| Authors: | Isabella Porter (OMS-II) and Benford Mafuvadze (PhD) |
| Background: | The use of combined estrogen and progestin hormone replacement therapy has been linked with increased risk of breast cancer recurrence and metastasis in postmenopausal women. Progestins increase cancer risk through induction of potent angiogenic factors that promotes neovascularization and blood vessel development within tumors among several other potential mechanisms. There is growing evidence suggesting that aberrant expression of microRNAs may significantly contribute to the growth, progression and metastasis of human cancers. Progestins and other sex steroids have been shown to alter expression of microRNAs in in some human cancers and different body tissues. Our understanding of how progestins regulate miRNAs that may control several genes that influence hormone-responsive breast cancer growth is limited. This study sought to determine the effects of different progestins on transcriptional expression of two microRNAs namely, pri-miR-190 and pri-miR-199 in two breast cancer cell lines that express progesterone receptors. |
| Design: | T47D and BT-474 breast cancer cells were exposed to medroxyprogesterone acetate (MPA) and other progestins for 24hour after which RNA was extracted and real-time PCR used to determine expression of pri-miR-190 and pri-miR-199. |
| Results: | Our results showed that MPA significantly increased transcriptional expression of pri-miR-199 in both T47D and BT-474 breast cells. Concurrent exposure of cells to Ru-486, a known progesterone receptor antagonist blocked this induction of pri-miR-199 by MPA. We observed different effects of MPA on pri-miR-190 expression between T47D and BT-474. While MPA significantly increased expression of pri-miR-190 in T47D, there was no significant effect in BT-474. Two synthetic progestins, norgestrol and norethindrone also significantly increased expression of both pri-miR-190 and pri-miR-199 in T47D cells. |
| Conclusion: | We concluded that progestins increase transcriptional expression of both pri-miR-190 and pri-miR-199 through processes that directly depend on the progesterone receptor (PR). |
| Authors: |
Lauren Farrell, BS; Audrey A. Vasauskas, PhD; Jonathan Brown, PhD |
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Background: |
Pulmonary arterial hypertension (PAH) is a disease characterized by an increase in pulmonary circulation pressure due to the abnormal differentiation of endothelial cells. Proliferation of endothelial cells leads to right-side heart failure in patients. Survival rates for patients diagnosed with PAH remain poor. Here we demonstrate that Foxo proteins are differentially expressed in endothelial cells harvested from our murine PAH model. |
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Design: |
Pulmonary arterial endothelial cells from wild-type (FR6) rats and the Sugen5416/hypoxia PAH model (SPAD) were provided by Dr. Troy Stevens at the University of South Alabama. Both FR6 and SPAD cells were plated in a 6-well plate at concentrations of 7x105 and 5x105 in 2mL D10F and incubated overnight at 37°C. Antibodies were purchased from Cell Signaling. Western blot analysis was performed with Licor Software. The supernate from both FR6 and SPAD was harvested, centrifuged, and stored at -20°C. |
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Results: |
Foxo3a is elevated in SPAD cells compared to FR6 while Foxo1 is diminished in SPAD cells compared to FR6 cells at resting states. Addition of SPAD media did not alter Foxo1 or Foxo3a expression. AKT has elevated phosphorylation at base levels in SPAD cells compared to FR6 cells but supernate from SPAD has minimal effect on phosphorylation levels. |
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Conclusion: |
Foxo1 and Foxo3 are differentially expressed in SPAD cells compared to FR6 cells. Interestingly, while Foxo3 is elevated in SPAD cells at resting states compared to FR6 cells, Foxo1 expression is lower. This indicates that SPAD cells utilize the Foxo proteins in different ways during disease progression. |
| Authors: |
A.A. Khan, BS, M.O. Ibiwoye, MD, MS, DTM&H, MPH, PhD, A.A. Vasauskas, PhD, J. Lyons, MD and J.D. Foster, PhD |
| Background: |
Cadmium is a heavy metal found in association with zinc, lead, and copper ores as natural components of the earth’s crust and its harmful effect is well known. The adverse health impact of cadmium is well known and its association with multi-organ damage throughout the body has been well documented. Cadmium pollutes the environment via diverse anthropogenic activities including rechargeable batteries production, smelting, electroplating, phosphate fertilizers, and high-volume waste incineration. Cadmium enters the air, soil, and water through soil erosion, wildfires, and volcanic eruptions. Studies have established that maternal exposure during pregnancy results in children manifesting growth retardation, memory disorders and other cognitive disabilities. The mechanism cadmium neurotoxicity is not completely understood, but the metal appears to exert its toxicity via reactive oxidants stress-induced morphological and functional perturbation of blood-brain barrier (BBB) structures. A rat BBB-specific marker has been previously identified and characterized by Sternberger. Nrf2 plays a key role in cellular antioxidant defense mechanisms against adverse consequences of oxidant exposure such as cadmium. |
| Design: |
We applied a standard two-step immunofluorescent protocol to detect and characterize the expression of Nrf2 and EBA within the hippocampus in paraformaldehyde-fixed coronal brain sections of six-week-old albino rats. |
| Results: |
We detected a reduced expression of Nrf2 and EBA in all regions of the hippocampal formation in Cd-exposed animals compared to normal controls. Sections from the experimental animals exhibited neuronal loss, degeneration and disorganization throughout the hippocampus especially in all segments of the Ammon’s horn. |
| Conclusion: | Thus, Nrf2 may be involved in cadmium-induced neurotoxicity in rodent model system. |
| Authors: |
Rajashree Hariprasad, OMS-II, Alabama College of Osteopathic Medicine; Benjamin Barwick, PhD, Emory University; David Alan Frank, MD, PhD, Emory University. |
| Background: |
Multiple myeloma arises due to complex alterations in cellular pathways that converge on ectopic MYC expression. The aberrant activation of the oncogenic transcription factor STAT3 has been of interest, given microenvironment IL6 secretion is known to activate STAT3 and confers therapeutic resistance. MYC translocations have led to an increased proximity of the MYC gene to super-enhancer regions, where histone acetyltransferase P300 is preferentially localized. |
| Objective: | This study aims to test the hypothesis that the combination of the P300 inhibitor GNE781 with the STAT3 inhibitor atovaquone or pyrimethamine, would synergize to inhibit MYC expression and viability of multiple myeloma cells. |
| Methods: | We utilized JJN3 myeloma cell lines, in which the endogenous MYC loci had a modified green fluorescent protein (GFP) with a two-hour half-life knocked-in using CRISPR/Cas9 (Steinberger et al, Cell Chemical Biology, 2019). Cells were treated with various dosages of EP300 and STAT3 inhibitors and were monitored over a 72-hour time course for GFP expression and cell viability. |
| Results: | Combining EP300 inhibitor at 25nM GNE781 and STAT inhibitor at 20uM atovaquone/5uM pyrimethamine results in decreased cell viability and GFP expression after 72 hours. This time frame is consistent with the transcriptional inhibitor's time requirement to inhibit transcription and for its target mRNA to decay. |
| Conclusion: | Atovaquone and pyrimethamine demonstrated viability and GFP downregulation at their lowest therapeutic doses, indicating a potential synergistic effect with the GNE781. To explore this further, additional combinations of both drugs should be tested and study the effects of different dosage combinations of these drugs on multiple myeloma cell lines. |
| Authors: | Courtney Harris OMS-II, Gordon G. MacGregor PhD, MBA |
| Background: | Infrared Sauna bathing is a passive activity that involves exposure to dry heat to simulate moderate intensity aerobic exercise. Recent studies have suggested a correlation between sauna use and increased health benefits, such as enhanced vasodilation abilities and improved blood pressure. To expand upon pre-existing knowledge of sauna-induced health benefits, this current study aims to determine the effects of sauna bathing on markers of physiological stress including, cortisol, endorphins, and heat shock proteins. |
| Design: | Twenty-five research subjects sat in an infrared sauna at 130 °F for up to 45 minutes. Blood pressure, weight, saliva, sweat, and urine were collected before and after the sauna session. Cortisol (Salimetrics 1-3002), Heat Shock protein (Enzo ADI-EKS-715) and Beta-endorphin (Abcam ab287794), were measured with commercial specific ELISA assays. |
| Results: | Saliva cortisol was 381.5 ± 131.3 ng/dl before sauna and decreased over 35% to 242.6 ± 131.3 ng/dl (p < 0.0001, n = 20) after 45 min of infrared sauna therapy. Cortisol was also secreted in the sweat at similar levels to saliva. There was no change in another two markers of increased physiological stress, saliva Beta-endorphin (p = 0.2475) and saliva HSP-70 levels (p = 0.9550). |
| Conclusion: | The lack of increase in cortisol, beta-endorphin, and HSP-70 after sauna bathing, suggests that infrared sauna therapy is not a physiological stressful experience. Infrared sauna bathing could be a safe therapy and potentially benefit patient populations to mimic exercise and in those who suffer from overproduction of cortisol. |
| Authors: | Julee A. Reitzel, OMS-II, Melinda Gevorgian, OMS-II, and Lawrence LeClaire, PhD |
| Background: | Dysregulation of kinase signaling is implicated in the uncontrolled growth and survival of cancer cells. Therefore, understanding the intricacies of these pathways is paramount for the development of targeted therapies. The mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a member of the germinal center kinase (GCK) family, has been identified as a key player in many signaling pathways associated with cancer from cell division, migration, invasion, and adhesion. MAP4K4 is encoded by a single gene, however, alternative splicing produces 55 known variants across all tissues. The role of each variant is not understood but may represent functional domains necessary for tissue specific signaling. In this study, we map these variable domains for future study. |
| Design: | MAP4K4 sequence was accessed using the NCBI gene viewer location 2q11.2. Sequence variant files were archived and analyzed using Clustal Omega. |
| Results and Conclusions: | The gene encoding MAP4K4 is located on chromosome 2 and covers ~200 kilobases. The sequence includes 33 exons, which are alternatively spliced to produce 55 variants. All variants of MAP4K4 share an N-terminal kinase domain (exons 2-11) and a C-terminal citron homology domain (CHD) (exons 25-33). Between the kinase and CHD is a variable interdomain with three varying regions of interest: 1) a charged arg/lys domain, 2) a proline-rich domain, and 3) a ser/thr region. |
| Conclusion: |
These data show that MAP4K4 alternative splicing produces unique gene products. Future experiments will seek to identify the functional role of each of these sequence variations. |
| Authors: | Melinda Gevorgian OMS - I, Julee A. Reitzel, OMS - II, and Lawrence LeClaire, PhD |
| Background: | Dysregulation of kinase signaling is implicated in the uncontrolled growth and survival of cancer cells. Therefore, understanding the intricacies of these pathways is paramount for the development of targeted therapies. Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a member of the germinal center kinase (GCK) family, has been implicated in signaling pathways associated with cancer from cell division, migration, invasion, and adhesion. MAP4K4 is a single gene expressed as 55 differentially spliced variants across human tissues and may be involved in tissue specific signaling pathways. Here, we describe a PCR-based system to identify tissue splice variants of MAP4K4. |
| Design: | Human MCF-10A (normal breast epithelial) and MDA-MB-231 (triple negative metastatic breast cancer) cell lines were cultured by standard procedures. RNA was purified with the miRNeasy Mini-Kit and quantified using a BioTek Synergy HTX Multimode Reader. cDNA was generated with the Applied Biosystems High-Capacity cDNA RT Kit. PCR was performed to amply MAP4K4 regions. PCR products were purified using an Invitrogen gel isolation kit and submitted for Sanger sequencing. |
| Results and Conclusions: | Primer sets were designed to amplify coding sequences for internal controls and variable MAP4K4 regions. Primers spanning exons 2-11 (kinase domain) are used to identify all transcripts. Primers to variants were designed to amplify exons 13-17, where 5 exons are alternatively spliced between poly-proline and arg/lys repeat regions. Primer set three span the conserved region of the interdomain between exons 18-20. |
| Conclusion: |
Together, these primer sets provide an assay to identify each variant from cell lines and isolated tissues. |
| Authors: | Alejandra Hernandez, Drew Gende OMS - I, Khushi Saigal, Cara Knasel OMS - III, Sean Meehan, Sanjoy Bhattacharya Ph.D. |
| Background: | A list of metabolites were identified by the team due to their upregulation during optic nerve regeneration. Cell Death: Measuring neuroblastoma cell death with different metabolites provides insight on which metabolite may improve cell survival. Axonal Outgrowth: Measuring axonal length of retinal ganglion cells (RGCs), whose axons make up the optic nerve, demonstrates which metabolites promote regeneration. Both analyses allow for selectivity of the most viable metabolites with potential to regenerate the optic nerve due to ocular neurodegenerative diseases. |
| Design: | The five metabolites we tested were Taurine, Histidine, Allantoin, alpha-Glycerophosphocholine, and Acetyl-L-carnitine. The SH-SY5Y neuroblastoma cell line was used. Twenty-four hours after seeding, normal media was switched with metabolite-specific media. Cell survival was evaluated on day five with a staining process using Propidium Iodide, which stains for cell death, and calcein AM, which stains for live cells. RGCs were isolated by immunopanning from p6 mouse’s retinas, seeded into a 96 well plate, and left to incubate and grow in metabolite media. After three days, the RGCs were fixed and stained by a neuronal marker, β-tubulin III. Laboratory imaging and analysis were accomplished by a Leica confocal microscope and Image J, respectively. |
| Results: | In comparison to the vehicle, none of the metabolites prevented cell death. All of the metabolites promoted neurite outgrowth. |
| Conclusion: | These results implicate that the metabolites used cannot be used for increased cell survival. However, all of them can be used for regenerating cells on the optic nerve to slow down or reverse degeneration. |