Applicability of new degradable hypericin-polymer-conjugates as photosensitizers: principal mode of action demonstrated by in vitro models.

Two series of water soluble novel conjugates of the photosensitizer hypericin were prepared and evaluated for their use as agents for photodynamic therapy, with covalently and non-covalently loaded hypericin on functionalised, hydrolytically degradable inorganic-organic hybrid polyphosphazenes. The conjugates showed excellent aqueous solubility and similar fluorescence spectra to pristine hypericin. Detailed in vitro investigations revealed that the substances were non-toxic in the dark over a wide concentration range, but displayed phototoxicity upon irradiation. Cell uptake studies showed rapid uptake with localization of hypericin observed in endoplasmic reticulum, Golgi complex and particularly in the lysosomes. Furthermore, a DNA fragmentation assay revealed that the photosensitizer conjugates are efficient inducers of apoptosis with some tumor cell selectivity caused by faster and enhanced accumulation in A431 than in HaCaT cells, and thus a moderately higher phototoxicity of A431 compared to HaCaT cells. These novel photosensitizer conjugates hence represent viable hydrolytically degradable alternatives for the advanced delivery of hypericin.

Back to the roots: photodynamic inactivation of bacteria based on water-soluble curcumin bound to polyvinylpyrrolidone as a photosensitizer.

Photodynamic inactivation (PDI), the light-induced and photosensitizer-mediated overproduction of reactive oxygen species in microorganisms, represents a convincing approach to treat infections with (multi-resistant) pathogens. Due to its favourable photoactive properties combined with excellent biocompatibility, curcumin derived from the roots of turmeric (Curcuma longa) has been identified as an advantageous photosensitizer for PDI. To overcome the poor water solubility and the rapid decay of the natural substance at physiological pH, we examined the applicability of polyvinylpyrrolidone curcumin (PVP-C) in an acidified aqueous solution (solubility of PVP-C up to 2.7 mM) for photoinactivation of Gram(+) and Gram(-) bacteria. Five micromolar PVP-C incubated for 5 minutes and illuminated using a blue light LED array (435 ± 10 nm, 33.8 J cm(-2)) resulted in a >6 log10 reduction of the number of viable Staphylococcus aureus. At this concentration, longer incubation periods result in a lower phototoxicity, most likely due to degeneration of curcumin. Upon an increase of the PVP-C concentration to 50 µM (incubation for 15 or 25 min) a complete eradication of Staphylococcus aureus can be achieved. As expected for a non-cationic photosensitizer, cell wall permeabilization with CaCl2 prior to addition of 50 µM PVP-C for 15 min is necessary to induce a drop in the count of the Gram(-) Escherichia coli for more than 3 log10. As both constituents of the formulation, curcumin (E number E100) and polyvinylpyrrolidone (E1201), have been approved as food additives, a PDI based on PCP-C might allow for a very sparing clinical application (e.g. for disinfection of wounds) or even for employment in aseptic production of foodstuffs.

Epidermolysis bullosa - a group of skin diseases with different causes but commonalities in gene expression.

Epidermolysis bullosa (EB) is a group of hereditary skin disorders. Although each subtype is caused by mutations in genes encoding differentially located components of the skin, the resulting phenotype is similar. In this study, we investigated similarities in the gene expression profiles of each subtype on mRNA level. Type XVI collagen (COL16A1), G0/G1 switch 2 (G0S2), fibronectin (FN1), ribosomal protein S27A (RPS27A) and low density lipoprotein receptor (LDLR) were shown to exhibit corresponding changes in gene expression in all three EB subtypes. While COL16A1, G0S2 and FN1 are up-regulated, LDLR and RPS27A mRNA levels are decreased. These data indicate that EB cells seem to take measures increasing their mechanical stability. Apoptosis is likely to be exacerbated, and migratory potential appears to be elevated. Protein degradation is hampered, and the release of fatty acids and glycerol is restricted, probably to save energy. These commonalities might benefit existing EB treatment strategies or could help to reveal new starting points for the treatment of EB in the future.

Fluorescence detection and depletion of T47D breast cancer cells from human mononuclear cell-enriched blood preparations by photodynamic treatment: Basic in vitro experiments towards the removal of circulating tumor cells.

OBJECTIVES: A major obstacle for permanent cancer eradication is the persistence of circulating tumor cells (CTCs) in blood, which often escape radio- or chemotherapy. Currently no efficient strategy to remove CTCs from peripheral blood in order to lower the risk of metastases or tumor recurrence exists. Photodynamic treatment (PDT) using aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) as photosensitizer offers an innovative approach to overcome this problem. This study aims at providing basic evidence towards fluorescence detection and photodynamic depletion of scattered cancer cells from blood preparations. METHODS: The breast cancer cell line T47D, endothelial GP8 cells, red blood cells (RBCs) and peripheral blood mononuclear cells (MNCs) have been tested for ALA-induced formation kinetics of PPIX by flow cytometry and microplate fluorescence analysis. The influence of the presence of RBCs on the PPIX-accumulation in cancer cells was evaluated by flow cytometry; the efficacy of PDT on cancer cells and MNCs has been tested by resazurin assay. Mixtures of T47D and GP8 cells and MNCs spiked with cancer cells were tested to determine the limit of fluorescence detection by flow cytometry and antibody co-staining. RESULTS: T47D cells accumulated significantly higher PPIX-amounts after ALA-incubation than any other cell type tested. The presence of RBCs had no impact on PPIX-formation in T47D cells. Experiments towards the fluorescence detection of cancer cells in blood revealed that the sensitivity of this method is yet limited. Viability testing after PDT showed that cancer cells where almost completely eradicated after illumination whereas MNCs were almost spared. CONCLUSION: We clearly demonstrate in vitro tumor cell selectivity of PPIX-accumulation over endothelial cells, MNCs and RBCs. Breast cancer cells are efficiently killed by PDT with minor depletion of MNCs. Our findings provide a basis for the PDT of blood samples for a future depletion of CTCs.

Antibacterial photodynamic therapy using water-soluble formulations of hypericin or mTHPC is effective in inactivation of Staphylococcus aureus.

Antibacterial photodynamic treatment (APDT) might prove valuable as an alternative to the application of chemoantibiotics for the treatment of staphylococcal infections. The rapid uptake of the photosensitizing agent into bacteria allows for selective killing of microorganisms whilst sparing the eukaryotic host tissue, but also requires removal of excessive dye after the incubation period. We tested water-soluble formulations of hypericin (PVP-hypericin) and m-tetrahydroxyphenylchlorin (Fospeg), which could be applied as aqueous sprays and removed easily by flushing with buffers, for their efficiency in killing Staphylococcus aureus. For both sensitizers, 100 nM of the photoactive substance incubated for 5 min and illuminated for 30 min at 75 mW cm(-2) lead to a 4-5 log unit reduction in bacterial count. At a concentration of 300 nM (incubation time 5 min), 30 min illumination at 25 mW cm(-2) is more effective than 10 min illumination at 75 mW cm(-2) (both resulting in the same fluence). We suggest both substances as promising candidates for treatment of staphylococcal infections in wounds with APDT.

Development of curcumin-loaded gemini surfactant nanoparticles: Synthesis, characterization and evaluation of anticancer activity against human breast cancer cell lines.

BACKGROUND: Curcumin, the polyphenolic constituent of turmeric, has been recognized as an effective anticancer agent in the treatment of breast cancer. However, the poor bioavailability of curcumin triggers finding of new approaches for elevating its therapeutic efficiency. PURPOSE: We aimed to use gemini surfactant nanocarriers for curcumin in order to overcome its limitations. STUDY DESIGN: We investigated the in vitro characterization of gemini surfactant-curcumin (Gemini-Cur) and examined its antiproliferative & apoptotic activities on breast cancer cell lines. METHODS: Gemini-Cur polymersomes were synthesized through nanoprecipitation method and characterized by dynamic light scattering (DLS), transmission and scanning electron microscopies, HPLC and X-ray diffraction (XRD). The anticancer effect of Gemini-Cur nanoparticles was studied on three different breast cancer cell lines including MCF-7, SkBr-3 and MDA-MB-231 through uptake kinetics, viability & cytotoxicity recordings and apoptotic assays. Furthermore, qRT-PCR was performed to evaluate the expression of apoptotic genes including p16INK4a, p14ARF, Bax and Bcl-2. RESULTS: According to physicochemical analysis, the average particle size, zeta potential value and drug entrapment efficiency for Gemini-Cur compound were recorded as 161 ±â€¯6.2 nm, +5.32 mV and 89.13% ±â€¯0.93, respectively. XRD analysis also confirmed the incorporation of curcumin in gemini surfactant micelles. Regarding the enhanced cellular uptake of sphere shaped Gemini-Cur, our data showed that this nano compound suppresses cancer cell proliferation via induction of apoptosis. Moreover, qRT-PCR analysis revealed that Gemini-Cur could effectively upregulate the expression of p16INK4a, p14ARF and Bax, while significantly decreasing the Bcl-2 expression in these breast cancer cells. CONCLUSION: Our data demonstrates the great potential of gemini surfactants for efficient delivery of curcumin and subsequently, the improvement of its anticancer effect. Therefore, it is sagacious to support the idea that Gemini-Cur nano compound might have the potential to be considered as an anticancer agent.

Mechanically Strong Silica-Silk Fibroin Bioaerogel: A Hybrid Scaffold with Ordered Honeycomb Micromorphology and Multiscale Porosity for Bone Regeneration.

Due to the synergic feature of individual components in hybrid (nano)biomaterials, their application in regenerative medicine has drawn significant attention. Aiming to address all the current challenges of aerogel as a potent scaffold in bone tissue engineering application, we adopted a novel synthesis approach to synergistically improve the pore size regime and mechanical strength in the aerogel. The three-dimensional aerogel scaffold in this study has been synthesized through a versatile one-pot aqueous-based sol-gel hybridization/assembly of organosilane (tetraethyl orthosilicate) and silk fibroin (SF) biopolymer, followed by unidirectional freeze-casting of the as-prepared hybrid gel and supercritical drying. The developed ultralight silica-SF aerogel hybrids demonstrated a hierarchically organized porous structure with interesting honeycomb-shaped micromorphology and microstructural alignment (anisotropy) in varied length scales. The average macropore size of the hybrid aerogel lied in ∼0.5-18 µm and was systematically controlled with freeze-casting conditions. Together with high porosity (91-94%), high Young's modulus (∼4-7 MPa, >3 order of magnitude improvement compared to their pristine aerogel counterparts), and bone-type anisotropy in the mechanical compressive behavior, the silica-SF hybrid aerogel of this study acted as a very competent scaffold for bone tissue formation. The results of in vitro assessments revealed that the silica-SF aerogel is not only cytocompatible and nonhemolytic but also acted as an open porous microenvironment to trigger osteoblast cell attachment, growth, and proliferation on its surface within 14 days of incubation. Moreover, to support the in vitro results, in vivo bone formation within the aerogel implant in the bone defect site was studied. The X-ray radiology and microcomputed tomography analyses confirmed that a significant new bone tissue density formed in the defect site within 25 days of implantation. Also, in vivo toxicology studies showed a zero-toxic impact of the aerogel implant on the blood biochemical and hematological parameters. Finally, the study clearly shows the potential of aerogel as a bioactive and osteoconductive open porous cellular matrix for a successful osseointegration process.

Reduction of cancer cell viability by synergistic combination of photodynamic treatment with the inhibition of the Id protein family.

The inhibitor of DNA binding and cell differentiation (Id) proteins are dominant negative regulators of the helix-loop-helix transcription factor family and play a key role during development as well as in vascular disorders and cancer. In fact, impairing the Id-protein activity in cancer cells reduces cell growth and even chemoresistance. Recently, we have shown that a synthetic Id-protein ligand (1Y) consisting of a cyclic nonapeptide can reduce the viability of the two breast cancer cell lines MCF-7 and T47D and of the bladder cancer cells T24 to about 50% at concentrations ≥100µM. Moreover, the cyclopeptide displays both proapoptotic and antiproliferative effects on MCF-7 cells. Herein, we show that the cyclopeptide does not induce cell death at the dose of 5µΜ, but it still inhibits MCF-7 and T24 cell proliferation, which correlates with an increased protein level of the cell-cycle regulator p27Kip1. Furthermore, 1Y-pretreated MCF-7, T47D, and T24 cells are more susceptible than untreated cells to the phototoxic effects of the three photosensitizers meta-tetra(hydroxyphenyl)chlorin, porfimer sodium, and hypericin, which are applied in photodynamic therapy (PDT). The combination of the Id-protein ligand with each of the light-activated photosensitizers shows synergistic effects on the reduction of cell viability. In conclusion, an Id-protein ligand with moderate cancer cell killing activity at concentrations ≥100µM can be applied at a 20-fold lower and barely toxic dose to raise the sensitivity of cancer cells towards phototoxicity associated with photodynamic treatment. This suggests the potential benefit of targeting the Id proteins in combined drug approaches for cancer therapy.

Influence of ALA-mediated photodynamic therapy on secretion of interleukins 6, 8 and 10 by colon cancer cells in vitro.

BACKGROUND: Photodynamic therapy has apart from a direct cytotoxic effect also immunomodulatory properties. The aim of our study was to investigate how photodynamic therapy with 5-aminolevulinic acid (ALA-PDT) in sublethal doses influences the secretion of interleukins 6, 8 and 10 from colon cancer cells in vitro. METHODS: We used two human colon cancer cell lines SW480 and SW620 of different malignancies which were treated with a sublethal PDT protocol. Determination of interleukins was carried out using the Bio- Plex Assay Pro™ kit on the Bio- Plex Suspension Array System. RESULTS: Sublethal ALA-PDT did not affect IL-6 secretion by SW480 cells, but caused a 40% decrease of IL-6 release by the SW620 cell line. It increased IL-8 secretion in both, the SW480 and SW620 cell lines, by 23% and 46%, respectively, and decreased the production of IL-10 (25% in SW480 and 32% in SW620 cells). CONCLUSIONS: ALA-PDT in sublethal doses might influence colon cancer cell's progression and invasion by reducing the secretion of IL-6, IL-10 and increasing the IL-8 concentration with higher values in the more malignant cell line.

Secretion of the angiogenic factor VEGF after photodynamic therapy with ALA under hypoxia-like conditions in colon cancer cells.

BACKGROUND: Photodynamic therapy (PDT), eliminates not only the tumor, but also modulates signaling factors release, e.g. vascular endothelial growth factor (VEGF), which plays a crucial role in cancer progression. Assessment of the VEGF-secreting activity of resistant colon cancer cells in different degree of malignancy: SW480 and SW620 under hypoxia-like conditions during δ- aminolevulinic acid (ALA) PDT was the objective of our study. METHODS: The colon cancer cell lines SW480 and SW620 were treated in sublethal doses with ALA PDT in hypoxia- like conditions with cobalt chloride (CoCl2). To assess cell viability, MTT assays were performed and the discrimination of the cell death mode was monitored via fluorescence microscopy. The cells cytotoxicity using LDH test was assessed. Determination of VEGF was carried out using the Bio- Plex Assay Pro™ kit on the Bio- Plex Suspension Array System. RESULTS: ALA PDT used in sublethal doses decreases release of VEGF in more aggressively growing SW620 colon cancer cell line in hypoxia-like conditions. In addition the level of secretion of VEGF in SW620 was much higher than in SW480 cells, which correlates with the grade of aggressive growth of colon cancer cells. CONCLUSION: Our outcomes offer evidence, that in hypoxia mimic condition sublethal ALA-PDT- mediated VEGF inhibition could be clinically relevant.

Gene expression profiling of the human carcinoma cell line A-431 after 5-aminolevulinic acid-based photodynamic treatment.

The photosensitizer protoporphyrin IX, endogenously accumulated from the precursor aminolevulinic acid (ALA) as well as other less photodynamically effective intermediates, is a successful agent in photodynamic therapy. Despite encouraging clinical results, the basic mechanisms leading to cell death are not fully understood. To elucidate these fundamentals, the alteration of the gene expression pattern in the squamous cell carcinoma cell line A-431 was studied at different time-points after photodynamic treatment with ALA by cDNA-array technique. Cells were incubated for 16 h with 100 microg/ml ALA and irradiated with a fluence of 3.5 J/cm(2) resulting in 50% survival until 8 h post treatment. RNA was isolated at 1.5, 3, 5 and 8 h post treatment and from 3 controls (untreated, light only and dark), radioactively labeled by reverse transcription with 33P-dCTP and hybridized onto macroarray PCR filters containing PCR products of 2135 genes, which were selected for relevance in carcinogenesis, stress response and signal transduction. Verification of observed expression changes was carried out by real-time RT-PCR. We found a strong induction of expression of the immediate early genes c-jun and c-fos as well as decreased expression of genes involved in proliferation such as myc, genes involved in apoptosis such as Fas associated via death domain (FADD) and the fibronectin gene for cell adhesion. An apoptosis induction rate of not more than 30% as proved by apoptosis detection assays and caused by PpIX localization in the membrane was reflected by the expression profile.

Fluorescence-based CdTe nanosensor for sensitive detection of cytochrome C.

Cytochrome c (Cyt c) is commonly used as intrinsic biomarker for several characteristics of the cell such as respiration, energy level and apoptosis. In the present study a simple colorimetric sensor should be developed and tested for the real-time detection of Cyt c in living cells. We synthesized cadmium telluride quantum dots (CdTe QDs) capped with thioglycolic acid (TGA) as a fluorometric Cyt c nanosensor. The synthesized TGA/CdTe QDs nanosensor was characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and absorption as well as fluorescence spectrophotometry. We investigated the developed TGA/CdTe QDs sensor with regard to its applicability in the fluorometric detection of Cyt c. Results showed that the TGA/CdTe QDs could be used as a sensitive fluorescence probe for the quantification of different concentrations of Cyt c ranging from 0.5 - 2.5µM. Increased binding of QDs to Cyt c results in decreasing fluorescence. The fluorescence of the QDs is inversely correlated to the Cyt c concentration. Based on these data, a standard curve up to 2.5µM Cyt c was established. Moreover, the developed nanosensor was applied in different concentrations on primary human dermal fibroblasts. Results showed that TGA/CdTe QDs were taken up by cells and could be visualized by fluorescence microscopy. Quantification of Cyt c within living cells via QDs is, however, influenced by various factors such as cell damage, QD aggregation or the level of reactive oxygen species, which have to be taken into account.

Targeting of a Helix-Loop-Helix Transcriptional Regulator by a Short Helical Peptide.

The Id proteins (Id1-4) are cell-cycle regulators that play a key role during development, in cancer and vascular disorders. They contain a conserved helix-loop-helix (HLH) domain that folds into a parallel four-helix bundle upon self- or hetero-association with basic-HLH transcription factors. By using such protein-protein interactions, the Id proteins inhibit cell differentiation and promote cell-cycle progression. Accordingly, their supporting role in cancer has been convincingly demonstrated, which makes these proteins interesting therapeutic targets. Herein we present a short peptide containing an (i,i+4)-lactam bridge and a hydrophobic (Φ) three-residue motif Φ(i)-Φ(i+3)-Φ(i+6), which adopts a helical conformation in water, shows Id protein binding in the low-micromolar range, penetrates into breast (MCF-7 and T47D) and bladder (T24) cancer cells, accumulates in the nucleus, and decreases cell viability to ∼50 %. Thus, this cyclopeptide is a promising scaffold for the development of Id protein binders that impair cancer cell viability.

NTRK2 (TrkB gene) variants and temporal lobe epilepsy: A genetic association study.

OBJECTIVE: The NTRK2 gene encodes a member of the neurotrophic tyrosine kinase receptor family known as TrkB. It is a membrane-associated receptor with signaling and cellular differentiation properties that has been involved in neuropsychiatric disorders, including epilepsy. We report here the frequencies of NTRK2 allele variants in patients with temporal lobe epilepsy (TLE) compared to controls without epilepsy and explore the impact of these polymorphisms on major clinical variables in TLE. METHODS: A case-control study comparing the frequencies of the NTRK2 gene polymorphisms beween 198 TLE Caucasian patients and 200 matching controls without epilepsy. In a second step, the impact of allelic variation on major clinical and electroencephalographic epilepsy variables was evaluated in the group of TLE patients. The following polymorphisms were determined by testing different regions of the NTRK2 gene: rs1867283, rs10868235, rs1147198, rs11140800, rs1187286, rs2289656, rs1624327, rs1443445, rs3780645, and rs2378672. To correct for multiple correlations the level of significance was set at p<0.01. RESULTS: Patients with TLE showed a statistical trend for increase of the T/T genotype in rs10868235 compared to control (O.R.=1.90; 95%CI=1.17-3.09; p=0.01). Homozygous patients for the A allele in rs1443445 had earlier mean age at onset of seizures, p=0.009 (mean age of 16.6 versus 22.4years). We also observed that the T allele in rs3780645 was more frequent in patients who needed polytheraphy for seizure control than in patients on monotherapy, (O.R.=4.13; 95%CI=1.68-10.29; p=0.001). This finding may reflect an increased difficulty to obtain seizure control in this group of patients. No additional differences were observed in this study. CONCLUSIONS: Patients with epilepsy showed a trend for a difference in rs10868235 allelic distribution compared to controls without epilepsy. NTRK2 variability influenced age at seizure onset and the pharmacological response to seizure control. As far as we know, this is the first study showing an association between NTKR2 allelic variants in human epilepsy. We believe that further studies in this venue will shade some light on the molecular mechanisms involved in epileptogenesis and in the clinical characteristics of epilepsy.

Cellular mechanisms and prospective applications of hypericin in photodynamic therapy.

During the last decades, Photodynamic Therapy (PDT) has been established as a powerful alternative approved by health agencies of several countries for treatment of various malignant and some non-malignant diseases. PDT makes use of the light-induced destruction of target cells by formation of cytotoxic products in the presence of a photosensitizing agent and oxygen. The light-dependent tumor destructive properties of Hypericin have drawn attention to its promising application as a photosensitizer in the frame of PDT. Hypericin is a naturally occurring secondary metabolite in plants of the Hypericum genus, with Hypericum perforatum (St. John's wort) as it is a commonly known representative. This review focuses on the cellular mechanisms of Hypericin-based phototoxicity and provides an outlook for future application of Hypericin as a fluorescing and photosensitizing agent for diagnosis and treatment of cancerous diseases, respectively.

Characterization of apoptosis induced by photodynamic treatment with hypericin in A431 human epidermoid carcinoma cells.

Hypericin is a naturally occurring metabolite extracted from Hypericum plants and is regarded as a promising photosensitizing agent for applications in the frame of photodynamic treatment (PDT). This treatment procedure is based on the light-induced formation of reactive oxygen species and subsequent destruction of target cells. We used an in vitro model system consisting of human epidermoid carcinoma cells (A431) and hypericin as a photosensitizer to study the time- and dose-dependent characteristics of hypericin-PDT-based induction of cytotoxicity and apoptotic cell death. The induction of apoptosis by hypericin-PDT was found to follow a strict dose-dependent manner with a transition to necrotic cell death at higher doses. Apoptosis was analyzed by characteristical biochemical and morphological markers (activation of caspases, nuclear fragmentation and membrane blebbing). Time-course analysis of an almost homogenous apoptotic population of cells (at 1.44 J/cm2) showed a rapid increase in nuclear fragmentation and activation of caspases reaching a maximum at 5 hr after irradiation. Using specific caspase substrates, significant activation of caspase-2, -3, -6, and -9 was found. Mitochondrial involvement during hypericin-PDT-induced apoptosis could be proven by a rapid reduction of the mitochondrial membrane potential; interestingly, the level of intracellular adenosine-5'-triphosphate (ATP) remains at control level for up to 6 hr post irradiation suggesting upregulation of glycolysis as a compensating mechanism of energy supply. Our data contribute to a deeper understanding of the processes involved in apoptotic cell death following photodynamic treatment with hypericin.

Boosting Tumor-Specific Immunity Using PDT.

Photodynamic therapy (PDT) is a cancer treatment with a long-standing history. It employs the application of nontoxic components, namely a light-sensitive photosensitizer and visible light, to generate reactive oxygen species (ROS). These ROS lead to tumor cell destruction, which is accompanied by the induction of an acute inflammatory response. This inflammatory process sends a danger signal to the innate immune system, which results in activation of specific cell types and release of additional inflammatory mediators. Activation of the innate immune response is necessary for subsequent induction of the adaptive arm of the immune system. This includes the priming of tumor-specific cytotoxic T lymphocytes (CTL) that have the capability to directly recognize and kill cells which display an altered self. The past decades have brought increasing appreciation for the importance of the generation of an adaptive immune response for long-term tumor control and induction of immune memory to combat recurrent disease. This has led to considerable effort to elucidate the immune effects PDT treatment elicits. In this review we deal with the progress which has been made during the past 20 years in uncovering the role of PDT in the induction of the tumor-specific immune response, with special emphasis on adaptive immunity.

A new biocompatible nanocomposite as a promising constituent of sunscreens.

Skin naturally uses antioxidants to protect itself from the damaging effects of sunlight. If this is not sufficient, other measures have to be taken. Like this, hydroxyapatite has the potential to be applied as an active constituent of sunscreens since calcium phosphate absorbs in the ultraviolet region (UV). The objective of the present work was to synthesize a hydroxyapatite-ascorbic acid nanocomposite (HAp/AA-NC) as a new biocompatible constituent of sunscreens and to test its efficiency with skin cell models. The synthesized HAp/AA-NC was characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, absorption spectrophotometry and X-ray diffraction analysis. The protective effect of the construct was tested with respect to viability and intracellular reactive oxygen species (ROS) generation of primary human dermal fibroblasts (SKIN) and human epidermal keratinocytes (HaCaT). Both cell lines were irradiated with UV light, λmax=254 nm with a fluence of 25 mJ cm(-2) to mimic the effect of UV radiation of sunlight on the skin. Results showed that HAp/AA-NC had a stimulating effect on the cell viability of both, HaCaT and SKIN cells, relative to the irradiated control. Intracellular ROS significantly decreased in UV irradiated cells when treated with HAp/AA-NC. We conclude that the synthesized HAp/AA-NC have been validated in vitro as a skin protector against the harmful effect of UV-induced ROS.

Photodynamic treatment with hexyl-aminolevulinate mediates reversible thiol oxidation in core oxidative stress signaling proteins.

Photodynamic therapy (PDT) is a highly selective two-step cancer treatment involving a photosensitizer and illumination with visible light in the presence of molecular oxygen. PDT is clinically approved worldwide for treating several premalignant conditions and cancer forms, especially endoscopically accessible tumors and dermatological malignancies. PDT-mediated cytotoxicity takes place via autophagy, apoptosis and necrosis, but the exact trigger mechanisms for various death-pathways are still unknown. PDT induces reactive oxygen species (ROS) through photochemical reactions. ROS can react with different macromolecules resulting in cellular damage, including oxidation of proteins. One of the known protein modifications is reversible oxidation of cysteine thiols (-SH), which in many cases constitute a redox switch to modulate protein activity and cellular signaling. Here we have examined the role of reversible oxidation of protein thiols as a potential mediator of cytotoxicity after hexylaminolevulinate-mediated photodynamic treatment (HAL-PDT) in the human epidermoid carcinoma cell line A431. Nearly 2300 proteins were found to be reversibly oxidized after HAL-PDT, of which 374 high-confidence proteins were further allocated to cellular compartments and functional networks. 115 of the high confidence proteins were associated with apoptosis and 257 have previously not been reported to be reversibly oxidized on cysteines. We find an enrichment of DNA damage checkpoint and oxidative stress response proteins. Many of these constitute potential signaling hubs in apoptosis, including ATM, p63, RSK1 p38, APE1/Ref-1 and three 14-3-3 family members. Our study represents the first comprehensive mapping of reversibly oxidized proteins subsequent to HAL-PDT. Several of the proteins constitute potentially novel redox-regulated apoptotic triggers as well as potential targets for adjuvants that may improve the efficacy of HAL-PDT and PDT using other photosensitizers.

Differential effects of glucose deprivation on the cellular sensitivity towards photodynamic treatment-based production of reactive oxygen species and apoptosis-induction.

Photodynamic treatment (PDT) employs a photosensitizer and the light-induced formation of reactive oxygen species--antagonized by cellular antioxidant systems--for the removal of harmful cells. This study addresses the effect of altered carbohydrate metabolism on the cellular antioxidant glutathione system, and the subsequent responses to PDT. It is shown that glucose-deprivation of 18 h prior to PDT causes a reduced level of intracellular glutathione and an increased cytotoxicity of PDT. These effects can be mimicked by inhibitors of glutathione synthesis (buthionine-sulfoximine) or its regeneration (1,3-bis-(2-chlorethyl)-1-nitrosourea). Inhibited glutathione metabolism shifts the apoptotic window to lower fluences, while glucose deprivation abolishes apoptosis as a result of ATP deficiency. Our results prove evidence for manipulation of the outcome of PDT through internal metabolic pathways.