tetracycline and Pancreatic-Neoplasms

Improved treatments for pancreatic cancer remain a clinical imperative. Sabutoclax, a small-molecule BH3 mimetic, inhibits the function of antiapoptotic Bcl-2 proteins. Minocycline, a synthetic tetracycline, displays antitumor activity. Here, we offer evidence of the combinatorial antitumor potency of these agents in several preclinical models of pancreatic cancer. Sabutoclax induced growth arrest and apoptosis in pancreatic cancer cells and synergized with minocycline to yield a robust mitochondria-mediated caspase-dependent cytotoxicity. This combinatorial property relied upon loss of phosphorylated Stat3 insofar as reintroduction of activated Stat3-rescued cells from toxicity. Tumor growth was inhibited potently in both immune-deficient and immune-competent models with evidence of extended survival. Overall, our results showed that the combination of sabutoclax and minocycline was highly cytotoxic to pancreatic cancer cells and safely efficacious in vivo.

Topics: Animals; Apoptosis; Cell Line, Tumor; Drug Synergism; Gene Expression Regulation, Neoplastic; Gossypol; Humans; Mice; Minocycline; Pancreatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tetracycline; Xenograft Model Antitumor Assays

Cyclin D1 is important for pancreatic cancer growth. Our aim was to determine the effects of cyclin D1 inhibition on the growth of established pancreatic tumors.. PANC-1 cells harboring cyclin D1 antisense cDNA in a tetracycline-inducible vector system were prepared. The effects of cyclin D1 inhibition after tumor development were characterized in a mouse model.. In vitro removal of tetracycline induced cyclin D1 antisense cDNA expression and inhibited cyclin D1 expression and cyclin D1-associated kinase activity as well as anchorage-dependent and -independent growth. After establishment of xenograft tumors in the presence of tetracycline (2 mg/mL) in the drinking water, animals were assigned to either control (tetracycline remained in the drinking water) or to the group without tetracycline for which tetracycline was removed from the drinking water. Tumor growth was significantly inhibited after removal of tetracycline. Microscopic analysis revealed that the area of central necrosis was significantly increased in the group without tetracycline paralleled by a reduction of the vital peripheral area of proliferating cells.. Our results confirmed that cyclin D1 plays an important role in the growth of pancreatic cancer cells and may be an attractive molecular target for the treatment of human pancreatic cancer.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA, Antisense; Female; Gene Expression Regulation; Genes, Reporter; Genetic Therapy; Humans; Mice; Mice, Nude; Necrosis; Pancreatic Neoplasms; Tetracycline; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Elevated aerobic glycolysis in cancer cells (the Warburg effect) may be attributed to respiration injury or mitochondrial dysfunction, but the underlying mechanisms and therapeutic significance remain elusive. Here we report that induction of mitochondrial respiratory defect by tetracycline-controlled expression of a dominant negative form of DNA polymerase γ causes a metabolic shift from oxidative phosphorylation to glycolysis and increases ROS generation. We show that upregulation of NOX is critical to support the elevated glycolysis by providing additional NAD+. The upregulation of NOX is also consistently observed in cancer cells with compromised mitochondria due to the activation of oncogenic Ras or loss of p53, and in primary pancreatic cancer tissues. Suppression of NOX by chemical inhibition or genetic knockdown of gene expression selectively impacts cancer cells with mitochondrial dysfunction, leading to a decrease in cellular glycolysis, a loss of cell viability, and inhibition of cancer growth in vivo. Our study reveals a previously unrecognized function of NOX in cancer metabolism and suggests that NOX is a potential novel target for cancer treatment.

Topics: Animals; Cell Survival; Enzyme Activation; Gene Knockdown Techniques; Genes, Neoplasm; Glycolysis; HEK293 Cells; Humans; Mice; Mice, Nude; Mitochondria; NADPH Oxidase 1; NADPH Oxidases; Oxidative Phosphorylation; Pancreatic Neoplasms; Plasmids; Reactive Oxygen Species; RNA, Small Interfering; Superoxide Dismutase; Superoxide Dismutase-1; Tetracycline; Transfection; Xenograft Model Antitumor Assays

To establish stable tetracycline-inducible pancreatic cancer cell lines.. Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised.. Use of the chicken (β-actin) promoter proved superior for both the production and maintenance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nucleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellular proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransferase and enolase, are altered upon CapG induction.. We have generated pancreatic-cancer derived cell lines in which gene expression is fully controllable.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Chickens; Doxycycline; Electrophoresis, Gel, Two-Dimensional; Humans; Microfilament Proteins; Molecular Sequence Data; Neoplasm Proteins; Nuclear Proteins; Pancreatic Neoplasms; Tetracycline

Activating mutations of the oncogene K-ras are found in one third of all human cancers. Much of our knowledge on K-ras signal transduction and its influence on tumor initiation and progression come from in vitro studies with cell lines. However, mouse models of human cancer allow a much more faithful recapitulation of the human disease, and the in vivo perspective is crucial for our understanding of neoplasia. In recent years, several new murine models for K-ras-induced tumorigenesis have been described. They allow new insights into the specific role that oncogenic K-ras proteins play in different solid tumors, and they permit the molecular dissection of the pathways that are initiated by somatic mutations in subsets of cells. Key advances have been made by the use of tissue-specific and inducible control of expression, which is achieved by the Cre/loxP technology or the tetracycline system. From these sophisticated models, a common picture emerges: the effects of K-ras on tumor initiation depend strongly on the cellular context, and different tissues vary in their susceptibility to K-ras transformation.

Topics: Animals; Crosses, Genetic; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Genes, ras; Genes, Tumor Suppressor; Intestinal Mucosa; Intestinal Neoplasms; Lung Neoplasms; Mice; Mice, Transgenic; Mutation; Neoplasms; Neoplastic Stem Cells; Organ Specificity; Pancreatic Neoplasms; Protein Synthesis Inhibitors; ras Proteins; Tetracycline; Transgenes

While Newcastle disease virus (NDV) causes serious infections in birds, it is apparently nonpathogenic in mammalian species, including humans. Previous observations and small-scale clinical trials indicated that NDV exerts oncolytic effects. Isolates of NDV were found to have selective affinity to transformed cells. We previously showed that the attenuated NDV strain MTH-68/H causes apoptotic cell death in cultures of PC12 rat pheochromocytoma cells. The aim of the present study was to extend MTH-68/H cytotoxicity testing with human tumor cell lines and to analyze certain biochemical aspects of its oncolytic effect. MTH-68/H was found to be able to kill a wide range of transformed cells by apoptosis. While caspase-8 and caspase-9 are not involved in MTH-68/H-induced apoptosis, activation of caspase-3 and caspase-12 was detected in virus-infected PC12 cells. A human glioblastoma cell line with repressible expression of the p53 protein did not show any difference in MTH-68/H sensitivity in its p53-expressing and p53-depleted states, indicating that the apoptotic process induced by MTH-68/H does not depend on p53. Apoptosis was accompanied by virus replication in two tumor cell lines tested (PC12 cells and HeLa human cervical cells), and signs of endoplasmic reticulum stress (phosphorylation of protein kinase R-like endoplasmic reticulum kinase and eIF2alpha) were also detected in transformed cells. In contrast, proliferation of nontransformed mouse and rat fibroblast cell lines and human primary fibroblasts was not affected by MTH-68/H treatment. MTH-68/H thus selectively kills tumor cell cultures by inducing endoplasmic reticulum stress leading to p53-independent apoptotic cell death.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Carcinoma; Caspase 12; Caspase 3; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Viral; Endoplasmic Reticulum; Enzyme Activation; Female; Glioblastoma; HCT116 Cells; HeLa Cells; HT29 Cells; Humans; Newcastle disease virus; Oxidative Stress; Pancreatic Neoplasms; PC12 Cells; Rats; Tetracycline; Tumor Suppressor Protein p53; Virus Replication

A line of double-transgenic mice that develop neoplasms arising primarily in the pancreas was established. In these mice, the oncogene SV40 T antigen (Tag) was detected in the pancreas with and without the control of Tet-on system. The transgenic mice that developed pancreatic tumors as early as 20 weeks of age showed hypoglycemia on a blood glucose test. Pathological and immunohistochemical characterizations demonstrated that the tumors belonged to neuroendocrine neoplasms arising from pancreatic islets. A change in IGFs/IGF-1R signaling pathway was detected using real-time PCR analysis. A potential association between the IGFs/IGF-1R system and SV40Tag was studied to further explain the cancerogenesis of the double-transgenic mice by Western blot analysis and immunoprecipitation experiments. The results suggest that a Tag transgenic mice model could be used to study the molecular mechanism of the tumorigenesis of islets.

Topics: Animals; Antigens, Polyomavirus Transforming; Blood Glucose; Disease Models, Animal; Insulin Receptor Substrate Proteins; Insulinoma; Mice; Mice, Transgenic; Neuroendocrine Tumors; Pancreatic Neoplasms; Phosphoproteins; Somatomedins; Tetracycline

In response to transforming growth factor beta (TGF-beta), Smad4 forms complexes with activated Smad2 and Smad3, which accumulate in the nucleus, where they both positively and negatively regulate TGF-beta target genes. Mutation or deletion of Smad4 is found in about 50% of pancreatic tumors and in about 15% of colorectal tumors. As Smad4 is a central component of the TGF-beta/Smad pathway, we have determined whether Smad4 is absolutely required for all TGF-beta responses, to evaluate the effect of its loss during human tumor development. We have generated cell lines from the immortalized human keratinocyte cell line HaCaT or the pancreatic tumor cell line Colo-357, which stably express a tetracyline-inducible small interfering RNA targeted against Smad4. In response to tetracycline, Smad4 expression is effectively silenced. Large-scale microarray analysis identifies two populations of TGF-beta target genes that are distinguished by their dependency on Smad4. Some genes absolutely require Smad4 for their regulation, while others do not. Functional analysis also indicates a differential Smad4 requirement for TGF-beta-induced functions; TGF-beta-induced cell cycle arrest and migration, but not epithelial-mesenchymal transition, are abolished after silencing of Smad4. Altogether our results suggest that loss of Smad4 might promote TGF-beta-mediated tumorigenesis by abolishing tumor-suppressive functions of TGF-beta while maintaining some tumor-promoting TGF-beta responses.

Topics: Cell Cycle; Cell Differentiation; Cell Line, Transformed; Cell Movement; DNA-Binding Proteins; Epithelial Cells; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Keratinocytes; Mesoderm; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Promoter Regions, Genetic; RNA, Small Interfering; Smad4 Protein; Tetracycline; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

There is a significant correlation between the occurrence of pancreatic islet amyloid and beta-cell failure in advanced type II diabetes mellitus. Islet amyloid is composed primarily of the fibrillar form of the pancreatic hormone, amylin. Using thioflavin-T fluorescence binding and radioprecipitation assays, we investigated whether or not a series of small tricyclic compounds, tetracycline or Congo Red could interfere with the conversion of synthetic human amylin into its insoluble amyloid form. Of the compounds investigated, incubation of human amylin with a 20-fold molar excess of either Congo Red or Acridine Orange resulted in significant inhibition in the rate of amyloid formation. With Congo Red, maximal inhibition effectively occurred at a 1:1 molar ratio or greater over human amylin, whereas inhibition by Acridine Orange was dose-dependent. A 20-fold molar excess of the compound tetracycline also decreased insoluble amyloid content after extended incubation periods of approx. 20 h. Amyloid fibril morphology in the presence of tetracycline, as measured by transmission electron microscopy, was characterized by short fragmented fibrils compared with the longer and denser appearance of fibrils formed by amylin alone. These findings show that polycyclic compounds can suppress the formation of amyloid by human amylin, providing support for an alternative approach to peptide-based strategies by which islet amyloid formation could be modulated.

Topics: Acridine Orange; Amyloid; Animals; Congo Red; Dose-Response Relationship, Drug; Humans; Insulinoma; Islet Amyloid Polypeptide; Microscopy, Electron; Molecular Structure; Pancreatic Neoplasms; Polycyclic Compounds; Rats; Solubility; Tetracycline; Tumor Cells, Cultured

We have analysed HER-2 expression and function in pancreatic cancer cells to determine whether HER-2 has a rate-limiting role for pancreatic cancer cell growth in vitro and in vivo. To specifically assess HER-2 function, we used HER-2-targeted ribozymes expressed under the control of the tet-off promoter system. Six out of 11 human pancreatic cancer cell lines expressed all four epidermal growth factor (EGF)-receptor family members (HER-1 (EGF-R), HER-2, HER-3, and HER-4), including Panc89 cells. Expression of the ribozymes quenched endogenous HER-2 mRNA levels in Panc89 cells by approximately 40-60% which was reflected by a 40-50% reduction of the HER-2 surface glycoprotein. HER-2 depletion inhibited the in vitro proliferation rate by approximately 40% and decreased in vivo tumour growth by approximately 60% (P<0.05). Our study demonstrates for the first time a rate-limiting role for HER-2 in pancreatic cancer cell proliferation and suggests HER-2 targeting as a potential approach in pancreatic cancer therapy.

Topics: Animals; Cell Division; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Promoter Regions, Genetic; Receptor, ErbB-2; RNA, Catalytic; Tetracycline; Trans-Activators; Tumor Cells, Cultured

Conditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic beta cells in transgenic mice. A fusion protein containing the tet repressor (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the repressor into a transcription activator, was produced in beta cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in beta cells activated TAg transcription, resulting in the development of beta-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable beta-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited beta-cell proliferation. These findings indicate that transformed beta cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of beta-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest.

Topics: Animals; Antigens, Viral, Tumor; Blood Glucose; Cell Division; Cell Line; Cell Transformation, Neoplastic; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Herpes Simplex Virus Protein Vmw65; Insulinoma; Islets of Langerhans; Mice; Mice, Transgenic; Neoplasms, Experimental; Pancreatic Neoplasms; Repressor Proteins; Simian virus 40; Tetracycline; Tetracycline Resistance

Topics: Cytodiagnosis; Evaluation Studies as Topic; Fluorescence; Gastric Lavage; Humans; Inhalation; Pancreatic Neoplasms; Secretin; Stomach Neoplasms; Tetracycline

Topics: Anemia; Anemia, Pernicious; Biomedical Research; Fluorescence; Humans; Neoplasms; Pancreatic Neoplasms; Saliva; Stomach Neoplasms; Stomach Ulcer; Tetracycline