Relative Gene Expression of Mortality Factor 4-Like 2 Rattus norvegicus Schwann Cells Following Activation of Protease Activated Receptor-1
Protease Activated Receptor-1 (PAR-1), a seven transmembrane G-protein coupled receptor localized to the membranes of motor neurons and is activated normally by thrombin or by the synthetic peptide, SFLLRNP. Activation of PAR-1 by either means results in motor neuron apoptosis; however, its role in Schwann cells is just beginning to be understood. Preliminary results from our lab show that PAR-1 activation on Schwann cells leads to apoptosis and an alteration of cell morphology. To examine the molecular changes that may be involved, microarray analysis was performed following PAR-1 activation by 100 nM SFLLRNP. Microarray analysis revealed a significant increase in gene expression of mortality factor 4-like 2 (morf4l2), a gene involved in cell survival, 12 hours after PAR-1 activation. Since microarray analysis often yields false positive results, q-RTPCR was conducted following PAR-1 activation at 0, 4, 8, and 12 hour time points. The results showed that there was no significant difference at any of the time points in the expression of morf4l2 suggesting that the microarray differences for this gene were a false positive and that Schwann cell apoptosis is not dependent on morf412. Despite these results, further research in our lab is focused on verifying other genes that were indicated as potential targets of PAR-1 activation.
Biology, Furman University, Greenville, SC.
P128 • Zachary C. Perdun, Katey M. Robinson, Dr. Diana Ivankovic, Dr. Donna Weinbrenner
Anticarcinogenic Properties of Extracts Derived From the Clusia rosea Tree Tested on Rat Derived Pheochromocytoma (PC12), ASPC-1 Pancreatic Cancer Cell Line and AGS Gastric Adenocarcinoma Cell Line
The purpose of this experiment was to assess any potential cytotoxic, antiproliferative or neurodegenerative properties of extracts from the tree Clusia rosea. Cell viability and proliferation of two morphologies of rat derived pheochromocytoma (PC12 cells), ASPC-1 pancreatic cancer cell line and AGS gastric adenocarcinoma cell line were all tested in the presence of crude extracts of Clusia rosea prepared using the soxhlet apparatus extraction method. The extraction solvents used were methanol, deionized distilled water and methylene chloride for duration of 20-24 hours in the apparatus. The extracts used were derived from the leaves and stems of both young and mature plants. The extracts were added to cell growth medium at varying concentrations, then applied to all cell lines. The PC12 cell line was differentiated with 100ng/mL neural growth factor (NGF) for 48 hours while being sub-cultured on rat tailed collagen coated growth vessels. Proteins distinctly associated with neuronal growth in the differentiated morphology were assessed using a western blot analysis, and an MTS cell viability assay was performed on the tumor morphology of the PC12 cell line, the ASPC-1 cell line and the AGS gastric adenocarcinoma cell line after the extracts were allowed to incubate for 48-96 hours.
Protein Expression Analysis of the AMPK Pathway in Human Melanoma: Identifying the Pros and Cons of Metformin Therapy
Metformin, an AMPK activator, is an anti-diabetic agent that has gained attention in the oncology field due to preclinical findings that suggest anti-tumor effects. Importantly, however, some studies have revealed alarming adverse consequences of using this drug for cancer therapy whereby metformin promotes tumor survival through induction of angiogenesis. Here, metastatic MDA-MB-435 human melanoma cells treated with metformin were evaluated using immunohistochemistry to determine whether the Metformin-AMPK activation process promoted or inhibited the tissue invasion survival phenotype. It was hypothesized that AMPK activation would ablate tumor invasion potential by inhibiting nuclear (active) NFκB and consequently inhibiting MMP-9 secretion. Since NFκB was found to be cytoplasmic and non-active in all samples it was concluded that the Metformin-AMPK activation process had no effect on its activity in this melanoma model. Interestingly, however, MMP-9 did demonstrate protein up-regulation as a result of metformin therapy. Since the protein regulation was not occurring through NFκB signaling, an alternative pathway that bridges the Metformin-AMPK activation process to MMP-9 production must exist. Future studies will focus on identifying this missing link and evaluating whether this link also contributes to the other survival pathways such as angiogenesis. Identification of such a protein could generate a biomarker signature that can be used in the clinic to identify patient candidates that may benefit from metformin therapy and eliminate risk for those who are contraindicated.
Wingate University
P130 • Brianna L. Taylor, Naomi Jackson, Benjamin J. Thornton
The Effects of Ascorbic Acid on Drosophila melanogaster Susceptibility to Permethrin and Suppression of Cytochrome P450 Activity
Permethrin, 3-Phenoxybenzyl (1RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate, is a widely used pesticide in the United States as well as other countries around the world. Because of the pervasive use many non-target species are exposed resulting in challenges to individuals and possibly populations. An organism’s defense against toxins in general, including permethrin, is via detoxification by ubiquitous enzyme systems, such as cytochrome P450s. Their role in the detoxification process is to oxidize xenobiotics, thereby increasing solubility in water and promoting excretion (Zhu et al. 2008). A number of studies have shown that dietary vitamin supplementation can enhance detoxification (Brodfuehrer & Zannoni 1986; Zannoni et al. 1982). The current study tested the susceptibility of Drosophila melanogaster to permethrin subsequent to exposure to various concentrations of ascorbic acid. Percent survivorship was determined revealing a sex-dependent response. Ascorbic acid supplementation had no observable effect on female susceptibility to permethrin. Males exposed to 15% ascorbic acid exhibited a significant increase in survivorship compared to controls. Lang and Liu (2013) showed that elevated P450 gene expression enhanced resistance to Permethrin in Culex auinquefasciatus. In this investigation, males exposed to 15% and 20% ascorbic acid showed an unexpected significant decrease in cytochrome P450 enzyme activity compared to the control. Females showed a similar change in P450 activity in the 20% exposure only. These documented changes in P450 activity do not explain the observed increased resistance to Permethrin in male D. melanogaster.
Biology and Allied Health, Southern Adventist University, Collegedale, TN
P131 • Lucas Newman, Paul A. Scott, Sarah Rosario, Amanda L. Duffus, Mustapha Durojaiye, Cathy Lee
