Effect Of Photodynamic Treatment Research Articles

Rapid tyrosine phosphorylation of HS1 in the response of mouse lymphoma L5178Y-R cells to photodynamic treatment sensitized by the phthalocyanine Pc 4.

The ability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to activate cellular signal transduction pathways in murine lymphoma L5178Y-R cells has been assessed by observing increases in protein tyrosine phosphorylation at early times post-PDT. Western blot analysis with an anti-phosphotyrosine antibody revealed a dramatic increase in phosphorylation of two major protein bands of Mr approximately 80,000 and approximately 55,000 in response to PDT. The increase was PDT dose-dependent, occurred as early as 20 s after initiation of light exposure of Pc 4-preloaded cells and was amplified by the presence of the protein tyrosine phosphatase inhibitor, sodium orthovanadate (NaVO4). By immunoprecipitation, one of the Mr approximately 80,000 phosphorylated proteins has been identified as HS1, a substrate of nonreceptor-type protein tyrosine kinases. Although vanadate greatly enhanced the level and extent of PDT-induced phosphorylation, it had no influence on overall photocytotoxicity or on the rate of apoptotic DNA fragmentation. Genistein, an inhibitor of protein tyrosine kinases, diminished tyrosine phosphorylation of the Mr approximately 80,000 and other proteins and dramatically potentiated cell killing induced by PDT but did not significantly affect PDT-induced apoptosis. The results suggest that PDT rapidly activates a membrane-associated src family kinase(s) in L5178Y-R cells, one substrate of which is HS1, and that protein tyrosine phosphorylation is part of a stress response, protecting a portion of the cells from the lethal effects of PDT but not altering the mechanism by which they die.

Photodynamic treatment of human endothelial cells promotes the adherence of neutrophils in vitro.

The effects of photodynamic treatment (PDT) on venules include vascular leakage accompanied by oedema formation, vasoconstriction and blood flow stasis. The goal of this study was to gain insight into the mechanism underlying these vascular events by studying one of the earliest observations after PDT, granulocyte adhesion, in an in vitro model. For this purpose human umbilical vein endothelial cells (HUVECs) preincubated with Photofrin II (PII) were illuminated with red light and incubated with neutrophils. PDT led to a dramatic change in the morphology of the endothelial cells. Clearly, neutrophils adhered to the subendothelial matrix and their adherence coincided with an increase in the percentage of exposed subendothelial matrix by the gradual contraction of endothelial cells. Furthermore, the increase in adherence was dependent on drug dose, illumination time and the time delay after PDT. The neutrophil adherence could be inhibited by anti-beta2-integrin antibodies, which suggests that the alphaL-, alphaM- or alphaX-beta2 receptors of the neutrophil mediated this phenomenon. At 4 degrees C or by preincubation of the neutrophils with staurosporin, their adherence to the subendothelial matrix exposed by PDT of endothelial cells could be prevented. Apparently, activation of the beta2-integrin receptor by interaction with the subendothelial matrix is necessary for the increased binding of neutrophils. Taken together, these in vitro findings suggest that the PDT-induced contraction of the endothelial cells permits neutrophil adherence to the subendothelial matrix. It is conceivable that a similar mechanism contributes to the initial adherence of granulocytes to the vessel wall as observed after PDT in vivo.

Effect of photodynamic treatment of human endothelial cells on cell volume and cell viability

Photodynamic therapy (PDT) has yielded promising results in the treatment of malignant tumors. However, the mechanisms leading to tumor destruction during PDT are still not completely understood. In addition to effects on the microcirculation, damage to cellular structures has been observed following exposure of cells to PDT. A phenomenon preceding these events might possibly be cell swelling. We therefore studied the influence of treatment with Photofrin(R) (PF) and laser light on volume changes and cell viability of endothelial cells. Endothelial cells were obtained from human umbilical cord veins (HUVEC) by an adaption of the method of Maruyama. After subcultivation the cells were harvested and transferred as a cell suspension into a specially designed incubation chamber. Cells received either PF in concentrations of 1.5 or 3.0 mu g/ml and laser illumination 60 min post incubation (630 nm; 40 mW/cm(2), 4 Joule), PF alone, or laser treatment only. Following start of PF incubation and after phototreatment cell samples were taken for volume measurements using flow cytometry, and for studies of cellular morphology using scanning electron microscopy. Simultaneously, cell viability was monitored by the trypan blue exclusion test and the colorimetric MTT assay. Both control groups, HUVEC receiving PF or laser treatment alone, revealed constant cell volumes and cell viability during the entire course of the experiment. After PDT (60 min post-incubation) with 1.5 and 3.0 mu g PF/ml cell volume of HUVEC was increased at 15 min to 122%+/-6% and 140%+/-10% of baseline (100%), at 60 min to 152%+/-9% and 134%+/-18%, respectively (p<0.01). The number of viable cells was significantly reduced of samples treated with 1.5 and 3.0 mu g PF/ml at 15 min after PDT to 81%+/-3% and 76%+/-10% of baseline (100%), at 60 min after PDT to 32%+/-14% and 20%+/-15%, respectively (p<0.01). Scanning electron microscopy of cells exposed to PDT following 60 min incubation with Photofrin (3.0 mu g/ml) revealed significant cell damage. At the highest PF concentration HUVEC showed loss of microvilli and formation of blebs on the cellular surface. Our study demonstrates that PDT induces a significant increase in endothelial cell volume and a loss of cell viability. We suggest that swelling and damage of endothelial cells following PDT is a primary event finally contributing to cessation of blood flow and subsequent necrosis of tumors.

INTRACELLULAR DAMAGE IN YEAST CELLS CAUSED BY PHOTODYNAMIC TREATMENT WITH TOLUIDINE BLUE

The positively charged photosensitizer toluidine blue (TB) can induce loss of clonogenicity in Kluyveromyces marxianus. Previous studies have revealed that, as a consequence of the localization of this dye at the cell surface, photodynamic action results in extensive damage at the level of the plasma membrane. In this paper, a study is reported on the effect of photodynamic treatment with TB on intracellular enzymes. It is shown that treatment with TB and light resulted in the inhibition of alcohol dehydrogenase, cytochrome c oxidase, glyceraldehyde-3-phosphate dehydrogenase and hexokinase. Photodynamic treatment also lowered the ATP levels. The ATP levels could be partially restored in the presence of glucose but not with ethanol. Toluidine blue binding experiments revealed that photodynamic treatment caused a rapid increase in the amount of cell-associated dye. Moreover, it also appeared that this treatment decreased the binding of TB to the cell surface. It is concluded that TB enters the cell during the first minutes of illumination, whereafter intracellular enzymes are inactivated. The data indicate that photodynamic damage of intracellular sites contributes to the loss of viability.

PHOTODYNAMIC TREATMENT OF YEAST WITH CHLOROALUMINUM‐PHTHALOCYANINE: ROLE OF THE MONOMERIC FORM OF THE DYE

AbstractThe effect of photodynamic treatment on the yeast Kluyveromyces marxianus with aluminum‐phthalocyanines has been studied. It was found that the nonsulfonated sensitizer caused light‐dependent loss of colony‐forming capacity, whereas the mono‐ and tetrasulfonated forms did not induce loss of clonogenicity. The effect of the nonsulfonated sensitizer increased with longer preincubation periods of cells with the dye. Formation of cellattached, mostly intracellularly localized monomelic sensitizer also increased with time. The amount of cell‐bound multimeric nonsulfonated phthalocyanine did not vary with time. Experiments designed to specifically increase the amount of cell‐attached monomers led also to an increased photoinactivation of the cells. It is therefore concluded that the photodynamic effect of the nonsulfonated Al‐phthalocyanine is mediated by the monomeric form of the dye.

Photodynamic therapy of C3H tumours in mice: Effect of drug/light dose fractionation and misonidazole

C3H mammary carcinomas transplanted to the feet of mice were treated with haematoporphyrin derivative (HPD) or Photofrin II(PII) and laser light at 630 nm. While fluence rates lower than 100 mW cm−2 gave minimal hyperthermic effects, a slight but significant growth delay was observed in unsensitized tumours exposed to a fluence rate of 150 mW cm−2 which induced tumour temperatures in the range 40–50°C. Different modes of fractionation of the light fluence and of the HPD dose were tested but were found to give poorer rather than better results than the application of a single light exposure 24 h after intraperitoneal injection of HPD. Different PII doses were applied together with different light fluences, keeping the product of the drug dose and light fluence constant. In the dose range 6.25–50 mg/kg body weight the resulting effect on tumours was constant, allowing for a slight effect of hyperthermia at the highest light fluences, and possibly a photodegradation of PII. Misonidazole given before photodynamic treatment (PDT) slightly reduced the effect of PDT on the tumour growth. When given after PDT, however, misonidazole improved the therapeutic results significantly.

The effect of photodynamic treatment with thiopyronine on yeast nDNA and mtDNA

In order to ascertain the main target of the photodynamic effect with the sensitizer thiopyronine (TP) and its interference with cellular DNA, the uptake of TP into the yeastSaccharomyces cerevisiae and the distribution of the dye within the cells were studied. After fractionation of the cellular components, about 1% of the TP was found to be bound to the nucleus in anaerobically grown yeast cells; in aerobically grown cells, about 7% could be detected at the mitochondria.

The effect of photodynamic treatment with thiopyronine on yeast DNA

The effect of photodynamic treatment with thiopyronine on yeast DNA

Thiopyronine-sensitized Photodynamic Effect on RNA Synthesis inSaccharomycesCellsin Vivo

The effect of photodynamic treatment with thiopyronine and visible light on RNA metabolism in yeast cells was investigated at different times during logarithmic growth. The results show that RNA synthesis in the nucleus of the cells is not directly inhibited photodynamically. In the endoplasmic reticulum of photodynamically treated cells one finds mRNA in about the same relative amounts and quality as in untreated control cells, but the binding of polysomes on membranes in the cytoplasm as the first step of protein synthesis is inhibited for a long time after treatment as well as the synthesis of ribosomes.

On the treatment with proflavine of herpes simplex or vaccinia infected GMK cells. An electron microscopic study.

Green monkey kidney (GMK) cells were inoculated with vaccinia or herpes simplex virus, and the effect of photodynamic treatment with proflavine and light was studied in the electron microscope. Whereas virions were abundant in all untreated cultures (incubated with virus for 15 hours or more), no virus particles could be found in the treated ones. Proflavine and light, in the amounts used, seemed to exert a certain degree of toxicity on the GMK cell cultures, but virus reproduction was effectively stopped by the treatment. Preliminary results on photodynamic treatment of human herpes keratitis are mentioned.