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Cell Biology International (2008) 32, 1024–1028 (Printed in Great Britain)
Mitochondria, endoplasmic reticulum and actin filament behavior after PDT with chloroaluminum phthalocyanine liposomal in HeLa cells
Maíra Maftoum‑Costaa, Karina Teixeira Navesa, Alexandre Lima Oliveiraa, Antonio Cláudio Tedescob, Newton Soares da Silvaa and Cristina Pacheco‑Soaresa*
aLaboratory of Cellular Culture and Tecidual Biology – Dynamics of Cellular Compartments, Universidade do Vale do Paraíba – UNIVAP, Av. Shishima Hifumi 2911, 12211-300 São José dos Campos, SP, Brazil
bDepartment of Chemistry, Faculty of Philosophy Science and Letters, Universidade de São Paulo – USP, Ribeirão Preto, SP, Brazil


Abstract

Photodynamic therapy (PDT) for cancer is a therapeutic modality in the treatment of tumors in which visible light is used to activate a photosensitizer. Cell membranes have been identified as an important intracellular target for singlet oxygen produced during the photochemical pathway. This study analyzed the cytotoxicity in specific cellular targets of a photosensitizer used in PDT in vitro. The photosensitizing effects of chloroaluminum phthalocyanine liposomal were studied on the mitochondria, cytoskeleton and endoplasmic reticulum of HeLa cells. Cells were irradiated with a diode laser working at 670nm, energy density of 4.5J/cm2 and power density of 45mW/cm2. Fluorescence microscopic analysis of the mitochondria showed changes in membrane potential. After PDT treatment, the cytoskeleton and endoplasmic reticulum presented basic alterations in distribution. The combined effect of AlPHCl liposomal and red light in the HeLa cell line induced photodamage to the mitochondria, endoplasmic reticulum and actin filaments in the cytoskeleton.


Keywords: PDT, Fluorescence microscopy, AlPHCl liposomal.

*Corresponding author. Instituto de Pesquisa & Desenvolvimento – UNIVAP, Av. Shishima Hifumi, 291 Urbanova, 12244-000 São José dos Campos, SP, Brazil. Tel.: +55 12 39471143; fax: +55 12 39471149.


1 Introduction

Photodynamic therapy (PDT) for cancer is a therapeutic modality in the treatment of tumors in which visible light is used to activate a photosensitizer (Juarranz et al., 2001). The precise mechanism of PDT on cells and tissues is not yet totally understood. However, singlet oxygen generated after exposing the sensitizer to an appropriate light wavelength has been identified as the cytotoxic agent most likely responsible for direct tumor cell damage or cell death (Weishaupt et al., 1976).

Phthalocyanines constitute a large class of compounds with high extinction coefficients in the red spectral region (630–800nm), and have been found to present excellent tumor-localizing properties and high photosensitizing efficiency (Ben-Hur, 1992). The metal phthalocyanines, Zn(II) and Al(III) complexes (zinc phthalocyanine, ZnPc and chloroaluminum phthalocyanine, AlPHCl), present the most favorable photophysical properties for application in PDT, i.e., relatively long-lived excited singlet state and long-lived triplet states (Nunes et al., 2004). However, hydrophobic photosensitizers, such as AlPHCl, strongly aggregate in aqueous media. This aggregation significantly reduces their photosensitizing efficacy, since only monomeric species are appreciably photoactive, in this case liposomal formulation can substantially decrease the extent of photosensitizer aggregation (Derycke and Witte, 2004).

The subcellular localization of a photosensitizer is of utmost importance, since it determines where primary damages are located (Moor, 2000), and is dependent upon the chemical properties of the sensitizer, such as hydrophobicity, charge and amphiphilic character (Berg and Moan, 1997).

Cell membranes have been identified as an important intracellular target, and many of their natural constituent macromolecules are readily susceptible to the organism, reacting with the singlet oxygen produced during the photochemical pathway, typically present in the PDT process. Such membranes include the plasma membrane surrounding the cell, the membranes of the endoplasmic reticulum distributed throughout the cytoplasm and the membranes of mitochondria and Golgi apparatus (Ferreira et al., 2004).

Apoptosis, also known as ‘programmed cell death’ or ‘cellular suicide’, is an active form of death with particular changes in cell morphology and protein activity. It is characterized by cell shrinking, surface membrane blebbing, chromatin condensation and DNA fragmentation. Apoptosis can be initiated in various manners, including PDT, and the common effector mechanism is to induce caspase-mediated cleavage of substrates (Gupta, 2003).

Initiator caspases are responsible for the first proteolytic events, e.g. cleavage of the cytoskeleton and related proteins including (van Engeland et al., 1997) actin (Kayalar et al., 1996), and fodrin (a membrane-associated cytoskeletal protein) (Huppertz et al., 1999; Greidinger et al., 1996). Amongst others, these early apoptotic events are thought to be responsible for the characteristic cell surface blebbing (McCarthy et al., 1997).

The purpose of the present investigation was to evaluate the primary therapy-induced damaged sites in HeLa cells. We have focused our attention on the effects on mitochondrial membrane potential (ΔΨm), endoplasmic reticulum membrane and actin filaments related to the cytoskeletal structure.

2 Material and methods

2.1 Cell culture

The human HeLa carcinoma line was obtained from the Institute of Biophysics, Carlos Chagas Filho, Brazil (UFRJ-RJ). Cells were routinely cultivated in 25cm2 flasks using minimum essential medium (MEM), containing 10% (vol/vol) fetal calf serum (FCS), 100IU/mL penicillin, 100mM/mL streptomycin at 37°C in humidified air atmosphere containing 5% of CO2 (all products were purchased from Gibco®).

2.2 Drug

Chloroaluminum phthalocyanine liposomal (AlPHCl liposomal) was provided by Prof. Dr. Antonio Carlos Tedesco from the Department of Chemistry, Faculty of Philosophy of Science and Letters, Brazil (USP-SP) and stored in the dark at room temperature.

2.3 Photodynamic therapy

Cells (5×104cells/mL) were cultivated in 24-well plates (NUNC®) containing round sterile coverslips. They were incubated with AlPHCl liposomal for 1h at concentration of 5mM and maintained at 37°C in a humidified air atmosphere containing 5% CO2. They were washed twice with PBS to remove the photosensitizer that had not been taken up by the cells, and subsequently irradiated. Irradiation was performed using a diode laser emitting at 670nm. Each well was exposed to 35mW of optical power, with energy density of 4.5J/cm2 and power density of 45mW/cm2. After PDT, cells were incubated for 24h.

2.4 Epifluorescence microscopy

After irradiation, cells were stained by incubation with dyes 3,3′dihexyloxacarbocyanine iodide – DiOC6(3) (10mM/15min), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide/chloride – JC-1 (5mM/30min) and rhodamine phalloidin (1:100/10min) (all dyes were purchased from Molecular Probes®). Probes were used to evaluate changes in the endoplasmic reticulum, mitochondria and actin filaments, respectively. The 5-min fix was in 4% paraformaldehyde. A Leica photomicroscope (DMLB) was used for microscopic observations and photographs.

3 Results

3.1 Mitochondrial membrane potential after PDT

The mitochondrial activity was determined with JC-1 24h after PDT. JC-1 is a potential-sensitizing cationic dye capable of entering selectively into the mitochondria, since it reversibly changes its color from green to orange as membrane potential increases. JC-1 fluorescence in control cells showed a heterogeneous mitochondrial distribution (Fig. 1A). The irradiated cells presented hyperpolarized mitochondria in the perinuclear region and fluorescence dissipation throughout the cytoplasm (Fig. 1B).


Fig. 1

Mitochondria of HeLa cells were stained with JC-1, 24h after treatment. (A) Control cells showed a heterogeneous mitochondrial distribution; (B) irradiated cells presented hyperpolarized mitochondria in the perinuclear region and fluorescence dissipation throughout the cytoplasm (scale bar: 20μm).


3.2 Endoplasmic reticulum membrane after PDT

To visualize the endoplasmic reticulum (ER) structure, cells were incubated with cationic fluorescent dye DioC6(3), a cell-permeant, green-fluorescent lipophilic dye, which, at higher concentrations, can be used to stain endoplasmic reticulum. Control cells presented net tubular elements distributed over the whole cytoplasm (Fig. 2A). Analysis of irradiated cells 24h after PDT (Fig. 2B), demonstrated alterations of tubular elements formed at numerous sites along the ER, located throughout the cytoplasm.


Fig. 2

Endoplasmic reticulum of HeLa cells was stained with DiOC6(3), 24h after treatment. (A) Control cells presented net tubular elements distributed over the whole cytoplasm; (B) irradiated cells demonstrated alterations of tubular elements formed at numerous sites along the ER, located throughout the cytoplasm (scale bar: 20μm).


3.3 Photodamage to actin filaments

The fluorescent study revealed an alteration in the structure of actin filament networks after PDT in the presence of rhodamine phalloidin. Fig. 3A shows the control pattern of actin filaments (organized fibers crossing the cytoplasm). However, after PDT (Fig. 3B), disruption in the filament structure and a variable disorganization were observed.


Fig. 3

Actin filaments of HeLa cells were stained with rhodamine phalloidin 24h after treatment. (A) Control cells showed organized fibers crossing the cytoplasm; (B) irradiated cells showed disruption in the filament structure and a variable disorganization (scale bar: 20μm).


4 Discussion

In JC-1 staining, polarized mitochondria are marked by punctuate orange-red fluorescent dye. Upon depolarization, the orange-red punctuate staining is replaced by diffuse green monomer fluorescence. Results using specific dye for mitochondrial membrane potential showed that the ΔΨm was dissipated in part of the mitochondrial population (Fig. 1B). Mitochondria are increasingly recognized as an important target organelle during photodamage to tumor cells (Mak et al., 2004). Numerous studies using photodynamic treatment show the collapse of ΔΨm after therapy (Ferreira et al., 2004; Mak et al., 2004; Huang et al., 2005; Kessel and Luo, 1999; Grebenová et al., 2003). Loss of ΔΨm might be due to the opening of a large channel, called mitochondrial permeability transition pore. This channel is not completely defined, but apparently consists of both inner and outer membrane proteins, and can open to depolarize the mitochondrial membrane (Moor, 2000) with release of pro-apoptosis elements such as cytochrome c, apoptosis-inducing factor (AIF), second mitochondria-derived activator of caspases (SMAC) and certain procaspases (Oleinick et al., 2002).

Using aluminium(III) phthalocyanine tetrasulfonate chloride as a photosensitizer for PDT, Platzer et al. (2002) have shown that a mitochondrial fraction might stay functional long enough to supply the energy required to execute apoptosis. In fact, the intracellular level of ATP has been shown to be one of the major determinants for apoptosis or necrosis (Platzer et al., 2002), since several steps in the induction and/or execution of apoptosis have been reported to depend on ATP (Castano et al., 2005).

Apoptosis, or type I cell death, is characterized by cell shrinkage, chromatin condensation and DNA fragmentation, membrane blebbing, caspase activation and phagocytosis by neighboring cells (Danial and Korsmeyer, 2004). It is well established that caspase activation in mammalian cells occurs mainly through death-receptor activation (extrinsic pathway) or through mitochondrial outer membrane permeabilization (intrinsic pathway) (Danial and Korsmeyer, 2004). Recent studies suggest that the ER acts as a critical control point in several apoptotic paradigms induced by cellular signals that cause Ca2+ overload or perturbation in the Ca2+ homeostasis (Demaurex and Distelhorst, 2003; Ahmad et al., 1998). In our study, fragmentation of the endoplasmic reticulum was observed 24h after PDT in HeLa cells. Ferreira et al. (2004) reported the same phenomenon in HeLa cells 24h after therapy using aluminium(III) phthalocyanine tetrasulfonate chloride as a photosensitizer. The authors proposed that this fragmentation resulted from a breakdown of the cytoskeleton, due to ATP loss caused by a ΔΨm dissipation that leads to bleb-like structures which are prone to shear stress.

However, an alternative pathway could be observed after PDT. Teiten et al. (2003) described ER as the primary damage site after PDT using Foscan® as photosensitizer in a MCF-7 cell line. Since ER is known to be the major intracellular Ca2+ store, PDT-induced ER stress may result in Ca2+ release from ER and confer cell sensitivity to mitochondria-mediated apoptotic cell death (Mak et al., 2004). Some authors have related the disturbance of ER calcium homeostasis to the activation of m-calpain and caspase 12 (McGinnis et al., 1999; Ahmad et al., 1998). Both proteins can activate procaspase 3 (Grebenová et al., 2003), an important component of the apoptosis cascade (Oleinick et al., 2002).

Depolymerization or cleavage of actin, cytokeratins, lamins and other cytoskeletal proteins has been found to be involved in cell preparation and execution of apoptosis (Bursch et al., 2000). Actin is a prominent substrate for caspases in vitro and in vivo (Janmey, 1995). PDT has been shown capable of inducing actin depolymerization and cleavage when using zinc(II) phthalocyanine and aluminium(III) phthalocyanine tetrasulfonate chloride in HeLa cells (Juarranz et al., 2001; Ferreira et al., 2004). Fluorescence microscopic analysis of actin filaments in HeLa cells after PDT using AlPHCl liposomal (Fig. 3B) revealed an aberrant cytoplasmatic distribution and loss of actin stress fibers. Nevertheless, these cells retain rhodamine phalloidin staining, thus demonstrating the presence of actin in its globular form (Bursch et al., 2000). Since the cytoskeleton plays a crucial role in numerous cell functions, such as signal transduction, division, motility and cell shape, the possibility of selective targeting of cytoskeletal proteins constitutes a very important objective for all cancer therapies, including PDT (Juarranz et al., 2001).

The present study revealed that the combined effect of AlPHCl liposomal and red light on HeLa cell line induced photodamage to the mitochondria, endoplasmic reticulum and actin filaments of the cytoskeleton, suggesting loss of cell viability.

Acknowledgements

This study was supported by research grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo and a scholarship from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).

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Received 10 December 2007/21 January 2008; accepted 2 April 2008

doi:10.1016/j.cellbi.2008.04.005


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)