Enzyme-assisted photosensitization with rose Bengal acetate induces structural and functional alteration of mitochondria in HeLa cells

Abstract

Rose Bengal acetate (RB-Ac) can be used as a fluorogenic substrate for photosensitization of cells both in vivo and in vitro: once inside the cells, RB-Ac is converted into photoactive rose Bengal (RB) molecules which redistribute dynamically in the cytoplasm and, upon irradiation by visible green light, can damage organelles such as the endoplasmic reticulum, the Golgi apparatus, and the cytoskeleton. Recently, evidence has been provided that mitochondria may also be affected. The aims of the present study were to describe RB-induced photodamage of mitochondria in single HeLa cells and to define, on a quantitative basis, the effects of photosensitization on their morphofunctional features. HeLa cell cultures were exposed to 10(-5) M RB-Ac for 60 min and then irradiated with a light emitting diode at 530 nm (total light dose, 1.6 J/cm2). After irradiation, the cells were transferred to a drug-free complete medium and allowed to grow for 24-72 h. Using conventional and confocal fluorescence microscopy, transmission electron microscopy, and flow cytometry, we demonstrate that, in photosensitized cells, mitochondria undergo structural and functional alterations which can lead cells to apoptosis. Interestingly, in our system some cells were able to survive 72 h post-treatment and to recover, exhibiting the same mitochondrial structure, distribution and inner membrane potential as those in untreated controls. Taking into account that the photoactive molecules redistribute dynamically inside the cell upon RB-Ac administration, it may be hypothesized that cells can be differently affected by irradiation, depending on the relative amount and organelle location of the photosensitizer.

Apoptosis in tumour cells photosensitized with Rose Bengal acetate is induced by multiple organelle photodamage

“…More recently, the mitochondria have also actually been found to undergo structural and functional alterations as a consequence of RB photosensitization (Panzarini et al 2006;Bottone et al 2007). …”

“…The marked phototoxic eVect of RBAc on cells after short incubation times suggests a rapid localization of activated RB to sensitive subcellular sites (Bottiroli et al 1997;Bottone et al 2007); for example, mitochondria have proved to be particularly damaged in HeLa cells (Panzarini et al 2006;Bottone et al 2007) although cytoskeletal alterations have also been described ). This suggests that multiple organelles are possibly involved in the induction of cell death.…”

State-of-the-art technologies, current opinions and developments, and novel findings: news from the field of histochemistry and cell biology

“…A number of recent original publications and reviews addressed various aspects of the induction, the prevention and the detection of diVerent forms of cell death, including necrosis, apoptosis, and autophagy (see above, and Bottone et al 2007;Jacob et al 2007;Soldani et al 2007;Spano et al 2007;Vejux et al 2007;Brandt et al 2008;Taatjes et al 2008a, b). One problem that is paramount in studies of processes leading to or occurring during cell death is the correct classiWcation of the death mode.…”

Rose Bengal Acetate photodynamic therapy-induced autophagy

“…Indeed, upon RBAc administration, the photoactive molecules of RB accumulate in perinuclear region and than diffuse dynamically throughout the cytoplasm. 20,[37][38][39][40] Endosomes, Golgi apparatus and the ER are the precocious intracellular sites of accumulation of RB. 37,40,41 The photodamage to ER exerted by the RBAc-PDT, as demonstrated by the drastic reduced incorporation of DiOC 6 and by the increased level of cleaved caspase-12, promotes autophagy.…”

“…20,[37][38][39][40] Endosomes, Golgi apparatus and the ER are the precocious intracellular sites of accumulation of RB. 37,40,41 The photodamage to ER exerted by the RBAc-PDT, as demonstrated by the drastic reduced incorporation of DiOC 6 and by the increased level of cleaved caspase-12, promotes autophagy. However, the molecular mechanism leading to the removal of the damaged ER through the formation of the autophagosomes and their following fusion with the lysosomes for degradation during PDT, is not yet fully elucidated.…”