Poster Abstract 50
Application of quantitative redox biology to basic research on the use of pharmacological ascorbate in cancer therapy

 

Garry R. Buettner, The University of Iowa
Claire M. Doskey, The University of Iowa
Visarut Buranasudja, The University of Iowa
Brett A. Wagner, The University of Iowa
Justin G. Wilkes, The University of Iowa
Juan Du, The University of Iowa
Joseph J. Cullen, The University of Iowa


Ascorbate functions as a versatile reducing agent in biology. At healthy, physiological concentrations (40 - 80 μM) it exhibits antioxidant properties and is essential in maintaining the function of many enzymes. However, when used at pharmacological doses (P-AscH- plasmalevels≈2 mM,achievedv iaIV),its oxidation can deliver a high flux of H2O2. This unique feature of P-AscH- is currently being investigated for use as an adjuvant to standard of care cancer therapy. A great deal of information on the potential mechanism of the action of P-AscH- is gathered in pre- clinical studies that employ cell culture. In these studies, we have found that expression of dose/ exposure to P-AscH- is better specified as mole of ascorbate per cell. We have also developed a kinetic assay to determine the rate constant for removal of extracellular hydroperoxide by cells (kcell) as well as a quantitative assay for catalase. We have demonstrated quantitatively that catalase is the major enzyme contributing to kcell. Rate constants for removal of H2O2 (kcell) and catalase activities were determined for 15 tumor and 10 normal cell lines of various tissue types. A differential in the capacity of cells to remove H2O2 was revealed, with the average kcell for normal cells being twice that of tumor cells. The ED50 (50 %clonogenic survival) of P-AscH- correlated directly with kcell and catalase activity. The response to pharmacological ascorbate in murine-models of pancreatic cancer paralleled the in vitro results, e.g. kcell, when these same cells were exposed to P-AscH-. Quantitative approaches increase efficiency of the research effort, increase rigor and reproducibility, and importantly yield more information from data. (Supported by NIH grants R01 CA169046, R01 CA184051, P30 CA086862, and P42 ES013661.)

 
Poster Abstract 51
4D quantitative image analysis of cancer cell invasion in a brain microenvironment using ImageJ software

 

John Gamble, Department of Biochemistry and Biophysics, Oregon State University
Robert Tanguay, Department of Environmental and Molecular Toxicology, Sinnhuber Aquatic Research Laboratory, Linus Pauling Institute, Oregon State University
Juliet A. Greenwood, Department of Biochemistry and Biophysics, Oregon State University

 

Acquisition of quantitative data of individual tumor cell invasion within live brain environment is currently limited. Individual cell monitoring is needed to understand cellular mechanisms for aggressive cancer behavior so that these mechanisms can be targeted or exploited for therapies. While numerous in vitro systems are well adapted to elucidate these mechanisms, they lack a dynamic environment that present numerous cell signals, mechanical forces and host cell interactions that combine to provide complex conditions that influence cell behavior. It is this complex microenvironment that cancer is found in humans and therefore it provides the optimal conditions for studying tumor cell invasion. To study glioblastoma (GBM), a very aggressive and deadly brain cancer, our lab has developed an in vivo model where fluorescently dyed human cancer cells are transplanted into the brain of larval zebrafish to monitor invasion of the tumor cells through time- lapse confocal imaging. Detailed 4D images of the entire tumor cell population in the brain microenvironment were produced. Using ImageJ software (Fiji), 4D images of the population of individual tumor cells in larva brains can be automatically tracked to provide a litany of quantifiable data. The resulting data contains information on the invasiveness of individual tumor cells in brain tissue. Data from this method can generate many different quantifiable tumor cell invasion statistics that can help in elucidating specific mechanisms used by cells to maneuver or invade through a brain microenvironment. These statistics can be used to help determine in what ways invasion is affected for tumor cells exposed to potential therapies or alterations in protein expression. Parameters for invasiveness can be set to create subpopulations of tumor cells where the subpopulations are seen to have different responses. All this leading to reliable data that can be more easily compared across studies.

 
Poster Abstract 52
Primary T-cell Lymphoblastic Leukemia (T-ALL) xenotransplant in zebrafish as a model for chemoprevention

 

Youngjoo Lee, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
Lindsey St. Mary, Department of Environmental and Molecular Toxicology, Oregon State University
Beth Siddens, Department of Environmental and Molecular Toxicology, Linus Pauling Institute, Oregon State University
Jane La Du, Department of Environmental and Molecular Toxicology, Oregon State University
Robert Tanguay, Department of Environmental and Molecular Toxicology, Sinnhuber Aquatic Research Laboratory Linus Pauling Institute, Oregon State University
Linda C. Stork, Division of Hematology and Oncology, Department of Pediatrics, Doernbecher Children's Hospital, Oregon Health & Science University
Bill W. Chang, Division of Hematology and Oncology, Department of Pediatrics, Doernbecher Children's Hospital, Oregon Health & Science University
David Williams, Department of Environmental and Molecular Toxicology, Linus Pauling Institute, Oregon State University

 

Acute Lymphoblastic Leukemia (ALL) is the most frequently diagnosed cancer in children and about 15% of ALL cases have origins in T-cells (T-ALL). Zebrafish develop most tumor types found in humans with comparable signaling pathways and morphology, making them a promising model for high throughput chemoprevention and therapy screening. The acid condensation products (RXM) of indole-3- carbinol (I3C), notably 3,3’-diindolylmethane (DIM), have shown chemopreventative and chemotherapeutic properties employing human T-ALL cells in vitro and mouse xenografts of human T-ALL as well as maternal diet supplementation with I3C in a mouse transplacental model. Current efforts aim to recapitulate this response with a T-ALL xenotransplant model in zebrafish. We have established a method for T-ALL xenotransplantation in zebrafish using commercially available T-ALL cells (HSB-2) from ATCC and then applied the same method using primary cells from pediatric patients at Oregon Health and Sciences University (OHSU). Casper strain (transparent) embryos at 6 hour post- fertilization (hpf) were dechorionated and exposed to 1 μM DIM, the predetermined NOAEL, until 48 hpf. At 48 hpf, embryos were injected with fluorescently labelled pediatric T-ALL cells, and xenotransplants were maintained at 33°C while being monitored and imaged over Rhodamine and Brightfield at 4 days post-injection (dpi) and 6 dpi. Fish that were treated with DIM in both DMSO vehicle and gel vehicle showed less T-ALL cells migrating in their vascular system compared to control groups. Next in model development is to expose the zebrafish to an I3C reaction mixture before injection and monitor cell migration and proliferation. This pilot project grant is supported by the Horizon Initiative, an OSU-OHSU Cancer Prevention Consortium.

 
Poster Abstract 53
The aryl hydrocarbon receptor mediates growth-inhibitory effects of SU5416 in hepatoma cells
 

Hyo Sang Jang, Environmental and Molecular Toxicology, Oregon State University
Edmond F. O’Donnell, Environmental and Molecular Toxicology, Oregon State University 
Martin Pearce, Environmental and Molecular Toxicology, Oregon State University 
Nancy I. Kerkvliet, Environmental and Molecular Toxicology, Oregon State University 
Siva Kumar Kolluri, Environmental and Molecular Toxicology, Linus Pauling Institute, Oregon State University

 

The aryl hydrocarbon receptor (AhR) is a potential clinical target for cancer and autoimmune dysfunction. Identifying selective AhR modulators that produce desirable clinical outcomes represents an opportunity for developing new anti-cancer agents. Repurposing clinically-used drugs with established safety profiles that activate the AhR represents a good starting place to pursue this goal. In this study, we characterized the AhR-dependent effects of SU5416 (Semaxanib) following its identification in a small-molecule library screen. SU5416 potently activated AhR-dependent reporter genes, induced AhR nuclear localization, facilitated AhR-DNA binding and increased expression of its endogenous target genes. SU5416 significantly inhibited proliferation of Hepa1 hepatoma cells in an AhR-dependent manner, but did not induce apoptosis. SU5416 also inhibited the growth of human HepG2 liver cancer cells. The effects of SU5416 correlated with an increased G1 population and increased expression of cell cycle inhibitor p21cip1/waf1 at both the mRNA and protein level. Increased expression of p21cip1/waf1 by SU5416 required expression of both AhR and Arnt. In addition, evidence for long-term activation of the AhR in vivo by a single dose of SU5416 was identified by analyzing published microarray data. Our results provide support for continued investigation of the AhR as therapeutic for cancers such as hepatoma. In addition, our findings raise the possibility that some of the previously observed anti-proliferative effects of SU5416 may be due to activation of the AhR.

 
Poster Abstract 54
Targeting therapy resistant cancer through Bcl-2 functional conversion

Martin Pearce, Environmental and Molecular Toxicology, Oregon State University
Prasad Kopparapu, Environmental and Molecular Toxicology, Oregon State University
John Gamble, Department of Biochemistry and Biophysics, Oregon State University
Siva K. Kolluri, Environmental and Molecular Toxicology, Linus Pauling Institute, Oregon State University

 

Emergence of tumor resistance to cancer therapeutics is a major cause of treatment failure which results in poor overall survival. Patients who have stopped responding to current treatments due to acquired resistance have a very poor prognosis and few alternative treatment options. Tackling this therapy resistance will not only improve prognosis for many but increase the impact of current therapies. Identifying molecular targets that are present in the therapy resistant cell population is essential to prevent resistance and treat patients that have developed acquired resistance to therapy. The B-cell lymphoma 2 (Bcl-2) family of proteins is associated with resistance mechanisms. In this study, we investigated mechanisms of resistance to chemotherapeutics and found upregulation of anti-apoptotic members of the Bcl-2 family in response to several chemotherapeutics. Cell lines resistant to chemotherapeutics were susceptible to Bcl-2 functional converters, which convert Bcl-2 from a protector to a killer of cancer cells. The combination of chemotherapeutics and Bcl-2 functional converters leads to synergistic induction of apoptosis in therapy resistant cancer cells. Thus, this study identifies a potential strategy to treat patients who have developed acquired resistance and are no longer responsive to current chemotherapeutics.

 
Poster Abstract 55
AhR-dependent induction of p27Kip1 by select AhR ligands

 

Veronica Puig-Sanvicens, Environmental and Molecular Toxicology, Oregon State University Jessica L. Phillips, Environmental and Molecular Toxicology, Oregon State University Siva K. Kolluri, Environmental and Molecular Toxicology, Linus Pauling Institute, Oregon State University

 

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that was firstly described as a mediator of toxicity of environmental pollutants such as dioxins. Upon ligand-binding, latent AhR localized in the cytosol translocates to the nucleus and heterodimerizes with AhR nuclear translocator (ARNT). The active transcription factor complex regulates the expression of responsive genes. Nonetheless, a novel anticancer role has recently been attributed to AhR due to its involvement in cell proliferation inhibition. Previous data showed that activation of AhR induces p27Kip1, a cell cycle regulatory gene that controls cell fate1. p27Kip1 inhibits cyclin-dependent kinases (CDKs) and regulates the entry of cells into the S phase. Recent evidence from Takahashi’s research group revealed an essential role for the AhR-induced p27Kip1 upon dioxin exposure on neuronal toxicity. In their study, up-regulation of p27Kip1 led to quiescence of neural progenitor cells and reduction in the number of neurons. Herein we hypothesize that activation of p27Kip1 by the AhR has therapeutic utility by suppressing cancer cell proliferation. Our laboratory has identified AhR modulators that activate AhR, induce up-regulation of p27Kip1 mRNA and protein levels and inhibit cancer cell viability. These compounds are termed as Selective Modulators of Ah Receptor Transcription (SMAhRTs) and are structurally distinct from dioxins. Our results demonstrate that SMAhRT or their analogs cause rapid up-regulation of p27Kip1 in an AhRdependent manner. In addition, SMAhRTs selectively inhibit cell viability of cancer cells through activation of the AhR. Future experiments aim to study the signaling pathways initiated after activation of AhR by SMAhRTs, and p27Kip1-mediated cell fate decisions.

 
Poster Abstract 56
Grain legume consumption may provide chemo-preventive effects on incident and prevalent colorectal adenoma and colorectal cancer: A meta-analysis of human studies

Thushanthi Perera, College of Public Health and Human Sciences, Linus Pauling Institute, Oregon State University
Yumie Takata, College of Public Health and Human Sciences, Oregon State University
Gerd Bobe, Department of Animal and Rangeland Science, Linus Pauling Institute, Oregon State University


Background:
Grain legumes are plants that are harvested solely for their dry grains. The American Institute for Cancer Research made no conclusions regarding the relation between grain legume consumption and colorectal cancer (CRC) in 2007 because of limited data available. As more data have been published since then, we re-evaluated the relation between grain legume consumption and colorectal adenoma (CRA) and CRC risk. Methods: We conducted a meta-analysis of human studies to evaluate the association between grain legume consumption and risk of CRA and CRC. Eleven prospective cohorts (1,533,527 participants including 12,274 cases) and 12 retrospective studies (42,473 controls and 12,408 cases) were included in the meta-analysis. The pooled risk ratios (RR) and 95% confidence interval (CI) for the highest (~45 g/d) comparing with the lowest (0 g/d) grain legume intake group were evaluated through a random effects model. Also, heterogeneity of estimates (I2), influential risk estimates, and publication bias were examined using funnel plots and Egger’s method.

Results: In cohort studies, high grain legume consumption was inversely associated with risk of incident CRA (RR=0.72; 95% CI: 0.60-0.87; I2=0%), prevalent CRA (RR=0.87; 95% CI: 0.75-1.01; I2=0%), and CRC (RR=0.89; 95% CI: 0.83-0.96; I2=8.9%) and no significant publication bias (P=0.13). In retrospective studies, similar associations were observed between grain legume intake and risk of prevalent CRA (RR=0.93; 95% CI: 0.84-1.03; I2=0%) and CRC (RR=0.77; 95% CI: 0.66-0.89; I2=53.3%), the heterogeneity of the latter can be explained by differences in CRC estimates between men (RR=0.79; 95% CI: 0.60-1.05; I2=0%) and women (RR=0.48; 95% CI: 0.34-0.69; I2=0%). When both study types were combined, high grain legume consumption was inversely associated with risk of CRA (RR=0.87; 95% CI: 0.81-0.94) and CRC (RR=0.82; 95% CI: 0.74-0.91).

Conclusions: Regular grain legume consumption (~45 g/d) may provide a chemo-preventive effect against colorectal neoplasia.

 
Poster Abstract 56
Chemopreventive response markers to navy bean extract in a mouse model of inflammation induced colorectal cancer

Thushanthi Perera, College of Public Health and Human Sciences, Linus Pauling Institute, Oregon State University
Matthew R. Young, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute - Frederick
Shakir M. Saud, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute - Frederick
Christopher R. Dextras, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute - Frederick
Yava L. Jones-Hall, Purdue University College of Veterinary Medicine Lilia Ileva, Small Animal Imaging Program, SAIC-Frederick, Inc.
Edward D. Karoly, Metabolon, Inc.
Bronte P. Sampe, Metabolon, Inc.
Young S. Kim, Nutritional Science Research Group, Division of Cancer Prevention, National Cancer Institute - Rockville
Nancy H. Colburn, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute - Frederick
Gerd Bobe, Department of Animal and Rangeland Sciences, Linus Pauling Institute, Oregon State University


Background: Consumption of dry beans and their fractions decrease colorectal neoplasia; however, the underlying molecular mechanisms are unclear. Aim of the study was to identify response indicators of dietary attenuation of colorectal tumorigenesis using metabolic, cytokine, and genomic profiling. Methods: After azoxymethane/dextran sodium sulfate (AOM/DSS) induction, mice were fed an AIN93G diet containing either 0 (Control) or 10% navy bean ethanol extract (BE) for 4, 41, or 85 days. Magnetic resonance imaging colonography of life mice was done to observe diet-induced differences in AOM/DSS-induced colorectal inflammation and tumorigenesis. Serum, colon tissue, and fecal samples were collected and analyzed for metabolite (serum, feces), cytokine (serum), and gene profiles (colon tissue).
Results: Dietary BE attenuated AOM/DSS-induced chronic colitis, aberrant epithelial cell proliferation, fecal blood (heme), and tumorigenesis in the colon. Response indicators were serum markers of fatty acid oxidation (carnitine, hexanoylcarnitine), and the pentose phosphate pathway (sedophetulose-7-phosphate) and fecal medium-chain fatty acids (hexanoate, octonoate) and plant phenolics (vanillate), the AOM/DSS-induced increase was attenuated by dietary BE. Moreover, dietary BE increased fecal markers of apoptosis (ribose, uracil, pseudouridine, xanthine, hypoxanthine) and attenuated the AOM/DSS-induced decrease in serum glycerophosphocholine and cytochrome P450 2c55 mRNA.
Conclusions: Dietary BE may inhibit survival and proliferation of premalignant colorectal epithelial cells by limiting their nutrient supply and resolving inflammation.