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.

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.

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.

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.

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.

Cytoreduction surgery reduces systemic myeloid suppressor cell populations and restores intratumoral immunotherapy effectiveness.

BACKGROUND: Multiple immunotherapy approaches have improved adaptive anti-tumor immune responses in patients with early stage disease; however, results have been less dramatic when treating patients with late stage disease. These blunted responses are likely due to a host of factors, including changes in the tumor microenvironment and systemic immunosuppressive features, which accompany advanced tumor states. We hypothesized that cytoreductive surgery could control these immunosuppressive networks and restore the potency of immunotherapy in advanced disease scenarios. METHODS: To test these hypotheses, two representative intratumoral immunotherapies (an adenoviral vector encoding a suicide gene, AdV-tk, or a type-I interferon, Ad.IFNα) were tested in murine models of lung cancer. Cytoreductive surgery was performed following treatment of advanced tumors. Mechanistic underpinnings were investigated using flow cytometry, in vivo leukocyte depletion methods and in vivo tumor neutralization assays. RESULTS: AdV-tk and Ad.IFNα were effective in treating early lung cancers, but had little anti-tumor effects in late stage cancers. Interestingly, in late stage scenarios, surgical cytoreduction unmasked the anti-tumor potency of both immunotherapeutic approaches. Immune mechanisms that explained restoration in anti-tumor immune responses included increased CD8 T-cell trafficking and reduced myeloid derived suppressor cell populations. CONCLUSION: This study demonstrates that surgical resection combined with immunotherapy may be a rational therapeutic option for patients with advanced stage cancer.

A positive-margin resection model recreates the postsurgical tumor microenvironment and is a reliable model for adjuvant therapy evaluation.

Up to 30% of cancer patients undergoing curative surgery develop local recurrences due to positive margins. Patients typically receive adjuvant chemotherapy, immunotherapy and/or radiation to prevent such relapses. Interestingly, evidence supporting these therapies is traditionally derived in animal models of primary tumors, thus failing to consider surgically induced tumor microenvironment changes that may influence adjuvant therapy efficacy. To address this consideration, we characterized a murine model of local cancer recurrence. This model was reproducible and generated a postoperative inflammatory tumor microenvironment that resembles those observed following human cancer surgery. To further validate this model, antagonists of two pro-inflammatory mediators, TGFß and COX-2, were tested and found to be effective in decreasing the growth of recurrent tumors. We appreciated that preoperative TGFß inhibition led to wound dehiscence, while postoperative initiation of COX-2 inhibition resulted in a loss of efficacy. In summary, although not an exact replica of all human cancer surgeries, our proposed local recurrence approach provides a biologically relevant and reliable model useful for preclinical evaluation of novel adjuvant therapies. The use of this model yields results that may be overlooked using traditional preclinical cancer models that fail to incorporate a surgical component.

Transcriptomic analysis comparing tumor-associated neutrophils with granulocytic myeloid-derived suppressor cells and normal neutrophils.

The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naïve neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC).In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-α, IL-1-α/ß), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages.This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations.

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.

Activation of mitogen-activated protein kinases by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) plays an important role in macrophage stimulation.

The small molecule anti-tumor agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA, now called Vadimezan) is a potent macrophage and dendritic cell activating agent that, in the murine system, results in the release of large amounts of cytokines and chemokines. The mechanisms by which this release is mediated have not been fully elucidated. The mitogen-activated protein kinase (MAPK) pathways play an important role in the regulation of proinflammatory cytokines, such as TNF-α, IL-1ß, as well as the responses to extracellular stimuli, such as lipopolysaccharide (LPS). The results of this study demonstrate that DMXAA activates three members of mitogen-activated protein kinase (MAPK) superfamily, namely p38 MAPK, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), and c-Jun N-terminal kinases (JNKs) via a RIP2-independent mechanism in murine macrophages. By using selective inhibitors of MAPKs, this study confirms that both activated p38/MK2 pathways and ERK1/2 MAPK play a significant role in regulation of both TNF-α and IL-6 protein production induced by DMXAA at the post-transcriptional level. Our findings also show that interferon-γ priming can dramatically augment TNF-α protein secretion induced by DMXAA through enhancing activation of multiple MAPK pathways at the post-transcriptional level. This study expands current knowledge on mechanisms of how DMXAA acts as a potent anti-tumor agent in murine system and also provides useful information for further study on the mechanism of action of this potential anti-tumor compound in human macrophages.

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).

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.

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.

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.

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.

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.

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.

(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.

CCL2 blockade augments cancer immunotherapy.

Altering the immunosuppressive microenvironment that exists within a tumor will likely be necessary for cancer vaccines to trigger an effective antitumor response. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immunoinhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the human papillomavirus E7 antigen expressed by a non-small cell lung cancer (NSCLC) line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus-expressing IFN-alpha to treat a nonimmunogenic NSCLC line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intratumoral CD8+ T cells that were more activated and more antitumor antigen-specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T regulatory cells. CCL2 seems to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T-cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.