Protein Kinase C θ Affects Ca2+ Mobilization and NFAT Activation in Primary Mouse T Cells

Protein kinase C (PKC)θ is an established component of the immunological synapse and has been implicated in the control of AP-1 and NF-κB. To study the physiological function of PKCθ, we used gene targeting to generate a PKCθ null allele in mice. Consistently, interleukin 2 production and T cell proliferative responses were strongly reduced in PKCθ-deficient T cells. Surprisingly, however, we demonstrate that after CD3/CD28 engagement, deficiency of PKCθ primarily abrogates NFAT transactivation. In contrast, NF-κB activation was only partially reduced. This NFAT transactivation defect appears to be secondary to reduced inositol 1,4,5-trisphosphate generation and intracellular Ca2+ mobilization. Our finding suggests that PKCθ plays a critical and nonredundant role in T cell receptor–induced NFAT activation

MUG-Mel2, a novel highly pigmented and well characterized NRAS mutated human melanoma cell line

NRAS mutation in melanoma has been associated with aggressive tumor biology and poor prognosis. Although targeted therapy has been tested for NRAS mutated melanoma, response rates still appear much weaker, than in BRAF mutated melanoma. While plenty of cell lines exist, however, only few melanogenic cell lines retain their in vivo characteristics. In this work we present an intensively pigmented and well-characterized cell line derived from a highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2. We present the clinical course, unique morphology, angiogenic properties, growth characteristics using in vivo experiments and 3D cell culture, and results of the exome gene sequencing of an intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis of an aggressive NRAS p. Q61R mutated melanoma. Amongst several genetic alterations, mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present. These mutations, known to reinforce DNA repair problems in melanoma, might serve as potential treatment targets. The aggressive and fast growing behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model for research in the field of NRAS mutated melanoma.Peer reviewe

Anti-tumor effects of shikonin derivatives on human medullary thyroid carcinoma cells

New treatment options are needed for medullary thyroid carcinoma (MTC), a highly metastasizing neuroendocrine tumor that is resistant to standard radiotherapy and chemotherapy. We show that the following shikonin derivatives inhibit cell proliferation and cell viability of the MTC cell line TT: acetylshikonin, β,β-dimethylacrylshikonin, shikonin and a petroleum ether extract of the roots of Onosma paniculata containing several shikonin derivatives. The unsubstituted shikonin derivative was found to be the most effective compound with an IC50 of 1.1 μM. The cell viability of normal human skin fibroblasts, however, was not affected by the tested substances, indicating that shikonin derivatives might be selectively toxic for cancer cells. We further report that migration and invasion of TT cells were inhibited at non-toxic concentrations. Finally, shikonin was tested in vivo using the chick chorioallantoic membrane assay, where it significantly reduced tumor growth by inhibiting cell proliferation and inducing apoptosis. In summary, our results suggest that shikonin derivatives have the potential for the treatment of medullary thyroid carcinomas

Lasp1 misexpression influences chondrocyte differentiation in the vertebral column

The mouse mutant wavy tail Tg(Col1a1-lacZ)304ng was created through transgene insertion and exhibits defects of the vertebral column. Homozygous mutant animals have compressed tail vertebrae and wedge-shaped intervertebral discs, resulting in a meandering tail. Delayed closure of lumbar neural arches and lack of processus spinosi have been observed; these defects become most prominent during the transition from cartilage to bone. The spina bifida was resistant to folic acid treatment, while retinoic acid administration caused severe skeletal defects in the mutant, but none in wild type control animals. The transgene integrated at chromosome 11 band D, in an area of high gene density. The insertion site was located between the transcription start sites of the RpI23 and Lasp1 genes. LASP1 (an actin binding protein involved in cell migration and survival) was found to be produced in resting and hypertrophic chondrocytes in the vertebrae. In mutant vertebrae, temporal and spatial misexpression of Lasp1 was observed, indicating that alterations in Lasp1 transcription are most likely responsible for the observed phenotype. These data reveal a yet unappreciated role of Lasp1 in chondrocyte differentiation during cartilage to bone transition

Testing the effects of photobiomodulation on angiogenesis in a newly established CAM burn wound model

Abstract Burn wounds are a common challenge for medical professionals. Current burn wound models hold several limitations, including a lack of comparability due to the heterogeneity of wounds and differences in individual wound healing. Hence, there is a need for reproducible in vivo models. In this study, we established a new burn wound model using the chorioallantoic membrane assay (CAM) as a surrogate model for animal experiments. The new experimental setup was tested by investigating the effects of the auspicious biophysical therapy, photobiomodulation (PBM), on the wound healing of an induced CAM burn wound with a metal stamp. PBM has been shown to positively influence wound healing through vascular proliferative effects and the increased secretion of chemotactic substances. The easily accessible burn wounds can be treated with various therapies. The model enables the analysis of ingrowing blood vessels (angiogenesis) and diameter and area of the wounds. The established model was used to test the effects of PBM on burn wound healing. PBM promoted angiogenesis in burn wounds on day 4 (p = 0.005). Furthermore, there was a not significant trend toward a higher number of vessels for day 6 (p = 0.065) in the irradiated group. Changes in diameter (p = 0.129) and the burn area (p = 0.131) were not significant. Our results suggest that CAM can be a suitable model for studying burn wounds. The novel experimental design enables reproducible and comparable studies on burn wound treatment

A Novel Artificial Intelligence-Based Approach for Quantitative Assessment of Angiogenesis in the Ex Ovo CAM Model

Angiogenesis is a highly regulated process. It promotes tissue regeneration and contributes to tumor growth. Existing therapeutic concepts interfere with different steps of angiogenesis. The quantification of the vasculature is of crucial importance for research on angiogenetic effects. The chorioallantoic membrane (CAM) assay is widely used in the study of angiogenesis. Ex ovo cultured chick embryos develop an easily accessible, highly vascularised membrane on the surface. Tumor xenografts can be incubated on this membrane enabling studies on cancer angiogenesis and other major hallmarks. However, there is no commonly accepted gold standard for the quantification of the vasculature of the CAM. We compared four widely used measurement techniques to identify the most appropriate one for the quantification of the vascular network of the CAM. The comparison of the different quantification methods suggested that the CAM assay application on the IKOSA platform is the most suitable image analysis application for the vasculature of the CAM. The new CAM application on the IKOSA platform turned out to be a reliable and feasible tool for practical use in angiogenesis research. This novel image analysis software enables a deeper exploration of various aspects of angiogenesis and might support future research on new anti-angiogenic strategies for cancer treatment

Exacerbated vein graft arteriosclerosis in protein kinase Cδ–null mice

Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCδ knockout mice and performed vein bypass grafts on these animals. PKCδ(–/–) mice developed normally and were fertile. Vein segments from PKCδ(–/–) mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCδ(+/+) vein grafts. Arteriosclerotic lesions in PKCδ(–/–) mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCδ(–/–) mice. SMCs derived from PKCδ(–/–) aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCδ(–/–) relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCδ maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis

15d-PGJ2 Promotes ROS-Dependent Activation of MAPK-Induced Early Apoptosis in Osteosarcoma Cell In Vitro and in an Ex Ovo CAM Assay

Osteosarcoma (OS) is the most common type of bone tumor, and has limited therapy options. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has striking anti-tumor effects in various tumors. Here, we investigated molecular mechanisms that mediate anti-tumor effects of 15d-PGJ2 in different OS cell lines. Human U2-OS and Saos-2 cells were treated with 15d-PGJ2 and cell survival was measured by MTT assay. Cell proliferation and motility were investigated by scratch assay, the tumorigenic capacity by colony forming assay. Intracellular ROS was estimated by H2DCFDA. Activation of MAPKs and cytoprotective proteins was detected by immunoblotting. Apoptosis was detected by immunoblotting and Annexin V/PI staining. The ex ovo CAM model was used to study growth capability of grafted 15d-PGJ2-treated OS cells, followed by immunohistochemistry with hematoxylin/eosin and Ki-67. 15d-PGJ2 substantially decreased cell viability, colony formation and wound closure capability of OS cells. Non-malignant human osteoblast was less affected by 15d-PGJ2. 15d-PGJ2 induced rapid intracellular ROS production and time-dependent activation of MAPKs (pERK1/2, pJNK and pp38). Tempol efficiently inhibited 15d-PGJ2-induced ERK1/2 activation, while N-acetylcystein and pyrrolidine dithiocarbamate were less effective. Early but weak activation of cytoprotective proteins was overrun by induction of apoptosis. A structural analogue, 9,10-dihydro-15d-PGJ2, did not show toxic effects in OS cells. In the CAM model, we grafted OS tumors with U2-OS, Saos-2 and MG-63 cells. 15d-PGJ2 treatment resulted in significant growth inhibition, diminished tumor tissue density, and reduced tumor cell proliferation for all cell lines. Our in vitro and CAM data suggest 15d-PGJ2 as a promising natural compound to interfere with OS tumor growth