Molecular response to hypericin-induced photodamage.

Hypericin (Hyp) is used as a powerful natural photosensitizer in photodynamic therapy (PDT). After selective accumulation in tumor tissue, vessels and matrix, and activated by visible light, it destroys the tumor mainly via generation of reactive oxygen species. After photoactivation, molecular biological mechanisms lead to different cellular endpoints: "biostimulation" (increased proliferation rate), repair of the damage leading to rescue of the cells, autophagy, apoptosis and necrosis. Growth stimulation after low-dose Hyp-PDT seems to be induced via the p38 or JNK survival pathways. Since both pathways are also activated by stress, modification of these pathways may also contribute to rescue mechanisms as well as to damage processing. By increasing PDT doses beyond sublethal damage, stress response pathways are activated such as the ER-stress pathway with disruption of Ca2+ homeostasis and unfolded protein response. This leads either to apoptosis or autophagic cell death, dependent on the availability of Bax/Bak. Apoptosis triggered directly at the mitochondria or by the ER-stress response is executed via the mitochondrial pathway, whereas in some cases, the receptor-mediated pathway is preferred. If the damage is too severe, the cellular energy level low and /or the cytoplasma membrane leaky, cells will die necrotically. The different modes of cellular responses depend mainly on the PDT-protocol, photosensitizer localisation, cellular damage protection and the available intracellular energy.

Photophysics and photochemistry of photodynamic therapy: fundamental aspects.

Photodynamic therapy (PDT) is a treatment modality for cancer and various other diseases. The clinical protocol covers the illumination of target cells (or tissue), which have been loaded with a photoactive drug (photosensitizer). In this review we describe the photophysical and primary photochemical processes that occur during PDT. Interaction of light with tissue results in attenuation of the incident light energy due to reflectance, absorption, scattering, and refraction. Refraction and reflection are reduced by perpendicular light application, whereas absorption can be minimized by the choice of a photosensitizer that absorbs in the far red region of the electromagnetic spectrum. Interaction of light and the photosensitizer can result in degradation, modification or relocalization of the drug, which differently affect the effectiveness of PDT. Photodynamic therapy itself, however, employs the light-induced chemical reactions of the activated photosensitizer (triplet state), resulting in the production of various reactive oxygen species, amongst them singlet oxygen as the primary photochemical product. Based on these considerations, the properties of an ideal photosensitizer for PDT are discussed. According to the clinical experience with PDT, it is proposed that the innovative concept of PDT is most successfully implemented into the mainstream of anticancer therapies by following an application-, i.e. tumor-centered approach with a focus on the actual clinical requirements of the respective tumor type.

A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening.

Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.

Analysis of altered gene expression profiles in retinoic acid or CpG-treated Sprague-Dawley rats with MNU-induced mammary adenocarcinoma by cDNA macro array.

In the present work the effects of 13-cis retinoic acid (RA) and CpG-containing oligodeoxynucleotides (CpG-ODN) on the gene expression profile of spleen and tumor tissue in a MNU-induced mammary gland carcinoma ratmodel were investigated by the use of a commercial cDNA macro array (Atlas rat toxicology array 1.2, Clontech). Treatment with these components, either alone or in combination, induced differences of the expression profiles between the distinct treatment groups in both tissues. The large number of genes with altered expression (> 200) points to a highly complex process in vivo.

Clinical and experimental evidence of Bcl-2 involvement in the response to photodynamic therapy.

PURPOSE: The role of apoptosis related proteins in the response of human malignancies to photodynamic therapy (PDT) is under investigation. The aim of the study was to examine the role of p53 and of bcl-2 protein expression in the response to PDT. MATERIALS AND METHODS: Paraffin-embedded material from 37 patients with early esophageal cancer treated with PDT (argon dye laser after intravenous injection of hematoporphyrine derivative) was studied immunohistochemically for p53 protein nuclear accumulation and bcl-2 cytoplasmic expression. Patients with residual disease after two rounds of PDT received definitive radiotherapy. In a subsequent in vitro study, W138 human lung fibroblasts and W138-SV-40 virus transformed were assessed for their sensitivity to PDT. The constitutive bcl-2 overexpression of the transformed cells vs. normal cells (assessed with RT-PCR) was 16-fold. RESULTS: Positive bcl-2 and p53 expression was noted in 10 out of 36 (27%) and 14 out of 36 (39%) patients, respectively. Seven out of 11 tumors (63%) with bcl-2 expression responded completely to PDT vs. 6 out of 26 (23%) of cases with no bcl-2 expression (p = 0.02). No association of p53, T-stage and of histology grade with response to PDT or PDT/RT was noted. The sensitivity to PDT of transformed human fibroblasts compared to normal ones was 4 times more at a fluence of 4.3 J/cm2 (4% vs. 1% cell kill) as well as at a fluence of 5.4 J/cm2 (8% vs. 2% cell kill). CONCLUSION: Bcl-2 protein expression is associated with favorable response to PDT and can be used as a predictor of cancer response to PDT. This finding can be explained by experimental studies showing that PDT induces selective degradation of the bcl-2 protein, leading to apoptosis by decreasing the bcl-2/bax ratio. Studies on PDT combination with agents targeting bcl-2 (i.e. taxanes) are on going to eventually assess a super-additive effect.

Vascular effects of photodynamic therapy.

Photodynamic therapy (PDT) of tumours is based on a dual selectivity, i.e. preferential uptake and retention of a photosensitiser by tumours and irradiation of the tumour area with light wavelengths specifically absorbed by the photosensitiser. The photoexcited sensitiser generates highly reactive oxygen species that induce irreversible damage to neoplastic cells and vessels. Following sensitiser accumulation and irradiation, damage to sensitive sites within the microvasculature, namely to endothelial cells and the vascular basement membrane, is induced and leads to the establishment of thrombogenic sites within the vessel lumen. This initiates a physiological cascade of responses including platelet aggregation, the release of vasoactive molecules, leukocyte adhesion, increases in vascular permeability and vessel constriction. These result in tumour destruction by vascular collapse, blood flow stasis and tissue hemorrhages. Several photosensitisers are able to induce severe vasculature damage, although by variously different mechanisms. Due to its efficient vascular interactions, photodynamic treatment is also increasingly used for non-cancerous lesions. Successful application of PDT mainly for vessel occlusion and thrombosis in intimal hyperplasia, restenosis, atherosclerotic plaques, corneal and choroidal neovascularisation and port-wine stains is reported.

Plasma membrane properties involved in the photodynamic efficacy of merocyanine 540 and tetrasulfonated aluminum phthalocyanine.

Merocyanine 540 (MC540)-mediated photodynamic damage to erythrocytes was strongly reduced when illumination was performed at pH 8.5 as compared to pH 7.4. This could be explained by high pH-mediated hyperpolarization of the erythrocyte membrane, resulting in decreased MC540 binding at pH 8.5. In accordance, the MC540-mediated photooxidation of open ghosts was not inhibited at pH 8.5. Photoinactivation of vesicular stomatitis virus (VSV) was not inhibited at pH 8.5. This suggests that illumination at increased pH could be an approach to protect red blood cells selectively against MC540-mediated virucidal phototreatment. With tetrasulfonated aluminum phthalocyanine (AIPcS4) as photosensitizer, damage to erythrocytes, open ghosts and VSV was decreased when illuminated at pH 8.5. A decreased singlet oxygen yield at high pH could be excluded. The AIPcS4-mediated photooxidation of fixed erythrocytes was strongly dependent on the cation concentration in the buffer, indicating that the surface potential may affect the efficacy of this photosensitizer. This study showed that altering the environment of the target could increase both the efficacy and the specificity of a photodynamic treatment.

Activation of macrophage tumoricidal activity by photodynamic treatment in vitro--indirect activation of macrophages by photodynamically killed tumor cells.

Macrophages constitute a major part of natural tumor defense by their capacity to destroy selectively a broad range of tumor types upon specific activation. In the last couple of years, these cells have also been implicated as effector cells in the destruction of tumors by photodynamic therapy. In the present work, the potential role of macrophage-mediated tumor cytotoxicity after photodynamic treatment in vitro has been investigated with respect to photodynamic activation of macrophages for tumoricidal effector functions. Our data show that photodynamic treatment of highly pure murine bone-marrow-derived macrophages with the hematoporphyrin derivative Photosan-3 does not result in activation of these cells for cytotoxicity against YAC-1 tumor cells or secretion of tumor necrosis factor and nitric oxide, irrespective of co-stimulation with interferon-gamma, a potent priming agent for macrophage antitumoral activity. On the contrary, treatment with higher photosensitizer doses is found to reduce markedly the viability of the macrophage effector cells. Thus, these results do not lend any support to the hypothesis of direct macrophage activation by photodynamic treatment. However, macrophages are found to be activated for tumoricidal effector functions indirectly by photodynamically killed tumor cells, in a way reminiscent of phagocytosis-inducing stimuli. It is thus suggested that recognition and phagocytosis of photodynamically destroyed tumor cells constitutes the major signal for local activation of macrophages in photodynamically treated tumor tissues, which may be crucial for final, specific eradication by the immune system of tumor cells surviving photodynamic treatment.

Cutting edge: differential effect of apoptotic versus necrotic tumor cells on macrophage antitumor activities.

Macrophages (Mphi) play essential roles both in tumor defense and normal tissue homeostasis by removal of transformed as well as damaged and disintegrating cells. Whereas tissue necrosis is known to provoke inflammatory responses, removal of apoptotic cells has been assumed to be immunologically inert. We now show that while Mphi exposure to necrotized tumor cells causes pronounced stimulation of Mphi antitumor activity, exposure of Mphi to apoptotic tumor cells in contrast results in impairment of Mphi-mediated tumor defense and even support of tumor cell growth. Given the fact that apoptosis is a consequence of various cancer treatment modalities, this may lead to a suppression of local antitumor reactions and thus actually counteract endogenous immune-mediated tumor defense mechanisms.

bcl-2 and c-erbB-2 proteins are involved in the regulation of VEGF and of thymidine phosphorylase angiogenic activity in non-small-cell lung cancer.

Tumour angiogenesis has been recently recognised as one of the most important prognostic factors in lung cancer. Although a variety of angiogenic factors have been identified, the angiogenesis process remains poorly understood. Bcl-2, c-erbB-2 and p53 are well-known oncogenes involved in non-small-cell lung cancer pathogenesis. A direct correlation of thymidine phosphorylase (TP) and of vascular endothelial growth factor (VEGF) with intratumoural angiogenesis has been reported. In the present study we investigated the possible regulatory role of bcl-2, c-erB-2 proteins in angiogenesis and in VEGF and TP expression in non-small-cell lung cancer. Two hundred sixteen specimens from T1,2-N0,1 staged patients treated with surgery alone were immunohistochemically examined. Bcl-2 and c-erbB-2 were significantly inversely related to each other (P = 0.04) and both were inversely associated with microvessel density (P < 0.02). High TP and VEGF reactivity was statistically related to loss of bcl-2 expression (P < 0.01). A significant co-expression of c-erbB-2 with TP was noted (P = 0.01). However, TP expression was related to high angiogenesis only in cases with absence of c-erB-2 expression (P < 0.0001). c-erbB-2 expression in poorly vascularised tumours was linked with poor outcome (P = 0.03). The present study provides strong evidence that the bcl-2 gene has a suppressive function over genes involved in both angiogenesis (VEGF and TP) and cell migration (c-erbB-2) in NSCLC. TP and c-erbB-2 proteins are significantly, and often simultaneously, expressed in bcl-2 negative cases. However, expression of the c-erbB-2 abolishes the TP-related angiogenic activity. Whether this is a result of a direct activity of the c-erbB-2 protein or a consequence of a c-erbB-2-related immune response remains to be further investigated.

Expression kinetics of the (proto) oncogenes c-myc and bcl-2 following photodynamic treatment of normal and transformed human fibroblasts with 5-aminolaevulinic acid-stimulated endogenous protoporphyrin IX.

In the same way as common tumour therapies can cause secondary tumour induction, photodynamic tumour therapy also shows a moderate mutagenicity. The oncogenes responsible for it can be distinguished from their proto-oncogenic precursors by an irreversible increase in their constitutive expression. Transient changes of the expression level of (proto) oncogenes can indicate the beginning of disturbances in the cell homeostasis: many of these genes have a normal function in proliferation or play a role in apoptosis. In this study, therefore, quantitative determination of the expression of the (proto) oncogenes c-myc and bcl-2 in normal and transformed human fibroblasts at different times following photodynamic treatment with 5-aminolaevulinic acid-stimulated endogenous protoporphyrin IX and low-dose irradiation has been carried out by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The aim is to investigate if irreversibly increased (proto) oncogene expression can be found, and if expression changes are involved in cell-cycle alterations (detected in a parallel study) and in initiation of apoptotic processes. The results show: (1) no mutagenic risk, since the over-expression of c-myc and bcl-2 is transient; (2) an interaction of bcl-2 and c-myc associated with an increase of the proliferative activity of the cell cycle of transformed cells; (3) a possible role of bcl-2 in counteracting processes that could be at least precursors for apoptosis induction; and (4) higher constitutive expression of both genes in transformed than in normal fibroblasts.

Effect of photodynamic pretreatment on the susceptibility of murine tumor cells to macrophage antitumor mechanisms.

In vitro photodynamic treatment of YAC-1 murine T-lymphoma cells with the hematoporphyrin derivative Photosan 3 and red light resulted in dose-dependent phototoxicity. Photodynamic pretreatment, however, did not render these cells more susceptible to macrophage-mediated tumor cytotoxicity or the cytotoxic effects of macrophage-derived antitumor mediators like tumor necrosis factor alpha (TNF-alpha) or interferon beta (IFN-beta). Independent of the degree of photosensitization used, the cytotoxicity values obtained with macrophages or the different mediators were shifted by the respective values for phototoxicity, suggesting these effects to be additive and thus not interdependent. These data show that while higher overall tumor cytotoxicity can be achieved by a combination of photodynamic treatment and macrophage-mediated tumor destruction, this apparently is not a result of enhanced sensitivity of photodynamically treated tumor cells to macrophage antitumor mechanisms in general.

Does the in-vitro efficiency of meso-tetrahydroxy-phenyl-chlorin depend on pre-treatment of sensitizer?

The efficiency of a new photosensitizer of the second generation, meso-tetra-hydroxyphenyl-chlorin (mTHPC), which has a strong absorption at 652 nm, was investigated by oxygen consumption measurements and membrane integrity testing. The experiments proved a great increase in the efficiency of mTHPC after preincubation at 37 degrees C for 24 hours. From these findings it can be assumed that tumor cells can be treated in an optimal way with PDT after a longer delay following drug administration.

Clinical effect of meso-tetrahydroxyphenylchlorine based photodynamic therapy in recurrent carcinoma of the ovary: preliminary results.

This article addresses the use of meso-tetrahydroxyphenylchlorin-based photodynamic therapy (m-THPC-PDT) to treat recurrent gynaecologic malignancies of the ovary. Photodynamic therapy is an experimental approach in the treatment of neoplasms and results indicate it is a highly tissue selective, relatively simple intervention with few side effects, therefore reducing the overall burden on the patient. Of the three patients involved in the initial study, two were treated solely with photodynamic therapy by laparoscopy, and one underwent additional palliative debulking surgery of metastatic tumours. After a post-operative period of more than two years all three women remained free of relapses.

Necrotizing skin lesions induced by low-molecular-weight heparin after total knee arthroplasty.

We report the unusual complication of focal necrotizing skin lesions accompanied by moderate thrombocytopenia in a female patient undergoing thromboprophylaxis with low-molecular-weight heparin after total knee arthroplasty. Heparin-induced thrombocytopenia was suspected and confirmed using the heparin-induced platelet activation assay. The skin lesions improved gradually after the discontinuation of heparin application. In addition to the description of this exceptionally rare adverse effect of low-molecular-weight heparin, a brief discussion of previously reported cases is provided.

In-vitro investigation of ALA-induced protoporphyrin IX.

Protoporphyrin IX (PpIX) induced endogenously by delta-aminolevulinic acid (ALA), can be used to destroy photodynamically tumor cells. The influence of several parameters on the PpIX formation of human skin fibroblasts was investigated by fluorescence spectrophotometry. The PpIX formation increases (1) with the pH value of ALA (2) with the ALA incubation time in a moderate sigmoidal manner, and (3) with the ALA concentration up to 700 micrograms ml-1. Other parameters, such as cell washing procedures, have no influence on the PpIX production. ALA has to be applied in a concentration 30 times higher than external protoporphyrin IX and Photosan 3 in order to produce the same cytotoxic damage. Protoporphyrin bleaching and photoproduct generation at 646 nm was observed. Additional information about intracellular PpIX formation kinetics and its topographically correlation to cell structures was gained by a CCD camera mounted on a fluorescence microscope. A few minutes after the onset of incubation with ALA, PpIX generation is observed in the mitochondria, followed by relocalization in the cytoplasm and the nuclear membrane.

Photodynamic treatment and radiotherapy: combined effect on the colony-forming ability of V79 Chinese hamster fibroblasts.

The effect of photodynamic tumor therapy with Photosan 3 (PS3) combined with gamma-radiation on the colony-forming ability of Chinese hamster fibroblasts was studied using a modified clonogenic assay. Only the combination of the lowest doses (5 micrograms/ml PS3 + 100 mJ/cm2 red light + 1 Gy gamma-radiation) shows a more than additive effect. Combinations of higher doses resulted in survival levels which are not significantly different from calculations based on single treatments. Additionally, it could be shown that the application of PS3 in the dark impairs the colony-forming ability of V79 cells already at sensitizer concentrations of 2 1g/ml in a concentration-independent manner.

Role of calcium in photodynamically induced cell damage of human fibroblasts.

Photodynamically induced changes in the cytoplasmic free calcium concentration ([Ca2+]i) and its role in cell damage were investigated in human skin fibroblasts using confocal laser microscopy. Fluorescence and absorbance spectrophotometry measurements indicate that the photosensitizer aluminum phthalocyanine tetrasulfonate (AIPcS4) binds to the plasma membrane and only after irradiation is able to enter the cells, causing massive morphologic alterations. Upon irradiation of sensitizer-treated cells, the increase in [Ca2+]i is related to the amount of light and extracellular [Ca2+]e. The increase in [Ca2+]i was substantially reduced in the absence of [Ca2+]a. Cell damage or death after photodynamic treatment was prevented and shifted toward higher fluence by increasing [Ca2+]i at high [Ca2+]e and was greater at low [Ca2+]e. Application of Ca2+ channel blockers, such as Co2+, Cd2+ or verapamil, could not prevent the increase of [Ca2+]i. Our results indicate that activation of the photosensitizer, AIPcS4, causes an influx of Ca2+, which protects cells from, photodamage. At low [Ca2+]e and high fluence values, release of Ca2+ from internal stores probably as a protective measure occurs in order to increase the [Ca2+]i.

A new approach to cancer therapy due to appropriate uptake and retention kinetics of meta-tetrahydroxy-phenylchlorin in a human fibroblast cell line.

Studies have shown that meta-tetrahydroxy-phenylchlorin is an efficient tumor targeting agent for laser photodynamic therapy. The effectiveness of this approach for cancer treatment depends on drug concentration, incubation time and extracellular protein. We studied uptake and retention kinetics of mTHPC in a human fibroblast cell line. Our results clearly demonstrate a difference in the amount of extracellular mTHPC at an incubation temperature of 37 degrees C compared to 20 degrees C and 4 degrees C. pH-values were always constant and not responsible for the increase. Furthermore, both absorption and fluorescence of mTHPC increase when incubated at normal human body temperature. Incubation of human fibroblast cells with mTHPC (10 micg/mL) showed that intracellular mTHPC increases in a linear manner reaching saturation after 24 hours and declining until 48 hours with concommitant increase of supernatant mTHPC. Therefore, we believe that tumor cells can be treated optimally with PDT following a delay > 24 hours after drug administration with a minimum of damage to surrounding normal tissues.

Does delta-aminolaevulinic acid induce genotoxic effects?

5-Aminolaevulinic acid (ALA) is a precursor of protoporphyrin IX (PpIX) in the biosynthetic pathway of haem. The presence of exogenous ALA bypasses the feedback control and may induce the accumulation of PpIX. Since haem-containing enzymes are essential for energy metabolism, every nucleated cell in the body must have at least a minimal capacity to synthesize PpIX. Photodynamic therapy (PDT), which is the treatment of malignant lesions with light following the administration of a tumour-localizing photosensitizer, leads to oxidative damage, including the formation of genotoxic membrane degradation products via lipid peroxidation. In addition, it has been demonstrated that ALA itself can form the reactive oxygen species Ox.-, H2O2 and OH. by auto-oxidation, suggesting that it could potentially induce DNA damage. Therefore cultures of rat hepatocytes, which have been demonstrated to be very sensitive indicators for genotoxic effects induced by the lipid peroxidation product 4-hydroxynonenal and analogous aldehydes, were examined for possible mutagenic effects after treatment with ALA in the absence of light. The cytogenetic endpoints determined were chromosomal aberrations and the induction of micronuclei. Compared with the controls, significantly elevated levels of chromosomal aberrations and micronuclei were observed at concentrations of 1 microgram ml-1 or greater. Chromosomal aberrations and micronuclei were found to increase up to a concentration of 100 micrograms ml-1 ALA. While micronuclei decrease at higher concentrations, chromosomal aberrations remain at the same level. The kinetics of PpIX formation after induction with 100 and 1000 micrograms ml-1 ALA appear to be the same for both concentrations, suggesting that the induction of chromosomal aberrations may be due to PpIX.