Brent S. Sorenson

ANTI-INFECTIVE PROTECTIVE PROPERTIES OF S100 CALGRANULINS.

The calgranulins are a subgroup of proteins in the S100 family (calgranulin A, S100A8; calgranulin B, S100A9 and calgranulin C, S100A12) that provide protective anti-infective and anti-inflammatory functions for the mammalian host. In this review, we discuss the structure-function relationships whereby S100A8 and S100A9, and for comparison, S100A12, provide intra- and extracellular protection during the complex interplay between infection and inflammation and how the calgranulins are regulated to optimally protect the host. Ideally located to support epithelial barrier function, calprotectin, a complex of S100A8/S100A9, is expressed in squamous mucosal keratinocytes and innate immune cells present at mucosal surfaces. The calgranulins are also abundantly produced in neutrophils and monocytes, whereas expression is induced in epidermal keratinocytes, gastrointestinal epithelial cells and fibroblasts during inflammation. The calgranulins show species-specific expression and function. For example, S100A8 is chemotactic in rodents but not in humans. In humans, S100A12 appears to serve as a functional chemotactic homolog to murine S100A8. Transition metal-binding and oxidation sites within calgranulins are able to create structural changes that may orchestrate new protective functions or binding targets. The calgranulins thus appear to adopt a variety of roles to protect the host. In addition to serving as a leukocyte chemoattractant, protective functions include oxidant scavenging, antimicrobial activity, and chemokine-like activities. Each function may reflect the concentration of the calgranulin, post-transcriptional modifications, oligomeric forms, and the proximal intracellular or extracellular environments. Calprotectin and the calgranulins are remarkable as multifunctional proteins dedicated to protecting the intra- and extracellular environments during infection and inflammation.

Triptolide therapy for neuroblastoma decreases cell viability in vitro and inhibits tumor growth in vivo

BACKGROUND Heat shock protein (Hsp)-70 is overexpressed in several human malignancies, and its inhibition has been shown to kill cancer cells. Our objectives were to assess the effectiveness of triptolide, an Hsp-70 inhibitor, in treating neuroblastoma in vitro and in vivo, and to measure the associated effects on Hsp-70 levels and apoptosis markers. METHODS After exposing N2a and SKNSH cell lines to triptolide, cell viability was assessed. Caspase-3 and -9 activities were measured and annexin staining performed to determine if cell death occurred via apoptosis. Hsp-70 protein and mRNA levels were determined using Western blot and real-time polymerase chain reaction. In an orthotopic tumor model, mice received daily triptolide injections and were humanely killed at study completion with tumor measurement. RESULTS Triptolide treatment resulted in dose- and time-dependent N2a cell death and dose-dependent SKNSH killing. Triptolide exposure was associated with dose-dependent increases in caspase activity and annexin staining. Triptolide decreased Hsp-70 protein and mRNA levels in a dose-dependent fashion. Mice receiving triptolide therapy had significantly smaller tumors than controls. CONCLUSION Triptolide therapy decreased neuroblastoma cell viability in vitro and inhibited tumor growth in vivo. Our studies suggest that triptolide killed cells via apoptosis and in association with inhibition of Hsp-70 expression. Triptolide may provide a novel therapy for neuroblastoma.

Inhibition of angiogenesis and suppression of colorectal cancer metastatic to the liver using the Sleeping Beauty Transposon System

BackgroundMetastatic colon cancer is one of the leading causes of cancer-related death worldwide, with disease progression and metastatic spread being closely associated with angiogenesis. We investigated whether an antiangiogenic gene transfer approach using the Sleeping Beauty (SB) transposon system could be used to inhibit growth of colorectal tumors metastatic to the liver.ResultsLiver CT26 tumor-bearing mice were hydrodynamically injected with different doses of a plasmid containing a transposon encoding an angiostatin-endostatin fusion gene (Statin AE) along with varying amounts of SB transposase-encoding plasmid. Animals that were injected with a low dose (10 μg) of Statin AE transposon plasmid showed a significant decrease in tumor formation only when co-injected with SB transposase-encoding plasmid, while for animals injected with a higher dose (25 μg) of Statin AE transposon, co-injection of SB transposase-encoding plasmid did not significantly affect tumor load. For animals injected with 10 μg Statin AE transposon plasmid, the number of tumor nodules was inversely proportional to the amount of co-injected SB plasmid. Suppression of metastases was further evident in histological analyses, in which untreated animals showed higher levels of tumor cell proliferation and tumor vascularization than animals treated with low dose transposon plasmid.ConclusionThese results demonstrate that hepatic colorectal metastases can be reduced using antiangiogenic transposons, and provide evidence for the importance of the transposition process in mediating suppression of these tumors.

S100A8/A9 (Calprotectin) Negatively Regulates G2/M Cell Cycle Progression and Growth of Squamous Cell Carcinoma

Malignant transformation results in abnormal cell cycle regulation and uncontrolled growth in head and neck squamous cell carcinoma (HNSCC) and other cancers. S100A8/A9 (calprotectin) is a calcium-binding heterodimeric protein complex implicated in cell cycle regulation, but the specific mechanism and role in cell cycle control and carcinoma growth are not well understood. In HNSCC, S100A8/A9 is downregulated at both mRNA and protein levels. We now report that downregulation of S100A8/A9 correlates strongly with a loss of cell cycle control and increased growth of carcinoma cells. To show its role in carcinogenesis in an in vitro model, S100A8/A9 was stably expressed in an S100A8/A9-negative human carcinoma cell line (KB cells, HeLa-like). S100A8/A9 expression increases PP2A phosphatase activity and p-Chk1 (Ser345) phosphorylation, which appears to signal inhibitory phosphorylation of mitotic p-Cdc25C (Ser216) and p-Cdc2 (Thr14/Tyr15) to inactivate the G2/M Cdc2/cyclin B1 complex. Cyclin B1 expression then downregulates and the cell cycle arrests at the G2/M checkpoint, reducing cell division. As expected, S100A8/A9-expressing cells show both decreased anchorage-dependent and -independent growth and mitotic progression. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increases growth and survival and reduces Cdc2 inhibitory phosphorylation at Thr14/Tyr15. The level of S100A8/A9 endogenous expression correlates strongly with the reduced p-Cdc2 (Thr14/Tyr14) level in HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B. S100A8/A9-mediated control of the G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may suggest new therapeutic approaches.

Modification of atrzine desorption during field incubation experiments

Abstract The effect of field aging on atrazine desorption was determined. 14 C-ring labelled atrazine was applied to the top of soil columns in field plots. The columns were periodically removed from the field for 16 months after application. The desorption isotherms of [ 14 C]atrazine residues were determined on the surface soil using the batch equilibration method with 0.01 M CaCl 2 solution. The distribution of atrazine residues in soil and liquid phases was determined by total 14 C analysis and HPLC with a radiactive flow detector to identify the [ 14 C]atrazine derivatives. In laboratory experiments, desorption coefficients were dependent on the absorbed atrazine concentrations; low concentrations were retained to a greater extent and were more difficult to desorb than the higher concentrations. For the field aged residues, desorption K f values increased slightly with aging. However, the values were lower than would be expected from the laboratory values if equal adsorbed concentrations are compared. It is not possible, however, to predict the desorption behavior of atrazine in the field samples from the simple adsorption-desorption laboratory studies. It is necessary to take into account the change in the atrazine-soil interactions with aging.

Attenuated Salmonella typhimurium with interleukin 2 gene prevents the establishment of pulmonary metastases in a model of osteosarcoma

PURPOSE The current management of osteosarcoma (OS) entails an aggressive preoperative and postoperative chemotherapeutic regimen with limb salvage surgery. Despite these efforts, relapse-free survival is less than 60% in patients with classic OS, whereas most patients relapse with pulmonary metastases. In these studies, we sought to prevent the establishment of pulmonary metastases from OS with a single oral dose of SalpIL2. METHODS Mice were administered attenuated Salmonella typhimurium with (SalpIL2) and without a gene for human interleukin 2 (Sal-NG) 7 days before challenge with 2 x 10(5) OS cells via tail vein. Three weeks after injection, mice were harvested for splenic lymphocytes and tumor enumeration. RESULTS Prophylaxis with attenuated SalpIL2 significantly reduces pulmonary metastases in number and volume (P < .0001 and P < .0001) with respect to saline controls. Furthermore, splenic natural killer cell populations were increased 396% with SalpIL2 (P < .0007) and 426% with Sal-NG (P < .0003) compared to nontreated groups. CONCLUSIONS Host natural killer response is greatly amplified and maybe partially responsible for the effective immune response against the formation of pulmonary metastases. A single oral dose of SalpIL2 may be a novel form of adjuvant therapy for patients after early detection of primary OS.

IL-1 receptor regulates S100A8/A9-dependent keratinocyte resistance to bacterial invasion.

Previously, we reported that epithelial cells respond to exogenous interleukin (IL)-1α by increasing expression of several genes involved in the host response to microbes, including the antimicrobial protein complex calprotectin (S100A8/A9). Given that S100A8/A9 protects epithelial cells against invading bacteria, we studied whether IL-1α augments S100A8/A9-dependent resistance to bacterial invasion of oral keratinocytes. When inoculated with Listeria monocytogenes, human buccal epithelial (TR146) cells expressed and released IL-1α. Subsequently, IL-1α-containing media from Listeria-infected cells increased S100A8/A9 gene expression in naïve TR146 cells an IL-1 receptor (IL-1R)-dependent manner. Incubation with exogenous IL-1α decreased Listeria invasion into TR146 cells, whereas invasion increased with IL-1R antagonist. Conversely, when S100A8/A9 genes were knocked down using short hairpin RNA (shRNA), TR146 cells responded to exogenous IL-1α with increased intracellular bacteria. These data strongly suggest that infected epithelial cells release IL-1α to signal neighboring keratinocytes in a paracrine manner, promoting S100A8/A9-dependent resistance to invasive L. monocytogenes.

PR_b functionalized stealth liposomes for targeted delivery to metastatic colon cancer

A gene delivery system was designed to carry a payload to integrin overexpressing cells. Branched-polyethyleneimine (bPEI) condensed plasmid DNA was encapsulated into targeted stealth liposomes, thereby combining the condensing and transfection properties of bPEI with the stealth and targeting properties of the liposomal carrier system. PR_b was used as a targeting ligand - a peptide we designed to bind specifically to the cancer cell surface marker α5β1 integrin - and such a robust receptor-ligand interaction achieved higher specificity than what has been previously reported for targeted delivery systems. In the process of formulating the PR_b functionalized gene delivery vehicle, we developed a protocol to fully encapsulate condensed DNA in liposomes and accurately quantify the total DNA in the system. We demonstrate that compared to non-targeted stealth liposomes and non-encapsulated condensed DNA, the PR_b functionalized stealth liposomes mediated improved in vitro transfection specifically to colon cancer cells overexpressing the α5β1 integrin. Furthermore, when administered in vivo to metastatic tumor bearing mice, PR_b functionalized stealth liposomes outperformed non-targeted liposomes and delivered genes specifically to the tumor site.

Attenuated Salmonella typhimurium prevents the establishment of unresectable hepatic metastases and improves survival in a murine model

PURPOSE The authors investigated the utility of attenuated Salmonella typhimurium for preventing the establishment of hepatic metastases in a murine model. METHODS A single, oral 10(8) cfu dose of attenuated S typhimurium was given 8 days before the establishment of a model of unresectable hepatic metastases. Animals were assessed for hepatic tumor number and volume, hepatic lymphocyte population analysis, and survival. RESULTS Pretreatment with Salmonella provided a 10-fold reduction in hepatic tumor burden compared with saline-treated controls. The antitumor effect is associated with markedly elevated natural killer (NK), CD8+ and CD4+ hepatic lymphocytes. Pretreatment with Salmonella provided a 90-day survival rate of 30%, whereas control animals were dead by 30 days. All long-term survivors were devoid of hepatic tumor. CONCLUSIONS Attenuated S typhimurium effectively prevents the establishment of hepatic metastases in a murine model, providing a clear survival benefit. Thus, it may represent a novel form of in vivo immunotherapy for the prevention of hepatic metastases for patients with locally advanced colorectal cancer.

Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

ABSTRACT To protect against invading bacteria, oral epithelial cells appear to use two effector antimicrobial peptides (AMPs): calprotectin (S100A8-S100A9 heterodimer [S100A8/A9]) in the cytosol and cathelicidin antimicrobial protein (CAMP) in endosomes. We sought to learn whether innate immunity might be augmented benignly to increase resistance against invasive bacteria. Epithelial cells were transiently transfected with mRNA constructs containing either the CAMP, S100A8, and S100A9 open reading frames, A8-IRES-A9 (fusion sequence), or A8-nIRES-A9 (fusion with native internal ribosome entry site [IRES] sequence). CAMP, S100A8, and S100A9 protein levels generally peaked between 16 and 44 h after mRNA transfection, depending on the construct; CAMP was processed to LL-37 over time. Following transfection with the respective mRNAs, CAMP and S100A8/A9 each independently increased resistance of epithelial cells to invasion by Listeria and Salmonella for up to 48 h; tandem S100A8/A9 constructs were also effective. Cotransfection to express S100A8/A9 and CAMP together augmented resistance, but synergy was not seen. Independent of the new proteins produced, transfection reduced cell viability after 48 h by 20%, with only 2% attributable to apoptosis. Taken together, these results suggest that epithelial cell resistance to invasive pathogens can be augmented by transient transfection of antimicrobial mRNAs into epithelial cells.