h or longer incubation. PpIX retained in the plasma membrane during efflux may be responsible for PDT-induced impairment of cell adhesion. On the other hand, MTR-PDT or ALA-PDT after 15-min incubation, when the newly synthesized PpIX should remain in mitochondria, also inhibited enzymatic cell detachment. Therefore, photodynamic targeting of mitochondria, remote from the cell surface where adhesion occurs, may disturb cell adhesion. Photodynamic inhibition of enzymatic cell detachment may be related to PDT-induced inhibition of tumour metastasis. D 2003 Elsevier B.V. All rights reserved. Keywords: ALA-PDT; MitoTracker Red; TPPS 2a ; Cell adhesion; Photosensitizer localization; Detachment 1. Introduction Photodynamic therapy (PDT) is based on light induced destruction of selectively stained pathological tissues in the presence of oxygen. Singlet oxygen and secondary free radicals generated by photoexcited dye molecules can de- stroy several cellular structures [1,2]. An important, but not well-studied effect of PDT is its influence on cell adhesion. Two aspects of photodynamic influence on cell adhesion processes have been described: (i) the effect on cell attach- ment to a plastic substratum [5,6], to a substratum coated with extracellular matrix proteins [7,8], or to other cells [5,6]. (ii) Effects on trypsin/EDTA-induced detachment of cultured cells from a substratum [5,9,10]. Hematoporphyrin deriva- tives (HPD) [5,6,9,10], benzoporphyrin derivative monoacid ring A (BPD-MA) [7,8], zinc phthalocyanine derivative and meso-tetra-hydroxyphenyl-chlorin (mTHPC) [10] were used as photosensitizers in these studies. These effects are impor- tant for cancer therapy since cell adhesion may play a significant role in cancer metastasis [11,12]. Photodynamic treatment has been found to reduce cancer metastases [13,14]. 5-Aminolevulinic acid (ALA), a biochemical precursor of the potent endogenous photosensitizer protoporphyrin IX (PpIX), is being successfully used for cancer treatment [1,3,4]. PpIX accumulates in rapidly proliferating cancer cells, thus providing selective destruction of tumours. In recent years the mechanisms of ALA-PDT have been thor- oughly studied on the cellular and tissue levels [3,4]. How- ever, the effect of ALA-PDT on adhesive properties of cultured cancer cells has not been studied so far. 0304-4165/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2003.10.013 Abbreviations: ALA, 5-aminolevulinic acid; BPD-MA, benzoporphyr- in derivative monoacid ring A; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetate; mTHPC, meso-tetra-hydroxyphenyl-chlorin; MTR, MitoTracker Red CMXRos; PBS, phosphate-buffered saline; PDT, photodynamic therapy; PpIX, protoporphyrin IX; TPPS 2a , disulfonated meso-tetraphenylporphyrine * Corresponding author. Department of Biophysics, Rostov State University, 194/1 Stachy ave., NIINK, 344090, Rostov-on-Don, Russia. Tel.: +7-8632-433577; fax: +7-8632-433588. E-mail address: uzd@krinc.ru (A. Uzdensky). www.bba-direct.com Biochimica et Biophysica Acta 1670 (2004) 1 – 11