3,3'-Diindolylmethane enhances apoptosis in docetaxel-treated breast cancer cells by generation of reactive oxygen species.

CONTEXT: A major problem in the treatment of cancer is the development of toxic side effects and resistance to chemotherapy. The use of plant compounds to overcome resistance and prevent toxicity is a potential strategy for treatment. OBJECTIVE: We evaluated whether 3,3'-diindolylmethane (DIM) enhanced the sensitivity of breast cancer cells to docetaxel (DOC). MATERIALS AND METHODS: MDA-MB231 and Sk-BR-3 cells were treated with and without 25 or 50 µM of DIM and 1 nM of DOC for 48 and 72 h, respectively. MTT assay was used to measure cell survival. Apoptosis and intracellular reactive oxygen species (ROS) were determined by flow cytometry. The expression of proteins regulating ROS production and apoptosis was evaluated by immunoblotting technique. RESULTS: Combining 25 µM of DIM with 1 nM DOC decreased cell survival by 42% in MDA-MB231 cells and 59% in Sk-BR-3 cells compared to control, DIM, or DOC (p ≤ 0.05). The combination treatment increased apoptosis over 20% (p ≤ 0.01) in both cell lines, which was associated with decreased Bcl-2, increased Bax, cleaved PARP and activated JNK (p ≤ 0.01). ROS production increased by 46.5% in the MDA-MB231 and 29.3% in Sk-BR-3 cells with the combination compared to DIM or DOC alone. Pretreating cells with N-acetyl-cysteine or Tiron abrogated the anti-survival effect of the combination. The increase in ROS was associated with a 54% decrease in MnSOD and 47% increase in NOX2 protein compared to the other groups. CONCLUSIONS: Our findings indicated that DIM enhances the sensitivity of breast cancer cells to DOC treatment by increasing ROS, which led to decreased cell survival and apoptosis.

Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis.

BACKGROUND: Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. METHOD AND RESULTS: Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1ß maturation and accumulation in human and mouse platelets NOD2 dependently. CONCLUSIONS: NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.

Changes in the local tumor microenvironment in recurrent cancers may explain the failure of vaccines after surgery.

Each year, more than 700,000 people undergo cancer surgery in the United States. However, more than 40% of those patients develop recurrences and have a poor outcome. Traditionally, the medical community has assumed that recurrent tumors arise from selected tumor clones that are refractory to therapy. However, we found that tumor cells have few phenotypical differences after surgery. Thus, we propose an alternative explanation for the resistance of recurrent tumors. Surgery promotes inhibitory factors that allow lingering immunosuppressive cells to repopulate small pockets of residual disease quickly. Recurrent tumors and draining lymph nodes are infiltrated with M2 (CD11b(+)F4/80(hi)CD206(hi) and CD11b(+)F4/80(hi)CD124(hi)) macrophages and CD4(+)Foxp3(+) regulatory T cells. This complex network of immunosuppression in the surrounding tumor microenvironment explains the resistance of tumor recurrences to conventional cancer vaccines despite small tumor size, an intact antitumor immune response, and unaltered cancer cells. Therapeutic strategies coupling antitumor agents with inhibition of immunosuppressive cells potentially could impact the outcomes of more than 250,000 people each year.

Metformin decreases growth of pancreatic cancer cells by decreasing reactive oxygen species: Role of NOX4.

BACKGROUND: Retrospective epidemiologic studies show that metformin reduced the incidence of pancreatic cancer in diabetic patients. One potential mechanism may be by altering reactive oxygen species (ROS) and apoptosis. In this in vitro study, we explore the role of ROS and apoptosis in mediating the growth-inhibitory effects of metformin in pancreatic cancer cells. METHODS: We cultured MIA PaCa and Panc1 pancreatic cancer cells in medium containing physiological concentrations of glucose (5 mM) or supra-physiological concentrations of glucose and then treated cells with metformin. Cell viability, ROS production, apoptosis, and protein levels of manganese superoxide dismutase (MnSOD), NADPH oxidase (NOX) 2, and NOX4 were measured. RESULTS: Metformin decreased viability of MIA PaCa and Panc1 cells under physiological glucose conditions in comparison to untreated cells; metformin did not have any effect on human pancreatic normal epithelial (HPNE) cells. The decrease in cell survival was associated with decreased intracellular ROS, increased protein levels of MnSOD, and decreased levels of NOX2 and NOX4 proteins in MIA PaCa and Panc1 cells but not HPNE cells. Transfecting MIA PaCa and Panc1 cells with pcDNA3NOX4 protected against the anti-survival effects of metformin. CONCLUSION: Our findings suggest that metformin decreases cell survival by reducing ROS production, in part through down regulation of NOX4 protein expression.

Treating tumors with a vaccinia virus expressing IFNß illustrates the complex relationships between oncolytic ability and immunogenicity.

Since previous work using a nonreplicating adenovirus-expressing mouse interferon-ß (Ad.mIFNß) showed promising preclinical activity, we postulated that a vector-expressing IFNß at high levels that could also replicate would be even more beneficial. Accordingly a replication competent, recombinant vaccinia viral vector-expressing mIFNß (VV.mIFNß) was tested. VV.mIFNß-induced antitumor responses in two syngeneic mouse flank models of lung cancer. Although VV.mIFNß had equivalent in vivo efficacy in both murine tumor models, the mechanisms of tumor killing were completely different. In LKRM2 tumors, viral replication was minimal and the tumor killing mechanism was due to activation of immune responses through induction of a local inflammatory response and production of antitumor CD8 T-cells. In contrast, in TC-1 tumors, the vector replicated well, induced an innate immune response, but antitumor activity was primarily due to a direct oncolytic effect. However, the VV.mIFNß vector was able to augment the efficacy of an antitumor vaccine in the TC-1 tumor model in association with increased numbers of infiltrating CD8 T-cells. These data show the complex relationships between oncolytic viruses and the immune system which, if understood and harnessed correctly, could potentially be used to enhance the efficacy of immunotherapy.

A trial of intrapleural adenoviral-mediated Interferon-α2b gene transfer for malignant pleural mesothelioma.

New therapeutic strategies are needed for malignant pleural mesothelioma (MPM). We conducted a single-center, open-label, nonrandomized, pilot and feasibility trial using two intrapleural doses of an adenoviral vector encoding human IFN-α (Ad.IFN-α2b). Nine subjects were enrolled at two dose levels. The first three subjects had very high pleural and systemic IFN-α concentrations resulting in severe "flu-like" symptoms necessitating dose de-escalation. The next six patients had reduced (but still significant) pleural and serum IFN-α levels, but with tolerable symptoms. Repeated vector administration appeared to prolong IFN-α expression levels. Anti-tumor humoral immune responses against mesothelioma cell lines were seen in seven of the eight subjects evaluated. No clinical responses were seen in the four subjects with advanced disease. However, evidence of disease stability or tumor regression was seen in the remaining five patients, including one dramatic example of partial tumor regression at sites not in contiguity with vector infusion. These data show that Ad.IFN-α2b has potential therapeutic benefit in MPM and that it generates anti-tumor immune responses that may induce anatomic and/or metabolic reductions in distant tumor. Clinical trial registered with www.clinicaltrials.gov (NCT 01212367).

Monocyte chemoattractant protein-1 blockade inhibits lung cancer tumor growth by altering macrophage phenotype and activating CD8+ cells.

The role of chemokines in the pathogenesis of lung cancer has been increasingly appreciated. Monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) is secreted from tumor cells and associated tumor stromal cells. The blockade of CCL2, as mediated by neutralizing antibodies, was shown to reduce tumorigenesis in several solid tumors, but the role of CCL2 in lung cancer remains controversial, with evidence of both protumorigenic and antitumorigenic effects. We evaluated the effects and mechanisms of CCL2 blockade in several animal models of non-small-cell lung cancer (NSCLC). Anti-murine-CCL2 monoclonal antibodies were administered in syngeneic flank and orthotopic models of NSCLC. CCL2 blockade significantly slowed the growth of primary tumors in all models studied, and inhibited lung metastases in a model of spontaneous lung metastases of NSCLC. In contrast to expectations, no significant effect of treatment was evident in the number of tumor-associated macrophages recruited into the tumor after CCL2 blockade. However, a change occurred in the polarization of tumor-associated macrophages to a more antitumor phenotype after CCL2 blockade. This was associated with the activation of cytotoxic CD8(+) T lymphocytes (CTLs). The antitumor effects of CCL2 blockade were completely lost in CB-17 severe combined immunodeficient mice or after CD8 T-cell depletion. Our data from NSCLC models show that CCL2 blockade can inhibit the tumor growth of primary and metastatic disease. The mechanisms of CCL2 blockade include an alteration of the tumor macrophage phenotype and the activation of CTLs. Our work supports further evaluation of CCL2 blockade in thoracic malignancies.

Pharmacologic activation of the innate immune system to prevent respiratory viral infections.

Drugs that can rapidly inhibit respiratory infection from influenza or other respiratory pathogens are needed. One approach is to engage primary innate immune defenses against viral infection, such as activating the IFN pathway. In this study, we report that a small, cell-permeable compound called 5,6-di-methylxanthenone-4-acetic acid (DMXAA) can induce protection against vesicular stomatitis virus in vitro and H1N1 influenza A virus in vitro and in vivo through innate immune activation. Using the mouse C10 bronchial epithelial cell line and primary cultures of nasal epithelial cells, we demonstrate DMXAA activates the IFN regulatory factor-3 pathway leading to production of IFN-ß and subsequent high-level induction of IFN-ß-dependent proteins, such as myxovirus resistance 1 (Mx1) and 2',5'-oligoadenylate synthetase 1 (OAS1). Mice treated with DMXAA intranasally elevate mRNA/protein expression of Mx1 and OAS1 in the nasal mucosa, trachea, and lung. When challenged intranasally with a lethal dose of H1N1 influenza A virus, DMXAA reduced viral titers in the lungs and protected 80% of mice from death, even when given at 24 hours before infection. These data show that agents, like DMXAA, that can directly activate innate immune pathways, such as the IFN regulatory factor-3/IFN-ß system, in respiratory epithelial cells can be used to protect from influenza pneumonia and potentially in other respiratory viral infections. Development of this approach in humans could be valuable for protecting health care professionals and "first responders" in the early stages of viral pandemics or bioterror attacks.

Activation of the nucleotide oligomerization domain signaling pathway by the non-bacterially derived xanthone drug 5'6-dimethylxanthenone-4-acetic acid (Vadimezan).

The cytosolic nucleotide-binding oligomerization domain 1 (NOD1)/CARD4 and NOD2/CARD15 proteins are members of NOD-like receptors recognizing specific motifs within peptidoglycans of both Gram-negative and Gram-positive bacteria. NOD1 and NOD2 signal via the downstream adaptor serine/threonine kinase RIP2/CARDIAK/RICK to initiate NF-kappaB activation and the release of inflammatory cytokines/chemokines. In this report, we show that 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a cell-permeable, small molecule that has anti-tumor activity, can also activate NOD1 and NOD2. This was demonstrated: 1) by using human embryonic kidney epithelial (HEK) 293 cells transfected with a NF-kappaB reporter plasmid in combination with NOD1 or NOD2 expression plasmids; 2) by inhibiting DMXAA-induced chemokine (CXCL10) mRNA and protein production in the AB12 mesothelioma cell line using a pharmacological inhibitor of RICK kinase, SB20358; and 3) by using small interfering RNA to knock down NOD2 and lentiviral short hairpin RNA to knock down RICK. These findings expand the potential ligands for the NOD-like receptors, suggesting that other xanthone compounds may act similarly and could be developed as anti-tumor agents. This information also expands our knowledge on the mechanisms of action of the anti-tumor agent DMXAA (currently in clinical trials) and may be important for its biological activity.

Chemotherapy delivered after viral immunogene therapy augments antitumor efficacy via multiple immune-mediated mechanisms.

The most widely used approach to cancer immunotherapy is vaccines. Unfortunately, the need for multiple administrations of antigens often limits the use of one of the most effective vaccine approaches, immunogene therapy using viral vectors, because neutralizing antibodies are rapidly produced. We hypothesized that after viral immunogene therapy "primed" an initial strong antitumor immune response, subsequent "boosts" could be provided by sequential courses of chemotherapy. Three adenoviral (Ad)-based immunogene therapy regimens were administered to animals with large malignant mesothelioma and lung cancer tumors followed by three weekly administrations of a drug regimen commonly used to treat these tumors (Cisplatin/Gemcitabine). Immunogene therapy followed by chemotherapy resulted in markedly increased antitumor efficacy associated with increased numbers of antigen-specific, activated CD8(+) T-cells systemically and within the tumors. Possible mechanisms included: (i) decreases in immunosuppressive cells such as myeloid-derived suppressor cells (MDSC), T-regulatory cells (T-regs), and B-cells, (ii) stimulation of memory cells by intratumoral antigen release leading to efficient cross-priming, (iii) alteration of the tumor microenvironment with production of "danger signals" and immunostimulatory cytokines, and (iv) augmented trafficking of T-cells into the tumors. This approach is currently being tested in a clinical trial and could be applied to other trials of viral immunogene therapy.

A phase I trial of repeated intrapleural adenoviral-mediated interferon-beta gene transfer for mesothelioma and metastatic pleural effusions.

We previously showed that a single intrapleural dose of an adenoviral vector expressing interferon-beta (Ad.IFN-beta) in patients with malignant pleural mesothelioma (MPM) or malignant pleural effusions (MPE) resulted in gene transfer, humoral antitumor immune responses, and anecdotal clinical responses manifested by modified Response Evaluation Criteria in Solid Tumors (RECIST) disease stability in 3 of 10 patients at 2 months and an additional patient with significant metabolic response on positron emission tomography (PET) imaging. This phase I trial was conducted to determine whether using two doses of Ad.IFN-beta vector would be superior. Ten patients with MPM and seven with MPE received two doses of Ad.IFN-beta through an indwelling pleural catheter. Repeated doses were generally well tolerated. High levels of IFN-beta were detected in pleural fluid after the first dose; however, only minimal levels were seen after the second dose of vector. Lack of expression correlated with the rapid induction of neutralizing Ad antibodies (Nabs). Antibody responses against tumor antigens were induced in most patients. At 2 months, modified RECIST responses were as follows: one partial response, two stable disease, nine progressive disease, and two nonmeasurable disease. One patient died after 1 month. By PET scanning, 2 patients had mixed responses and 11 had stable disease. There were seven patients with survival times longer than 18 months. This approach was safe, induced immune responses and disease stability. However, rapid development of Nabs prevented effective gene transfer after the second dose, even with a dose interval as short as 7 days.

Advanced glycation end products on stored red blood cells increase endothelial reactive oxygen species generation through interaction with receptor for advanced glycation end products.

BACKGROUND: Recent evidence suggests that storage-induced alterations of the red blood cell (RBC) are associated with adverse consequences in susceptible hosts. As RBCs have been shown to form advanced glycation end products (AGEs) after increased oxidative stress and under pathologic conditions, we examined whether stored RBCs undergo modification with the specific AGE N-(carboxymethyl)lysine (N(ε) -CML) during standard blood banking conditions. STUDY DESIGN AND METHODS: Purified, fresh RBCs from volunteers were compared to stored RBCs (35-42 days old) obtained from the blood bank. N(ε) -CML formation was quantified using a competitive enzyme-linked immunosorbent assay. The receptor for advanced glycation end products (RAGE) was detected in human pulmonary microvascular endothelial cells (HMVEC-L) by real-time polymerase chain reaction, Western blotting, and flow cytometry. Intracellular reactive oxygen species (ROS) generation was measured by the use of 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester-based assays. RESULTS: Stored RBCs showed increased surface N(ε) -CML formation when compared with fresh RBCs. HMVEC-L showed detectable surface RAGE expression constitutively. When compared to fresh RBCs, stored RBCs triggered increased intracellular ROS generation in both human umbilical vein endothelial cells and HMVEC-L. RBC-induced endothelial ROS generation was attenuated in the presence of soluble RAGE or RAGE blocking antibody. CONCLUSIONS: The formation of the AGE N(ε) -CML on the surface of stored RBCs is one functional consequence of the storage lesion. AGE-RAGE interactions may be one mechanism by which transfused RBCs cause endothelial cell damage.

(Bis{2-[3-(2,4,6-trimethyl-benz-yl)imid-azolin-2-yliden-1-yl-κC]-4-methyl-phenyl}amido-κN)chloridopalladium(II).

The coordination geometry about the Pd centre in the title compound, [Pd(C(40)H(42)N(5))Cl], is approximately square-planar. The CNC pincer-type N-heterocyclic carbene ligand binds to the Pd atom in a tridentate fashion by the amido N atom and the two carbene atoms and generates two six-membered chelate rings, completing the coordination.

The vascular disrupting agent, DMXAA, directly activates dendritic cells through a MyD88-independent mechanism and generates antitumor cytotoxic T lymphocytes.

5,6-Di-methylxanthenone-4-acetic acid (DMXAA) is a small molecule in the flavanoid class that has antitumor activity. Although classified as a "vascular disrupting agent," we have recently conducted studies showing that DMXAA has remarkable efficacy in a range of tumors, working primarily as an immune modulator that activates tumor-associated macrophages and induces a subsequent CD8(+) T-cell-mediated response. To more completely analyze the effect of DMXAA on CD8(+) T-cell generation, we treated mice bearing tumors derived from EG7 thymoma cells that express the well-characterized chicken ovalbumin neotumor antigen. Treatment with DMXAA led to cytokine release, tumor cell necrosis, and ultimately reduction in tumor size that was lymphocyte dependent. Within 24 h of administration, we observed dendritic cell activation in tumor-draining lymph nodes (TDLN). This was followed by a rapid and marked increase in the number of tetramer-specific CD8(+) T cells in the spleens of treated animals. In contrast, the vascular disrupting agent combretastatin A4-phosphate, which caused a similar amount of immediate tumor necrosis, did not activate dendritic cells, nor induce an effective antitumor response. Using in vitro systems, we made the observation that DMXAA has the ability to directly activate mouse dendritic cells, as measured by increased expression of costimulatory molecules and proinflammatory cytokine release via a pathway that does not require the Toll-like receptor adaptor molecule MyD88. DMXAA thus has the ability to activate tumor-specific CD8(+) T cells through multiple pathways that include induction of tumor cell death, release of stimulatory cytokines, and direct activation of dendritic cells.

Paradoxical effects of statins on aortic valve myofibroblasts and osteoblasts: implications for end-stage valvular heart disease.

OBJECTIVES: We evaluated the effects of statins on aortic valve myofibroblasts (AVMFs) and osteoblast calcification in vitro. METHODS AND RESULTS: Cultured porcine AVMFs and M2-10B4 cells were treated with simvastatin and pravastatin. Mevalonate, a 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase metabolite, was added in parallel experiments. Manumycin A, which inhibits protein prenylation, was added to cultures in the absence of statins. Calcification was assessed by counting the number of calcific nodules formed and measuring alkaline phosphatase activity (APA). Statins inhibited calcific nodule formation (P<0.01) and APA (P<0.01) in AVMFs. Mevalonate reversed the statin effect on nodule formation (P<0.05) and APA (P<0.01). Manumycin A had no effect on either parameter. M2-10B4 cells treated with simvastatin formed more calcific nodules compared with controls (P<0.01), although pravastatin had no effect. Both statins, however, resulted in increased APA in M2-10B4 cells (P<0.01). Mevalonate had no impact on nodule numbers or APA in M2-10B4 cells. CONCLUSIONS: Statins inhibit calcification in AVMFs by inhibiting the cholesterol biosynthetic pathway, independent of protein prenylation, but paradoxically stimulate bone cell calcification. Because 15% of patients with end-stage valvular heart disease exhibit mature bone in their aortic valves, statins may differentially regulate calcification within a valve, limiting dystrophic calcification but promoting ossification of formed bone.

Role of pescadillo in the transformation and immortalization of mammalian cells.

The murine and human homologs of the zebrafish pescadillo protein (Pes1 and PES1, respectively) play important roles in ribosome biogenesis and DNA replication. We investigated the effect of Pes1 on the growth of mouse embryo (3T3-like) fibroblasts and conditionally immortalized human fibroblasts expressing the SV40 T antigen (AR5 cells). Increased expression of Pes1 causes transformation of mouse and human fibroblasts in culture (colony formation in soft agar). Although Pes1 can replace the SV40 T antigen in inducing colony formation in soft agar, it cannot substitute the T antigen in the immortalization of human fibroblasts, indicating that it distinguishes between the two functions. As the biological effects of Pes1 are similar to those of the insulin receptor substrate-1 (IRS-1), we investigated the interactions of Pes1 with IRS-1 itself and with the SV40 T antigen. The Pes1 protein (which localizes to the nuclei and nucleoli of cells) interacts with both IRS-1 and the SV40 T antigen, and markedly decreases the interaction of T antigen with p53. Taken together, these results suggest mechanisms for the ability of Pes1 to transform cells, and its failure to immortalize them.

Role of cAMP, PKA and Rap1A in thyroid follicular cell survival.

Cyclic AMP (cAMP) rescues cells from apoptosis stimulated by diverse insults. We examined the role of cAMP as a survival factor, and the signaling pathways through which cAMP affords protection. Rat thyroid cells were selected for these studies given the predominant role of cAMP in thyrotropin (TSH)-stimulated proliferation and as an oncogene in thyroid cells. Wistar rat thyroid (WRT) cells perished via apoptosis following sodium nitroprusside (SNP) treatment. Elevations in cAMP following treatment with forskolin, 8BrcAMP or IBMX rescued cells from SNP-induced cell death. Notably, TSH prevented apoptosis, implicating an important role for this hormone as a survival factor. Cyclic AMP activates multiple signaling pathways including those mediated through PKA, PI3K, p70S6k and the Ras-related small G protein, Rap1. Intriguingly, multiple pathways modulate thyroid cell survival. Interference with cAMP-stimulated p70S6k, but not PI3K, activity abrogated cell survival. Treatment with PKA inhibitors was sufficient to stimulate apoptosis in hormone-deprived cells and markedly enhanced cell death in response to SNP. Cells expressing an activated Rap1A mutant exhibited an enhanced sensitivity to SNP-induced apoptosis, while those expressing dominant negative Rap1A were resistant to SNP-initiated cell death. Together, these findings establish an important role for PKA and Rap1 in the control of thyroid cell survival.

Ras stimulates aberrant cell cycle progression and apoptosis in rat thyroid cells.

Abundant evidence supports the ability of Ras to stimulate thyroid cell proliferation. Stable expression of activated Ras enhances the sensitivity of thyroid cells to apoptosis. We report that apoptosis is a primary and general response of rat thyroid cells to acute expression of activated Ras in the absence or presence of thyrotropin, insulin, and serum, survival factors for thyroid cells. Ras induced apoptosis in quiescent and cycling cells. Concomitantly, Ras stimulated S phase entry in quiescent cells and enhanced G1/S transition in cycling cells. Ras effects on the cell cycle were characterized by delayed progression through S phase and an apparent failure to proceed through G2/M phase. Unlike thyroid cell mitogens, Ras markedly decreased cyclin D1 expression. Although acute expression of Ras decreased cyclin D1 protein levels, cells selected to survive chronic Ras expression exhibited a selective increase in cyclin D1 expression. In summary, thyroid cells harbor an apoptotic program activated by Ras that outstrips the protective effects of thyrotropin, insulin, and serum. Apoptosis is accompanied by dysregulated cell cycle progression, suggesting that cell death may arise, at least in part, as a consequence of inappropriate proliferative cues.

Adenoviral-based immunotherapy provides local disease control in an orthotopic murine model of esophageal cancer.

Despite recent advances in the development of novel therapies, esophageal carcinoma remains an aggressive cancer associated with a poor prognosis. The lack of a high throughput, reproducible syngeneic animal model that replicates human disease is partly responsible for the paucity of novel therapeutic approaches. In this report, we present the first successful syngeneic, orthotopic model for esophageal cancer. This model was used to test an established adenoviral-based tumor vaccine. We utilized a murine esophageal cancer cell line established from the ED-L2-cyclin D1;p53 mouse that was transduced to express a viral tumor antigen, the Human Papilloma Virus (HPV) E7 protein. The tumor was established in its natural microenvironment at the gastroesophageal junction. Tumor growth was consistent and reproducible. An adenoviral vaccine to E7 (Ad.E7) induced an E7-specific population of functionally active CD8 T cells that trafficked into the tumors and retained cytotoxicity. Ad.E7 vaccination reduced local tumor growth and prolonged overall survival. These findings suggest that orthotopic tumor growth is a reasonable preclinical model to validate novel therapies.

Refractoriness of interferon-beta signaling through NOD1 pathway in mouse respiratory epithelial cells using the anticancer xanthone compound.

AIM: To explore the possibility that nucleotide oligomerization domain 1 (NOD1) pathway involved in refractoriness of interferon-ß signaling in mouse respiratory epithelial cells induced by the anticancer xanthone compound, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). METHODS: C10 mouse bronchial epithelial cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 units/mL penicillin, 100 g/mL streptomycin. Pathogen-free female BALB/c mice were used to explore the mechanisms of refractoriness of interferon-signaling. Mouse thioglycollate-elicited peritoneal macrophages, bone marrow derived macrophages and bone marrow derived dendritic cells were collected and cultured. The amount of interferon (IFN)-inducible protein-10 (IP10/CXCL10), macrophage chemotactic protein (MCP1/CCL2) and interleukin (IL)-6 secreted by cells activated by DMXAA was quantified using enzyme-linked immunosorbent assay kits according to the instructions of the manufacturers. Total RNA was isolated from cells or nasal epithelium with RNeasy Plus Mini Kit, and cDNA was synthesized. Gene expression was checked using Applied Biosystems StepOne Real-Time Polymerase Chain Reaction System. Transfection of small interfering RNA (siRNA) control, NOD1 duplexed RNA oligonucleotides, and high-mobility group box 1/2/3 (HMGB1/2/3) siRNA was performed using siRNA transfection reagent. RESULTS: DMXAA activates IFN-ß pathway with high level of IFN-ß dependent antiviral genes including 2', 5'-oligoadenylate synthetase 1 and myxovirus resistance 1 in mouse thioglycollate-elicited peritoneal macrophages, bone marrow derived macrophages and bone marrow derived dendritic cells. Activation of IFN-ß by DMXAA involved in NOD1, but not HMGB1/2/3 signal pathway demonstrated by siRNA. NOD1 pathway plays an important role in refractoriness of IFN-ß signaling induced by DMXAA in mouse C10 respiratory epithelial cells and BALB/c mice nasal epithelia. These data indicate that DMXAA is not well adapted to the intrinsic properties of IFN-ß signaling. Approaches to restore sensitivity of IFN-ß signaling by find other xanthone compounds may function similarly, could enhance the efficacy of protection from influenza pneumonia and potentially in other respiratory viral infections. CONCLUSION: NOD1 pathway may play an important role in refractoriness of IFN-ß signaling in mouse respiratory epithelial cells induced by DMXAA.