Longitudinal Analysis of Circulating Tumor Cells in Colorectal Cancer Patients by a Cytological and Molecular Approach: Feasibility and Clinical Application

Introduction Liquid biopsies allowing for individualized risk stratification of cancer patients have become of high significance in individualized cancer diagnostics and treatment. The detection of circulating tumor cells (CTC) has proven to be highly relevant in risk prediction, e.g., in colorectal cancer (CRC) patients. In this study, we investigate the clinical relevance of longitudinal CTC detection over a course of follow-up after surgical resection of the tumor and correlate these findings with clinico-pathological characteristics. Methods In total, 49 patients with histologically proven colorectal carcinoma were recruited for this prospective study. Blood samples were analyzed for CTC presence by two methods: first by marker-dependent immunofluorescence staining combined with automated microscopy with the NYONE® cell imager and additionally, indirectly, by semi-quantitative Cytokeratin-20 (CK20) RT-qPCR. CTC quantification data were compared and correlated with the clinico-pathological parameters. Results Detection of CTC over a post-operative time course was feasible with both applied methods. In patients who were pre-operatively negative for CTCs with the NYONE® method or below the cut-off for relative CK20 mRNA expression after analysis by PCR, a statistically significant rise in the immediate post-operative CTC detection could be demonstrated. Further, in the cohort analyzed by PCR, we detected a lower CTC load in patients who were adjuvantly treated with chemotherapy compared to patients in the follow-up subgroup. This finding was contrary to the same patient subset analyzed with the NYONE® for CTC detection. Conclusion Our study investigates the occurrence of CTC in CRC patients after surgical resection of the primary tumor and during postoperative follow-up. The resection of the tumor has an impact on the CTC quantity and the longitudinal CTC analysis supports the significance of CTC as a prognostic biomarker. Future investigations with an even more extended follow-up period and larger patient cohorts will have to validate our results and may help to define an optimal longitudinal sampling scheme for liquid biopsies in the post-operative monitoring of cancer patients to enable tailored therapy concepts for precision medicine

Small Molecules in the Treatment of Squamous Cell Carcinomas: Focus on Indirubins

Skin cancers are the most common malignancies in the world. Among the most frequent skin cancer entities, squamous cell carcinoma (SCC) ranks second (~20%) after basal cell carcinoma (~77%). In early stages, a complete surgical removal of the affected tissue is carried out as standard therapy. To treat advanced and metastatic cancers, targeted therapies with small molecule inhibitors are gaining increasing attention. Small molecules are a heterogeneous group of protein regulators, which are produced by chemical synthesis or fermentation. The majority of them belong to the group of receptor tyrosine kinase inhibitors (RTKIs), which specifically bind to certain RTKs and directly influence the respective signaling pathway. Knowledge of characteristic molecular alterations in certain cancer entities, such as SCC, can help identify tumor-specific substances for targeted therapies. Most frequently, altered genes in SCC include TP53, NOTCH, EGFR, and CCND1. For example, the gene CCND1, which codes for cyclin D1 protein, is upregulated in nearly half of SCC cases and promotes proliferation of affected cells. A treatment with the small molecule 5'-nitroindirubin-monoxime (INO) leads to inhibition of cyclin D1 and thus inhibition of proliferation. As a component of Danggui Longhui Wan, a traditional Chinese medicine, indirubins are used to treat chronic diseases and have been shown to inhibit inflammatory reactions. Indirubins are pharmacologically relevant small molecules with proapoptotic and antiproliferative activity. In this review, we discuss the current literature on indirubin-based small molecules in cancer treatment. A special focus is on the molecular biology of squamous cell carcinomas, their alterations, and how these are rendered susceptible to indirubin-based small molecule inhibitors. The potential molecular mechanisms of the efficacy of indirubins in killing SCC cells will be discussed as well

Cannabinoid Compounds as a Pharmacotherapeutic Option for the Treatment of Non-Cancer Skin Diseases

The endocannabinoid system has been shown to be involved in various skin functions, such as melanogenesis and the maintenance of redox balance in skin cells exposed to UV radiation, as well as barrier functions, sebaceous gland activity, wound healing and the skin’s immune response. In addition to the potential use of cannabinoids in the treatment and prevention of skin cancer, cannabinoid compounds and derivatives are of interest as potential systemic and topical applications for the treatment of various inflammatory, fibrotic and pruritic skin conditions. In this context, cannabinoid compounds have been successfully tested as a therapeutic option for the treatment of androgenetic alopecia, atopic and seborrhoeic dermatitis, dermatomyositis, asteatotic and atopic eczema, uraemic pruritis, scalp psoriasis, systemic sclerosis and venous leg ulcers. This review provides an insight into the current literature on cannabinoid compounds as potential medicines for the treatment of skin diseases

Cannabidiol Promotes Endothelial Cell Survival by Heme Oxygenase-1-Mediated Autophagy

Cannabidiol (CBD), a non-psychoactive cannabinoid, has been reported to mediate antioxidant, anti-inflammatory, and anti-angiogenic effects in endothelial cells. This study investigated the influence of CBD on the expression of heme oxygenase-1 (HO-1) and its functional role in regulating metabolic, autophagic, and apoptotic processes of human umbilical vein endothelial cells (HUVEC). Concentrations up to 10 µM CBD showed a concentration-dependent increase of HO-1 mRNA and protein and an increase of the HO-1-regulating transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). CBD-induced HO-1 expression was not decreased by antagonists of cannabinoid-activated receptors (CB1, CB2, transient receptor potential vanilloid 1), but by the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC). The incubation of HUVEC with 6 µM CBD resulted in increased metabolic activity, while 10 µM CBD caused decreased metabolic activity and an induction of apoptosis, as demonstrated by enhanced caspase-3 cleavage. In addition, CBD triggered a concentration-dependent increase of the autophagy marker LC3A/B-II. Both CBD-induced LC3A/B-II levels and caspase-3 cleavage were reduced by NAC. The inhibition of autophagy by bafilomycin A1 led to apoptosis induction by 6 µM CBD and a further increase of the proapoptotic effect of 10 µM CBD. On the other hand, the inhibition of HO-1 activity with tin protoporphyrin IX (SnPPIX) or knockdown of HO-1 expression by Nrf2 siRNA was associated with a decrease in CBD-mediated autophagy and apoptosis. In summary, our data show for the first time ROS-mediated HO-1 expression in endothelial cells as a mechanism by which CBD mediates protective autophagy, which at higher CBD concentrations, however, can no longer prevent cell death inducing apoptosis

Paracetamol and cyclooxygenase inhibition: is there a cause for concern?

Paracetamol is recommended as first-line therapy for pain associated with osteoarthrosis and is one of the most widely used over-the-counter analgesic drugs worldwide. Despite its extensive use, its mode of action is still unclear. Although it is commonly stated that paracetamol acts centrally, recent data imply an inhibitory effect on the activity of peripheral prostaglandin-synthesising cyclooxygenase enzymes. In this context paracetamol has been suggested to inhibit both isoforms in tissues with low levels of peroxide by reducing the higher oxidation state of cyclooxygenase enzymes. Two recent studies have also demonstrated a preferential cyclooxygenase 2 (COX-2) inhibition by paracetamol under different clinically relevant conditions. This review attempts to relate data on paracetamol's inhibitory action on peripheral cyclooxygenase enzymes to the published literature on its anti-inflammatory action and its hitherto underestimated side-effects elicited by cyclooxygenase inhibition. As a result, a pronounced COX-2 inhibition by paracetamol is expected to occur in the endothelium, possibly explaining its cardiovascular risk in epidemiological studies. A careful analysis of paracetamol's cardiovascular side-effects in randomised studies is therefore strongly advised. On the basis of epidemiological data showing an increased gastrointestinal risk of paracetamol at high doses or when co-administered with classic cyclooxygenase inhibitors, paracetamol's long-term gastrointestinal impact should be investigated in randomised trials. Finally, paracetamol's fast elimination and consequently short-lived COX-2 inhibition, which requires repetitive dosing, should be definitely considered to avoid overdosage leading to hepatotoxicity

Reduction of visual stimulus artifacts using a spherical tank for small, aquatic animals

Abstract Delivering appropriate stimuli remains a challenge in vision research, particularly for aquatic animals such as zebrafish. Due to the shape of the water tank and the associated optical paths of light rays, the stimulus can be subject to unwanted refraction or reflection artifacts, which may spoil the experiment and result in wrong conclusions. Here, we employ computer graphics simulations and calcium imaging in the zebrafish optic tectum to show, how a spherical glass container optically outperforms many previously used water containers, including Petri dish lids. We demonstrate that aquatic vision experiments suffering from total internal reflection artifacts at the water surface or at the flat container bottom may result in the erroneous detection of visual neurons with bipartite receptive fields and in the apparent absence of neurons selective for vertical motion. Our results and demonstrations will help aquatic vision neuroscientists on optimizing their stimulation setups

The Combination of Δ⁹-Tetrahydrocannabinol and Cannabidiol Suppresses Mitochondrial Respiration of Human Glioblastoma Cells via Downregulation of Specific Respiratory Chain Proteins

Phytocannabinoids represent a promising approach in glioblastoma therapy. Previous work has shown that a combined treatment of glioblastoma cells with submaximal effective concentrations of psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD) greatly increases cell death. In the present work, the glioblastoma cell lines U251MG and U138MG were used to investigate whether the combination of THC and CBD in a 1:1 ratio is associated with a disruption of cellular energy metabolism, and whether this is caused by affecting mitochondrial respiration. Here, the combined administration of THC and CBD (2.5 µM each) led to an inhibition of oxygen consumption rate and energy metabolism. These effects were accompanied by morphological changes to the mitochondria, a release of mitochondrial cytochrome c into the cytosol and a marked reduction in subunits of electron transport chain complexes I (NDUFA9, NDUFB8) and IV (COX2, COX4). Experiments with receptor antagonists and inhibitors showed that the degradation of NDUFA9 occurred independently of the activation of the cannabinoid receptors CB1, CB2 and TRPV1 and of usual degradation processes mediated via autophagy or the proteasomal system. In summary, the results describe a previously unknown mitochondria-targeting mechanism behind the toxic effect of THC and CBD on glioblastoma cells that should be considered in future cancer therapy, especially in combination strategies with other chemotherapeutics

Influence of Test Specimen Geometry and Water Soaking on the In Vitro Cytotoxicity of Orthocryl^®, Orthocryl^® LC, Loctite^® EA 9483 and Polypropylene

Depending on their composition, plastics have a cytotoxic potential that needs to be evaluated before they are used in dentistry, e.g., as orthodontic removable appliances. Relevant guidelines set out requirements that a potential new resin in the medical field must meet, with a wide scope for experimental design. In the present study, test specimens of different geometries consisting of varying polymers (Orthocryl®, Orthocryl® LC, Loctite® EA 9483, Polypropylene) were soaked for different periods of time, then transferred to cell culture medium for 24 h, which was subsequently used for 24-h cultivation of A549 cells, followed by cytotoxicity assays (WST-1, Annexin V-FITC-propidium iodide (PI) flow cytometry). In this context, a reduction in the cytotoxic effect of the eluates of test specimens prepared from Orthocryl® LC and Loctite® EA 9483 was particularly evident in the Annexin V-FITC-PI assay when the soaking time was extended to 48 h and 168 h, respectively. Consistent with this, a reduced release of potentially toxic monomers into the cell culture medium, as measured by gas chromatography-mass spectrometry, was observed when the prior soaking time of test specimens of all geometries was extended. Remarkably, a significant increase in cytotoxic effect was observed in the WST-1 assay, which was accompanied by a higher release of monomers when the thickness of the test sample was increased from 0.5 to 1.0 mm, although an elution volume adapted to the surface area was used. However, further increasing the thickness to 3.0 mm did not lead to an increase in the observed cytotoxicity or monomer release. Test specimens made of polypropylene showed no toxicity under all test specimen sizes and soaking time conditions. Overall, it is recommended to perform toxicity studies of test specimens using different geometries and soaking times. Thereby, the influence of the different specimen thicknesses should also be considered. Finally, an extension of the test protocols proposed in ISO 10993-5:2009 should be considered, e.g., by flow cytometry or monomer analysis as well as fixed soaking times