Cancer
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Item SURVEY OF ATTITUDES AND BELIEFS OF ADULTS OF VARIOUS ETHNIC BACKGROUNDS REGARDING COLORECTAL CANCER SCREENING IN OCHILTREE COUNTY(2014-03) Davies, Tabitha A.; Chiapa-Scifres, Ana; Bowling, John; Brown, L. StevenWith barriers to health care in rural areas in mind, the purpose of this study was to evaluate the existing attitudes about colorectal cancer (CRC) screening and to determine the association with screening compliance. Purpose (a): With barriers to health care in rural areas in mind, the purpose of this study was to evaluate the existing attitudes about colorectal cancer (CRC) screening and to determine the correlation with screening compliance and behavior. Methods (b): A survey was used that included questions from the Group-Based Medical Mistrust Scale (GBMMS), colorectal cancer screening behaviors, and demographic information. Inclusion criteria included age ≥ 35 years, residence or employment within Ochiltree County, and ability to answer questions in English or Spanish. The GBMMS consists of three sub-scales: Suspicion, Group Disparities in Healthcare, and Lack of Support from Healthcare Providers. Results (c): A total of 74 surveys were used for analysis: respondents included 67.6% women, 78.4% Caucasian, and 21.6% Hispanic. The average age of respondents was 53.4 ± 10.5 years. Hispanics scored consistently higher on all three sub-scales measuring medical mistrust, indicating they are more suspicious and feel a lack of support from their healthcare providers. While 48.7% of Caucasians over the age of 50 have received colonoscopy or sigmoidoscopy, only 12.5% of Hispanics admitted to having ever been screened for CRC using these methods . Though not significant, Hispanics also had higher raw scores on the Predetermination sub-scale of Fatalism, or a greater sense that obtaining medical care does not play a significant role in one’s ability to live a healthy life. Conclusions (d): Hispanics had lower levels of CRC screening, which may be related to their higher levels of medical mistrust and feelings of ethnic disparities. This group may benefit from CRC screening and prevention education, and efforts should be focused to increase screening compliance.Item CARDIAC FUNCTION IN CHILDHOOD CANCER SURVIVORS TREATED WITH ANTHRACYCLINES: THE ROLE OF ECHOCARDIOGRAPHIC AND ELECTROCARDIOGRAPHIC SCREENING(2014-03) Minter, Melodie M.; Bowman, W. Paul; Bashore, LisaPurpose (a): Anthracyclines have been a mainstay in cancer treatment because of their proven effectiveness in many children with acute leukemia, but have a dose limiting toxicity on cardiac function, in particular cardiomyopathy and potential arrhythmias. This cardiotoxicity is correlated with age at treatment, total cumulative dose of anthracyclines administered, and delivery of radiation therapy to the mediastinum. Current Children’s Oncology Group (COG) treatment guidelines recommend that childhood cancer survivors who received anthracyclines be monitored for long-term cardiotoxic effects using echocardiograms (ECHO) and electrocardiograms (ECG). To date there has been little research on whether following COG guidelines prevent any morbidity or mortality in these cancer survivors. Methods (b): A retrospective chart review of the anthracycline treated survivors seen in the Cook Children’s Life After Cancer Program (LACP) who received cardiac screening ECHOs and ECGs between January 1, 2011, through June 30, 2013, was performed in order to examine the clinical utility of screening ECHOs and ECGs. Results (c): Initial results from this retrospective chart review study showed that most Acute Lymphoblastic Leukemia survivors displayed no signs of cardiotoxicity on ECHOs or ECGs. Only three subjects required further cardiac evaluation from the results of their cardiac screening. Of those three, only one subject was advised to undergo interventional therapy. Conclusions (d): Preliminary results from this study suggest that these survivors who show little change in their cardiac function could benefit from less frequent screening, which would result in less time away from school and/or work and prevent extra medical cost.Item EFFECT OF 4-HYDROXYNONENAL ON MIGRATION AND INVASION ENHANCER PROTEIN 1 (MIEN1) IN COLORECTAL CANCER(2014-03) Raychaudhuri, Urmimala; Vishwanatha, Jamboor K.MIEN1 could be a potential target for therapy in colorectal cancer. Purpose (a): Colorectal cancer (CRC) is the second leading cause of death in the United States. It is believed that the intestinal mucosa is constantly challenged with diet- and bacterial-derived oxidants and carcinogens. Chronic exposure of such challenging conditions may lead to the generation of reactive oxygen species (ROS). ROS initiate an autocatalytic chain of lipid peroxidation (LPO) of polyunsaturated fatty acids, resulting in the formation of large amounts of toxic electrophilic species and free radicals that may play important roles in various human diseases, including carcinogenesis. Consequently, even a minimal transient exposure of cells to ROS causes substantial lipid peroxidation, leading to a significant rise in the level of LPO end product, 4-hydroxynonenal (4-HNE), which is considered to be one of the most abundant cytotoxic aldehydes. HNE reacts not only with DNA but also with proteins and other molecules containing thiol and other nucleophilic groups and can alter the protein structure and functions. We have identified a novel protein called Migration and Invasion Enhancer protein 1 (MIEN1), is highly overexpressed in cancer cells and modulates the AKT activity as a membrane bound adaptor protein. Ectopic expression of MIEN1 activates Akt mediated downstream signaling through NF-kB pathway and induces the expression of several migratory and invasive proteins. However, 4-HNE has also been reported to induce the expression of various proteins involved in cell proliferation and migration. We hypothesize that 4-HNE mediated oxidative stress plays an important role in the etiology of colorectal cancer by modulating the expression and function of MIEN1. In the present studies, we have addressed this question by investigating the effect of 4-HNE on MIEN1 expression in colorectal cancer cell lines SW480 and HT29. Methods (b): Colorectal cancer cell lines, SW480 and HT29 were grown in RPMI-1640 medium containing 10% fetal bovine serum, in a humidified incubator at 37°C with 5% CO2. The toxicity of 4HNE in SW480 cells was determined by MTT assay. The effect of 4HNE on MIEN1 expression was determined by Western blotting in HT29. The effect of 4HNE on SW480 cell migration was examined by scratch wound assay. The LigandFit docking program available in the Accelrys molecular modeling software – Discovery studio, was used to carry out a docking study for the protein MIEN1 and substrate 4HNE. The 3D structure of the protein was obtained from PDB. Results (c): Our results demonstrated that exposure of 4HNE to SW480 cells is toxic. 4HNE concentrations ranging from 0 to 250 μM gradually decreased cell viability in SW480 cells corresponding to an IC50 value of 160 μM. Furthermore, our Western blot analysis demonstrated that treatment of 4HNE increased the expression of MIEN1 at the protein level in HT-29 cells. The scratch wound healing assay showed an increase in migration after treatment with low doses of 4HNE. The docking study produced 10 top scoring(dock score) poses. The poses indicate a possible interaction between the protein binding sites and the substrate (4HNE) including formation of hydrogen bonds between them. Conclusions (d): Together, these results suggest that 4HNE induced cell migration could be mediated via MIEN1.Item NOVEL USE OF PROLIFERATING CELL NUCLEAR ANTIGEN AS A BIOMARKER OF METASTATIC CANCER(2014-03) Horton, Nathan; Mathew, Porunelloor A.Novel biomarkers to identify metastatic tumor cells are needed to better identify these cells and to appropriately choose therapeutic measures. Cancer stem cells are believed to be responsible for metastasis and relapse after therapy. We have identified novel expression of Proliferating Cell Nuclear Antigen on the cell surface of tumors and characterized these cells as potential cancer stem cells. This research may facilitate the generation of novel immune based cancer therapies to specifically identify and target metastatic tumor cells. Purpose (a): Primary tumors account for 10% of cancer related deaths. Thus, identifying novel biomarkers on tumor cells which resist treatment or potentially become metastatic is vital. Proliferating Cell Nuclear Antigen (PCNA) has traditionally been used as a biomarker to identify and grade tumor biopsies based on PCNA's involvement in DNA replication. Typically located intracellularly, we have recently identified PCNA at the cell surface. When recognized by the Natural Killer (NK) cell receptor, NKp44, PCNA inhibits NK cell effector functions, allowing tumor cells to escape immunosurveillance. We have characterized tumor cells expressing cell surface PCNA to evaluate the use of cell surface PCNA as a potential marker of cancer stem cells, believed to be responsible for relapse and metastasis. Methods (b): We analyzed extracellular PCNA expressing tumor cells for expression of vimentin by confocal microscopy and expression of CD44 and CD24 by flow cytometry, which mark cancer stem cells. We also analyzed these cells for expression of genes which can induce formation of cancer stem cells or maintain stem cell characteristics by real time PCR. Finally, since stem cells are often quiescent, we analyzed cell cycle progression of these cells using propidium iodide and flow cytometry analysis. Results (c): Expression of vimentin is exclusive to cells expressing extracellular PCNA. These cells also express intermediate levels of CD44, which marks metastatic cells in vivo, and differentially express genetic markers of cancer stem cells. Populations of tumor cells expressing PCNA at the cell surface were enriched for cells in the G2/M phase of the cell cycle. Conclusions (d): Extracellular PCNA may be a marker for metastatic cancer stem cells based on intermediate expression of CD44, concomitant expression of vimentin, and expression of genetic markers. Alternatively, extracellular PCNA marks cells in the G2/M phase. Further studies in mouse models will be needed to confirm extracellular PCNA as a marker for cancer stem cells.Item GENOMICS-GUIDED DISCOVERY OF POTENT ANTICANCER NATURAL PRODUCTS FROM EXOTIC BACTERIAL SPECIES(2014-03) Liu, Xiangyang; Zhu, Hui; Thapa, Santosh; Cheng, Yi-QiangNatural products are small chemical molecules produced by bacteria, fungi or plants. Natural products have made great contributions to medicine, particularly as anticancer drugs or anti-infective drugs. Currently there is a renaissance of natural product discovery due to the development of new discovery technologies and unmet medical needs. We hypothesized that exotic Gram-negative bacterial species can be a good source of diverse natural products. We analyzed the genomes of two bacterial species originally isolated from central Thailand or north Australia, and found that each genome contains multiple natural product biosynthetic gene clusters. Those information facilitated our discovery of two groups of new natural products, named thailandepsins and thailanstatins, that were found to possess potent antiproliferative activities against an array of human cancer cell lines. Additional studies are being conducted in animal models through collaborations. We thus concluded that Gram-negative bacterial species is a good source of diverse natural products, and genomics-guided discovery approach is effective and particularly suitable for small research laboratories with limited resources. Purpose (a): We hypothesize that exotic Gram-negative bacterial species can be a good source of diverse natural products. The purpose of the research is thus to discover new bioactive natural products from exotic bacterial species. Methods (b): Burkholderia thailandensis E264, a Gram-negative beta-proteobacterium strain originally isolated from a rice paddy in central Thailand, was purchased from the American Type Culture Collection (ATCC); Burkholderia thailandensis MSMB43, another Gram-negative beta-proteobacterium strain originally isolated from a water source in north Australia, was obtained from the US Centers for Disease Control (CDC). Bacterial genome analysis and natural product discovery and identification were performed according to standard procedures. Results (c): Mining the genome of B. thailandensis E264 revealed a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS–PKS) biosynthetic gene cluster that resembles that of FK228 (romidepsin, drug name Istodax) in Chromobacterium violaceum No. 968, which led us to discover thailandepsins A–F, natural analogues of FK228, and potent histone deacetylase inhibitors and antiproliferative agents with GI50 values in the sub-nM range. Mining the genome of B. thailandensis MSMB43 revealed at least 13 biosynthetic gene clusters. Among them one hybrid NRPS–PKS gene cluster is highly homologous to that of FR901464 (a prototype spliceosome inhibitor) in Pseudomonas sp. No. 2663, which led us to discover thailanstatins A–D, natural and more stable analogues of FR901464, and potent pre-mRNA splicing inhibitors and antiproliferative agents with GI50 values in the low nM range. Selected members of those natural products are under intensive collaborative investigations as anticancer drug candidates, and preliminary results are encouraging. Metabolic engineering approach is being undertaken to increase the yield of those potent compounds that are often produced in minute amounts by the wild-type bacteria. Conclusions (d): Potent new anticancer natural products have been discovered from exotic bacterial species via a genomics-guided discovery approach, which is effective and particularly suitable for small research laboratories with limited resources. We seek additional collaborations for identifying the best possible use of our small collection of potent natural products; we also seek to establish a “Texas Network for Collaborative Natural Product Discovery and Development” for sharing the resources, risks and rewards.Item TETRANDRINE INDUCES ROS-DRIVEN CASPASE-DEPENDENT APOPTOSIS OF PROSTATE CANCER CELLS VIA MITOCHONDRIAL AND CELL DEATH RECEPTOR PATHWAY(2014-03) Chaudhary, Pankaj; Vishwanatha, Jamboor K.Androgen deprivation is still the standard systemic therapy for prostate cancer, but patients invariably relapse with a more aggressive form of prostate cancer termed hormone refractory, androgen independent, or castration resistant prostate cancer. Once prostate cancer becomes castration-resistant, metastasis is a significant problem and treatment options are limited. Therefore, identification of novel agents that can selectively kill tumor cells with no additional toxicity to normal tissue would have significant impact on prostate cancer therapy. Purpose (a): Tetrandrine, a bisbenzylisoquinoline alkaloid, isolated from the root of Stephania tetrandra is used in traditional Chinese medicine as an anti-rheumatic, anti-inflammatory, and anti-hypertensive agent for the past several years. During recent years, increasing number of studies have focused on the potential of tetrandrine in cancer therapy. Despite its great potential as an anti-cancer agent, the effect of tetrandrine in prostate cancer has not been studied. Therefore, in the present study, we demonstrate the cytotoxic efficacy of tetrandrine in human androgen-independent prostate cancer cells, PC3 and DU145, and delineate the mechanism of this effect. Methods (b): Prostate cancer cell lines, PC3 and DU145, and normal prostate PWR-1E cells were cultured in ATCC recommended medium. The toxicity of tetrandrine was analyzed by MTT assay and Vybrant Apoptosis Assay Kit. Western blotting was used to detect the expression of proteins involved in apoptosis. Results (c): Our results indicate that tetrandrine selectively inhibits the growth of PC3 and DU145 cancer cells compared to normal prostate PWR-1E cells. Treatment of cancer cells with tetrandrine caused the upregulation of Fas and Bax, downregulation of Bcl-2, cleavage of Bid, and release of cytochrome c, which were accompanied by activation of caspases-9, -3 and -8 and subsequently poly(ADP-ribose) polymerase cleavage. Pre-incubation with caspase-8 inhibitor significantly blocked the tetrandrine-induced Bid cleavage, reduction in mitochondrial membrane potential, and activation of caspase 3, and cell death. Together, these results suggest that the mitochondrial pathway is primarily involved in tetrandrine-induced apoptosis. Additionally, our results demonstrated that tetrandrine-induced apoptosis was caused by the generation of reactive oxygen species (ROS) and most of the signaling effects were attenuated with the preincubation of cells with N-acetylcysteine, thereby further confirming the involvement of ROS in these events. Conclusions (d): Our results demonstrated that treatment of prostate cancer cells with tetrandrine induces caspase-dependent apoptosis via Fas-mediated Bid cleavage and cytochrome c release.Item PREPARATION OF A CISPLATIN PRODRUG FOR USE AGAINST NON-SMALL CELL LUNG CANCER(2014-03) Redfearn, Warren; Shi, Yi; Koneru, Bhuvaneswari; Di Pasqua, AnthonyBackground: Lung cancer is the leading cause of cancer-related death in the United States, and non-small cell lung cancer (NSCLC) the most common type. NSCLC is extremely difficult to treat, resulting in a poor prognosis for patients. Platinum (Pt) anti-cancer agents, such as cisplatin, remain a mainstay in the clinic; however, these Pt complexes act nonspecifically, and thus result in serious side-effects. Development and delivery of a Pt complex with an improved therapeutic index would be highly advantageous to the fight against NSCLC. We prepared trans, cis, cis-bis(heptanoato)amine(cyclohexylamine)dichloridoplatinum(IV), referred to here as PtC, and studied its DNA binding and toxicity toward normal lung and NSCLC cells. Methods: A lipophilic Pt(IV) complex, PtC, was synthesized and characterized, and its binding to DNA and toxicity toward various cancer and normal cell lines determined. Results: We confirmed that the synthesized Pt complex binds to DNA in a manner similar to that of cisplatin, which suggests that it is cisplatin prodrug; however, probably due to its lipophilic nature and improved stability, PtC is much more toxic toward NSCLC cell lines than is cisplatin, and has a much improved therapeutic index. Conclusions: PtC shows promise as a therapeutic agent against NSCLC, and, furthermore, its lipophilic nature allows for us to incorporate it into mesoporous silica nanoparticles for fine-controlled release and the targeting of tumors.Item REGULATION OF MIEN1 IN PROSTATE CANCER(2014-03) Rajendiran, Smrithi; Parwani, Anil; Hare, Richard; Treuren, Timothy Van; Vishwanatha, JamboorMigration and Invasion ENhancer 1 (MIEN1) is a novel gene located in the 17q12 region of the human chromosome. While there is minimal expression of MIEN1 in multiple normal tissues and cells, it is abundantly elevated in many human cancers including the breast, prostate, gastrointestinal and oral. MIEN1 is a membrane bound signaling molecule that triggers downstream signaling through the AKT/NF-κB pathway (common oncogenic pathways) by up-regulating key proteases (thus aiding cancer progression). MIEN1 has also been shown to have a role in migration and invasion (key processes in cancer spread) of cancer cells by enabling filopodia formation (extensions that enable a cell to move). Thus, by various known and unknown mechanisms, MIEN1 promotes prostate cancer progression. While the cellular functions of MIEN1 have been deciphered, the reasons for its aberrant increased expression in cancer cells are still unclear. Understanding the mechanism(s) involved in the regulation of MIEN1 will aid in developing diagnostic marker(s) or in designing effective therapeutic approach(es) to treat prostate cancer patients. Purpose (a): The overall objective of this study is to identify the deregulated mechanisms leading to the differential regulation of MIEN1 between normal and cancer cells. Commonly deregulated mechanisms encompass alterations at DNA (chromosome) to destabilization at protein (translational) levels. Our study focuses on regulation by microRNA (miR) and methylation. Our hypothesis is that MIEN1 is post-transcriptionally regulated by a specific miR and its proximal putative promoter region is hypermethylated in normal cells. Deregulation of these mechanisms together explain the aberrant increased expression of MIEN1 in cancer. Methods (b): To validate the role of miR in MIEN1 regulation, we have performed various in vitro studies. To determine the global role of the miR, we ectopically expressed it in cancer cells. Additionally, we have used human tissue and serum samples to predict the use of miR-MIEN1 as biomarkers. To demonstrate the importance of methylation in the regulation of MIEN1, we performed global methylation inhibition and specific methyltransferase knockdown. Results (c): Our data indicate that MIEN1 is post-transcriptionally regulated by a specific miR which is highly expressed in normal cells compared to various cancer cells, inversely correlating with MIEN1. Ectopic expression of the miR led to decrease in MIEN1 expression, migratory and invasive potential and anchorage dependent growth of cells and impeded mesenchymal transition. Additionally, the miR expression was higher in the normal glands of prostate tissue compared to the tumor; while the secreted/circulating miR was higher in serum from cancer patients, much like PSA expression patterns; but with more significance than PSA. Inhibition of methylation by pharmacological inhibitors or by individually knocking down the methyltransferases increased MIEN1 in normal cells, indicating the role of methylation in the regulation of this gene. Conclusions (d): After proving our results in a larger cohort of patient specimen, this miR could be a useful non-invasive diagnostic biomarker. Additionally, understanding the regulation of MIEN1 by methylation will provide reasons to revisit the current strategies of methylation inhibition for cancer treatment. Overall, since the importance of MIEN1 as a key signaling molecule in cancer is well established, understanding the mechanisms involved in the regulation will aid in designing more effective therapeutic strategies to treat cancer patients.Item TETRANDRINE INDUCED INHIBITION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION (STAT) 3 CAUSES THE REDUCTION OF CELL SURVIVAL, PROLIFERATION, AND ANGIOGENESIS IN TNBC(2014-03) Gibbs, Lee D.; Chaudhary, Pankaj; Vishwanatha, JamboorNatural agents may be promising to combat aggressive behavior of the triple-negative breast cancer (TNBC). STAT3 is a protein that is highly expressed in breast cancer tissues compared to non-malignant breast tissues. Our objective for the present study is to analyze the anti-tumorogenic effects of a Chinese herbal drug, tetrandrine, in TNBC progression through inhibition of STAT3 phosphorylation. Purpose (a): The most successful therapies for breast cancer target the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her-2). Hormonal therapies are not useful in combating triple negative breast cancer (TNBC), which lacks these targeted hormonal receptors. In fact, some of these patients that undergo hormone deprivation and/or Herceptin therapy acquire resistance. The triple negative breast cancer (TNBC) phenotype, which lacks the presence of Her-2, ER, and PR are even more aggressive and resistant. Therefore, there is an urgent clinical need to identify novel agents that can kill tumor cells with no additional toxicity to normal cells and this would have great impact on treatment of such patients. Tetrandrine, a bis-benzylisoquinoline alkaloid isolated from the root of Stephania tetrandra, is a calcium channel blocker used in Chinese medicine for the treatment of silicosis and arthritis. Studies have shown that tetrandrine also has anti-tumor and anti-growth activities. Our objective is to study the effects of tetrandrine on STAT3 signaling that plays an important role in cell proliferation, survival, chemoresistance and angiogenesis. STAT3 protein is highly expressed in breast cancer tissues compared to non-malignant breast tissues. We hypothesize that tetrandrine treatment inhibits the phosphorylation of STAT3 and its associated downstream signaling lead to the reduction of cell survival, proliferation, and angiogenesis in TNBC cells. Methods (b): TNBC cell lines, MDA-MB-231 and HCC70, and non-tumorigenic epithelial cell line MCF-10A were cultured in ATCC recommended medium. MTT assays were carried out to determine the effect of tetrandrine on cell viability. Additionally, cells were subjected to various concentrations of tetrandrine and Western blotting was performed for analysis of protein expression and phosphorylation. Results (c): Our data indicate that tetrandrine selectively inhibits the growth of MDA-MB-231 and HCC70 cells compared to non-tumorigenic MCF-10A cells. In the MTT assay, Tetrandrine concentrations ranging from 0 to 40μM gradually decreased MDA-MB-231, HCC70 and MCF-10A cell viability, corresponding to IC50 values of 25, 20 and 75 μM (n = 8), respectively, after 48 hours of treatment. Our results show that tetrandrine inhibited the phosphorylation of STAT3 in a concentration dependent manner. Furthermore, the inhibition of STAT3 activation by tetrandrine led to the suppression of proteins involved in proliferation (cyclin D1), survival (Bcl-2, Bcl-xL, and Mcl-1), and angiogenesis (VEGF). This effect correlated with the inhibition of proliferation and apoptosis in TNBC cells. Conclusions (d): Our preliminary results suggest that tetrandrine inhibits the proliferation of TNBC cells through inhibition of constitutive STAT3 phosphorylation and it’s associated down stream signaling and has therapeutic potential in the treatment of TNBC.Item MIMICKING INFECTION FOR IMMUNOTHERAPY AGAINST BREAST CANCER-FOOLING THE IMMUNE SYSTEM(2014-03) Kokate, Rutika; Thamake, Sanjay; Chaudhary, Pankaj; Mott, Brittney; Vishwanatha, Jamboor K.; Jones, Harlan P.Immunotherapy represents a potential and innovative means to combat cancer. It essentially harnesses the body’s immune system to fight against cancer. Previous literature suggests that cancer vaccines designed against a specific tumor antigen have been efficiently utilized to trigger immune responses against tumor cells. Despite the preliminary evidence in animal models, low immunogenicity is one of the major hurdles in the development of vaccines in humans. In order to surmount this obstacle, several approaches including the use of an “ideal” tumor antigen, appropriate delivery techniques, immune boosting strategies with co-stimulatory molecules are being explored. Purpose (a): The purpose of this study was to develop “bacteriomimetic nanoparticles” to enhance adaptive cell-mediated immune responses (CD4+ and CD8+ T cell responses) against tumor antigen as a therapeutic option for cancer treatment. Methods (b): NPs were prepared by modified solid/oil/water solvent evaporation method using an ultrasonic processor UP200H system (Hielscher Ultrasonics GmbH, Germany). We used membrane preparations of the 4T1 mouse mammary cancer cell line as a tumor antigen and CpG ODN’s as a “bacteriomimetic” stimulant. Fourteen days before tumor challenge BALB/c female mice (6-8 weeks) were pre-immunized with CpG followed by secondary immunization using respective NPs encapsulated with the membrane antigen preparation. Subsequently, mice (n=4) were challenged with 105 tumor cells intravenously (IV). Mice were sacrificed and tumors were harvested at days 3, 7 and 14 respectively. CD4+ and CD8+ T cell responses were measured in lower respiratory node and spleen using flow cytometry. In another experimental set, following the same immunization schedule as mentioned above, mice (n=5) were challenged subcutaneously (SC) with 105 tumor cells. Primary tumor size was monitored using vernier caliper and bioluminiscence imaging (Caliper Life Sciences Inc., MA, USA). Mice were sacrificed on day fourteen after tumor challenge; spleen cells were used for flow cytometric analysis and primary tumor tissue was used to evaluate CD4+ and CD8+ T cell via immunohistochemistry. Results (c): We found significant reduction in progression of tumor growth in mice immunized with CpG coated NPs containing tumor antigen (CpG-NP-Tag). Histological analysis confirmed that tumors in CpG-NP-Tag mice were relatively well differentiated and of lower grade in contrast to CpG-Blank tumors. Immunofluorescence (IF) data further revealed that CpG-NP-Tag tumors had lesser proliferation and higher apoptotic activity. Tumor CD4+ T cell infiltration as well as T cell response in spleen was found be higher in CpG-NP-Tag NP immunized mice as compared to the controls. Conclusions (d): Primary tumor size, IHC, IF and flow cytometry analysis indicate that CpG-NP-Tag NPs were successfully employed to boost the immune response against tumor cells.Item CLEAR CELL "SUGAR" TUMOR OF THE LUNG: BENIGN OR MALIGNANT?(2014-03) Rodriguez, Abraham; Yurvati, AlbertClear cell “sugar” tumors (CCST) of the lung are rare pulmonary tumors. This case study illustrates a patient who was found to have a persistent nodule in the left upper lobe of the lung. Positron Emission Tomographic scanning (PET) showed mild-moderate FDG (18Fluorodeoxyglucose) uptake. Based on these findings, a video assisted resection of the tumor was undertaken. The mass was identified histologically, as a clear cell “sugar” tumor of the lung. This case report discusses the benign versus malignant nature of this rare tumor. Purpose (a): Purpose of this study was to present a case study describing the presentation of a very rare lung tumor, a sugar cell clear cell tumor. There have only been around 50 reported cases. Our hope is to educate primary care physicians about this tumor. Methods (b): The main method used was conducting full literature search to look for the same tumor and compare and contrast the different presentations. We also searched for articles describing how benign and malignant lung tumors present so that we can differentiate this tumor from others. Results (c): Our results found that the patient's tumor is overall benign, but because sugar cell tumors are so hard to diagnose and differentiate from malignant tumors, the best treatment is surgical excision. Conclusions (d): Sugar cell tumors are rare benign lung tumors. Due to the difficulty with diagnosing this particular type of tumor, surgical excision is both diagnostic and curative.Item REMOVING BARRIERS TO BREAST CANCER SCREENING AMONG ETHNIC MINORITIES IN TEXAS(2014-03) Oyewole, Olusegun; Linnear, Kim; Cardarelli, Kathryn; Martin, Marcus; Petties, Karin; Williams, Angela; Lafayette, Camille; Martinez, Erika; Harris, PhyllisPurpose (a): This project seeks to reduce breast cancer mortality among ethnic minorities in Dallas County, Texas through an integrated breast cancer prevention program that includes outreach and education, delivery of screening services, follow-up navigation and screening behavior maintenance. While perceived susceptibility, perceived severity, perceived benefits and cues to action are important predictors of health-seeking behaviors, removal of perceived barriers has been found to be the most important factor in moving people from inactivity to action. This research seeks to answer the question: Does this program significantly reduce the perceived barriers to mammography screening among the participants? Methods (b): Participants had a pre-survey assessing their knowledge, attitude and behavior about breast cancer determinants and prevention as well as their perceived severity of breast cancer, perceived susceptibility to it, perceived benefit of regular screening and perceived barriers to regular screening. This was followed by up to 8 weeks of education and a post-survey. McNemar’s tests were done to compare the pre- and post-surveys on questions relating to perceived barriers to screening and mammogram use. Results (c): A significant reduction in perceived barrier to breast cancer screening was found among study participants. There was also a significant improvement in mammogram use among them during the intervention. Conclusions (d): The integrated breast cancer prevention program leads to a significant reduction in perceived barriers to screening with consequent improvement in mammogram use in study participants.Item MIEN1 PROMOTES CANCER CELL MIGRATION AND INVASION THROUGH ENHANCED ACTIN DYNAMICS(2014-03) Kpetemey, Marilyne; Dasgupta, Subhamoy; Vishwanatha, JamboorSurgical resection and adjuvant therapies have so far, only incrementally improved patient survival; as metastatic disease remains incurable. Knowledge has been in part limited because metastasis is a ‘hidden’ process that occurs inside the body and is inherently difficult to observe. Migration and invasion are critical parameters in the dissemination of cancer cells and the formation of distant metastases. Hence identifying migratory and invasive genes and their action mechanisms may provide new insights into the pathogenesis and management of tumor metastasis. In the present investigation, we report functional studies of one of the prime regulators of cancer cell migration and invasion, which is also prenylated and contains an ITAM like the atypical Rho GTPase, RhoH. Migration and invasion enhancer1 (MIEN1) also known as C35, C17orf37, RDX12, and MGC14832, is frequently amplified and overexpressed in breast tumors. Purpose (a): Migration and Invasion Enhancer 1 (MIEN1), previously known as C35, C17orf37, RDX12 and MGC14832, is a novel gene located in the chromosomal region 17q12-21. While absent or low in normal tissues, MIEN1 is abundantly expressed in multiple cancers; including breast, prostate, oral and gastrointestinal carcinomas. A membrane-bound signaling adaptor, MIEN1 localizes to the leading edge of migrating cells and promotes migration and invasion by increasing filopodium formation. MIEN1 contains several functional motifs including a prenylation motif and an immunoreceptor tyrosine-based activation motif (ITAM). While prenylation of MIEN1 is shown to be important for its functions, little is known about the importance of its ITAM. The overall goal of the present study is to dissect the mechanisms by which MIEN1 regulates breast cell motility and whether the ITAM is important. Methods (b): Using site-directed mutagenesis, we introduced point mutations in amino acid sequences in MIEN1-ITAM domains. We established NIH3T3 stable cell lines over-expressing the wild type or mutant proteins. We performed immunofluorescence, migration and invasion assays using the established stable cell lines and breast cancer cells to investigate the mechanisms by which different domains of MIEN1 potentiate cell motility. Results (c): Analyses of in vitro migration and invasion assays showed that stable cells over-expressing MIEN1 phosphorylation mutants failed to induce significant migration and invasion compared to cells over-expressing MIEN1 wild type protein. Immunofluorescence staining with rhodamine conjugated-phalladoin confirmed that MIEN1 induced- migration is associated with actin filaments; and post-translational modifications at the ITAM domains is critical for eliciting MIEN1 functions. Conclusions (d): Our results confirm that MIEN1 regulates cancer cell migration and invasion through filopodia formation. Furthermore we showed that MIEN1 is involved in cell-cell adhesion, a process required for cell motility. MIEN1 is a prime regulator of cancer cell motility; hence understanding the molecular mechanisms by which it is aiding the invasion-metastasis cascade will enable the design of novel and effective treatments for metastatic tumors.Item CHARACTERIZATION AND OPTIMIZATION OF MRNA ENTRAPPED PEPTIDE NANOPARTICLES FOR TARGETED GENE DELIVERY(2014-03) Conjeevaram Nagarajan, Bhavani Saranya; Sabnis, Nirupama; Lacko, Andras G.The mRNA entrapped nanoparticles have the potential to be used in targeted gene delivery. These nanoparticles are used in treating Cancer and neurological disorders. Purpose (a): Generally, mRNA is considered to be very labile and unstable and has not been significantly used for therapeutic purposes. However, compared to DNA based gene expression, mRNA is safer as it is does not integrate with the host genome, and it does not require nuclear localization. The main aim of this project is to entrap the mRNA inside a targeted nanoparticle to increase its stability and the tissue specificity of the gene delivery. Methods (b): The particles were assembled using heat denatured mRNA and a cationic oligomer or detergent to neutralize the negative charge of the polynucleotide. Subsequently phospholipid and a protein/peptide component are added to form the stable mRNA nanoparticle. In order to minimize the size of the particles, the preparation was carried out with several cationic detergents, including Hexadecyltrimethyl ammonium bromide (HTAB), Tetrabutyl ammonium hydroxide (TBAH), and DOTAP. The peptide/protein components were 10-100µg of either Apolipoprotein A-I, or A-I mimetic peptide Myr-5A/ 5A. The incorporation efficiency of the polynucleotide is determined by separating the unincorporated mRNA using OligodT beads and lysing the particles using Trizol reagent to release the entrapped mRNA. Results (c): The yield of the entrapped mRNA analyzed using Ribogreen assay was 17-20%. Based upon the size analysis measurements made using Dynamic Light Scatterer, it was observed that the particles prepared with 5A peptide (10µg) and DOTAP (10µg) resulted in 48% of 268nm particles. Conclusions (d): Further optimization of this formulation may be achieved to produce more homogenous nanoparticles with higher mRNA incorporation efficiency, using DOTAP as the neutralizer and 5A as the peptide.