MicroRNAs (miRNAs) are small non-coding 18-22 long RNAs, which act as key mediators in many cellular processes involved in tumorigenesis including proliferation, differentiation, invasion, and apoptosis. miRNAs repress gene expression by inhibiting mRNA translation or by promoting mRNA degradation. Due to the importance of miRNAs as master regulators of gene expression many studies have emerged to distinguish both miRNA targets and regulatory mechanisms governing miRNA translation and maturation. Uncovering mechanisms that govern miRNA expression and function in cellular biology gives greater insight into pathways facilitating multiple cellular processes involved in tumor initiation and progression. miRNAs are altered in breast cancer and these alterations can lead to changes in signaling pathways involved in cancer progression. The purpose of this work is to determine the effects of miRNA expression on the cancer phenotype and to examine the roles of insulin-like growth factor 1 (IGF-1) and estrogen receptor (ER) signaling on miRNA expression and function in ER+ breast cancer cell systems. Our results demonstrate the intricate relationship between miRNAs and cell signaling networks. Through uncovering miRNA regulating networks such as IGF-1 signaling and ER induced miRNA maturation and star strand selection we have added greater insight to the complex mechanisms involved in miRNA function. miRNAs function through inhibition of mRNA targets, though the possible targets for a single miRNA can be in the thousands. Additionally, mRNA isoform variability and loss of 3’UTR greatly enhance the complexity of these networks. / acase@tulane.edu

MicroRNAs (miRNAs) are a group of small, noncoding RNAs that modulate the translation of genes into proteins by binding to specific target sites in messenger RNAs. This study investigated the biological function and molecular mechanism of microRNA-21 (miR-21) in human cholangiocarcinoma. In situ hybridization analysis of human cholangiocarcinoma tissues showed increased miR-21 in cholangiocarcinoma cells compared to the noncancerous biliary epithelial cells. Forced overexpression of miR-21 by lentivirus transduction enhanced human cholangiocarcinoma cell growth and clonogenic efficiency in vitro, whereas inhibition of miR-21 decreased these parameters. MiR-21 overexpression also promoted cholangiocarcinoma growth in a tumor xenograft model. The NAD+-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme that converts the pro-tumorigenic prostaglandin E2 (PGE2) to biologically inactive metabolite, was identified as a direct target of miR-21 in cholangiocarcinoma cells. In parallel, cyclooxygenase-2 (COX-2) overexpression and PGE2 treatment increased miR-21 expression and enhanced miR-21 promoter reporter activity in human cholangiocarcinoma cells. Our results disclose a novel cross-talk between COX-2/PGE2 and miR-21 signaling pathways that converges at 15-PGDH which is crucial in cholangiocarcinogenesis and tumor progression. The enhancer of zeste homolog 2 (EZH2) is the catalytic subunit in the PRC2 complex catalyzing the trimethylation of histone3 lysine27 (H3K27) and mediates gene silencing of the target genes. The biological function of EZH2 in cholangiocarcinoma was investigated in this study. Immunohistochemistry staining of EZH2 on human cholangiocarcinoma tissues showed increased EZH2 expression in cholangiocarcinoma cells. Pharmacologically inhibition of EZH2 by EZH2 inhibitors decreased cholangiocarcinoma growth and induced G1 arrest. The CD133, one of the putative cancer stem cell markers, was found to express in the cholangiocarcinoma cell lines we used. Inhibition of EZH2 decreased CD133+ population and the sphere forming ability of cancer cells. Our results indicate that EZH2 may represent a promising target for targeting the tumor-initiating cell population and future cholangiocarcinoma therapy. / acase@tulane.edu

acase@tulane.edu

Nitric oxide (NO) is the principal mediator of erectile function. NO is released from the nerves and endothelium of small arteries in the penis and diffuses into surrounding smooth muscle to vasodilate through activation of soluble guanylate cyclase (sGC). Erectile dysfunction (ED) occurs in 50% of men between the ages of 40 and 70. It is likely that the pathology of ED results from impairment of NO formation or bioavailability in penile tissue. Iatrogenic nerve damage occurring during prostatectomy can attenuate neurotransmission and release of vasodilators from cavernosal nerves. Oxidative stress from chronic conditions such as diabetes and cardiovascular disease generates reactive oxygen species that can oxidize NO and decrease the molecule's bioavailability. The "gold standard" treatment for ED involves use of oral PDE-5 inhibitors that rely on an intact NO-signaling mechanism for efficacy. Although these therapies are easy to use, they are not effective in many patients suffering from ED associated with pathological conditions of decreased NO bioavailability. Rho-kinase inhibitors, sGC stimulators and sGC activators offer three new interventions that may demonstrate efficacy in treating ED associated with low NO bioavailability. Our results suggest that erectile responses to Rho-kinase inhibitors are not modulated by muscarinic receptor blockade, soluble guanylate cyclase inhibition or cavernosal nerve injury in the rat and that Rho-kinase inhibitors are additive and do not potentiate the endogenous NO-mediated erectile response. Our results with BAY 41-8543 show that this sGC stimulator has significant erectile activity and can potentiate erectile responses to low levels of exogenous and endogenously released NO. These results suggest that BAY 41-8543 would be useful in the treatment of ED occurring following nerve damage from prostatectomy. The sGC activator BAY 60-2770 has very potent erectile activity that is enhanced significantly in conditions of oxidative stress when erectile responses to endogenous NO or sGC stimulators are severely diminished. In oxidizing conditions erectile activity of sGC activators may be enhanced further with concomitant PDE-5 inhibitor therapy, providing evidence that sGC activators may be used alone and in combination with existing treatments to improve erectile function in patients who are non-responsive to standard therapeutic options for ED. / acase@tulane.edu

acase@tulane.edu

No description available.

In the work presented here we have used a function-based approach to isolate 12 novel cell-penetrating peptides from a 10,000+ member peptide library of rational design. Our unique high-throughput screen differentiates non-membranolytic from membranolytic translocation of peptides across lipid bilayers, thus allowing the selection of potential cell-penetrating peptides over potential antimicrobial peptides or peptide toxins. The 12 residue framework of the peptide library, designed with translocation in mind, is a series of 9 combinatorial sites followed by a C-terminal α-1-chymotrypsin cleavage site that is integral to the screen. The resulting residue in each of the combinatorial sites is one of 2 - 4 variable amino acids, with a hydrophobic or cationic residue available in each position. The sequences of nonpore-forming translocating peptides pulled from the screen have a 3 residue motif, Leu-Leu-Arg (p=10-5), and an overall under representation of basic residues in favor of hydrophobic amino acids. Upon characterization, these novel peptides were shown to behave akin to known cell-penetrating peptides found in nature. Ex vivo studies, in mammalian tissue cultures, revealed that the peptides translocate across the cell membrane without toxicity to the cell. In addition, structural studies showed a lack of convergence regarding a structure- function relationship, a continued trend seen among membrane-active peptides. In the course of the screen and the ex vivo studies, the peptides carried small polar molecules across lipid bilayers and biological membranes respectively; suggesting that, in addition to being cell-penetrating peptides, they could be put to use as effective therapeutic agents. The discovery of these novel cell-penetrating peptides by use of our screen supports function-based screening of peptide libraries as the best way to arrive at de novo membrane-active peptides with specific functions of interest. / acase@tulane.edu

Background: Chronic wounds are a serious medical condition affecting over 6 million people in the United States. Biofilms, which are communities of bacteria attached to a surface and protected by a polysaccharide coating, are intimately associated with the development of chronic wounds. They alter the host immune response and establish a microenvironment that prevents wound healing. Current treatment options do not target biofilms. One novel treatment solution for chronic wounds involves the paracrine factors from mesenchymal stem cells (MSCs), which have been shown to stimulate wound healing in non-infected wounds. The aim of this dissertation was to examine the effects of reprogrammed MSCs and their paracrine factors on ameliorating infection and accelerating wound closure in biofilm infected wounds. Methods: MSCs were reprogrammed by 3 strategies: seeding on an extracellular matrix (ECM), as spheroids in static culture, and as spheroids in a bioreactor. The paracrine factors were analyzed using a 14-plex cytokine assay to confirm changes from baseline. The paracrine factors were applied to mature P. aeruginosa biofilms in vitro and the number of viable bacteria were quantitated. They were also used to stimulate RAW 264.7 murine macrophages and analyzed for the presence of CD206, a marker of anti-inflammatory macrophage phenotype. BALB/cJ mice were wounded and infected with P. aeruginosa biofilms and the paracrine factors from reprogrammed MSCs were applied topically. The wound area and CFU counts of the treatment group were compared to the control and untreated groups. Results: MSCs grown as spheroids in a bioreactor produced significant increases in IL-6 and IL-8 after 3 and 7 days (p<0.05 and p<0.0001). The paracrine factors from MSCs grown on ECM were found to reduce P. aeruginosa biofilm growth significantly (p<0.01). Spheroids grown statically and in a bioreactor increased the amount of macrophages expressing CD206. Mice wounds receiving the paracrine factors from MSCs grown on ECM had lower bacterial counts and an increased rate of wound closure compared to non-treated mice wounds. Conclusions: The results indicate that paracrine factors from reprogrammed MSCs accelerated wound healing and reduced the bacterial burden in biofilm infected wounds. / acase@tulane.edu

acase@tulane.edu

Docetaxel (DTX)-based regimen is the mainstay treatment against castration resistant prostate cancer (CRPC). However, significant side-effects of DTX mandate that strategies to chemosensitize CRPC cells be utilized. We investigated whether physiologically achievable concentrations of nelfinavir (NFR) and curcumin (CUR), known to target the endoplasmic reticulum (ER) stress and AKT pathways, can increase DTX cytotoxicity. A significant reduction (~70%) in survival of a CRPC cell line, C4-2B, was evident within 24 hrs post-exposure to a combination of DTX (10 nM), NFR (5 µM) and CUR (5 µM), as compared to DTX alone (~34%). This rapid cytotoxicity was not seen in non-tumorigenic RWPE-1 cells as well as in primary prostate epithelial cells (PrEC) and bone-marrow mesenchymal stem cells (BM-MSC). A significant increase in apoptosis was seen in C4-2B cells but not RWPE-1 cells, as indicated by DNA-fragmentation, caspase-3 assay, and PARP cleavage. A significant reduction in C4-2B-derived colony forming units (CFU) was observed following exposure to DTX-NFR-CUR combination (92%), as compared to DTX alone (34%). In C4-2B cells, immunodetection and real-time PCR studies showed that exposure to 3-drug combination drastically reduced AKT activation, increased unfolded protein response (UPR) markers, such as XBP-1 mRNA and phosphorylated eIF-2α, and increased ER-stress induced pro-apoptotic markers such as CHOP, ATF4 and TRIB3. In RWPE-1 cells, upregulation of CHOP was observed with DTX-NFR-CUR combination, but no increase in ATF-4 and TRIB3 were observed. In vivo studies using C4-2B tumor xenografts showed a significant reduction in tumor volume following 4 week exposure to the 3-drug combination, as compared to DTX alone. Immunohistochemistry (IHC) of tumor sections revealed decreased Ki-67 staining indicating reduced cell proliferation and increased TUNEL staining indicating apoptosis, in DTX-NFR-CUR treated mice as compared to DTX alone. Therefore, our studies show that NFR and CUR can provide a promising approach as an adjuvant therapy to chemosensitize CRPC to DTX therapy. / acase@tulane.edu