• Chronic Consumption of DNOP Induces an Epithelial-to-mesenchymal Transition State in Mouse Liver

      Amin, Monisha; Department of Biological Sciences (2017-03)
      Hepatocellular carcinoma is the cancer of the liver cells that is developed over time by the evolution of pre-neoplastic lesions. Di-n-octylphthalate (DNOP) is a plasticizer used to keep plastics flexible. If mice are exposed to DNOP, it causes an increase in pre-neoplastic hepatic lesions. Previously, our group found that DNOP increased the expression of transforming growth factor β (tgf-β) in AML-12 cells. Because tgf-β induces an epithelial-to-mesenchymal transition (EMT) state in mouse hepatocyte in vitro, our goal was to study the extent to which DNOP induces an EMT state in mouse liver. Two antibodies were used: anti-albumin antibody (a hepatocyte marker), and anti-vimentin (a mesenchymal cell maker). We first treated AML-12 cells with 0.1 % DNOP for 24, 48 and 72 h. No changes in the expression of albumin was seen. Because the limited time of 72 h may not have allowed sufficient time for a change in the phenotype, mice were fed diet containing 0.1 % DNOP for a month. We found that DNOP decreased the levels of albumin, whereas increased the levels of vimentin. In conclusion, chronic consumption of DNOP induces an EMT state in mouse liver. This mechanism may be involved in formation of hepatic pre-neoplastic lesions.
    • Di-N-Octylphthalate Acts as a Proliferative Agent in Murine Cell Hepatocytes by Regulating the Levels of Pro-Apoptotic Proteins

      Pruitt, Allison; Miller, Laurence; Wiley, Faith; Department of Biological Sciences (2017-03)
      Hepatocellular carcinoma (HCC) is the fifth most common cancer in the US. Its development is thought to be associated with inactivation of tumor suppressors by methylation. Di-n-octylphthalate (DNOP), a common plasticizer, is believed to cause hepatic pre-neoplastic lesions. Because a number of tumor suppressors are shown to be not expressed in HCC, our goal was to identify tumor suppressor genes methylated upon treatment with 0.1 % DNOP at 24, 48, 72 h in mouse hepatocytes cell line AML-12 and isolated primary cultured mouse hepatocytes. None of tumor suppressors experienced a change in the methylation status in presence of DNOP. Because we found that DNOP causes an increase in cell proliferation, we studied whether the effect is paralleled to a suppression of apoptosis. We found that DNOP causes a decrease in pro-apoptotic proteins and no change in anti-apoptotic proteins. We studied the physiological effects of DNOP in mouse liver. Mice were treated with 0.1 % DNOP for a month. DNOP caused a decrease in bile secretion and an increase in the hepatic levels of bile acids and glutathione. Avoiding the use of DNOP as a plasticizer in products for human consumption can reduce the incidence of diseases related to its hepatotoxicity.
    • Does Leptin Treatment Decrease Alpha-Adrenergic Receptor Expression in Mouse Renal And Mesenteric Arteries?

      Momtahan, Mina; Department of Biological Sciences (2016-03)
      Obesity often leads to hypertension. Previous work from my lab demonstrated that the adipocyte-derived hormone leptin reduces the ability of the aorta to contract in response to adrenergic stimulation, likely due to a decreased expression of the aorta alpha-adrenergic receptors. However, it is not known whether leptin decreases the expression of alpha-adrenergic receptors in such arteries like the mesenteric and renal arteries that play a key role in the control of blood pressure. To determine whether leptin decreases alpha-adrenergic receptor expression in renal and mesenteric arteries, I infused leptin (10ug/day) by implanting subcutaneous mini-pumps in five male C57bl/6 mice. Five mice did not receive leptin and served as controls. After seven days of treatment I euthanized nine mice as one mouse died. Renal and mesenteric arteries were taken from the mice and mRNa was extracted from the arteries. Reverse Transcription (RT) was completed in order to induce the transcription of mRNA into cDNA. After checking the concentration of cDNA, real-time PCR (qPCR) conducted on the arteries revealed high CT values for a1D-receptor concluding that leptin-mediated increases in sympathetic tone decreased a1D-receptor expression. This data is supportive of my hypothesis that leptin- decreases adrenergic receptor expression in renal and mesenteric arteries.
    • Epidermal Growth Factor Mediates Di-N-Octyl Phthalate-Induced Hepatocyte Proliferation

      Buckner, Shelby; Department of Biological Sciences (Augusta University, 2016-05)
      Certain chemicals used in manufacturing plastics are linked to severe, negative health effects such as cancer. Di-N-Octylphthalate (DNOP) is a phthalate found in many plastics and has been linked with hepatocellular carcinoma. The aim of this research project was to study the effect of DNOP on both proliferation and differentiation of normal mouse hepatocytes. The expression of several growth factors and their receptors (epidermal growth factor (egf), epidermal growth factor receptor (egfr), insulin-like growth factor 1 (igf1), insulin-like growth factor 2 (igf2), insulin-like growth factor 1 receptor (igf1r), hepatocyte growth factor (hgf), and transforming growth factor-β (tgf-β )) was assessed in the normal hepatocyte AML-12 cell line by RT-PCR and qPCR. The rate of cell proliferation of AML-12 cells was measured using an MTT cell proliferation assay kit. Changes in cytoskeletal reorganization were assessed by immunostaining filamentous-actin using phalloidin staining. We found that DNOP at 0.1% caused an increase in expression of egf at 48h and tgf-β at 72h. This result was confirmed by Western blot. DNOP did not cause changes in any of the other studied genes. The rate of cell proliferation increased in those cells treated with 0.1% DNOP at 24, 48, and 72h. DNOP induced reorganization of the filamentous actin from basolateral to perinuclear. Our observation suggests that DNOP, through an increase in the expression of egf, acts as a proliferative agent in normal mouse hepatocytes, and through the expression of tgf-β, induces a reorganization of the filamentous-actin. These observations could be a mechanism by which DNOP leads to the development of hepatocellular carcinoma.
    • Impact of Myeloid Cell NF-κB Signaling on Glioblastoma Growth

      Venugopal, Natasha; Howard, Shelby; Achyut, Bhagelu; Jain, Meenu; Arbab, Ali; Bradford, Jennifer W.; Department of Biological Sciences (2017-03)
      Cancer consists of malignant tumor cells as well as supporting, non-cancerous cells that make up the tumor stroma. Tumor-associated macrophages (TAMs), a critical component of the stroma, can be present in very large numbers in a variety of cancers, and can lead to tumor progression through promotion of tumor inflammation, angiogenesis, invasion, and metastasis. Canonical nuclear factor-kappaB (NF-κB) pathway activity is very important in normal immune function, synaptic plasticity, and memory, and aberrant NF-κB activity is associated with autoimmune disease, and importantly, cancer. Previous studies have been reported about the importance of tumor cell associated NF-κB signaling in cancers. As myeloid cell NF-κB signaling may also be important in promoting cancers, we have been utilizing the p65fl/fl/LysMCre transgenic animal model, which lacks p65 protein in cells of the myeloid lineage, to study the impact of myeloid cell derived NF-κB signaling in glioblastoma (GBM), an extremely aggressive brain cancer. This transgenic model has a very efficient deletion of p65 protein and drastically reduced NF-κB signaling in bone marrow derived macrophages (BMDMs), but brain residing microglia do not have significantly lower p65 levels as compared to control microglia. Even with this finding, p65fl/fl/LysMCre mice implanted with syngeneic GBM cells have significantly reduced GBM tumor burden than LysMCre control mice, as measured by magnetic resonance imaging. This result underscores the potential importance of bone marrow cells that migrate to the tumor site and significantly contribute to GBM growth. This work also indicates the potential benefits of targeting myeloid specific NF-κB signaling in GBM patients.
    • Purification of Recombinant Human Histone Methyltransferases for Inhibition Studies

      Jahan, Asmat; Shaikh, Zahid; Department of Chemistry & Physics (2017-03)
      Resistance to cellular apoptotic pathways is a major contributing factor in the metastasis of cancer. One such cell-intrinsic pathway which induces programmed cell death is ligand cell surface death receptors, including Fas protein. In primary colorectal cancer, Fas expression is often diminished. The silencing of Fas expression is perhaps a mechanism by which human colorectal cancer cells evade apoptosis. The decrease in Fas expression levels is associated with hypermethylation of histone 3 lysine 9 catalyzed by histone methyltransferases (HMTases) such as SUV39H1 and SUV39H2. Because of the role of methylation in silencing Fas expression, HMTase inhibitors represent potential anti-cancer agents. Verticillin A, a natural compound extracted from wild mushroom, is a broad-based HMTase inhibitor and has been shown to cause toxicity in mice. Our goal in this study is to identify specific SUV39H1 and SUV39H2 inhibitors that are less toxic than Verticillin A. To investigate this, human SUV39H1 was sub-cloned into an expression vector, transformed into an E.coli expression strain and purified using affinity chromatography. Gel electrophoresis and Western blot analysis confirmed the presence of partially purified human SUV39H1. The next phase of the project will involve testing the activity of the purified protein in an in vitro assay.
    • Retinopathy of Prematurity: Role of MicroRNA-21 in Pathological Newvascularization

      Rajpurohit, Shubhra; Bartoli, Manuela; Department of Biological Sciences (2017-03)
      Retinopathy of prematurity (ROP) is a disease that occurs in premature infants weighing 1250g or less. ROP causes abnormal blood vessels to grow in the retina. This growth can cause the retina to detach from the back of the eye, leading to blindness in severe cases. ROP affects approximately 14,000 infants, and 90% of those affected have only mild disease. However, 1,100-1,500 children develop disease severe enough to require medical treatment, and 400-600 infants each year in the U.S. become legally blind from ROP. To study the mechanisms of abnormal vascularization in the retina we use a mouse model of oxygen induced retinopathy (OIR), which is a standard experimental model for ROP. In mice the retinal vasculature normally starts to develop around birth and is fully mature around 3 weeks after birth. To trigger OIR, we expose 7-day-old mouse pups to hyperoxia (70% O2), which results in regression of the vasculature. When the animals are returned to room air (21% O2) after five days in oxygen, a neovascular response is triggered. MicroRNAs, short noncoding RNAs that inhibit gene expression through the post-transcriptional repression of their target mRNAs, emerged as key regulators of diverse biologic processes. In this study we will determine the role of miR-21, a specific microRNA that has been involved in pathological angiogenesis, by blocking its activity using in vivo delivery of miR-21 inhibitors (antagomiR) in a mouse model of OIR and in vitro using isolated retinal microvascular cells subjected to hypoxia. The obtained results from western blots and qPCR suggest miR-21 expression is increased and tissue inhibitor of metalloprotease-3 (TIMP3), a target gene of miR-21, expression is decreased in retinal endothelial cells exposed to hypoxia.