Modulation of 5-Aminolevulinic acid mediated photodynamic therapy induced cell death in a human lung adenocarcinoma cell line

Photodynamic therapy (PDT) is a cancer treatment involving the administration of a photosensitising drug which selectively accumulates in tumor tissue, followed by irradiation with appropriate wavelength light. It triggers photochemical reactions inducing reactive oxygen species (ROS) production with the consequent cellular damage, which ultimately leads to cell death. Porphyrins are the only photosensitizers (PSs) endogenously synthesized by means of administration of the biological precursor, 5- aminolevulinic acid (ALA). Several antioxidants and ROS scavenger agents: reduced glutathione (GSH), mannitol (Man), l-tryptophan (Trp), ascorbate (Asc) and trolox (Trx), were assayed to determine their ability to modulate ALA-based PDT (ALA-PDT); it was performed on A549 human lung adenocarcinoma cells, by incubating with 1mM ALA for 3 hr and followed by irradiation with or without 1 hr pre-incubation with the modulators. They were previously tested for possible cytotoxicity/ photoactivity in concentrations ranging from 0.01 to 20 mM. The ratio between cell survival after ALA-PDT in the presence and in the absence of the scavenger agent (protection grade: PG) was determined, and the concentration showing no cytotoxicity/ photoactivity and providing the highest PG was used in the subsequent experiments. ALA-PDT alone induced a high percentage of apoptotic cell death (98.4 ± 3.5%) as revealed by acridine orange/ethidium bromide staining and AnnexinV-FITC/propidium iodide labelling. Pre-incubation with the modulators at their highest PG concentration significantly reduced apoptotic cells to 48.3 ± 2.7% (Asc), 58.8 ± 4.2 (Trx), 78.5 ± 3.1% (GSH), 64.3 ± 1.6% (Man), 74.6 ± 2.3% (Trp). ROS involvement in early cell death induction after ALA-PDT was tested by flow cytometry using the fluorescent probes dihydro-dichlorofluorescein diacetate (H2-DCFDA) and methoxyvinylpyrene (MVP) for detection of peroxides and singlet oxygen, respectively. ROS production increased after ALA-PDT (H2-DCFDA positive cells, control: 1.1 ± 0.1 %; 10 min-PDT: 69.3 ± 5.6%; MVP positive cells, control: 0.65 ± 0.35%; 10 min-PDT: 83.5 ± 1.9%). Asc prevented peroxide formation (H2-DCFDA positive cells: 50.7 ± 2.8%) and mostly prevented singlet oxygen increase (MVP positive cells: 25.4 ± 5.2%) whereas Trx limited peroxides formation (H2-DCFDA positive cells: 20.8 ± 0.5%), but did not significantly affected singlet oxygen production (MVP positive cells: 73.6 ± 3.4%). Selective scavenger mediated protection against PDT-induced cell death, and direct detection of specific pro-oxidative agents, entail the strong involvement of ROS in ALA-PDT-mediated tumor eradication, suggesting that undesired photodamage to normal tissue might be attenuated by administration of antioxidant agents.Fil: Teijo, Maria Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Diez, Berenice Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Battle, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Fukuda, Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Overview on topical 5-ALA photodynamic therapy use for non melanoma skin cancers

Ultraviolet radiation (UV) contributes to a variety of skin diseases including inflammation, degenerative aging, and cancer. Historically, humans have been exposed to UV radiation mainly through occupational exposure; recreational UV exposure, however, has increased dramatically in recent years, because of outdoor leisure activities and to purposely tan for cosmetic purposes. Both UVB and UVA radiation have been shown to cause DNA damage and immunosuppression, the important forms of biological damage that lead to NMSC. Nonmelanoma skin cancer (NMSC) is the most common malignancy, whose public health significance is often unrecognized which continues to grow at an alarming rate, becoming an occupational disease. Available treatments alternative to surgery include photodynamic therapy, electrochemotherapy, cryotherapy, ablative lasers, 5-fluorouracil, imiquimod, ingenol mebutate, and diclofenac. Among these, photodynamic therapy is a noninvasive technique with excellent cosmetic outcome and good curative results, when used in initial stages of skin cancers for superficial lesions. It is administered under numerous and significantly varied regimens and there are a wide range of cure rates reported, permitting treatment of large and multiple lesions with excellent cosmetic results. This is an overview of photodynamic applications especially for the treatment of NMSC, with a short focus on daylight modality. © 2014 Carmen Cantisani et al

Mechanisms of growth inhibition of primary prostate epithelial cells following gamma irradiation or photodynamic therapy including senscence, necrosis, and autophagy, but not apoptosis

In comparison to more differentiated cells, prostate cancer stem-like cells are radioresistant, which could explain radio-recurrent prostate cancer. Improvement of radiotherapeutic efficacy may therefore require combination therapy. We have investigated the consequences of treating primary prostate epithelial cells with gamma irradiation and photodynamic therapy (PDT), both of which act through production of reactive oxygen species (ROS). Primary prostate epithelial cells were cultured from patient samples of benign prostatic hyperplasia and prostate cancer prior to treatment with PDT or gamma irradiation. Cell viability was measured using MTT and alamar blue assay, and cell recovery by colony-forming assays. Immunofluorescence of gamma-H2AX foci was used to quantify DNA damage, and autophagy and apoptosis were assessed using Western blots. Necrosis and senescence were measured by propidium iodide staining and beta-galactosidase staining, respectively. Both PDT and gamma irradiation reduced the colony-forming ability of primary prostate epithelial cells. PDT reduced the viability of all types of cells in the cultures, including stem-like cells and more differentiated cells. PDT induced necrosis and autophagy, whereas gamma irradiation induced senescence, but neither treatment induced apoptosis. PDT and gamma irradiation therefore inhibit cell growth by different mechanisms. We suggest these treatments would be suitable for use in combination as sequential treatments against prostate cancer

Laser-induced generation of singlet oxygen and its role in the cerebrovascular physiology

For over 55 years, laser technology has expanded from laboratory research to widespread fields, for example telecommunication and data storage amongst others. Recently application of lasers in biology and medicine presents itself as one of the emerging areas. In this review, we will outline the recent advances in using lasers for the generation of singlet oxygen, traditionally used to kill tumour cells or induce thrombotic stroke model due to damage vascular effects. Over the last two decade, completely new results on cerebrovascular effects of singlet oxygen generated during photodynamic therapy (PDT) have been shown alongside promising applications for delivery of drugs and nanoparticles into the brain for therapy of brain cancer. Furthermore, a "gold key” has been found to overcome the limitations of PDT, such as low light penetration and high toxicity of photosensitizers, by direct generation of singlet oxygen using quantum-dot laser diodes emitting in the near infrared (NIR) spectral range. It is our motivation to highlight these pioneering results in this review, to improve understanding of the biological role of singlet oxygen and to provide new perspectives for improving clinical application of laser based therapy in further research

Immobilized photosensitizers for antimicrobial applications

Photodynamic antimicrobial chemotherapy (PACT) is a very promising alternative to conventional antibiotics for the efficient inactivation of pathogenic microorganisms; this is due to the fact that it is virtually impossible for resistant strains to develop due to the mode of action employed. PACT employs a photosensitizer, which preferentially associates with the microorganism, and is then activated with non-thermal visible light of appropriate wavelength(s) to generate high localized concentrations of reactive oxygen species (ROS), inactivating the microorganism. The concept of using photosensitizers immobilized on a surface for this purpose is intended to address a range of economic, ecological and public health issues. Photosensitising molecules that have been immobilized on solid support for PACT applications are described herein. Different supports have been analyzed as well as the target microorganism and the effectiveness of particular combinations of support and photosensitiser

Treatment of head and neck cancer with photodynamic therapy: results after one year

Photodynamic therapy (PDT) is a new and promising treatment modality for the treatment of malignant disease. This paper reports the preliminary experience of our group in the use of this therapy for the treatment of tumours arising in the head and neck. The majority of treatments in these cases have used a second generation systemic photosensitizer, meta-tetrahydroxyphenylchlorin (m-THPC). Two other cases were treated with either Photofrin 2 (a first generation systemic sensitizer) or with the topical photosensitizer, delta-aminolaevulinic acid (delta-ALA).The initial results have been encouraging with good clinical responses evident in patients presenting with a variety of differing tumour types. We feel there is now sufficient evidence of the efficacy of this treatment to warrant a multicentre prospective study into the treatment of early head and neck cancer with PDT

Effect of photosensitizers photosens, photodithazine and hypericin on glioma cells and primary neuronal cultures : a comparative analysis

The aim of the study was to compare the effect of photosensitizers photosens, photodithazine, and hypericin on primary brain cell cultures, and assess their toxic effect on tumor and normal nervous cells in order to choose the optimal photodynamic agent for glioma therapy. Materials and Methods. The cytotoxicity of photosens (NIOPIK, Russia), photodithazine (Veta-grand, Russia) and hypericin (Merck KGaA; Sigma-Aldrich, Germany) was assessed on primary brain cell cultures obtained from C57BL/6 mice (gestation day 18). On day 14 of cultivation, the tested photosensitizers were added to a culture medium at concentrations of 0.1, 1, 10, 50, and 100 mu M. Then the cultures were placed in a CO2-incubator in the dark. The viability of primary neuronal cultures was estimated on days 3 and 7 after photosensitizer application. Using confocal microscopy, we analyzed the rate of entry and subcellular localization of the tested agents in the primary neuronal cells. Statistical analysis was performed in SigmaPlot 11.0 (Systat Software Inc., USA) using ANOVA. Results. We analyzed the absorption and fluorescence spectra of the tested photosensitizers. Photosens and photodithazine showed the presence of absorption maximum in short- and long-wave spectral ranges. Hypericin was characterized by a complex spectrum with many peaks in both blue-violet and orange-red spectral ranges. Cell viability analysis revealed that high concentrations of photosensitizers caused a pronounced toxic effect on nervous cells. The most marked effect was shown for photodithazine. Photosens exhibited the lowest accumulation rate in primary neuronal cells. Photosens and hypericin were found to have a high phototoxic effect on glioma, and demonstrated low dark toxicity for normal brain cells. Conclusion. The photosensitizers hypericin and photosens are the least toxic for nervous tissue, though effectively penetrating in tumor cells. These properties enable to consider them as prospective photodynamic agents for clinic