Managing Inflammation in Chronic Lymphocytic Leukemia Treatment
Clinical trials for lenalidomide as a treatment for chronic lymphocytic leukemia (CLL) were halted by the FDA due to safety concerns involving higher death rates. Tumor flare inflammatory reactions occurred in around 30% of patients in one study. In patients that did not respond to lenalidomide treatment, IL-6 was significantly increased. In contrast, lenalidomide, approved for use with multiple myeloma, decreases IL-6. In the experiment reported here, natural killer (NK) cells and chronic lymphocytic leukemia cells were treated with lenalidomide and QNZ which inhibits NF-κB, a transcription factor that enhances transcription of IL-6. A potential new therapeutic option in CLL patients may be to administer lenalidomide and QNZ to treat the cancer while inhibiting NF-κB which would lead to decreased levels of IL-6. Results of this study demonstrate a statistically significant decrease in IL-6 in a NK and CLL co-culture as compared to treatment with lenalidomide alone (p<0.05). In non-treated co-cultures, there was a statistically significant decrease in viable cells compared to separate culture, indicating cytotoxicity (p<0.05). NK cell viability determined through using CD56 fluorescent markers showed a significant increase when cells were treated with both lenalidomide and QNZ as opposed to lenalidomide alone (p<0.05).
Novel ghrelin-activated mechanism in the intestines: solution to type-2 diabetes
Ghrelin is a hormone that stimulates hunger, glucose consumption, and glucagon-like-peptides (GLP-1 and GLP-2) which are hormones that regulate insulin production. GLP-1 has a significant role in type-2 diabetes, as it mediates hyperglycemia by inducing insulin production while GLP-2 antagonizes and, therefore, regulates GLP-1 activated insulin-production. These hormones are commonly modulated by food consumption, implying a interdependence between GLP-1 and GLP-2 which has not been fully investigated. The research presented here investigated whether there exists a direct relationship between ghrelin and intestinal GLP-1 secretion. This research also aimed to investigate the involvement of glucose transporters in this GLP-1 secretion, specifically GLUT2 and GLUT5. Intestinal NCI-H716 cells, which are able to secrete GLP-1, were treated with ghrelin (0.1 - 100 nM). ELISAs were performed to measure GLP-1, GLUT2, GLUT5, and GLP-2 production following ghrelin treatments. Treatment with 0.1 nM and 1.0 nM ghrelin caused a significant increase in GLP-1 secretion by NCI-H716 cells. Treatment with 10 nM and 100 nM ghrelin caused a significant increase in GLUT2. Treatment with 0.1 nM -100 nM caused a significant decrease in GLUT5. Finally, treatment with 10 nM and 100 nM ghrelin caused a significant increase in GLP-2. The elucidation of this novel pathway, causally linking ghrelin, the glucose transporters, and the glucagon-like peptides, suggests the therapeutic potential of ghrelin-based therapies for type-2 diabetes patients.
Multidisciplinary investigation of novel estrogenic regulation of breast cancer metabolism
A novel pathway was elucidated in which 17β-estradiol was found to induce apoptosis and metabolic disruption via the post-translational protein acetylation of HIF-1α. This pathway was found to be regulated by HATs and HDACs in estrogen receptor positive (ER+) MCF-7 breast cancer cells. 17β-estradiol significantly downregulated HDAC4, increased acetylation of HIF-1α and subsequently decreased the quantity of HIF-1α protein (p≤0.05). Contrary to its negative effect on HIF-1α expression, 17β-estradiol had significant positive effects on PFK and PKM2 protein expression (p≤0.05), suggesting a compensation mechanism induced by metabolic disruption. This pathway was exploited to introduce a novel combinational therapy (17β-estradiol and sodium butyrate) for ER+ breast cancers. Although 17β-estradiol and sodium butyrate at the selected concentrations were not able to significantly affect cell growth individually, when combined, they significantly decreased cell viability in MCF-7 most likely through the acetylation and degradation of HIF1-α protein (p≤0.05). Bioinformatics analysis supported this pathway. 17β-estradiol significantly increased RNA expression of PKM2, PFK and HAT genes; ESR1, the estrogen receptor 1 gene, was significantly correlated to HIF-1α, PFK, HAT and various HDAC genes (p≤0.05). A novel genetic profile, including genes implicated in this investigation and other major genes involved in cancer metabolism, was used to construct a SVM model to predict ER+ breast cancer recurrence. This model performed similarly to those reported in scientific literature regardless of p53 status, lymph node status, history of endocrine treatment and tumor size. This indicates that this proposed mechanism has significant implications as a high-potential therapeutic and prognostic target.
A Novel Method to Manipulate the Osteoblast Differentiation Process
Zeolite is a microporous, aluminosilicate mineral known for its variety of benefits. The molecule carries a negative charge which attracts positively charged metals, toxins and ions (Zeoponix, 2000). In this study the osteoblast cells were treated with the chelator, zeolite, in various concentrations ranging from 0.1 ug/mL to 100 ug/mL. Zeolite proved to induce a dose-dependent increase in cell proliferation. A potential mechanism for this increase in viability is due to the zeolite s ability to increase DNA synthesis of normal osteoblast cells. The results of this study suggested that proliferation of the cells was notable in the concentrations of 0.1u/mL, 1.0 u/mL, 10 u/mL and 100 u/mL with a significance of p<0.05 or p<0.01. Alkaline phosphatase staining on the cells was also done to show qualitative visual evidence of maturation (differentiation). Furthermore, the staining showed quantitative evidence of a decrease in alkaline phosphatase (ALP) as the concentrations of zeolite increased. Past literature has stated that the maturation phase, the second phase, of the three-step differentiation process is characterized by maximal expression of ALP (PromoCell, 2013). Enzyme-linked immunosorbent assays (ELISAs) were done to determine that the osteoblast cells dedifferentiated when treated with zeolite. Transmission Electron Microscope and Scanning Electron Microscope were used to establish the incapability of the zeolite to enter the cell and allowed for an elemental analysis of the zeolite, respectively. The zeolite s structure allows for a high affinity for adsorption, the adhesion of a substance to a surface. It can be used as an adsorbent medium for separation of phospholipids and their mixtures in affinity chromatography (Shrivastava & Prakash, 1986). Zeolite s framework, composed of a three-dimensional network of AlO4 and SiO4 tetrahedra, is deduced to aid in the adherence of the zeolite to phospholipids. The composition of zeolite through the elemental evaluation indicates possible mechanisms by which the elements in the particle interact with the cell membrane. The results of this study provide support for zeolite s potential as an effective mitogen and its ability to dedifferentiate osteoblast cells allows for prospective osteoporosis therapy and regeneration.
Dong Bien Kwon
The Effect of Catechin on Insulin Sensitivity of Hep G2 Cells
Insulin resistance, the physiological condition by which cells do not respond effectively to the insulin hormone, is a primary characteristic of diabetes mellitus type 2. In many studies, it has been suggested that catechins, a family of flavonoids, may increase insulin sensitivity through an increase in the proteins of the insulin signaling pathway; however, their mechanism of action is not completely understood. In this study, HepG2 liver carcinoma cells were cultured under diabetic conditions and treated with various concentrations of vitexin-2-O-rhamnoside, a previously uninvestigated flavonoid. Treatment with vitexin-2-O-rhamnoside alone resulted in a significant increase in the insulin receptor protein (INSR), however when challenged with insulin the receptor protein concentration decreased (p<0.05). The concentration of phosphorylated phosphatidylinositol 3-kinase (pPI3K) increased following the insulin challenge indicative of increased insulin sensitivity (p<0.05). The phosphorylation and inactivation of glycogen synthase kinase 3 beta (GSK-3β) at Ser9 decreased with and without insulin treatment (p<0.05). Modulations in the levels of these proteins are indicators of the ability of vitexin-2-O-rhamnoside to mediate insulin sensitivity in HepG2 cells.
The Cellular Pathways Mediating the Anti-Tumor Effects of Paeonia suffruticosa on Human Breast Cancer Cells
Extracts of Paeonia suffruticosa, traditionally used in herbal medicine to activate blood flow, have also demonstrated anti-tumor, anti-inflammatory and analgesic effects of unknown mechanisms. The present investigation studied and elucidated potential apoptotic mechanisms of aqueous extract of P. suffruticosa (0.3-8mg/mL) on triple negative breast cancer cells (MDA-MB-231). Analysis of cell viability revealed a biphasic dose-response with proliferation at low concentrations(<0.6mg/mL) and decreased viability at high concentrations(p<0.01). IL-6, IL-2, and TNF-alpha secretion levels were inversely related to viability(p<0.05). Cell cycle arrest at G0/G1 was also demonstrated with potential to induce apoptosis. IL-24, a tumor suppressor protein shown to be correlated with increased IL-2 expression, increased at 2.5mg/mL and 4.0mg/mL (p<0.05), indicating a significant cellular mechanism. The decrease in Bcl-2:Bax ratio(p<0.01) indicated the induction of apoptosis. Decrease in Fas Ligand production and increase in caspase 3/7 and 9 signified an intrinsic mechanism of action of P. suffruticosa (p< 0.05). The reduction of pAKT (p<0.05) suggested the extract s mechanism of cell death through JNK1/2 pathway. Evaluation of HPLC results revealed penta-O-galloyl-beta-D-glucose (PGG), benzoic acid and paeonol as the active constituents. Their effects on viability showed that PGG and paeonol may be the active ingredients prompting apoptosis in MDA-MB-231. The whole extract of P. suffruticosa and PGG did not decrease viability in human keratinocyte, demonstrating its selective effect on cancer cells. It was concluded that the aqueous extract of P. suffruticosa, and/or its active components might be a promising agent for triple negative breast cancer treatment or as an adjunct to conventional therapies.
A targeted method of reducing the adverse effects of chemotherapy
A problem with chemotherapy is the damage done to healthy tissue. In vitro studies suggest that increasing adhesion to extracellular matrix (EM) components, such as fibronectin, protects healthy cells from the adverse effects of chemotherapy, however potentially rendering cancer cells protected as well. This study investigated the potential use of the Gonadotropin Releasing Hormone Receptor (GnRHR) as a target for increasing EM adhesion in only healthy ovarian tissue, thus providing targeted protection. While healthy ovarian cells express the GnRH receptor, approximately 20% of ovarian cancers are GnRHR-negative. Therefore, the effect of GnRH on both GnRHR-positive and GnRHR-negative cell lines was investigated. Adhesion to fibronectin was measured after pretreatment with GnRH in both OVCAR-3, a GnRHR+ cell line, and SK-OV-3, a GnRHR- cell line. In OVCAR-3 there was statistically significant increase in adhesion to fibronectin (p<0.05), and an increase in the beta1-integrin component of the fibronectin receptors when measured through ELISA (p<0.05). To investigate protection from chemotherapy, cells were pretreated with GnRH, given 2h to adhere to fibronectin, and then treated with cisplatin. Viability was measured after 24h (MTS assay) and increased in the GNRHR + cell line, OVCAR-3, following the same trend as adhesion to fibronectin (p<0.05). In SK-OV-3 survival did not significantly increase from pretreatment with GnRH, demonstrating the efficacy of this method in protecting only GnRHR+ cells (p<0.05). When plated onto plastic, survival did not increase in OVCAR-3 suggesting that protection from cisplatin is mediated by a cellular pathway activated through integrin binding to fibronectin.
CXCR4/SDF-1α Signaling as a Target of Microenvironmental Regulation of Metastasis in Non-Small Cell Lung Cancer
The metastatic nature of non-small cell lung cancer (NSCLC) is often associated with treatment failure at advanced stages when it is typically diagnosed. Many treatments focus on cell-intrinsic mechanisms implicated in metastasis, but crosstalk in the microenvironment between tumor and nonmalignant cells also modulates progression. Addressing intracellular and extracellular mechanisms of metastasis is critical to improve treatment efficacy. This study investigated roles of CXCR4/SDF-1a signaling in microenvironmental regulation of metastatic potential and pro-tumor crosstalk in NSCLC. An off-label use of CXCR4 antagonist AMD3100 as a treatment for NSCLC (A549) was proposed. MMP-9, responsible for extracellular matrix remodeling for invasion, decreased with treatment (p<0.05). NFkB, involved in MMP-9 expression also decreased (p<0.05). AMD3100 (1.25-320ug/mL) inhibited A549 adhesion to marrow stromal cells (M2-10B4) (p<0.05), necessary for SDF-1a promotion of cancer cell survival, and adhesion to vascular cell adhesion protein (VCAM-1) (p<0.05) possibly by reducing CXCR4-mediated activation of integrin VLA-4 in A549 (p<0.05). VLA-4/VCAM-1 signaling was identified as a potential regulator of CXCR4 mediated cell adhesion in NSCLC. AMD3100 reduced VEGF, important in angiogenesis (p<0.05). Also investigated was the role tumor associated macrophages (TAMs) may play in promoting cancer progression, specifically invasion, in CXCR4+ NSCLC. Results suggest TAMs may play a greater role in promoting cell invasion regardless of CXCR4/SDF-1a signaling, indicating the significance of CXCR4-mediated TAM crosstalk as a target of tumor cell invasion. Findings suggest the therapeutic potential of disrupting CXCR4/SDF-1a signaling with AMD3100, which may act as a multi-targeted treatment approach for NSCLC by disrupting CXCR4 microenvironmental regulation of metastasis.
The Effect of Metformin on MDA Breast Cancer Cells
Recent studies have shown that metformin, a drug used by patients with type 2 diabetes, may possess antitumor properties through the activation of AMP-activated protein kinase. However, there are other pathways regulating cellular proliferation, leading to uncertainty regarding its mechanism of action, specifically in breast cancer. In this research, MDA-MB-231 breast cancer cells were targeted with metformin, and processes, such as autophagy and apoptosis, were investigated. An MTS assay and measurement of caspase 3/7 demonstrated that metformin significantly reduced cell proliferation (p<0.05) and induced programmed cell death (p<0.05). The detection of autophagic vacuoles demonstrated that metformin led to a significant increase in autophagy in MDA-MB-231 cells (p<0.05), potentially mediating this cell death. An increase in AMP-activated protein kinase (p<0.05) with mTOR only increasing at the highest concentration (p<0.05) suggested that metformin may induce apoptosis through an mTOR-independent AMPK pathway to autophagy. Furthermore, the inhibition of Akt (p<0.05), a mediator in an alternate pathway regulating cell growth, suggests metformin could also lead to apoptosis through additional mechanisms. The measurement of ERK showed no significant trend demonstrating that it is not contributing to metformin s mechanism of action. The downstream targets of AMPK, such as p27 and p53, may also play a role in metformin s mechanism of action with regard to inducing autophagy. Determining the mechanisms through which metformin decreases cell proliferation and exploiting them may offer a viable treatment for breast cancer.
High Dose Estrogen Therapy: A Novel Mechanism of Action in Treating Estrogen Receptor Negative Breast Cancers
Triple negative breast cancer (TNBC) is a highly aggressive breast cancer that is difficult treat, since it lacks estrogen, progesterone, and human epidermal growth factor receptors. Clinical trials with high dose estrogen therapy have shown that it has potential to become a chemotherapeutic treatment. However, researchers are hesitant to use it, since its mechanism of action is unknown. The goal of this study was to elucidate the mechanism of action of high dose estrogen therapy in reducing proliferation and inducing apoptosis in TNBC. In this study, the effect of high dose estrogen therapy was studied in relation to protein disulfide isomerase family A, member 3 (PDIA3), an endoplasmic reticulum (ER) protein whose is involved in folding proteins. PDIA3 was inhibited by high dose estrogen therapy (p<0.05), thus preventing the activation of downstream targets, such as the mammalian target of rapamycin complex 1 (p<0.05), which is involved in inducing cellular proliferation. Furthermore, the inhibition of PDIA3 led to a significant increase in unfolded proteins in the ER (p<0.05), thus causing ER stress. Heightened levels of ER stress forced the cell to activate caspase 9 (p<0.05), which is a death enzyme involved in apoptosis induced through an intrinsic mechanism. These results provide support for high dose estrogen therapy as a potential chemotherapy and the importance of targeting PDIA3 in TNBC.
Inhibiting eEF2K-Mediated Autophagy and its Effect on PANC-1 Cell Proliferation
Metabolic adaptation is crucial for cancer cell survival during nutrient deprivation (ND). In normal cells, ND leads directly to endoplasmic reticulum stress and induces both autophagy and apoptosis unless adaptive mechanisms are undertaken. This study elucidated a survival mechanism employed by the pancreatic cancer cell line, PANC-1, and evaluated the potential of targeting the adaptive molecule produced during ND. eEF2 kinase, one such protein expressed in cancer cells, has previously been shown to promote survival during ND by inhibiting eEF2 which is responsible for protein elongation, an energy demanding process. NH125 is a molecule that inhibits eEF2 kinase and was therefore evaluated as a potential inhibitor of the cancer cell survival mechanism. PANC-1 viability was significantly reduced under ND deprivation and was further reduced when ND was combined with NH125 (p < 0.05). Caspase 3/7 also increased over increasing concentrations of NH125 (p < 0.05). eEF2 kinase was inhibited across the concentrations of NH125 in a dose response fashion (p<0.05). In addition, a significant decrease in autophagy as a function of p62 levels was shown over increasing concentrations of NH125 (p < 0.05) demonstrating that eEF2 kinase is essential for inducing autophagy used for survival in PANC-1 cells and for determining their fate under ND. Thus, this study indicated that eEF2k is a necessary protein produced by PANC-1 cancer cells and can be targeted to cause an increased apoptotic response by reducing autophagy, thereby potentiating the effectiveness of ER stress-inducing agents against cancer.
Jerome Fratello & Christopher Capasso
The Effect of Tetrachloroethylene Bioremediation by Pseudomonas Putida on Vigna Radiata
This experiment aims to evaluate the biodegradation of the chlorocarbon, Tetrachloroethylene (PCE) by Pseudomonas putida, a bacteria strand with metabolic processes that serve to dechlorinate chlorocarbons. Reductive dechlorination of PCE involves the step-wise replacement of individual chlorine atoms with hydrogen atoms, resulting in the formation of breakdown products. Vigna radiata was used as the monitor for determining how tetrachloroethylene and its degradation products impact plant growth. In addition to an analysis of growth versus exposure, this experiment sought to evaluate the benefits and risks of PCE biodegradation by comparing the number of open and closed stomata among plant samples with scanning electron microscopy. Stomata were considered because abscisic acid induced stomatal closure reduces transpiration to prevent further water loss or limit the intake of harmful pollutants. The bacteria were first cultured in LB broth, and were inoculated on a biweekly basis. Spectroscopic assays and microplate readings were performed to determine the proliferation and death of bacteria at varying concentrations of PCE. Upon the completion of preliminary testing, bacteria samples were exposed to varying concentrations of PCE, and the concentration of free chlorine was measured using DPD free chlorine testing. The plants were exposed to PCE contaminated water after two weeks of uninterrupted development and measurements were performed daily. Finally, headspace gas chromatography was used to confirm the breakdown of tetrachloroethylene. The results of this project indicate that Pseudomonas putida degrades tetrachloroethylene and the breakdown products of tetrachloroethylene have a negative impact on the growth of Vigna radiata.
Cellulose Binding Domains and Free Glucose Availability for Biofuel Production
The dwindling supply of fossil fuels is one of the foremost issues plaguing our society. Biofuels remain an option, but without improvement of either the process or the supply of raw materials used to produce them, biofuels are not a viable option as a replacement. This study investigated the potential of a novel protein, cellulose binding domain (CBD), as an enhancer of free glucose availability for biofuel production in the model system V. radiata, or the common mung bean. The biomechanical and morphological consequences of CBD were measured. V. radiata was grown hydroponically in concentrations of CBD over a one-week period. On days five and eight beans were massed and measured length-wise. Free glucose concentration per unit mass was determined and increased significantly in a dose response fashion over the growth period (p<0.05). CBD in high doses caused an increase of over 550% in total free glucose ratio as compared to the control (p<0.05). Material stiffness was measured with a three-point flexural test and was found to be modulated significantly by CBD (p<0.05). SEM and TEM imaging demonstrated that CBD caused a qualitative morphological decrease in crystallinity in cellulose fibers. Eureqa Formulize was used to generate reliable mathematical models of the data. CBD also caused a protective effect on V. radiata in a dose response fashion (p<0.05) when plants were subjected to sublethal concentrations of silver nitrate solution as a model toxin. In conclusion, CBD represents a viable method of improving cellulosic materials for the production and sustainability of biofuels.
Hee Jae (Ana) Song
A Novel Approach to Traumatic Brain Injury: Antidepressant Induced Neuroprotection
Antidepressants have the potential to improve the condition of neuroglia injured during traumatic brain injury (TBI). Sertraline is an antidepressant used to treat depression and other psychiatric disorders. In this study, injured murine astrocytes, macroglia that play a vital role in the repair and scarring process of the brain and spinal cord following TBI, were treated with sertraline prior to injury. Increased levels of MMP-9 (p<0.05) and GFAP (p<0.01) were found after treatment, demonstrating astrocytic response to cellular reconstruction. Decreased surface area of the injury in scratched astrocytes at optimal concentrations of sertraline demonstrated repair efficiency (p<0.05). Levels of GDNF and BDNF, which contribute to neuroprotection and proliferation, demonstrated an increased expression after treatment (p<0.05). Levels of VEGF, which contribute to repair, significantly increased (p<0.05) in injured/uninjured cell cultures. A proliferative effect (p<0.05) was neutralized in injured/uninjured astrocytes across all concentrations when AAL-993 (VEGF receptor inhibitor) or AZD-4547 (FGFR2 inhibitor) were administered, demonstrating antidepressant function through these receptors. The upregulation of cAMP response element-binding protein (CREB) in injured astrocytes (p<0.05) after treatment showed direct correlation with levels of BDNF, GDNF, and VEGF. Results suggest that a previously unrecognized link between antidepressant treatment, CREB, and angioglioneurins may demonstrate neuroprotection and regeneration.