Lipid Droplets Protect Aging Mitochondria and Thus Promote Lifespan in Yeast Cells.

Besides their role as a storage for neutral lipids and sterols, there is increasing evidence that lipid droplets (LDs) are involved in cellular detoxification. LDs are in close contact to a broad variety of organelles where protein- and lipid exchange is mediated. Mitochondria as a main driver of the aging process produce reactive oxygen species (ROS), which damage several cellular components. LDs as highly dynamic organelles mediate a potent detoxification mechanism by taking up toxic lipids and proteins. A stimulation of LDs induced by the simultaneously overexpression of Lro1p and Dga1p (both encoding acyltransferases) prolongs the chronological as well as the replicative lifespan of yeast cells. The increased number of LDs reduces mitochondrial fragmentation as well as mitochondrial ROS production, both phenotypes that are signs of aging. Strains with an altered LD content or morphology as in the sei1∆ or lro1∆ mutant lead to a reduced replicative lifespan. In a yeast strain defective for the LON protease Pim1p, which showed an enhanced ROS production, increased doubling time and an altered mitochondrial morphology, a LRO1 overexpression resulted in a partially reversion of this "premature aging" phenotype.

Gold nanoparticles (AuNPs) impair LPS-driven immune responses by promoting a tolerogenic-like dendritic cell phenotype with altered endosomal structures.

Dendritic cells (DCs) shape immune responses by influencing T-cell activation. Thus, they are considered both an interesting model for studying nano-immune interactions and a promising target for nano-based biomedical applications. However, the accentuated ability of nanoparticles (NPs) to interact with biomolecules may have an impact on DC function that poses an unexpected risk of unbalanced immune reactions. Here, we investigated the potential effects of gold nanoparticles (AuNPs) on DC function and the consequences for effector and memory T-cell responses in the presence of the microbial inflammatory stimulus lipopolysaccharide (LPS). Overall, we found that, in the absence of LPS, none of the tested NPs induced a DC response. However, whereas 4-, 8-, and 11 nm AuNPs did not modulate LPS-dependent immune responses, 26 nm AuNPs shifted the phenotype of LPS-activated DCs toward a tolerogenic state, characterized by downregulation of CD86, IL-12 and IL-27, upregulation of ILT3, and induction of class E compartments. Moreover, this DC phenotype was less proficient in promoting Th1 activation and central memory T-cell proliferation. Taken together, these findings support the perception that AuNPs are safe under homeostatic conditions; however, particular care should be taken in patients experiencing a current infection or disorders of the immune system.

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.

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.

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.

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.

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.

Oscillations of ultra-weak photon emission from cancer and non-cancer cells stressed by culture medium change and TNF-α.

Cells spontaneously emit photons in the UV to visible/near-infrared range (ultra-weak photon emission, UPE). Perturbations of the cells' state cause changes in UPE (evoked UPE). The aim of the present study was to analyze the evoked UPE dynamics of cells caused by two types of cell perturbations (stressors): (i) a cell culture medium change, and (ii) application of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α). Four types of human cell lines were used (squamous cell carcinoma cells, A431; adenocarcinomic alveolar basal epithelial cells, A549; p53-deficient keratinocytes, HaCaT, and cervical cancer cells, HeLa). In addition to the medium change, TNF-α was applied at different concentrations (5, 10, 20, and 40 ng/mL) and UPE measurements were performed after incubation times of 0, 30, 60, 90 min, 2, 5, 12, 24, 48 h. It was observed that (i) the change of cell culture medium (without added TNF-α) induces a cell type-specific transient increase in UPE with the largest UPE increase observed in A549 cells, (ii) the addition of TNF-α induces a cell type-specific and dose-dependent change in UPE, and (iii) stressed cell cultures in general exhibit oscillatory UPE changes.

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.

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.

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.

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.

The interrelation between a pro-inflammatory milieu and fluorescence diagnosis or photodynamic therapy of human skin cell lines.

BACKGROUND: As carcinogenesis is often accompanied by inflammation, fluorescence diagnosis (FD) of tumours reportedly leads to false positive results. However, an influence of inflammation on the efficiency of photodynamic therapy (PDT) is not known. OBJECTIVE: Therefore we investigated whether LPS-induced expression of the pro-inflammatory cytokine interleukin-6 (IL-6) influences hypericin and protoporphyrin IX (PpIX) accumulation, and their photocytotoxicity. As in vitro models served normal and malignant human skin cell lines. Additionally we analyzed whether low-dose photodynamic treatment as found during FD and sublethal PDT could modulate the expression of IL-6 and other cytokines as low-dose PDT has the potential for treatment of inflammatory skin diseases. METHODS: Cells were pre-incubated with LPS to induce a pro-inflammatory milieu. Photosensitizer accumulation and toxicity were measured by a microplate reader, IL-6 via Sandwich ELISA and the other cytokines via a Multiplex Immunoassay. RESULTS AND CONCLUSIONS: Neither hypericin uptake nor PpIX accumulation was influenced by the proinflammatory state. The false positive classification in FD of patients could rather be due to sensitizer-loaded immune cells. On the other hand, low dose PDT is able to significantly reduce the level of IL-6 in all cell lines. Under differing PDT-conditions, PDT can enhance IL-6 in malignant cells, which could elicit a specific anti-tumour immune response. Furthermore, hypericin alone is able to up-regulate the anti-inflammatory cytokines IL-4, -5 and -10, which could be useful to reduce excessive inflammation. LPS-induction has no additional influence in most cases. Some of these findings could contribute to abate inflammatory diseases on skin or promote wound healing.

In vitro analysis of photosensitizer accumulation for assessment of applicability of fluorescence diagnosis of squamous cell carcinoma of epidermolysis bullosa patients.

Epidermolysis bullosa (EB) is a group of inherited skin disorders characterized by blistering following mechanical trauma. Chronic wounds of EB patients often lead to tumors such as squamous cell carcinoma (SCC). Early diagnosis may prevent its invasive growth--frequently the reason of premature mortality of EB-patients. Early detection of tumors is achieved by fluorescence diagnosis (FD), where photosensitizers localize selectively in tumors and fluoresce upon illumination. Excessive accumulation of photosensitizers in inflamed areas, as occasionally found at chronic wounds and tumors due to inflammatory processes, leads to false-positive results in FD. This study analyzed accumulation kinetics of the photosensitizers hypericin and endogenous protoporphyrin IX (PpIX) in different skin cell lines including the three EB subtypes under normal and proinflammatory conditions (stimulated with TNF-alpha). The aim was to assess the applicability of FD of SCC in EB. All cell lines accumulate hypericin or PpIX mostly increasing with incubation time, but with different kinetics. SCC cells of recessive dystrophic EB (RDEB) accumulate less hypericin or PpIX than nonmalignant RDEB cells. Nevertheless, tumor selectivity in vivo might be existent. Non-EB cell lines are more active concerning photosensitizer enrichment. Proinflammatory conditions of skin cell lines seem to have no major influence on photosensitizer accumulation.

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.

Low dose hypericin-PDT induces complete tumor regression in BALB/c mice bearing CT26 colon carcinoma.

BACKGROUND: Successful tumor eradication with photodynamic therapy (PDT) in vivo depends on the optimal combination of treatment parameters. (Low-dose) PDT may additionally induce antitumoral immune responses. Since the naturally occurring hypericin (Hyp) is a promising photosensitizer for PDT, the aim of the study was to investigate phototoxic and immunologic effects of a low-dose Hyp-PDT on murine tumors in contrast to commonly used Hyp-PDT conditions. METHODS: BALB/c mice bearing CT26 colon carcinoma received hypericin intravenously and were irradiated with red light 0.5-4h later. Tumor development was recorded. Mice were then re-challenged 60 days after the first tumor cell inoculation to investigate an antitumoral immune response. RESULTS: Different results of tumor/host responses were obtained, ranging from mice exitus over delayed tumor growth to complete tumor regression according to different treatment protocols. PDT with common doses and a 4h drug-light-interval resulted in a four times delayed tumor growth compared to the control groups. PDT with relatively low doses and a drug-light-interval of 0.5h led to 100% tumor eradication. Re-challenge of these mice with CT26 mouse colon carcinoma cells prevented new tumor growth. CONCLUSIONS: Not only drug concentrations and light doses seem to determine the efficiency of tumor eradication, but also the localization of hypericin at the time of irradiation. Targets in our low-dose PDT protocol are exclusively the vessels. The advantage of this low-dose PDT beside less drug and light exposure of the animals is reduced skin damage, faster healing of the lesions and induction of an antitumoral immune response.

TGFß-signaling in squamous cell carcinoma occurring in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary skin disorder characterized by mechanical fragility of the skin, resulting in blistering and chronic wounds. The causative mutations lie in the COL7A1 gene. Patients suffering from RDEB have a high risk to develop aggressive, rapidly metastasizing squamous cell carcinomas (SCCs). Cutaneous RDEB SCCs develop preferentially in long-term skin wounds or cutaneous scars. Albeit being well differentiated, they show a more aggressive behavior than UV-induced SCCs. These findings suggest other contributing factors in SCC tumorigenesis in RDEB. OBJECTIVE: To analyze factors contributing to RDEB tumorigenesis, we conducted a comprehensive gene expression study comparing a non-malignant RDEB (RDEB-CL) to a RDEB SCC cell line (SCCRDEB4) to achieve an overview on the changes of the gene expression levels in RDEB related skin cancer. METHODS: We applied cDNA arrays comprising 9738 human expressed sequence tags (EST) with various functions. Selected results were verified by Real-time RT PCR. RESULTS: Large-scale gene expression analysis revealed changes in the expression level of transforming growth factor ß1 (TGFß1) and several genes under the control of TGFß for RDEB and SCCRDEB4 cell lines. Even untransformed RDEB keratinocytes show elevated levels of TGFß1. CONCLUSION: Our findings demonstrate a prominent role of TGFß-signaling in RDEB-related skin cancer. Once activated, TGFß signaling either in response to wounding or in order to influence type VII collagen expression levels could facilitate cancer development and progression. Moreover, TGFß signaling might also represent a potentially useful therapeutic target in this disease.

Antibacterial effect of some benzopyrone derivatives.

The Schiff base derivatives of benzopyran-4-one are characterized by imine groups which have biological activities such as antimicrobial, antifungal and antitumoral. The aim of this study was therefore to evaluate the toxicity of Schiff bases towards bacterial cells. Escherichia coli as Gram-negative bacteria and Staphylococcus capitis as Gram-positive bacteria were exposed to different concentrations of Schiff bases. For assessment of toxicity a 96-well turbidimetric procedure, capable of testing the antimicrobial properties on a single microplate, was used. Analysis of the growth curves showed that the antibacterial effect of these Schiff bases on Gram-negative bacteria was higher than that on Gram-positive. Furthermore the presence of hydroxylic groups is correlated with an increased antibacterial effect.

Time-resolved gene expression profiling of human squamous cell carcinoma cells during the apoptosis process induced by photodynamic treatment with hypericin.

Hypericin is used as a powerful naturally occurring photosensitizer in photodynamic therapy (PDT). Activated by visible light, it kills tumour cells and tissues via generation of reactive oxygen species (ROS). Depending on the protocol, apoptotic cell death can be achieved very effectively by hypericin-PDT. To analyze the fundamental molecular mechanisms leading to apoptosis induced by photodamage especially with regard to human skin cancer cells, we studied the alteration of the gene expression pattern in the human squamous cell carcinoma cell line A-431 at 1.5, 3, 5 and 8 h after hypericin-PDT by cDNA-macroarray technique. Radioactively labelled samples were hybridized onto macroarray filters containing PCR products of 9738 ESTs of the Incyte Human UniGEM Microarray clone set. In total, 168 genes were found to be differentially upregulated and 45 down-regulated. Verification of expression changes of 45 genes of interest was performed by quantitative real-time PCR. Due to the observed significant expression changes the following can be concluded: lipoprotein receptor-mediated endocytosis could play a role in the uptake of lipophilic hypericin. Extracellular signal transduction to the cell is reduced, cell detachment facilitated, changes of the morphology, cytoskeleton and formation of apoptotic bodies occur. The promotion of p38MAPK, ERK, JNK and Ras signalling pathways supports survival and/or apoptosis. Switches between life and death could be the strongly upregulated transcription factors c-jun and FOSB as well as the MAPK-phosphatase 1 DUSP-1, possibly activated via H3 histone modifications. ROS activate ER-stress pathways or adaptive response, and provoke damage protection against ROS, partly in a cell-type specific way.