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Macrophages are active contributors to the innate immune defense system. As macrophage activation is clearly affected by the surrounding microenvironment, the present study investigated the effect of sulforaphane (SFN) on the bactericidal activity of macrophages and the underlying molecular mechanisms involved in this process. Human THP‑1‑derived macrophages, primary human peripheral blood mononuclear cell‑derived macrophages, and primary mouse bone marrow derived‑macrophages (BMDMs) pretreated with SFN or DMSO were utilized in a model of Staphylococcus aureus infection. The results suggested that SFN pretreatment of macrophages effectively repressed the intracellular survival of S. aureus through modulation of p38/JNK signaling and decreased S. aureus‑induced caspases‑3/7‑dependent cell apoptosis, potentially through downregulation of microRNA (miR)‑142‑5p and miR‑146a‑5p. As SFN is a well‑known activator of nuclear factor erythroid 2‑related factor 2 (Nrf2), Nrf2‑/‑ BMDMs were used to demonstrate that the SFN‑mediated inhibitory effect was independent of Nrf2. Nevertheless, an increase in intracellular bacterial survival in Nrf2‑deficient macrophages was observed. In addition, SFN pretreatment suppressed S. aureus‑induced transcriptional expression of genes coding for the proinflammatory cytokines interleukin (IL)‑1β, IL‑6, and tumor necrosis factor‑α (TNF‑α), as well as for the M1 markers C‑C motif chemokine receptor 7, IL‑23 and inducible nitric oxide synthase (iNOS). Western blot analysis indicated that S. aureus challenge activated p38 mitogen‑activated protein kinase (MAPK) (p38) and c‑Jun N‑terminal kinase (JNK) MAPK signaling pathways, while SFN pretreatment prevented p38 and JNK phosphorylation. Pretreatment with 2 specific inhibitors of p38 and JNK, SB203580 and SP600125, respectively, resulted in a decrease in S. aureus‑induced proinflammatory gene expression levels compared with those observed in the SFN‑pretreated macrophages. Furthermore, THP‑1‑derived macrophages pretreated with SB203580 or SP600125 prior to bacterial infection exhibited a significant inhibition in intracellular S. aureus survival. In conclusion, we hypothesize that concomitant targeting of the p38/JNK‑inflammatory response and the S. aureus‑induced apoptosis with SFN may be a promising therapeutic approach in S. aureus infection.

The innate immune system, including phagocytic cells, is the first line of defense against pathogens. During infection by microorganisms such as viruses, bacteria, or parasites, phagocytic cells produce an excess of oxidants, a crucial process for the clearance of pathogens. This increase in oxidants creates an imbalance between oxidants and endogenous antioxidants. Left unchecked, this acute or chronic oxidative stress can lead to apoptotic cell-death and oxidative stress-induced diseases including neurodegenerative and cardiovascular disorders, premature aging, secondary infections, and cancer. The activation of nuclear factor E2-related factor 2 (Nrf2) is an efficient antioxidant defensive mechanism used by host cells to counteract oxidative stress. The transcription factor Nrf2 has been identified as the master regulator of several hundred of genes involved in the antioxidant defense response. The review objectives were to collect recent findings on the contribution of oxidative stress to complications of infection, and to highlight the beneficial impact of antioxidants in reducing inflammation and oxidant-related tissue damage. Furthermore, a direct relationship between infection and decline in Nrf2 activity has been demonstrated. Thus, an interesting therapeutic approach in disease prevention and treatment of stress-related diseases may consist in optimizing antibiotic or antiviral therapy with a combination of Nrf2 inducer treatment.

Mycobacterium abscessusis a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF).Mycobacterium abscessusis known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show thatM. abscessusS and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome–cytosol communication. In conclusion,M. abscessusS exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.

Keratin 19 is a member of the cytokeratin family that is critical for maintenance of cellular architecture and organization, especially of epithelia. The pancreas has three distinct cell types, ductal, acinar, and islet, each with different functions. Embryologically, the pancreatic and duodenal homeobox 1 (PDX1) homeodomain protein is critical for the initiation of all pancreatic lineages; however, the later differentiation of the endocrine pancreas is uniquely dependent upon high PDX1 expression, whereas PDX1 is down-regulated in the ductal and acinar cell lineages. We find that this down-regulation may be required for normal ductal expression of cytokeratin K19. The K19 promoter-reporter gene assay demonstrates that ectopic PDX1 inhibits K19 reporter gene activity in primary pancreatic ductal cells. This is reinforced by our findings that retrovirally mediated stable transduction of PDX1 in primary pancreatic ductal cells suppresses K19 expression, and short interfering RNA to PDX1 in Min6 insulinoma cells results in the induction of normally undetectable K19. Complementary functional and biochemical approaches led to the unexpected finding that a multimeric complex of PDX1 and two members of the TALE homeodomain factor family, MEIS1a and PBX1b, regulates K19 gene transcription through a specific cis-regulatory element (-341 to -325) upstream of the K19 transcription start site. These data suggest a unifying mechanism whereby PDX1, myeloid ecotropic viral insertion site (MEIS), and pre-B-cell leukemia transcription factor 1 (PBX) may regulate ductal and acinar lineage specification during pancreatic development. Specifically, concomitant PDX1 suppression and MEIS isoform expression result in proper ductal and acinar lineage specification. Furthermore, PDX1 may inhibit the ductal differentiation program in the pancreatic endocrine compartment, particularly beta cells.

The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.

Pancreatic ductal adenocarcinoma is the most common pancreatic neoplasm. There are approximately 33,000 new cases of pancreatic ductal adenocarcinoma annually in the United States with approximately the same number of deaths. Surgery represents the only opportunity for cure, but this is restricted to early stage pancreatic cancer. Pancreatic ductal adenocarcinoma evolves from a progressive cascade of cellular, morphological and architectural changes from normal ductal epithelium through preneoplastic lesions termed pancreatic intraepithelial neoplasia (PanIN). These PanIN lesions are in turn associated with somatic alterations in canonical oncogenes and tumor suppressor genes. Most notably, early PanIN lesions and almost all pancreatic ductal adenocarcinomas involve mutations in the K-ras oncogene. Thus, it is believed that activating K-ras mutations are critical for initiation of pancreatic ductal carcinogenesis. This has been proven through elegant genetically engineered mouse models in which a Cre-activated K-RasG12D allele is knocked into the endogenous K-Ras locus and crossed with mice expressing Cre recombinase in pancreatic tissue. As a result, mechanistic insights are now possible into how K-Ras contributes to pancreatic ductal carcinogenesis, what cooperating events are required, and armed with this knowledge, new therapeutic approaches can be pursued and tested.

SummaryTo define the genetic requirements for pancreatic ductal adenocarcinoma (PDA), we have targeted concomitant endogenous expression of Trp53R172H and KrasG12D to the mouse pancreas, revealing the cooperative development of invasive and widely metastatic carcinoma that recapitulates the human disease. The primary carcinomas and metastases demonstrate a high degree of genomic instability manifested by nonreciprocal translocations without obvious telomere erosion—hallmarks of human carcinomas not typically observed in mice. No mutations were discovered in other cardinal tumor suppressor gene pathways, which, together with previous results, suggests that there are distinct genetic pathways to PDA with different biological behaviors. These findings have clear implications for understanding mechanisms of disease pathogenesis, and for the development of detection and targeted treatment strategies.

Cell adhesion can be divided into cell-cell adhesion mediated by three major structures, tight junctions, adherens junctions, desmosomes, and cell-matrix adhesion, which is for the most part integrin-based. Cell-cell interactions play an important role in the maintenance and integrity of tissues and organs but similar to cell-matrix complexes may also transduce cell signaling. While this is the basics for understanding cell adhesion and its function, the role of cell adhesion proteins in disease, especially tumorigenesis, is far more diverse. A summary is given in the paper presented by Dr. Andl in this issue where she also focuses on recently discovered cell adhesion components and the regulation of cell adhesion molecules by miRNAs. In cancer, cell adhesion molecules are generally thought to be deactivated in order to aid in cancer cell dissemination and dispersal. However, aside from the loss of function, various gain-of-function events occur during induction and progression of cancer that can be mediated by cell adhesion molecules. Many of the molecules involved in these processes, such as the components of desmosomes, were initially identified as targets of autoimmune diseases. This special issue outlines the latest findings in research and review articles. Tight junctions mediate apical cell-cell adhesion and regulate epithelial cell polarity. Members of the claudin family are involved in a number of diseases including cancer; however, little is known regarding the mechanisms behind their pathophysiological function. Drs. Singh, Sharma, and Dhawan give an overview of the current state of knowledge in this issue. To identify cell transformation mechanisms other than previously described, Lynch et al. investigated the effect of MMP-7 mediated cleavage of E-cadherin. They demonstrate that loss of cell-cell adhesion is induced following E-cadherin processing, which results in increased cell migration, loss of polarization, and activation of RhoA. Interactions of cell adhesion molecules with their environment are central to their regulation and to cell migration and invasion. In the tumor context, the array of integrin receptors expressed causes the cells to differentially respond to external signals, resist the effects of cytotoxic drugs, and facilitate growth and invasion through a range of different tissue environments encountered by invasive cancer cells. In particular, the regulation of integrin receptor interaction with the extracellular matrix is thought to be critical for those cancers that invade and metastasize using a mesenchymal mode of migration. Jossen et al. show a novel prostate cancer bone metastatic model to assay drug sensitivity in three-dimensional cultures that mimic the preclinical or clinical setting more accurately. In here, they show that prostate cells revert to more epithelial cells, mesenchymal-epithelial transition (MET) in the presence of bone stroma modeling later stages of metastatic colonization, and are sensitized to radiation treatment when blocking E-cadherin or α-integrin. Mesenchymal migration as a therapeutic target for glioblastoma is discussed by Zhong et al. Another potential therapeutic agent, a Src inhibitor, is described by Yaseem et al. Comparison of SKI-606 and Iressa effects, Src/Abl and EGFR inhibitors, respectively, on cervical cancer cell lines shows inhibition of cell proliferation. Additionally, SKI-606 decreases cell migration and invasion through MET accompanied by the re-expression of E-cadherin and the recruitment of β-catenin to the cell membrane. Hironobu Yamashita et al. demonstrate that lysophosphatidic acid, a phospholipid growth factor, induces the stimulation of lamellipodia formation and enhanced cell migration through the upregulation of the TGFβ1 target gene Laminin-322. The role of focal adhesion kinase (FAK) phosphorylation in the dynamic regulation of integrin-based adhesions and cellular migration is shown in a paper by Hamadi et al. Upon pervanadate-induced phosphorylation, FAK delocalizes from focal adhesions to newly formed membrane ruffles in a src-dependent event. Finally, advances in nanotechnology that allow the study of cellular transformation in engineered cellular environments are introduced by Siti Hawa Ngalmi et al. Such cross-disciplinary approaches have begun to advance our knowledge of how adhesion signaling is organized and how cells relay information regarding the composition and structure of the external environment into biological activity. In particular, these advances are being driven by novel microscopy technologies and the continuing development of new techniques that allow us to visualize the intricate workings of adhesion-dependent signaling processes. This synopsis gives a glimpse at the current leading edge science published in this special issue of the Journal of Oncology. We hope that these papers inspire interest in multidisciplinary approaches as well as in the identification of novel therapeutics. Claudia D. Andl Ala-Eddin Al Moustafa Therese B. Deramaudt Geraldine M. O'Neill

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.

In a recent issue of Cell Death and Discovery, we described a new mechanism by which sulforaphane decreased mycobacterial burden in an in vitro model of THP-1-derived macrophage infection by Mycobacterium abscessus.1 This sulforaphane-mediated diminution was due to the induction of a caspase-independent cell apoptosis and necessitated activation of both nuclear factor E2-related factor 2 (Nrf2) and p38 mitogen-activated protein kinases (MAPK) signaling pathways. M. abscessus is a nontuberculous mycobacterium that belongs to the group of rapid growing mycobacteria. This emerging pathogen is able to cause skin, bone and soft tissue infections, and more generally exacerbations of lung diseases.2 Its high tolerance to antibiotics, which limits patient treatment, has become a public health concern worldwide. M. abscessus has been increasingly involved in patients with cystic fibrosis and in immunosuppressed patients,3 enhancing their risk of developing chronic airway infections and leading to a fatal outcome. Bacterial infection induces an imbalance between oxidants and antioxidants, triggering an oxidative burst that is well known for its bactericidal effect. Yet, some selected mycobacteria are able to thrive in this oxidative environment. It is then unsurprising that the use of oxidant scavengers such as MnTE-2-PyP or N-acetyl-L-cysteine has been shown to decrease M. abscessus load by activating bacterial killing in phagolysosomes.4 In our study, we decreased the oxidative environment in THP-1-derived macrophages by activating Nrf2, the key transcription factor that controls the cascade of cytoprotective and antioxidant defense mechanisms, and the maintenance of the redox homeostasis. Upon oxidative stress or infection, cytoplasmic Nrf2 is released from its inhibitor Keap-1, translocates into the nucleus, and forms a cofactor complex that binds to specific antioxidant responsive elements (ARE) found in promoters of phase II antioxidant and detoxifying enzymes. Nrf2 signaling pathway has been shown to have an important role whether beneficial or detrimental, in microbial infections.5 M. abscessus infection of THP-1-derived macrophages activates Nrf2 signaling pathway, and induces the expression of heme oxygenase-1 and NADPH quinone oxidoreductase-1, two downstream targets of Nrf2. In addition, M. abscessus induced reactive oxygen species (ROS) production in infected macrophages and blocked phagosomal acidification thus allowing its intracellular proliferation. Sulforaphane, a well-established activator of Nrf2 was used to pretreat macrophages before mycobacterial infection. The results showed an inhibition of mycobacterial growth 7 days postinfection in sulforaphane pretreated macrophages compared with vehicle pretreated ones. This sulforaphane-induced mycobacterial growth inhibition was neither caused by phagosomal pH acidification nor increase in cell phagocytosis, but rather induction of cell apoptosis. Contrary to the well-known cyprotective effect of sulforaphane and Nrf2 signaling pathway,6 we demonstrated that pre-stimulation of Nrf2 signaling pathway by sulforaphane before M. abscessus infection triggered a caspase-independent cell apoptosis. Pretreatment of macrophages with sulforaphane alone had no significant effect on cell death. To our knowledge, this is the first study showing sulforaphane-induced Nrf2 triggers a caspase3/7-independent and p38 MAPK-dependent cell apoptosis in mycobacteria infected macrophages (Figure 1). Figure 1 Infection of THP-1 derived macrophages by M. abscessus elicits caspase3/7-dependent cell apoptosis. Pretreatment of macrophages with an Nrf2 activator before mycobacterial challenge initiates a caspase-independent cell apoptosis Cell death in macrophages has a critical role in the host response to mycobacterial infection. Cell apoptosis is an effective mechanism used by the innate defense system to fight bacterial infection. This energy-dependent mechanism generates apoptotic bodies that will facilitate T-dependent antigen response and induce mycobacterial killing by uninfected neighboring macrophages.7 As M. abscessus has only been recently identified as a separate species, very little is known in the dynamics between M. abscessus and macrophage cell survival/cell death. In our in vitro model, M. abscessus induced cell necrosis in untreated cells but not cell apoptosis. This phenomenon has been observed in M. tuberculosis that has the ability to inhibit cell apoptosis in favor of cell necrosis.8 Recent highlights on caspase-independent forms of apoptosis triggered by infection have been described in the literature. Necroptosis, also known as programmed necrosis, can be initiated by various agents including microbial infection, and is characterized by activation of the receptor-interacting protein (RIP) kinases RIP1 and RIP3-dependent signaling pathway. This programmed cell death is a critical antiviral process in the host innate immune defense system. Phagocytes infected with viruses that are able to inhibit the classical caspase-dependent apoptosis pathway can undergo necroptosis to facilitate viral clearance by the neighboring immune system.9, 10 Moreover, Roca and Ramakrishnan have demonstrated a tumor-necrosis factor (TNF)-induced necroptosis in a M. tuberculosis infected zebrafish model. In the early stages of mycobacteria infection, injection of an excess of TNF in infected macrophages induced mitochondrial ROS production through a RIP1/RIP3-dependent kinase pathway, process necessary for the bacterial clearance. However, ROS-mediated necroptosis rapidly led to release and propagation of M. tuberculosis in the extracellular microenvironment.11 Another caspase 3/7-independent apoptosis is the inflammation-mediated cell death known as pyroptosis. This particular programmed cell death participates in the host cell defense system against pathogens and requires activation of caspase 1, IL-1β and IL-18.12 Recently, Bai et al.13 have shown that M. tuberculosis-mediated production of IL-32γ in THP-1-derived macrophages induced caspase 1-dependent pyroptosis which help reduce intracellular M. tuberculosis burden. In conclusion, additional studies are required to specifically identify the caspase-independent form(s) of apoptosis initiated by the combination of Nrf2 activation and M. abscessus infection. Nevertheless, our findings strongly suggest that Nrf2 activators may be of great interest as future therapeutic treatments in supplement to the actual lengthy multi-drug therapies used in patients diagnosed with mycobacterial infection.

Background & Aims: The Ki-RAS oncogene is altered in pancreatic ductal neoplasms. Pancreatic ductal cells (PDCs) were purified from cytokeratin 19 (K19)- Ki-RAS G12V transgenic mice and control littermates to identify properties of Ki-Ras activation in a cell-type-specific context. Because Ki-RAS mutation has prognostic significance in patients treated with radiation, we studied the influence of Ki-RAS status on radiation survival. Methods: Pancreatic ductal fragments from mice with Ki-RAS G12V mutation or wild-type (WT)- Ki-RAS were cultured. Growth curves, electron microscopy, flow cytometry, and analysis of signaling and cell-cycle proteins were established. Farnesyltransferase inhibitor (FTI) treatment with R115777 before and after irradiation was used to determine the effect of Ki-Ras farnesylation on cell survival. Results: PDCs from WT and K19-Ki-RAS G12V mice had features of ductal cells with formation of 3-dimensional structures on collagen without differences in morphology, growth, and cell-cycle distribution. This may result from up-regulation of p16INK4 and p27 Kip1 and lack of hyperstimulation of the mitogen-activated protein kinase pathway in Ki-RAS G12V PDCs. No differences in radiation survival between Ki-RAS G12V PDCs and WT PDCs were observed. However, Ki-RAS G12V PDCs expressing mutant p53 V143A had enhanced survival compared with WT PDCs transduced with p53 V143A . R115777 treatment sensitized Ki-RAS G12V PDCs and Ki-RAS G12V /p53 V143A PDCs, but not WT PDCs. Conclusions: Novel characterization of murine WT PDCs and Ki-RAS G12V PDCs is described. Induction of cell-cycle regulators and lack of mitogen-activated protein kinase hyperstimulation likely are responsible for constraining activated Ki-RAS G12V -mediated proliferation. Because its activation was required for sensitization by an FTI, R115777 may be useful against pancreatic tumors expressing oncogenic Ki-Ras.

Heme oxygenase-1 (HO-1) is a stress protein, and its induction has been suggested to participate in defense mechanisms against agents that promote oxidative injury such as endotoxins and heme. We have shown that the inflammatory cytokines, interleukin-6 (IL-6) and heme-induced HO-1 gene expression, were suppressed by dexamethasone (Dex) in a sustained manner. We examined the mechanism by which the anti-inflammatory agent, Dex, inhibits IL-6 and heme-induced HO-1 expression in rabbit coronary endothelial cells. Endothelial cells treated with heme (10 microM) and IL-6 (25 ng/ml), increased HO-1 mRNA 15- and 60-fold, respectively. The activity of HO was increased 3-fold after such treatment. Although Dex failed to inhibit heme-mediated HO-1 mRNA and HO activity, it was able to reverse IL-6-stimulated HO activity. Several human HO-1 promoter-drive chloramphenicol acetyltransferase (CAT) constructs were examined to analyze IL-6 and Dex-mediated modulation of the HO-1 gene in endothelial cells. CAT assays revealed that the HO-1 promoter region between -180 and -1500 might contain a Dex-mediated negative regulator. Gel mobility shift assays using nuclear extracts from IL-6-treated endothelial cells showed a binding to the synthetic 21 base pairs of the HO-1 sequence that contains the putative STAT3 sequence. STAT3-specific probe inhibited nuclear binding protein to the putative HO-1-STAT3 sequence. This suggests that IL-6 induction of human HO-1 is mediated via the JAK-STAT pathway and that Dex inhibition of gene expression is carried out by activation of a transcriptional protein in competition with the STAT3 binding site.