Epigenetic regulation affects N-myc downstream-regulated gene 1 expression indirectly in pancreatic cancer cells

N-myc downstream-regulated gene 1 (NDRG1), important in tumor growth and metastasis, has recently gained interest as a potential therapeutic target. Loss of NDRG1 expression is generally associated with poor clinical outcome in pancreatic cancer (PaCa) patients. As the NDRG1 gene possesses a large promoter CpG island, we sought to determine whether its repression is epigenetically mediated in PaCa cells.

Glucagonlike peptide-1 receptor: an example of translational research in insulinomas: a review

Glucagonlike peptide-1 receptors (GLP-1R) play an increasingly important role in endocrine gastrointestinal tumor management. In particular, virtually all benign insulinomas express GLP-1R in high density. Exendin-4 is a GLP-1 analog that has a longer half-life than GLP-1. Targeting GLP-1R by (111)In-DOTA-exendin-4 or (111)In-DPTA-exendin-4 offers a new approach that permits the successful localization of small benign insulinomas. It is likely that this new noninvasive technique has the potential to replace the invasive localization by selective arterial stimulation and venous sampling.

Tumor necrosis factor sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. CONCLUSION: Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid.

A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation

Sphingosine kinase 1 (SK1) is a key enzyme in the generation of sphingosine 1-phosphate (S1P) which critically regulates a variety of important cell responses such as proliferation and migration. Therefore, inhibition of SK-1 has been suggested to be an attractive approach to treat tumor growth and metastasis formation.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

One-Step (18)F-Labeling of Carbohydrate-Conjugated Octreotate-Derivatives Containing a Silicon-Fluoride-Acceptor (SiFA): In Vitro and in Vivo Evaluation as Tumor Imaging Agents for Positron Emission Tomography (PET)

The synthesis, radiolabeling, and initial evaluation of new silicon-fluoride acceptor (SiFA) derivatized octreotate derivatives is reported. So far, the main drawback of the SiFA technology for the synthesis of PET-radiotracers is the high lipophilicity of the resulting radiopharmaceutical. Consequently, we synthesized new SiFA-octreotate analogues derivatized with Fmoc-NH-PEG-COOH, Fmoc-Asn(Ac?AcNH-?-Glc)-OH, and SiFA-aldehyde (SIFA-A). The substances could be labeled in high yields (38 ± 4%) and specific activities between 29 and 56 GBq/?mol in short synthesis times of less than 30 min (e.o.b.). The in vitro evaluation of the synthesized conjugates displayed a sst2 receptor affinity (IC?? = 3.3 ± 0.3 nM) comparable to that of somatostatin-28. As a measure of lipophilicity of the conjugates, the log P(ow) was determined and found to be 0.96 for SiFA-Asn(AcNH-?-Glc)-PEG-Tyr³-octreotate and 1.23 for SiFA-Asn(AcNH-?-Glc)-Tyr³-octreotate, which is considerably lower than for SiFA-Tyr³-octreotate (log P(ow) = 1.59). The initial in vivo evaluation of [¹?F]SiFA-Asn(AcNH-?-Glc)-PEG-Tyr³-octreotate revealed a significant uptake of radiotracer in the tumor tissue of AR42J tumor-bearing nude mice of 7.7% ID/g tissue weight. These results show that the high lipophilicity of the SiFA moiety can be compensated by applying hydrophilic moieties. Using this approach, a tumor-affine SiFA-containing peptide could successfully be used for receptor imaging for the first time in this proof of concept study.

Synergistic induction of cell death in liver tumor cells by TRAIL and chemotherapeutic drugs via the BH3-only proteins Bim and Bid

Although death receptors and chemotherapeutic drugs activate distinct apoptosis signaling cascades, crosstalk between the extrinsic and intrinsic apoptosis pathway has been recognized as an important amplification mechanism. Best known in this regard is the amplification of the Fas (CD95) signal in hepatocytes via caspase 8-mediated cleavage of Bid and activation of the mitochondrial apoptosis pathway. Recent evidence, however, indicates that activation of other BH3-only proteins may also be critical for the crosstalk between death receptors and mitochondrial triggers. In this study, we show that TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic drugs synergistically induce apoptosis in various transformed and untransformed liver-derived cell lines, as well as in primary human hepatocytes. Both, preincubation with TRAIL as well as chemotherapeutic drugs could sensitize cells for apoptosis induction by the other respective trigger. TRAIL induced a strong and long lasting activation of Jun kinase, and activation of the BH3-only protein Bim. Consequently, synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs was dependent on Jun kinase activity, and expression of Bim and Bid. These findings confirm a previously defined role of TRAIL and Bim in the regulation of hepatocyte apoptosis, and demonstrate that the TRAIL-Jun kinase-Bim axis is a major and important apoptosis amplification pathway in primary hepatocytes and liver tumor cells.

Tumor lymphangiogenesis and metastasis to lymph nodes induced by cancer cell expression of podoplanin

The membrane glycoprotein podoplanin is expressed by several types of human cancers and might be associated with their malignant progression. Its exact biological function and molecular targets are unclear, however. Here, we assessed the relevance of tumor cell expression of podoplanin in cancer metastasis to lymph nodes, using a human MCF7 breast carcinoma xenograft model. We found that podoplanin expression promoted tumor cell motility in vitro and, unexpectedly, increased tumor lymphangiogenesis and metastasis to regional lymph nodes in vivo, without promoting primary tumor growth. Importantly, high cancer cell expression levels of podoplanin correlated with lymph node metastasis and reduced survival times in a large cohort of 252 oral squamous cell carcinoma patients. Based on comparative transcriptional profiling of tumor xenografts, we identified endothelin-1, villin-1, and tenascin-C as potential mediators of podoplanin-induced tumor lymphangiogenesis and metastasis. These unexpected findings identify a novel mechanism of tumor lymphangiogenesis and metastasis induced by cancer cell expression of podoplanin, suggesting that reagents designed to interfere with podoplanin function might be developed as therapeutics for patients with advanced cancer.

Isolated GH deficiency type II: knockdown of the harmful Delta3GH recovers wt-GH secretion in rat tumor pituitary cells

Isolated GH deficiency type II (IGHD II) is the autosomal dominant form of GHD. In the majority of the cases, this disorder is due to specific GH-1 gene mutations that lead to mRNA missplicing and subsequent loss of exon 3 sequences. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH (Delta3GH) isoform that reduces the accumulation and secretion of wild-type-GH. At present, patients suffering from this type of disease are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the Delta3GH mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. We developed a strategy involving Delta3GH isoform knockdown mediated by expression of a microRNA-30-adapted short hairpin RNA (shRNA) specifically targeting the Delta3GH mRNA of human (shRNAmir-Delta3). Rat pituitary tumor GC cells expressing Delta3GH upon doxycycline induction were transduced with shRNAmir-Delta3 lentiviral vectors, which significantly reduced Delta3GH protein levels and improved human wild-type-GH secretion in comparison with a shRNAmir targeting a scrambled sequence. No toxicity due to shRNAmir expression could be observed in cell proliferation assays. Confocal microscopy strongly suggested that shRNAmir-Delta3 enabled the recovery of GH granule storage and secretory capacity. These viral vectors have shown their ability to stably integrate, express shRNAmir, and rescue IGHD II phenotype in rat pituitary tumor GC cells, a methodology that opens new perspectives for the development of gene therapy to treat IGHD patients.

Disabling c-Myc in childhood medulloblastoma and atypical teratoid/rhabdoid tumor cells by the potent G-quadruplex interactive agent S2T1-6OTD

We investigated here the effects of S2T1-6OTD, a novel telomestatin derivative that is synthesized to target G-quadruplex-forming DNA sequences, on a representative panel of human medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) childhood brain cancer cell lines. S2T1-6OTD proved to be a potent c-Myc inhibitor through its high-affinity physical interaction with the G-quadruplex structure in the c-Myc promoter. Treatment with S2T1-6OTD reduced the mRNA and protein expressions of c-Myc and hTERT, which is transcriptionally regulated by c-Myc, and decreased the activities of both genes. In remarkable contrast to control cells, short-term (72-hour) treatment with S2T1-6OTD resulted in a dose- and time-dependent antiproliferative effect in all MB and AT/RT brain tumor cell lines tested (IC(50), 0.25-0.39 micromol/L). Under conditions where inhibition of both proliferation and c-Myc activity was observed, S2T1-6OTD treatment decreased the protein expression of the cell cycle activator cyclin-dependent kinase 2 and induced cell cycle arrest. Long-term treatment (5 weeks) with nontoxic concentrations of S2T1-6OTD resulted in a time-dependent (mainly c-Myc-dependent) telomere shortening. This was accompanied by cell growth arrest starting on day 28 followed by cell senescence and induction of apoptosis on day 35 in all of the five cell lines investigated. On in vivo animal testing, S2T1-6OTD may well represent a novel therapeutic strategy for childhood brain tumors.

Fully Automatic Segmentation of Brain Tumor Images using Support Vector Machine Classiffication in Combination with Hierarchical Conditional Random Field Regularization

Delineating brain tumor boundaries from magnetic resonance images is an essential task for the analysis of brain cancer. We propose a fully automatic method for brain tissue segmentation, which combines Support Vector Machine classification using multispectral intensities and textures with subsequent hierarchical regularization based on Conditional Random Fields. The CRF regularization introduces spatial constraints to the powerful SVM classification, which assumes voxels to be independent from their neighbors. The approach first separates healthy and tumor tissue before both regions are subclassified into cerebrospinal fluid, white matter, gray matter and necrotic, active, edema region respectively in a novel hierarchical way. The hierarchical approach adds robustness and speed by allowing to apply different levels of regularization at different stages. The method is fast and tailored to standard clinical acquisition protocols. It was assessed on 10 multispectral patient datasets with results outperforming previous methods in terms of segmentation detail and computation times.

Multi-Scale Modeling for Image Analysis of Brain Tumor Studies

Image-based modeling of tumor growth combines methods from cancer simulation and medical imaging. In this context, we present a novel approach to adapt a healthy brain atlas to MR images of tumor patients. In order to establish correspondence between a healthy atlas and a pathologic patient image, tumor growth modeling in combination with registration algorithms is employed. In a first step, the tumor is grown in the atlas based on a new multi-scale, multi-physics model including growth simulation from the cellular level up to the biomechanical level, accounting for cell proliferation and tissue deformations. Large-scale deformations are handled with an Eulerian approach for finite element computations, which can operate directly on the image voxel mesh. Subsequently, dense correspondence between the modified atlas and patient image is established using nonrigid registration. The method offers opportunities in atlasbased segmentation of tumor-bearing brain images as well as for improved patient-specific simulation and prognosis of tumor progression.

Segmentation of brain tumor images based on atlas-registration combined with a Markov-Random-Field lesion growth model

We present an automatic method to segment brain tissues from volumetric MRI brain tumor images. The method is based on non-rigid registration of an average atlas in combination with a biomechanically justified tumor growth model to simulate soft-tissue deformations caused by the tumor mass-effect. The tumor growth model, which is formulated as a mesh-free Markov Random Field energy minimization problem, ensures correspondence between the atlas and the patient image, prior to the registration step. The method is non-parametric, simple and fast compared to other approaches while maintaining similar accuracy. It has been evaluated qualitatively and quantitatively with promising results on eight datasets comprising simulated images and real patient data.