Karin Kunzi-Rapp

Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer

Histone deacetylase inhibitors (HDACs) are known to exhibit antiproliferative effects on various carcinoma cells. In this study, the in vivo efficiency of two HDACs, sodium butyrate and tributyrin, on prostate cancer growth inhibition were investigated. To gain an insight into the possible underlying pathways, cell culture experiments were performed focusing on the expression of p21, Rb and c-myc. For in vivo testing, prostate cancer cell lines (PC3 and TSU-Pr1) were seeded on the chorioallantois membrane (CAM) and implanted in a xenograft model using nude mice. Standard Western blot analysis was performed for protein expression of p21, Rb and c-myc in HDAC-treated vs untreated prostate cancer cells. Both sodium butyrate and tributyrin had a considerable treatment effect on microtumours on the chicken egg at already very low concentrations of 0.1 mM. Tributyrin-treated tumours showed the strongest effect with 38% apoptotic nuclei in the prostate cancer cell line PC3. In the mouse model, there was almost no difference between sodium butyrate and tributyrin. In untreated animals the tumours were almost double the size 4 weeks after implantation. Tumours of the treatment groups had a significantly lower percentage of Ki-67-positive-stained nuclei. As demonstrated by Western blot analysis, these effects seem to be independent of p53 status and a pathway via p21–Rb–c-myc is possibly involved. In this study we have demonstrated a substantial in vivo treatment effect, which can be induced by the application of sodium butyrate or the orally applicable tributyrin in human prostate cancer. The given results may provide the rationale to apply these drugs in well-controlled clinical trials in patients being at high risk of recurrence after specific therapy or in patients with locally or distant advanced prostate cancer.

CHORIOALLANTOIC MEMBRANE ASSAY: VASCULARIZED 3-DIMENSIONAL CELL CULTURE SYSTEM FOR HUMAN PROSTATE CANCER CELLS AS AN ANIMAL SUBSTITUTE MODEL

PURPOSE Chorioallantoic membranes have been used as a reliable biomedical assay system for many years. Chicken eggs in the early phase of breeding are between in vitro and in vivo systems but may provide an immunodeficient, vascularized test environment. We tested this model as an in vivo system for prostate cancer research. MATERIALS AND METHODS Single cell suspensions of LNCaP, PC-3 and Tsu-Pr1 human prostatic cancer cell lines as well as 2 immortalized normal human prostate epithelial cell lines were inoculated on the chorioallantoic membrane of fertilized chicken eggs on day 5 or 6 of breeding. Tumor growth and viability of the embryo was evaluated by stereo microscopy. At day 10 the membranes were removed and embedded in paraffin. Cell morphology was assessed after hematoxylin and eosin staining. Cellular expression of cytokeratin, prostate specific antigen and androgen receptor as well as apoptosis induction was confirmed by immunohistochemistry. RESULTS Three days after tumor cell inoculation on the extraembryonic vascular system of the chorioallantoic membrane cell growth and formation of 3-dimensional tumors became apparent in 100% of inoculated membranes. Strong neo-angiogenesis was detected next to the established tumors and tumor cells invading the stroma of the chorioallantoic membrane. Cytokeratin expression as well as prostate specific antigen and androgen receptor in LNCaP cells confirmed the human prostate tumor origin. Assessment of quantitative in vivo apoptosis induction in LNCaP cells after intravenous injection of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate confirmed the model as a versatile in vivo system. CONCLUSIONS The well vascularized chorioallantoic membrane of bred chicken eggs is a suitable system for early in vivo cancer research. Reliable growth of prostate cancer cell lines is feasible and allows the evaluation of proliferation and apoptosis induction after intravascular or topic application of anticancer drugs. Exploitation of this assay enables a substantial reduction in or substitution for subsequent animal experiments.

Optical Coherence Tomography: Clinical Applications in Dermatology

Summary Optical Coherence Tomography (OCT), a promising new and non invasive technology for diagnosis of tissue is on the cusp of being introduced in clinical dermatology. OCT instrumentations are commercially available (e.g. ISIS Optronics GmbH, Mannheim) which provide two dimensional images of high resolution (lateral 3μm, axial 5μm) of morphological microstructures of the skin. Compared to high frequency sonography, OCT images are more detailed in structural information and contrast. The limitation, however, is the penetration depth of the light into the skin. Due to the strong scattering of skin tissue, imaging is possible to a depth of about 1 mm. But for many cases this is enough to identify alterations in the structure of the dermal layers down to the upper dermis. In this study we report on examples of pathological skin lesions such as bullous diseases, inflammations and different types of skin cancer. OCT imaging as a non-invasive diagnostic method is also well suited to evaluate laser treatment effects by monitoring of therapeutic progress over time. Treatment of port-wine stain and wound healing can be followed on-line as a method of therapy control. Examples show the variety of OCT applications in monitoring skin diseases and the potency of this new technique.

Characterization of the chick chorioallantoic membrane model as a short-term in vivo system for human skin.

Abstract We report on the cultivation and characterization of human skin on the chorioallantoic membrane of chicken eggs with the aim of replacing animals in short-term investigations in dermatology. Adult human split-thickness skin was grafted onto the chorioallantoic membrane of 5-day chick embryos. Grafts and surrounding host tissue were examined daily by in vivo stereomicroscopy and in histological sections and were characterized using a panel of monoclonal antibodies. The skin grafts were completely incorporated into the chorioallantoic membrane 2 days after transplantation. A remarkable angiogenesis occurred towards the grafts. Skin tissues revascularized within 2 or 3 days by reperfusion of the existing graft vasculature. Anastomosis of host and graft blood vessels occurred and the transplanted skin was nourished by the host blood supply as indicated by nucleated chick erythrocytes in the skin vessels. The skin grafts on the chorioallantoic membrane preserved an almost entire human phenotype. Besides a fully differentiated human epidermis and dermis containing all the cellular and extracellular constituents such as skin immune cells, capillary vessels composed of human endothelial cells were enclosed by a basement membrane of human origin. The integrin expression pattern formed in human skin transplants 5 days after grafting was identical to that of human skin controls before grafting.

Nonlinear Dynamics of Intracellular Methylene Blue During Light Activation of Cell Cultures

Abstract— Methylene blue (MB+) is a well‐known dye in medicine and has been discussed as an easily applicable drug for topical treatment in photodynamic therapy (PDT). Methylene blue can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT. The kinetics of this process was analyzed on a subcellular level with confocal laser scanning microscopy. BKEz‐7 endothelial cells were incubated 4 h with 1 μM MB+. The fluorescence dynamics of MB+ during irradiation with 633 nm light was observed with subcellular resolution. Images were acquired at 0.5 s intervals (frame rate 1 image/0.5 s). Fluorescence was observed in the red channel of the laser scanning microscope. Synchronously, the phase‐contrast image was visualized with the green channel. Morphological changes could therefore be correlated with the dynamics of MB+. In addition, the light‐dose‐dependent phototoxicity at 633 nm irradiation was determined by viable cell counting. After an induction period (phase I), fast fluorescent spikes could be observed in the whole cytoplasm, which decayed with a time constant of about 20 s (phase II), followed by a period of nearly constant fluorescence intensity (phase III) and exponential photobleaching (phase IV). Phase II exhibits highly nonlinear kinetics, which is hypothesized to correlate probably with a nonlinear quantal production of reactive oxygen species (ROS). Morphological cell changes were not observed during phase II. During phase III, a pycnotic cell nucleus developed. From the determination of viable cells we can conclude that a light dose applied within phase II was only sublethal in correlation with morphological observations. Overproduction of ROS leading finally to cell killing during phases III and IV is discussed.

Comparative in vitro and in vivo measurements of hydrophilic and hydrophobic porphyrins

The photosensitizers uroporphyrin III (UP III), coproporphyrin III (CP III), and protoporphyrin IX (PP IX) were examined with respect to their composition (by different molecular species), uptake and distribution in cells and tissues, intracellular pH value, cytotoxicity and formation of photoproducts. Whereas hydrophilic UP III and CP III were characterized by low cytotoxicity and preferential accumulation in lysosomes, hydrophobic PP IX was found to be rather cytotoxic and mainly localized in cellular membranes. Rapid uptake in tissues and some preferential location in tumor cells were detected for hydrophobic photosensitizers.

In vivo uptake and biodistribution of lipophilic and hydrophilic photosensitizers

Uptake and biodistribution of photosensitizers are crucial parameters for evaluating the efficacy of photodynamic therapy (PDT). We used the tumor baring chorioallantoic membrane (CAM) as an in vivo model system to study biodistribution of hydrophilic and lipophile compounds of natural porphyrins and pthalocyanines using confocal laser scanning microscopy (LSM) and time-gated microspectrometry. Simultaneously we observed tumor vessels and tumor cells after intravascular application at different incubation times. Differences in biodistribution of hydrophilic and lipophilic sensitizers could be observed. Hydrophilic compounds were characterized by selective accumulation in tumor cells. In contrast, more lipophilic sensitizers are accumulated in endothelial cells at short incubation times as well as in tumor cells. These facts are in correlation with our studies in tumor cells and endothelial cells in vitro, where hydrophilic sensitizers are characterized by low uptake and low phototoxicity towards endothelial cells. In contrast more lipophilic sensitizers were rapidly taken up and showed high phototoxicity.

Uptake, subcellular localization, and phototoxicity of photosensitizing porphyrins

Cytotoxicity, uptake, intracellular location and phototoxicity were compared for the naturally occurring porphyrins uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP), as well as for tetra-, tri-, di- and monosulphonated meso-tetraphenyl porphyrins (TPPSn with n equals 4, 3, 2a, 1) in cultivated endothelial cells from calf aorta (BKEz-7). In addition, the uptake of CP and PP in the chick chorioallantoic membrane (CAM) was studied after intravenous application. Hydrophilic porphyrins (UP, CP, TPPS4 and TPPS3) exhibit low cellular uptake as well as low cyto- and phototoxicity as compared with the more hydrophobic compounds. UP, CP, TPPS4 and TPPS3 were mainly located in lysosomes, whereas PP, TPPS2a and TPPS1 were preferentially observed in the cytoplasm -- supposed to be located in cellular membranes of organelles -- and exhibited pronounced light-induced reactions. A rapid uptake by the CAM vasculature (5 min. - 30 min. after application) and some delayed uptake by the CAM matrix (30 min. - 6 h with a maximum at 60 min.) were measured for CP and PP.

Real-time fluorescence microscopy monitoring of porphyrin biodistribution

In vivo uptake of the natural porphyrins, uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP), was monitored by fluorescence microscopy. Experiments were performed using the chick chorioallantoic membrane (CAM) model, which allowed video documentation of fluorescence both in real time and after integration over a chosen time interval (usually 2 s). Sensitizers at a concentration of 50 (mu) M (100 (mu) L) were injected into a medium-sized vein (diameter approximately 40 micrometer) using an ultra-fine 10 micrometer diameter needle. Fluorescence images were quantitated by subtracting the fluorescence intensity of surrounding CAM tissue (Fmatrix) from the intravascular fluorescence intensity (Fintravascular), after transformation of the video frames into digital form. The differential fluorescence intensity, Fintravascular - Fmatrix, is a measure of the biodistribution. Real time measurements clearly showed that CP and UP fluorescence is associated with moving erythrocytes and not with endothelial cells of the vessel wall. Fluorescence intensity was monitored, up to 60 minutes after injection, by averaging the fluorescence over time intervals of 2 s and recording the integrated images. The fluorescence intensity reached its maximum in about 20 - 30 min after injection, presumably after monomerization inside erythrocyte membranes. The results are interpreted in terms of physical-chemical characteristics (e.g. hydrophilicity) and correlated with the photodynamically induced hemostasis in CAM blood vessels.

Optical coherence tomography versus ultrasound for diagnostics in dermatology

High resolution optical coherence tomography (OCT) has become a new diagnostic tool for the evaluation of structural changes of the skin. The instrumental resolution (3 μm lateral and 5 μm axial) was determined by the point spread function of 1nm particles. OCT now offers diagnosis of inflammation processes in deeper skin layers, not detectable by conventional 20MHz ultrasound applications. Therefore, therapy monitoring of patients will become possible.