FA 5 Progress Report WV-INBRE - Joan C. Edwards School of ...

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Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 121CYP2C9 mutants. Dr. Aguilar has recently taken steps to use chaperones to assist in protein folding as tominimize the toxicity of the mutated proteins, and has been able to express several of the mutations and isworking on getting the chaperones into those that do not express. The current list of proteins that have beenexpressed are N474I, T304A and S209A. He is working on E300V currently and has expressed R307Lwhich has an impact on this project.The purification of the expressed protein is being carried out according to protocol received from the lab ofDr. Timothy S. Tracy. Dr. Aguilar has successfully purified the wild type form of the protein and two of theexpressed mutated forms. Purification is a week long process that takes approximately 8 hours of steadywork to reach a stopping point and he is considering hiring a part time technician. With the success ofexpression and now purification he is in the process of beginning the kinetic analysis.The molecular modeling part of the experiment has been completed in terms of looking at correlations ofdistances and hydrogen bonding. He is still trying to determine a viable way to address stacking. MM-PBSAhas been run for the majority of the mutations in silico. He has been successful and an additional centrifuge,obtained from a WV-INBRE equipment grant, will allow him to speed up the processes andfinish the kinetics and put out publications. He plans to submit an AREA grant to the NIH before theFebruary 25th deadline.Dr. Robert Kreisberg, West Liberty University, “Mechanism of action of statins on endothelial cell function”Dr. Kreisberg’s project is designed to uncover the molecular mechanisms of endothelial cell andmacrophage activation by minimally-modified LDL (MM-LDL) and to determine whether statins inhibit thisactivation. He has been very active in working with undergraduate students over the years with over 25undergraduate students involved in his project. His laboratory received a grant (Rosuvastatin) fromAstraZeneca to look at compare rosuvastatin to lovastatin in its ability to inhibit IL8 production by HumanAortic Endothelial Cells. He has indicated that has some very interesting results but unfortunately, he isunable to disclose progress at this time due to the confidentiality statement he signed. With the success ofthe rosuvastatin experiments, he is currently in discussions with AstraZeneca to use his current laboratorytechniques to look at one of their new platelet drugs. He is also repeating some IL6 experiments (cleaningup data) so that a manuscript can be submitted for publication.Dr. Robert Shurina, Wheeling Jesuit University, “Homer2 as a suppressor of cell invasion and podosomeformation”Dr. Shurina’s project is designed to address the mechanism by which Homer2 abrogates podosomeformation. He has shown that Homer2 is expressed in a wide variety of human cancer cell lines, includingHEK-293T cell line; the ovarian cancer CaOV3 cell line; the prostate cancer PC3 cell line; the neuroblastomaSY5Y cell line; and the epithelial cancer UMSSC-1 cell line. He has also demonstrated that Homer2 andAFAP-110 co-localize to lamellipodia in A7r5 tumor cell lines that are stimulated with phorbol myristateacetate (PMA). He has recently served as mentor for eight undergraduate students that were awardedscholarships through the NASA-West Virginia Space Grant Consortium. He also recently formed a researchcollaboration with Dr. Gregory Merrick at the Schiffler Cancer Center of the Wheeling Hospital (Wheeling,WV) to investigate whether proteins involved in the AFAP-110/Src signaling pathway can be used asprognostic indicators of prostate cancer.Dr. Yi Charlie Chen, Alderson-Broaddus College, “PI3K, AKT, and ERR-alpha pathways inflavonoid-inhibiting tumorigenesis”Dr. Chen has been examining the role of specific signaling pathways as a mechanistic approach todetermining how a flavonoid, kaempferol, can inhibit the growth of ovarian cancer cells. He hassuccessfully completed the specific aims proposed in his previous proposal. (1) Identify the roles andmolecular mechanisms of flavonoids in inhibiting EGFR, PI3K, AKT, and ERRα in ovarian cancer cells. Hefound that the flavonoid kaempferol inhibited PI3K/AKT, and ERRα regulated VEGF expression in ovariancancer cells. He has published these results. (2) Determine flavonoid-inhibiting signaling pathways thataffect ovarian tumor growth. He found that both HIF dependent (PI3K/AKT) pathway and HIF independentPHS 2590 (Rev. 06/09)Continuation Format Page
Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 122(ERRα and cMyc) pathways were involved in the ovarian tumor growth inhibition. He has published theseresults. (3) Determine whether kaempferol inhibit ovarian tumor angiogenesis, and identify which signalingmolecules mediate kaempferol-inhibiting tumor angiogenesis. He found that kaempferol inhibits ovariantumor angiogenesis through inhibiting HIF-1α, VEGF, cMyc and ERRα signaling molecules. He alsopublished these results in peer-reviewed scientific journals. He has published three manuscripts and hasone in press since the last APR. He has also submitted an R15 application that was not funded. He hasrevised and resubmitted his application. Overall, Dr. Chen has been the most productive of the PUIinvestigators to date.Dr. Gerald Hankins, West Virginia State University, “ Sex steroid hormones and epigenetics inmeningiomas”Dr. Hankin’s central hypotheses are: (1) that meningioma tumorigenesis is driven in part by actions offemale steroid hormones and (2) that the tumorigenesis may be mediated in part by progesterone andestrogen receptor containing chromatin-modifying complexes. This past year one graduate student and anundergraduate student have focused on fibroblast growth factors and their receptors and havedemonstrated that both fibroblast growth factors and their receptors are expressed by meningioma cells.Further, growth factor message levels are modulated by steroid hormones. Treatment of meningioma cellswith antagonists of FGF receptors decreases the proliferation of the cells and the phosphorylation of ERK1and 2. Together, these results provide more evidence that steroid hormones may affect the expression offibroblast growth factors (FGF2 and FGF9) in meningiomas and also that an FGF autocrine loop plays a rolein meningioma cell proliferation, partially by signaling through ERK1/2.He has also found that the expression of the cyclin dependent kinase p27 kip1 (CDKN1B) is modulated inmeningioma cells by both the progesterone antagonist Mifepristone and the histone deacetylase inhibitorbutyric acid. The transcriptional repressor KLF10 (Krueppel-like factor 10 or TIEG-1) was down regulatedby a factor of 32 by the estrogen receptor antagonist, ZK164015 in more aggressive lines of meningioma,while Mifepristone had no effect. Since PR is typically higher in lower grades of meningioma while ERincreases in higher grades, he is examining this in cells from lower grade tumors.Although SLC20A2 (GLVR-2, Pit2) was not highlighted in the grant, he found that its expression wassignificantly down regulated by Mifepristone. Expression was also modulated by HDAC inhibitor treatment.The work of one graduate student who graduated during the past year was published during the summer(Manohar S et al. Cell Cycle 10 (15): 2529-2539, 2011). Dr. Hankin’s mentor Dr. Mayion Park has left MUand Dr. Travis Salisbury has replaced her as his mentor. However, he is still collaborating with her onstudies on chromatin modifying proteins. Dr. Salisbury’s research on the aryl hydrocarbon receptorcomplements the grant research given the parallels and crosstalk between AHR and steroid receptors.Dr. Tesfaye Belay, Bluefield State College, “Effect of stress on pathogenesis and immunity duringchlamydia genital infection”Dr. Belay’s hypothesis is that cold-stress increases the severity of chlamydia genital infection anddevelopment of complications by modulating the immune response against Chlamydia.To assess theinfluence of stress on distribution pattern of immune cells in different regions of the genital tract duringChlamydia trachomatis infection, Dr. Belay has used the mouse stress model and flow cytometry analysis. Ingeneral, the total number of immune cells in stressed mice was reduced; however, no statistically significantdifference between stressed and non-stressed was obtained. Increased infiltration of leukocytes into thegenital tract of stressed or non-stressed infected mice was obtained. Further flow cytometry experimentsare underway to localize neutrophils, lymphocytes and dendritic cells or adhesion molecules in the regions ofthe genital tract of stressed mice during chlamydia infection.Dr. Belay has begun to assess the effect of cold-stress on Chlamydia-induced infertility in the mouse model.His initial results suggest that stress may increase complications of immunopathogenesis resulting from C.trachomatis genital infection and infertility. Further experimentation is underway to determine the effect andmechanism of stress in modulation of fertility in mice. (Abstract submitted to the General Meeting ofAmerican Society for Microbiology, San Francisco, CA, June 2012).Dr. Belay is also determining the effect of cold-induced stress on the histopathological changes of thePHS 2590 (Rev. 06/09)Continuation Format Page
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FA 5 Progress Report WV-INBRE - Joan C. Edwards School of ...

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