Rose Bengal Acetate Research Articles

High Melanin Content in Melanoma Cells Contributes to Enhanced DNA Damage after Rose Bengal Photosensitization.

Melanoma is a type of tumor that originates from melanocytes. Irradiation of melanin with UVA and visible light can produce reactive oxygen species (ROS) such as singlet molecular oxygen (1 O2 ). The objective of this study was to examine DNA damage in melanoma cells (B16-F10) with different melanin contents, subjected to 1 O2 generation. To this end, we used the photosensitizer Rose Bengal acetate (RBAc) and irradiation with visible light (526 nm) (RBAc-PDT). We used the modified comet assay with the repair enzymes hOGG1 and T4 endonuclease V to detect the DNA damage associated with 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers lesions, respectively. We observed increased formation of hOGG1- and T4endoV-sensitive DNA lesions after light exposure (with or without RBAc). Furthermore, 18 h after irradiation, hOGG1-sensitive DNA lesions increased compared to that at the initial time point (0 h), which shows that a high melanin content contributes to post-irradiation formation of them, mainly via sustained oxidative stress, as confirmed by the measurement of ROS levels and activity of antioxidant enzymes. Contrastingly, the number of T4endoV-sensitive DNA lesions decreased over time (18 h). Our data indicate that in melanoma cells, a higher amount of melanin may affect DNA damage levels when subjected to RBAc-PDT.

Enzyme-mediated photoinactivation of Enterococcus faecalis using Rose Bengal-acetate

Rose Bengal-acetate (RB-Ac) is a pro-photosensitizer claimed to diffuse into target cells, where the acetate groups are hydrolyzed and the photosensitizing properties of Rose Bengal (RB) are restored. Despite promising results on tumor cells, the interaction of RB-Ac with bacteria has never been investigated. This study aimed to assess the interaction of RB-Ac with Enterococcus faecalis and to evaluate its potential use in antimicrobial photodynamic therapy (aPDT).Spectrofluorometry was used to assess the ability of E. faecalis to hydrolyze the RB-Ac compound. Fluorescence microscopy was employed to observe the distribution and to evaluate the cellular uptake of the RB produced. The antibacterial efficiency of RB-Ac-mediated aPDT was assessed by flow cytometry in combination with the LIVE/DEAD® staining.Results showed that RB-Ac was successfully hydrolyzed in the presence of E. faecalis cells. The RB produced appeared to incorporate the membrane of bacteria. Higher concentrations of RB-Ac resulted in higher incorporation of RB. The blue-light irradiation of RB-Ac-treated samples significantly reduced bacterial viability. Less than 0.01% of E. faecalis survived after incubation with 200 μM RB-Ac during 900 min and blue-light activation.The current report indicates that E. faecalis cells can hydrolyze the RB-Ac compound to produce active RB. The use of RB-Ac did not appear to allow cytoplasmic internalization of the RB produced, which rather incorporated the membrane bilayers of E. faecalis. The use of RB-Ac did not provide additional advantages over RB in terms of PS localization. Nonetheless, sufficient RB was produced and incorporated into the membranes of bacteria to elicit effective aPDT.

Rose Bengal Acetate PhotoDynamic Therapy (RBAc-PDT) Induces Exposure and Release of Damage-Associated Molecular Patterns (DAMPs) in Human HeLa Cells

The new concept of Immunogenic Cell Death (ICD), associated with Damage Associated Molecular Patterns (DAMPs) exposure and/or release, is recently becoming very appealing in cancer treatment. In this context, PhotoDynamic Therapy (PDT) can give rise to ICD and to immune response upon dead cells removal. The list of PhotoSensitizers (PSs) able to induce ICD is still short and includes Photofrin, Hypericin, Foscan and 5-ALA. The goal of the present work was to investigate if Rose Bengal Acetate (RBAc), a powerful PS able to trigger apoptosis and autophagy, enables photosensitized HeLa cells to expose and/or release pivotal DAMPs, i.e. ATP, HSP70, HSP90, HMGB1, and calreticulin (CRT), that characterize ICD. We found that apoptotic HeLa cells after RBAc-PDT exposed and released, early after the treatment, high amount of ATP, HSP70, HSP90 and CRT; the latter was distributed on the cell surface as uneven patches and co-exposed with ERp57. Conversely, autophagic HeLa cells after RBAc-PDT exposed and released HSP70, HSP90 but not CRT and ATP. Exposure and release of HSP70 and HSP90 were always higher on apoptotic than on autophagic cells. HMGB1 was released concomitantly to secondary necrosis (24 h after RBAc-PDT). Phagocytosis assay suggests that CRT is involved in removal of RBAc-PDT generated apoptotic HeLa cells. Altogether, our data suggest that RBAc has all the prerequisites (i.e. exposure and/or release of ATP, CRT, HSP70 and HSP90), that must be verified in future vaccination experiments, to be considered a good PS candidate to ignite ICD. We also showed tha CRT is involved in the clearance of RBAc photokilled HeLa cells. Interestingly, RBAc-PDT is the first cancer PDT protocol able to induce the translocation of HSP90 and plasma membrane co-exposure of CRT with ERp57.

In vitroandin vivoclearance of Rose Bengal Acetate-PhotoDynamic Therapy-induced autophagic and apoptotic cells

This study focuses on the clearance of Rose Bengal Acetate (RBAc)-PhotoDynamic Therapy (PDT)-generated apoptotic and autophagic HeLa cells by murine and human macrophages. Indeed, phagocytosis of dead cells drives the therapeutic efficacy of PDT through both efficient removal of dead/dying cells and macrophages response evoked during engulfment and, up to now, clearance of dying photosensitized cells has been less investigated than PDT mechanisms of cell death induction. RBAc-PDT ensures a long onset of cytotoxicity and a time-related cell death of HeLa cells by signals originating from or converging on almost all intracellular organelles. On this basis, to clarify whether the efficacious cell death commitment is followed by an efficient clearance mechanism, we primarily focused on the analysis of 'eat me' signals exposure and 'find me' signals release, and then investigated the migration, recognition, engulfment and response of murine Raw 264.7 and human blood isolated macrophages. Dead cells secreted 'find me' signals, i.e. fractalkine and Heat Shock Protein 70 (HSP 70), to recruit macrophages and promote their fast phagocytosis. Macrophages phagocytosed apoptotic and autophagic PDT-treated cells more efficiently than the respective positive controls, i.e. puromycin-induced apoptotic and Earle's balanced salt solution-starved autophagic cells. Phagocytosis depends on the glycans exposed on dead cells. The macrophages internalization of photokilled cells elicits the production of Interleukin-10, Transforming Growth Factor-β and Tumour Necrosis Factor-α by macrophages. TNFα production, along with HSP70 release and plasma membrane translocation on dead cells, suggest an immunogenic impact of RBAc-PDT. In fact, macrophages, activated fibroblasts and endothelial cells colonized the inoculum site of photosensitized cells in rat calf muscles, endorsing the hypothesis of immunogenic elicitation of RBAc-PDT.

Ultrastructural detection of photosensitizing molecules by fluorescence photoconversion of diaminobenzidine

Photodynamic therapy is a moderately invasive therapeutic procedure based on the action of photosensitizers (PSs). These compounds are able to absorb light, and dissipate energy through photochemical processes leading to the production of oxidizing chemical species (singlet oxygen, free radicals or reactive oxygen species) which can damage the cell molecular structures eventually inducing cell death. To increase the entering through the plasma membrane, a PS with suitable chemical structure can be modified by addition of chemical groups (e.g., acetate or phosphate): this affects both the fluorescence emission and of the photosensitizing properties of the native PS. The modified compounds behave as fluorogenic substrates (FSs), since inside the cell the bound groups can be enzymatically removed and the fluorescence and photosensitizing properties of the native molecules are restored. With the aim to detect the subcellular localization of photoactive molecules at transmission electron microscopy, we loaded cultured HeLa cells with two different FSs, Rose Bengal acetate (RB-Ac) or Hypocrellin B acetate (HypB-Ac), and took advantage of the photophysical properties of the intracellularly restored PS molecules to obtain the photoconversion of diaminobenzidine (DAB) into an electrondense product. We demonstrated that RB-Ac and HypB-Ac are mostly internalized by endocytosis, and are converted into the native PSs already at the cell surface. Endocytosed PS molecules apparently follow the endosomes-lysosome route, being found in endosomes, lysosomes and multivescicular bodies; PS molecules were also detected in the cytosol. This ultrastructural localization of the photoactive molecules is fully consistent with the multiorganelle photodamage observed after irradiation in culture of RB-Ac- or HypB-Ac-loaded cells. Due to the very short half-life of the oxidizing chemical species and their limited mobility, DAB deposits do localize in close proximity of the very place where photoactive molecules elicited the production of reactive oxygen species upon light irradiation. Therefore, DAB photoconversion promises to be a suitable tool for directly visualizing in single cells the PS molecules at high resolution, helping to elucidate their mode of penetration into the cell as well as their dynamic intracellular redistribution and organelle targeting.

Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy

Rose Bengal acetate photodynamic therapy (RBAc–PDT) induced multiple cell death pathways in HeLa cells through ROS and ER stress. Indeed, apoptosis was the first preferred mechanism of death, and it was triggered by at least four different pathways, whose independent temporal activation ensures cell killing when one or several of the pathways are inactivated. Apoptosis occurred as early as 1 h after PDT through activation of intrinsic pathways, followed by activation of extrinsic, caspase-12-dependent and caspase-independent pathways, and by autophagy. The onset of the different apoptotic pathways and autophagy, that in our system had a pro-death role, was timed by determining the levels of caspases 9, 8, 3 and 12; Bcl-2 family; Hsp70; LC3B; GRP78 and phospho-eIF2α proteins. Interestingly, inhibition of one pathway, that is, caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting that the independent onset of the different apoptotic pathways and autophagy did not occur in a subordinated manner. Altogether, our data indicate RBAc as a powerful photosensitiser that induces a prolonged cytotoxicity and time-related cell death onset by signals originating from or converging on almost all intracellular organelles. The fact that cancer cells can die through different mechanisms is a relevant clue in the choice and design of anticancer PDT.

Overview of Cell Death Mechanisms Induced by Rose Bengal Acetate-Photodynamic Therapy

Photodynamic Therapy (PDT) is a non-invasive treatment for different pathologies, cancer included, using three key components: non-toxic light-activated drug (Photosensitizer, PS), visible light, and oxygen. Their interaction triggers photochemical reactions leading to Reactive Oxygen Species (ROS) generation, that mediate cytotoxicity and cell death. In the present paper, the most important findings about the synthetic dye Rose Bengal Acetate (RBAc), an emerging photosensitizer for its efficient induction of cell death, will be reported with the aim to integrate RBAc phototoxicity to novel therapeutic PDT strategies against tumour cells. After its perinuclear intracellular localization, RBAc causes multiple subcellular organelles damage, that is, mitochondria, Endoplasmic Reticulum (ER), lysosomes, and Golgi complex. Indeed, RBAc exerts long-term phototoxicity through activation of both caspase-independent and- dependent apoptotic pathways and autophagic cell death. In particular, this latter cell death type may promote cell demise when apoptotic machinery is defective. The deep knowledge of RBAc photocytotoxicity will allow to better understand its potential photomedicine application in cancer.

Rose Bengal Acetate photodynamic therapy-induced autophagy

Photodynamic therapy (PDT), an anticancer therapy requiring the exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength, induces cell death by the efficient induction of apoptotic as well as non-apoptotic mechanisms, such as necrosis and autophagy, or a combination of all three mechanisms. However, the exact role of autophagy in photodynamic therapy is still a matter of debate. To understand the role of autophagy in PDT, we investigated the induction of autophagy in HeLa cells photosensitized with Rose Bengal Acetate (RBAc). After incubation with Rose Bengal Acetate (10-5 M), HeLa cells were irradiated for 90 seconds (green LED DPL 305, emitting at 530 +15 nm to obtain 1.6 J/cm2 as the total light dose) and allowed to recover for 72 h. Induction of autophagy and apoptosis were observed with peaks at 8 h and 12 h after irradiation, respectively. Autophagy was detected by biochemical (Western Blotting for the LC3B protein) and morphological criteria (TEM, cytochemistry). In addition, the pan-caspase inhibitor, z-VAD, was unable to completely prevent cell death. The simultaneous onset of apoptosis and autophagy following Rose Bengal Acetate PDT is of remarkable interest in light of the findings that autophagy can result in the class II presentation of antigens and thus, explain why low dose PDT can yield anti-tumor immune responses.

Photodynamic Therapy‐Induced Apoptosis of HeLa Cells

Photodynamic therapy (PDT), which is a treatment for cancer and certain noncancerous conditions, requires exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength. By using Rose Bengal Acetate (RBAc) as the photosensitizer and an innovative green light-emitting diode, we investigated the efficiency with which apoptosis is induced in HeLa cells, focusing our study on mitochondria alteration and cytochrome c release. Indeed, RBAc is a very efficient fluorogenic substrate and easily enters the cells where the original photoactive molecule is restored by specific esterases. HeLa cells after PDT underwent a consistent rate of apoptosis (peaked at 12 h of recovery post-PDT). Necrosis was observed at the longest times of recovery as a result of secondary necrosis. PDT gave rise to a series of shape modifications, mainly referable to apoptotic-related changes (i.e., extensive blebs formation) involving both F-actin and tubulin networks. Soon after PDT, mitochondria lose their potential membranes and release large quantities of cytochrome c.

The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate

The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc) and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vesicle traffic (i.e., microtubules) might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24 hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72 h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells.

Enzyme-assisted photosensitization activates different apoptotic pathways in Rose Bengal acetate treated HeLa cells

Photosensitization of tumor cells after incubation with Rose Bengal acetate (RB-Ac) induces multiple organelle photodamage followed by apoptotic cell death. We used immunocytochemical techniques in multicolor fluorescence microscopy to elucidate whether this occurs through the simultaneous activation of different apoptotic pathways, in HeLa cells. We detected in situ the activated forms of caspases 9 and 3, and the translocation from the mitochondria to the nucleus of the apoptosis inducing factor; DNA electrophoretic techniques were also used to assess the occurrence of nuclear DNA cleavage into either high- or low-molecular-weight fragments. Both the caspase-dependent and caspase-independent apoptotic pathways are activated. The genomic DNA is degraded into high molecular weight molecules only, without the formation of oligonucleosome-sized fragments. The ability of RB-Ac to induce the simultaneous release of apoptogenic signals from different photodamaged organelles makes it an especially powerful cytotoxic agent.

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

Rose Bengal (RB) is a very efficient photosensitizer which undergoes inactivation of its photophysical and photochemical properties upon addition of a quencher group-i.e. acetate-to the xanthene rings. The resulting RB acetate (RB-Ac) derivative behaves as a fluorogenic substrate: it easily enters the cells where the native photoactive molecule is restored by esterase activities. It is known that the viability of RB-Ac-loaded cells is strongly reduced by light irradiation, attesting to the formation of intracellular RB. The aim of this study was to identify the organelles photodamaged by the intracellularly formed RB. RB-Ac preloaded rat C6 glioma cells and human HeLa cells were irradiated at 530 nm. Fluorescence confocal imaging and colocalization with specific dyes showed that the restored RB molecules redistribute dynamically through the cytoplasm, with the achievement of a dynamic equilibrium at 30 min after the administration, in the cell systems used; this accounted for a generalized damage to several organelles and cell structures (i.e. the endoplasmic reticulum, the Golgi apparatus, the mitochondria, and the cytoskeleton). The multiple organelle damage, furthermore, led preferentially to apoptosis as demonstrated by light and electron microscopy and by dual-fluorescence staining with FITC-labelled annexin V and propidium iodide.

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

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 induction and mitochondria alteration in human HeLa tumour cells by photoproducts of Rose Bengal acetate

The aim of this work was to investigate the apoptosis induction and mitochondria alteration after photodamage exherted by incubation of HeLa cells with Rose Bengal acetate-derivative (RBAc) followed by irradiation for a total dose of 1.6 J/cm 2. This treatment was previously demonstrated to reduce cell viability under mild treatment conditions, suggesting the restoration of the photoactive molecule in particularly sensitive cell sites. Indeed, Rose Bengal (RB) is a very efficient photosensitizer, whose photophysical properties are inactivated by addition of the quencher group acetate. The RBAc behaves as a fluorogenic substrate by entering easily the cells where the original, photoactive molecule is restored by specific esterases. Different intracellular sites of photodamage of RB are present. In particular, fluorescence imaging of Rodamine 123 and JC-1 labelled cells showed altered morphology and loss of potential membrane of mitochondria. MTT and NR assays gave indications of alteration of mitochondrial and lysosomal enzyme activities. These damaged sites were likely responsible for triggering apoptosis. Significant amount of apoptotic cell death (about 40%) was induced after light irradiation followed RBAc incubation as revealed by morphological (modification of cell shape and blebs formation), cytochemical (FITC-Annexin-V positive cells) and nuclear fragmentation assays.