Potential role of the bitter taste receptor T2R14 in the prolonged survival and enhanced chemoresponsiveness induced by apigenin.

Bitter taste receptors (T2Rs) are G protein­coupled receptors originally detected in the gustatory system. More recently, T2Rs have been shown to be expressed in extra­oral cells eliciting non­gustatory functions. Emerging evidence has suggested a potential role for T2R signaling in diverse pathophysiological conditions, including cancer. -The aim of the present study was to evaluate the expression of T2R14 in pancreatic ductal adenocarcinoma (PDAC) and to assess its involvement in the anticancer effects induced by apigenin, a natural ligand of T2R14. For this purpose, T2R14 expression was explored in PDAC tumor tissue and tumor­derived cell lines. Using the cell lines expressing the highest levels of T2R14, its effects on chemoresponsiveness and migration upon activation with apigenin were investigated in vitro. To the best of our knowledge, the present study was the first to confirm the expression of the T2R family member T2R14 in PDAC. Patients with relatively high levels of T2R14 expression exhibited significantly prolonged overall survival compared with that of patients with low T2R14 expression. Furthermore, novel functions for apigenin were revealed; notably, apigenin was shown to elicit cytotoxic, anti­migratory and chemosensitizing effects to 5­fluoruracil (5­FU) and to 5­FU, leucovorin, irinotecan and oxaliplatin in pancreatic cancer cells. In conclusion, the present study extended the evidence for the anticancer effects of apigenin and strongly indicated the functional relevance of T2R14 in PDAC, even though their respective underlying pathways appear to be independent of each other.

The collagenase of the bacterium Clostridium histolyticum does not favor metastasis of breast cancer.

BACKGROUND: Breast cancer is the most common malignancy among women worldwide. As survival rates increase, breast reconstruction and quality of life gain importance. Of all women undergoing breast reconstruction, approximately, 70% opt for silicone implants and 50% of those develop capsular contracture, the most prevalent long-term complication. The collagenase of the bacterium Clostridium histolyticum (CCH) showed promising results in the therapy of capsule contracture; however, its influence on residual cancer cells is unknown. The aim of this study was to investigate whether CCH-treatment negatively impacts breast cancer cells in vitro and in vivo. METHODS: MDA-MB-231 and MCF-7 cells were used in this study. In vitro, we tested the influence of CCH on proliferation, wound healing, migration and cell cycle by MTT-assay, scratch-assay, transwell-migration-assay, and flow cytometry. In vivo, solid tumors were induced in immune-deficient mice. CCH was injected into the tumors and tumor growth and metastasis formation was monitored by caliper measurement, in vivo bioluminescence imaging and histology. Gene expression analysis was performed by microarray including 27,190 genes. RESULTS: CCH-incubation led to a dose-dependent reduction in proliferation for both cell lines, while wound healing was reduced only in MDA-MB-231 cells. No morphological alterations were monitored in cell cycle or apoptosis. In vivo, bioluminescence imaging and histology did not show any evidence of metastasis. Although CCH led to changes in gene expression of breast cancer cells, no relevant alterations in metastasis-related genes were monitored. CONCLUSION: CCH has no impact on tumor growth or metastasis formation in vitro and in vivo. This paves the way for first clinical trials.

The effect of surgical suture material on osteoclast generation and implant-loosening.

Background: Implant loosening - either infectious or aseptic- is a still a major complication in the field of orthopaedic surgery. In both cases, a pro-inflammatory peri-prosthetic environment is generated by the immune system - either triggered by bacteria or by implant wear particles - which leads to osteoclast differentiation and osteolysis. Since infectious cases in particular often require multiple revision surgeries, we wondered whether commonly used surgical suture material may also activate the immune system and thus contribute to loss of bone substance by generation of osteoclasts. Methods: Tissue samples from patients suffering from infectious implant loosening were collected intraoperatively and presence of osteoclasts was evaluated by histopathology and immunohistochemistry. Further on, human monocytes were isolated from peripheral blood and stimulated with surgical suture material. Cell supernatant samples were collected and ELISA analysis for the pro-inflammatory cytokine IL-8 was performed. These experiments were additionally carried out on ivory slices to demonstrate functionality of osteoclasts. Whole blood samples were incubated with surgical suture material and up-regulation of activation-associated cell surface markers CD11b and CD66b on neutrophils was evaluated by flow cytofluorometry analysis. Results: We were able to demonstrate that multinucleated giant cells form in direct vicinity to surgical suture material. These cells stained positive for cathepsin K, which is a typical protease found in osteoclasts. By in vitro analysis, we were able to show that monocytes differentiated into osteoclasts when stimulated with surgical suture material. Resorption pits on ivory slices provided proof that the osteoclasts were functional. Release of IL-8 into cell supernatant was increased after stimulation with suture material and was further enhanced if minor amounts of bacterial lipoteichoic acid (LTA) were added. Neutrophils were also activated by surgical suture material and up-regulation of CD11b and CD66b could be seen. Conclusion: We were able to demonstrate that surgical suture material induces a pro-inflammatory response of immune cells which leads to osteoclast differentiation, in particular in combination with bacterial infection. In conclusion, surgical suture material -aside from bacteria and implant wear particles- is a contributing factor in implant loosening.

Membranous nephropathy followed by anti-glomerular basement disease: A case report and review of clinical presentation and treatment.

Membranous nephropathy is a common cause of nephrotic syndrome in adults and can be primary or secondary through autoimmune disease, medication, infection, or malignancy. Rapidly progressive glomerulonephritis with crescent formation is rare in patients with membranous nephropathy. Thus, in cases with rapid decline in renal function, after excluding complications such as malignant hypertension, acute hypersensitivity interstitial nephritis, and bilateral renal vein thrombosis, the simultaneous occurrence of a superimposed glomerulonephritis should be considered. We report a 55-year-old man suffering from a biopsy-confirmed primary membranous nephropathy, who developed rapidly progressive glomerulonephritis with anti-glomerular basement membrane antibodies after being affected with membranous nephropathy for 8 years. The kidney biopsy revealed a concurrence of membranous nephropathy and anti-glomerular basement membrane disease. Clinical presentation and treatment of membranous nephropathy followed by anti-glomerular basement membrane disease are discussed based on our observation with promising follow-up.

Sensing developing biofilms: the bitter receptor T2R38 on myeloid cells.

Quorum-sensing molecules, also known as autoinducer, are essential for bacterial biofilm formation. Our focus is on N-(3-oxododecanoyl)-L-homoserine lactone (AHL-12), because it is also known as an 'interkingdom signalling molecule', which means that it also interacts with mammalian cells. AHL-12 activates defence-relevant functions of phagocytic cells, including enhancement of phagocytosis, increased expression of adhesion receptors and induction of chemotaxis. This leads to the hypothesis that early recognition of developing biofilms might be the key to a successful host defence against biofilm infection. In that context we studied activation of phagocytic cells by AHL-12, and found that phagocytes are activated via a rather specialized receptor that was not previously described on myeloid cells, the bitter taste receptor T2R38. Taste receptors are commonly associated with cells of the gustatory system. The extragustatory expression, however, suggests an additional role, namely the sensing of the onset of bacterial biofilm infection.

Tasting Pseudomonas aeruginosa Biofilms: Human Neutrophils Express the Bitter Receptor T2R38 as Sensor for the Quorum Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone.

Bacteria communicate with one another via specialized signaling molecules, known as quorum sensing molecules or autoinducers. The Pseudomonas aeruginosa-derived quorum sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (AHL-12), however, also activates mammalian cells. As shown previously, AHL-12-induced chemotaxis, up-regulated CD11b expression, and enhanced phagocytosis of polymorphonuclear neutrophils. Circumstantial evidence concurred with a receptor for AHL-12, which has been elusive so far. We now investigated the bitter receptor T2R38 as a potential candidate. Although identified as a taste receptor, extragustatory cells express T2R38, for example, epithelial cells in the lung. We now detected T2R38 in peripheral blood neutrophils, monocytes, and lymphocytes. T2R38 is not only found on the cell membrane but also intracellular. In neutrophils, T2R38 was located in vesicles with characteristics of lipid droplets, and super-resolution microscopy showed a co-localization with the lipid droplet membrane. Neutrophils take up AHL-12, and it co-localized with T2R38 as seen by laser scan microscopy. Binding of AHL-12 to T2R28 was confirmed by pull-down assays using biotin-coupled AHL-12 as bait. A commercially available antibody to T2R38 inhibited binding of AHL-12 to neutrophils, and this antibody by itself stimulated neutrophils, similarly to AHL-12. In conclusion, our data provide evidence for expression of functional T2R38 on neutrophils, and are compatible with the notion that T2R38 is the receptor for AHL-12.

The macrophage inflammatory proteins MIP1α (CCL3) and MIP2α (CXCL2) in implant-associated osteomyelitis: linking inflammation to bone degradation.

Bacterial infections of bones remain a serious complication of endoprosthetic surgery. These infections are difficult to treat, because many bacterial species form biofilms on implants, which are relatively resistant towards antibiotics. Bacterial biofilms elicit a progressive local inflammatory response, resulting in tissue damage and bone degradation. In the majority of patients, replacement of the prosthesis is required. To address the question of how the local inflammatory response is linked to bone degradation, tissue samples were taken during surgery and gene expression of the macrophage inflammatory proteins MIP1α (CCL3) and MIP2α (CXCL2) was assessed by quantitative RT-PCR. MIPs were expressed predominantly at osteolytic sites, in close correlation with CD14 which was used as marker for monocytes/macrophages. Colocalisation of MIPs with monocytic cells could be confirmed by histology. In vitro experiments revealed that, aside from monocytic cells, also osteoblasts were capable of MIP production when stimulated with bacteria; moreover, CCL3 induced the differentiation of monocytes to osteoclasts. In conclusion, the multifunctional chemokines CCL3 and CXCL2 are produced locally in response to bacterial infection of bones. In addition to their well described chemokine activity, these cytokines can induce generation of bone resorbing osteoclasts, thus providing a link between bacterial infection and osteolysis.

Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma.

BACKGROUND & AIMS: Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear. METHODS: By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients. RESULTS: Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not ß-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer. CONCLUSIONS: The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).