Reference #: DUS-1017-238068
Submit Date: 03/27/2002 07:48:36-0500

Photodynamic therapy with toluidine blue in K562 leukemic cells.

ABSTRACT:
Toluidine blue (TBO) is a thiazine dye which has been used for many years in the diagnosis of oral cancer. The aim of this project was to study the potential of TBO as a photodynamic therapy photosensitizer for the elimination of leukemic cells. Intracellular TBO accumulation was measured by spectrofluorometry for K562 leukemic cells and bone marrow cells from non-leukemic donors at different times after incubation with 2 g/mL of TBO. Subcellular localisation of TBO was studied by fluorescence microscopy using organelle dyes such as MitoTracker, LysoTracker and BODIPY-FL C₅-Ceramide. Clonogenic assays were performed to evaluate the survival of K562 cells following PDT with TBO at 11,68 J/cm² of light from an unfiltered Kodak Carousel 650H slide projector. K562 cells accumulate 5,7 times more TBO than normal BM cells after a one hour incubation with 2 g/mL of TBO. TBO accumulation by K562 cells was already maximal at 10 minutes and this level was maintained for up to 2 hours. Co-localization studies showed that TBO does not localize to the mitochondria or the Golgi apparatus. TBO-PDT induces a significant decrease in survival of K562 cells with concentrations as low as 0,15 g/mL. Clonogenic assays showed a reduction in survival of K562 cells of more than 10000 fold with TBO-PDT performed with 2 g/mL of TBO. In conclusion, TBO accumulates preferentially in leukemic cells and its activation by visible light causes a significant decrease of survival of leukemic cells.

Keywords: Photodynamic Therapy, Toluidine Blue, Leukemia

Reference #: ZHO-1017-105564
Submit Date: 03/25/2002 18:46:11-0500

Analysis of Cellular Respiration Changes In the RIF-1 Tumor in response to Photodynamic Therapy with Verteporfin

ABSTRACT:
Cellular respiration changes due to the mitochondrial damage occur rapidly in photodynamic therapy. Detection of this change in metabolism may be a useful method to directly monitor tissue damage in PDT in vivo. In our study, we investigate the changes in NADH fluorescence, oxygen consumption rate, blood flow and tissue oxygen partial pressure (pO₂) during PDT with verteporfin, delivered to RIF-1 tumor cells. Results of the MTT assay show changes in cell metabolism immediately after treatment indicating that the damage to the cells induces an immediate loss of metabolic function. In general, the fluorescence emission centered at 450 nm, due to excitation at 325 nm, always decreases with delivery of photodynamic therapy. This is thought to be attributed to NADH loss as the mitochondrial membranes loose their integrity. In the same manner, the oxygen consumption rate of the cells is decreased. Measurements in vivo agree with these observations and also indicate that tumor pO₂ can increase significantly towards the end of PDT treatment. These observations, taken together, would indicate that cellular respiration is halted acutely in response to PDT with verteporfin.

Keywords: mitochondria, NADH fluorescence, oxygen consumption rate, Benzoporphyrin Derivative

Reference #: VAN-1017-268892
Submit Date: 03/27/2002 16:21:24-0500

Monitoring real-time tumor response in the rat during PDT using positron emission tomography.

ABSTRACT:
Positron emission tomography (PET), using fluorodesoxyglucose (FDG) labeled with F-18 (t1/2 = 20 min) as a tracer, is a powerful tool to visualize in vivo metabolic activity. Viable tumor cells readily retain FDG and FDG-PET is used routinely in clinic to detect and stage tumors. Using an animal PET camera PDT response in an animal tumor model can be followed by FDG-PET scans to quantify tumor response within one week after treatment. By placing the animal during PDT in a PET scanner while infusing FDG, tumor response can be followed in real time allowing not only to measure the extend of tumor cell inactivation but also to detect differences in action mechanisms. We compared the effect on FDG tumor uptake during Photofrin-PDT, which initial action is largely at the vascular level, with that of phthalocyanine-PDT (AlPcS2a), which mainly acts directly at the cellular level. Characteristic differences in metabolic regression pattern were observed. With Photofrin (5 mg/kg) there was initially a slight increase in FDG uptake at the onset of PDT, followed by irreversible suppression. This reflects inflammation and edema preceding the collapse of the tumor vascular system and the resulting extensive tissue damage. With the phthalocyanine (1 mg/kg), FDG uptake declined shortly (1-2 min) after initiating PDT, but partially recovered after the light treatment. This is consistent with initial loss of cellular hexokinase activity, followed by partial recovery of the metabolic activity reflecting the level of tumor cell survival immediately after treatment. This study shows that FDG-PET imaging in a rodent tumor model can be used to investigate both the efficacy of a treatment protocol as well as certain aspects of the action mechanisms of tumor response during PDT.

Keywords: photodynamic therapy, positron emission tomography, photosensitizer, fluorodesoxyglucose

Reference #: HUN-1017-964897
Submit Date: 04/04/2002 17:43:45-0500

Dendritic cells and PDT

ABSTRACT:
Dendritic cells (DC) are antigen-presenting cells typically present at relatively low frequencies in most tissues. DC likely play an important role in the initiation and perpetuation of autoimmune disease as well as in the immune response against tumours. By virtue of their abundant expression of major histocompatibility complex (MHC), adhesion and co-stimulatory molecules as well as production of specific immuno-regulatory cytokines, DC are potent inducers of immune responses. These bone marrow-derived cells undergo activation/maturation following uptake of antigenic material and the receipt of appropriate molecular signals in their immediate environment. PDT has been shown to inhibit human and mouse DC expression of a number of immunoregulatory receptors and inhibit the immunostimulatory activity of these cell types in vitro. Conceivably, such an effect could contribute to a dampening of certain immune-mediated responses with PDT. It has been found that optimal anti-tumour PDT requires an involvement of the immune system. Since DC are essential for the generation of primary immune responses it is likely that DC play a role in anti-tumour immunity associated with PDT. Strategies to increase DC numbers and/or their activation state might augment the anti-tumour effectiveness of PDT. Of specific interest, is the influence of the mode of tumour cell death (apoptosis versus necrosis) induced by PDT on the subsequent activation of DC. Such potential differential responses can be evaluated with in vitro systems. Further, it is possible to dramatically increase DC numbers in normal and tumour-bearing mice by the administration of specific hematopoietic growth factors. The present discussion will review what is known regarding the direct effect of PDT on DC. In addition, more recent experiments examining the contribution of DC on the anti-tumour effect of PDT using the photosensitizer QLT0074 will be summarised.

Keywords: dendritic cells, QLT0074, tumour immunity, apoptosis

Reference #: 103326
Submit Date:

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Reference #: JOR-1016-641243
Submit Date: 03/20/2002 09:44:48-0500

Phthalocyanines as phototherapeutic agents for the treatment of microbial infections

ABSTRACT:
Infectious diseases are widespread all over the world, often have a lethal outcome and represent a major cost for public heath systems. The situation is aggravated by the extensive use of antibiotics, a main factor in the emergence of resistance. Thus, therapeutic alternatives based on different strategies are needed. Photodynamic therapy (PDT) with porphyrins and phthalocyanines appears to be a promising approach especially for the treatment of localized infections. Actually, the mechanism of action of photodynamic sensitizers on microbial cells is substantially different from that typical of antibiotic drugs; moreover, the photosensitivity of microorganisms is generally independent of their antibiotic resistance spectrum. Lastly, a careful control of the parameters involved in the PDT protocol allows one to achieve a very high differential phototoxicity between microbial and human cells. Amphiphilic phthalocyanines bearing positive charges due to the presence of pyridyl, piperidinyl or anilinium substituents with quaternarized nitrogens appear to be particularly active, since they photosensitise a 4-5 log decrease in the survival of Gram-positive and Gram-negative bacteria, as well as of yeasts and mycoplasmas, upon short irradiation with red light (ca. 700 nm, fluence-rates around 10 mW/cmsq)in the presence of phthalocyanine concentrations lower than 0.5 uM. These photosensitizers essentially localize in the cytoplasmic membrane so that the photoprocess does not lead to detectable damage of the genetic material. Apparently, the phthalocyanine-photosensitised inactivation of microbial cells does not induce the selection of photoresistant strains. Typical examples will be provided using selected pathogens, such as Staphylococcus aureus (including methicillin-resistant strains), Escherichia coli, Pseudomonas aeruginosa, Candida albicans and bacteria responsible for periodontal diseases.

Reference #: GIR-1017-086458
Submit Date: 03/25/2002 13:17:09-0500

Photosensitized lipid peroxidation and apoptotic killing in 5-aminolevulinate-treated COH-BR1 cells: protective effects of mitochondrial GPX4 overexpression.

ABSTRACT:
In 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), ALA enters tumor cells and is metabolized to protoporphyrin IX (PpIX), a cytotoxic photosensitizer which accumulates initially in mitochondria, where terminal steps of heme synthesis take place. Little is known about (i) the mechanism of singlet oxygen-mediated cells death in ALA-PDT and how it is influenced by ALA treatment conditions or (ii) cellular defenses against photokilling. We have addressed both issues, using a breast tumor line deficient in the lipid hydroperoxide (LOOH)-detoxifying selenoperoxidase GPX4 and a transfectant clone (7G4) exhibiting ~85-fold greater GPX4 activity, ~80% of which is in mitochondria. Vector control (VC) and 7G4 cells accumulated PpIX at the same rate and, after broad-band visible irradiation, succumbed via apoptosis when the porphyrin at a fixed intracellular concentration was mainly in mitochondria, but via necrosis when it was allowed to diffuse to other sites, including plasma membrane. Compared with VC controls, 7G4 cells were strongly protected against photoinduced apoptosis, but not against necrosis, indicating that mitochondrial GPX4 effectiveness was site-specific. 7G4 cells accumulated cholesterol-derived LOOHs at a substantially lower rate than VC during apoptotic photokilling, but at the same, much greater rate during necrotic killing, consistent with the known preponderance of cholesterol in the plasma membrane and lack of GPX4 in this compartment. These findings support the hypothesis that mitochondrial GPX4, by inactivating apoptosis-signaling LOOHs, plays a key role in tumor resistance to ALA-PDT. (Supported by NIH: CA72630)

Keywords: 5-aminolevulinic acid, phototherapy, apoptosis, selenoperoxidase