Identifying the role of caveolin-1 and caveolin-2 in cancer cell proliferation and cancer drug resistance [abstract]

Abstract only availableFaculty Mentor: Dr. Grzegorz Sowa, Medical Pharmacology and PhysiologyCaveolae are flask-shaped invaginations of the plasma membrane. Caveolins are the major protein component building these caveolae. Caveolins are involved in cell signaling and play a role multiple subcellular processes, such as endocytosis. Of the three types of caveolins (numbered 1, 2, and 3), most research has been performed on caveolin-1. For example, in the field of cancer, it has been found that caveolin-1 has the ability to either suppress or encourage growth of cancer cells (depending on the type of cancer). Interestingly, it has been reported that the level of both caveolin-1 and -2 increases in drug resistant cancer cell lines. However, it is unknown if such increases are coincidental or actually play an important role in the development of drug resistance. The major goal of this study is to better understand the role of caveolins-1 and -2 in the growth and drug resistance of A549 lung adenocarcinoma. For this purpose, the levels of caveolin-1 and -2 were dramatically reduced (knocked down) using small interfering RNA (siRNA) stably expressed in A549 cells. The efficient reduction of caveolin-1 and -2 levels by siRNA has been confirmed by Western Blot. By using colorimetric assay and cell count, we are currently determining the possible differences in growth of A549 cell populations expressing control siRNA and caveolin-1 or -2 siRNA in the absence or presence of different concentrations of two anticancer drugs, i.e., taxol and etoposide. We hypothesize that in the absence of drugs, cells with a knockdown of caveolin-1 or -2 will grow equally as fast as or faster than control cells. Conversely, we expect that knockdown of caveolins will increase sensitivity of A549 to anti-cancer drugs and limit the possibility of developing drug resistance. Such results would be a first direct evidence that caveolin-1 and/or -2 are involved in acquiring drug resistance. If the latter is true, targeting caveolins in lung adenocarcinomas and other cancers could limit or even prevent the development of drug resistance and thus increase the efficacy of anti-cancer drugs

Development of a stably transfected cell line to screen for potential endocrine disrupting chemicals [abstract]

Abstract only availableIntro Estrogens are steroid hormones that diffuse across cell membranes to interact with estrogen receptors (ER) in the nucleus. An estrogen-bound ER acts as a transcription factor to regulate gene transcription in a cell. Synthetic chemicals with estrogenic activity (called xenoestrogens) also interact with the estrogen receptor (ER), interfering with the body's delicate balance of hormones. There is a great need to characterize these chemicals as well as screen for until now unknown xenoestrogens. Methods We developed a stably transfected MCF-7 human breast cancer cell line that, in combination with reporter gene assays, will aid in screening for and characterization of these chemicals. MCF-7 cells were transfected with DNA containing two copies of an estrogen response element (ERE) from the promoter of the vitellogenin gene. The ERE was linked to either LacZ or the firefly luciferase gene (both reporter genes). When a chemical interacts with the ER, the ER will associate with the ERE to begin the transcription of the reporter gene that will encodes for a quantifiable protein. Cells were co-transfected with a plasmid containing the neomycin gene, conferring resistance to the antibiotic G418 to allow for selection of stable transformants. Following transfection, cells were treated with G418 and grown for 18-25 days until colonies formed. Colonies were selected and a dose response of estradiol, the body's most common estrogen, was performed on the clones. Those populations showing the greatest sensitivity to estrogen were chosen for future use. Ongoing Studies Future experiments will include using reporter gene assays to screen common chemicals, such as the pesticide carbaryl, for estrogenic activity both alone and in environmentally relevant combinations of other xenoestrogens. Secondly, bioassays on clinical samples of peritoneal fluid from women with an estrogen-dependent disease will be performed and compared to control samples to determine total estrogenic activity.Life Sciences Undergraduate Research Opportunity Progra

Peptide profiling: Correlating estrogen receptor conformation with biological response

Abstract only availableChemicals found in the environment have been found to behave like the body's natural estrogen, estradiol. These exogenous estrogen-mimicking compounds have been termed xenoestrogens. Both estradiol and xenoestrogens can bind two estrogen receptors (ERs), ER alpha and ER beta, to elicit biological responses. The receptors are ligand inducible transcription factors that exhibit unique biological actions. While estradiol binds both receptors equally, some xenoestrogens have been shown to bind ER beta preferentially. When the ER is bound, the ligand induces a unique ER shape and in turn causes an array of tissue-specific biological responses. For example, the ligand tamoxifen, a commonly used breast cancer pharmaceutical, exhibits an ER antagonist response in the breast and an ER agonist response in the bone. This dual ligand quality characterizes what is now known as a selective estrogen receptor modulator (SERM). Peptide profiling, a novel ER ligand screening assay, is a method that can potentially identify SERMS by correlating in vitro ER conformation with in vivo biological response. Each ligand is screened using a two-hybrid fusion protein reporter gene assay. Upon ligand binding, the ER assumes a conformation; with this induced shape, some ER-interacting peptides will be able to bind while others will not. After screening a ligand against a library of fifteen different peptides, a unique peptide profile will figuratively illustrate the induced ER conformation. Eight xenoestrogens were screened in this experiment: estradiol, a natural physiological estrogen; resveratrol and genistein, two phytoestrogens; MPP, bisphenol A, and 4-hydroxytamoxifen, all synthetic estrogens; α-endosulfan and methoxychlor, both insecticides used on crops. Each ligand was found to have a unique peptide profile and, implicitly, a distinct ER conformation. The next step will be to determine each ligand's tissue specific activity and identify the unique peptide fingerprint that predicts its in vivo biological response. By correlating a ligand's tissue specific estrogenic activity with its unique ER conformation, peptide profiling will not only further elucidate tissue-specific ER activity differences but could also be used as a high-throughput screening tool for other potential environmental xenoestrogens and identify novel therapeutic SERMs.Life Sciences Undergraduate Research Opportunity Progra

Glucose-Insulin Therapy, Plasma Substrate Levels and Cardiac Recovery After Cardiac Ischemic Events

INTRODUCTION: The potential usefulness of glucose-insulin therapy relies to a large extent on the premise that it prevents hyperglycemia and hyperlipidemia following cardiac ischemic events. METHODS: In this review we evaluate the literature concerning plasma glucose and free fatty acids levels during and following cardiac ischemic events. RESULTS: The data indicate that hyperlipidemia and hyperglycemia most likely occur during acute coronary ischemic syndromes in the conscious state (e.g. acute myocardial infarction) and less so during reperfusion following CABG reperfusion. This is in accordance with observations that glucose-insulin therapy during early reperfusion post CABG may actually cause hypolipidemia, because substantial hyperlipidemia does not appear to occur during that stage of cardiac surgery. DISCUSSION: Considering recent data indicating that hypolipidemia may be detrimental for cardiac function, we propose that free fatty acid levels during reperfusion post CABG with the adjunct glucose-insulin therapy need to be closely monitored. CONCLUSION: From a clinical point of view, a strategy directed at monitoring and thereafter maintaining plasma substrate levels in the normal range for both glucose (4-6 mM) and FFA (0.2-0.6 mM) as well as stimulation of glucose oxidation, promises to be the most optimal metabolic reperfusion treatment following cardiac ischemic episodes. Future (preclinical and subsequently clinical) investigations are required to investigate whether the combination of glucose-insulin therapy with concomitant lipid administration may be beneficial in the setting of reperfusion post CAB

Cause of Death and Predictors of All-Cause Mortality in Anticoagulated Patients With Nonvalvular Atrial Fibrillation : Data From ROCKET AF

M. Kaste on työryhmän ROCKET AF Steering Comm jäsen.Background-Atrial fibrillation is associated with higher mortality. Identification of causes of death and contemporary risk factors for all-cause mortality may guide interventions. Methods and Results-In the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF) study, patients with nonvalvular atrial fibrillation were randomized to rivaroxaban or dose-adjusted warfarin. Cox proportional hazards regression with backward elimination identified factors at randomization that were independently associated with all-cause mortality in the 14 171 participants in the intention-to-treat population. The median age was 73 years, and the mean CHADS(2) score was 3.5. Over 1.9 years of median follow-up, 1214 (8.6%) patients died. Kaplan-Meier mortality rates were 4.2% at 1 year and 8.9% at 2 years. The majority of classified deaths (1081) were cardiovascular (72%), whereas only 6% were nonhemorrhagic stroke or systemic embolism. No significant difference in all-cause mortality was observed between the rivaroxaban and warfarin arms (P=0.15). Heart failure (hazard ratio 1.51, 95% CI 1.33-1.70, P= 75 years (hazard ratio 1.69, 95% CI 1.51-1.90, P Conclusions-In a large population of patients anticoagulated for nonvalvular atrial fibrillation, approximate to 7 in 10 deaths were cardiovascular, whereasPeer reviewe