Maria P. Lambros

The Cystine/Glutamate Antiporter System xc− in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities

The antiporter system x(c)(-) imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system x(c)(-) is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x(c)(-), including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x(c)(-). Moreover, the roles of system x(c)(-) in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system x(c)(-) inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System x(c)(-) is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x(c)(-) in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS.

New high-performance liquid chromatographic method for amphotericin B analysis using an internal standard.

A simple and reproducible HPLC method for the analysis of amphotericin B (AmB) in serum, lung and liver using natamycin as the internal standard was developed. AmB and natamycin were extracted from serum, lung and liver and were separated using an isocratic elution from C18 reversed-phase column. The mobile phase consisted of acetonitrile-10 mM acetate buffer pH 4.0 (37:63, v/v). The HPLC system had two detectors in series. One was set at 303 nm and the other at 383 nm for the detection of natamycin and AmB, respectively. The retention times of AmB and natamycin were 15 and 6 min, respectively. The recovery efficiency was 96%-70%. The limit of quantification was 0.1 microgram/ml. The assay was reproducible, the within-day coefficient of variation (n = 6) was < 8% for serum, lungs and liver. The between-day variability (n = 6) was < 7.7% for serum, liver and lungs at 1 microgram/ml or 1 microgram/g tissue concentration. The assay was linear within the range 1-40 micrograms/ml (r2 = 0.99).

Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first step towards the development 3-D tissue as a screening tool.

INTERACTION OF A SYNTHETIC PEPTIDE BASED ON THE NEUTROPHIL-DERIVED ANTIMICROBIAL PROTEIN CAP37 WITH DIPALMITOYL-PHOSPHATIDYLCHOLINE MEMBRANES

CAP37, a cationic antimicrobial protein of Mr 37 kDa is constitutively expressed in human neutrophils. A synthetic peptide, CAP37 P20-44, corresponding to amino acid residues 20 through 44 of the native CAP37 molecule has been shown to mimic the antimicrobial activity of the native protein. An analog of peptide CAP37 P20-44 was synthesized in which the cysteine residues at positions 26 and 42 were replaced with serine residues (CAP37 P20-44Ser). This resulted in a peptide that no longer exhibited bactericidal activity. The effect of different concentrations of the active CAP37 peptide, CAP37 P20-44, and its inactive analog, CAP37 P20-44Ser, on artificial lipid membranes composed of dipalmitoyl phosphatidylcholine (DPPC) was studied using small-angle X-ray scattering and differential scanning calorimetry. The results indicated that CAP37 P20-44 perturbs the periodicity of the lamellar structure as shown by small angle X-ray diffraction, while the effect of the inactive peptide is not as strong. Differential scanning calorimetry further confirms that CAP37 P20-44 interacts with lipid membranes as indicated by increased width of the transition and decreased peak height. Moreover, it completely abolishes the pretransition temperature of the DPPC membranes. The effect of the inactive peptide, CAP37 P20-44Ser on the thermotropic properties of DPPC was small. These studies suggest that CAP37 perturbs the lamellar structure of lipid bilayers and further suggests that the antibiotic action of the molecule may be through its interactions with the lipid components of the Gram negative bacterial membrane.

Molecular Signatures in the Prevention of Radiation Damage by the Synergistic Effect of N-Acetyl Cysteine and Qingre Liyan Decoction, a Traditional Chinese Medicine, Using a 3-Dimensional Cell Culture Model of Oral Mucositis

Qingre Liyan decoction (QYD), a Traditional Chinese medicine, and N-acetyl cysteine (NAC) have been used to prevent radiation induced mucositis. This work evaluates the protective mechanisms of QYD, NAC, and their combination (NAC-QYD) at the cellular and transcriptional level. A validated organotypic model of oral mucosal consisting of a three-dimensional (3D) cell tissue-culture of primary human keratinocytes exposed to X-ray irradiation was used. Six hours after the irradiation, the tissues were evaluated by hematoxylin and eosin (H and E) and a TUNEL assay to assess histopathology and apoptosis, respectively. Total RNA was extracted and used for microarray gene expression profiling. The tissue-cultures treated with NAC-QYD preserved their integrity and showed no apoptosis. Microarray results revealed that the NAC-QYD caused the upregulation of genes encoding metallothioneins, HMOX1, and other components of the Nrf2 pathway, which protects against oxidative stress. DNA repair genes (XCP, GADD45G, RAD9, and XRCC1), protective genes (EGFR and PPARD), and genes of the NFκB pathway were upregulated. Finally, tissue-cultures treated prophylactically with NAC-QYD showed significant downregulation of apoptosis, cytokines and chemokines genes, and constrained damage-associated molecular patterns (DAMPs). NAC-QYD treatment involves the protective effect of Nrf2, NFκB, and DNA repair factors.

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

Genome wide expression after different doses of irradiation of a three-dimensional (3D) model of oral mucosal

We evaluated a three-dimensional (3D) human oral cell culture that consisted of two types of cells, oral keratinocytes and fibroblasts as a model of oral mucositis which is a debilitating adverse effect of chemotherapy and radiation treatment. The 3D cell culture model was irradiated with 12 or 2 Gy, and total RNA was collected 6 h after irradiation to compare global gene expression profiles via microarray analysis. Here we provide detailed methods and analysis on these microarray data, which have been deposited in Gene Expression Omnibus (GEO): GSE62395.

Abstract 2191: Imaging the kinetics of targeted nanoparticles to pancreatic cancer

Purpose: The purpose of this work is to image the kinetics of targeted lipid nanoparticles to an SCL transporter of pancreatic cancer using a near infrared (NIR) fluorescent dye DyLight® 747 and a mouse orthotopic model of fluorescent pancreatic cancer. Methods: Initially and before the in vivo experiments, the uptake of nanoparticles by human pancreatic cancer cells was evaluated in vitro using fluorescent microscopy and flow cytometry. A quantity of 1.5 x10 5 PANC-1, MIA PaCa, or BxPC-3 cells/ml was placed in each well of a six-well plate and incubated for 5 hours with different formulations of fluorescent lipid targeted and non-targeted nanoparticles encapsulation daunorubicin which has natural fluorescence. After incubation, the growth media were removed and the cells were washed with PBS. The uptake of the fluorescent lipid nanoparticles by the cancer cells was evaluated an EVOS® microscope. All the in vitro experiments were done in triplicate. For the in vivo targeting studies, nude mice (Balb/c Ola Hsd-Fox1 nu) were implanted orthotopically (in the tail of the pancreas) with 3 x10 6 PANC-1 cells expressing GFP. The injection volume for the implantation was 50 microliters and contained 3 x10 6 PANC-1-GFP cells mixed with serum free media and HC Matrigel (1:1). One month after the orthotopic implantation, the implanted pancreatic tumor was visualized in live, anesthetized animals using UVP Scientia (UVP, Upland, CA). The animals were separated into three groups (three animals in each group) and injected via the tail vein as follows: Group A, was injected with only the fluorescent dye, Dylight 747; group B was injected with fluorescent non-targeted nanoparticles; and group C was injected with fluorescent targeted nanoparticles. Results: Both the flow cytometry and fluorescence microscopy data show a significantly higher uptake of the targeted lipid nanoparticles compared to the non-targeted ones. The imaging of the fluorescent lipid nanoparticles in live mice shows that both targeted and nontargeted nanoparticles reach the pancreatic tumor. The nontargeted nanoparticles reside less than 48 hours on the tumor, whereas the targeted nanoparticles can reside for 96 hours on the tumor. The non-encapsulated fluorescent dye did not reach the tumor and was excreted in the urine. Conclusion: The results show that Dylight® 747 can be used to image the kinetics of a drug delivery system in a live animal. Furthermore while both lipid nanoparticles reach the tumor, the targeted lipid nanoparticles reside significantly longer on the tumor and this may translate to a better therapeutic outcome. Citation Format: Jonathan Moreno, Qin qin Fei, Maria P. Lambros. Imaging the kinetics of targeted nanoparticles to pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2191.

Interaction of Resveratrol with Lipid Membranes

Background: Resveratrol is a phytoalexin synthesized by plants. It has antioxidant properties and is a popular nutritional supplement. Beneficial properties of resveratrol, such as, anticancer and anti-inflammatory properties have been reported. Resveratrol is a constituent of red wine and found in the skin of red grapes. In order to understand the interaction between resveratrol and biological membranes, we evaluated the effect of resveratrol on model lipid membranes using differential scanning calorimetry (DSC) and computational studies. Methods: Phospholipids such as, Dilauroylphosphatidylcholine (DLPC), Dimyristoyphosphatidylcholine (DMPC), Dipalmitoylphosphatidyl choline (DPPC), Distearoylphosphatidylcholine (DSPC), and 1-palmitoyl-2-oleyl phosphatidylcholine (POPC) were purchased from Avanti Polar Lipids (Alabaster, Alabama). Each phospholipid was mixed with resveratrol at different molar ratios, phospholipid: resveratrol, 10:1, 10:3 and 10:5. Computational simulations were also performed to evaluate the interactions of DSPC and resveratrol.Results: Resveratrol abolishes only the transition of the DLPC, which is the shortest phospholipid of those tested. It reduces the transition temperature for all the other phospholipids even at the lowest ratio tested, phospholipid: resveratrol, 10:1. Resveratrol reduces the transition temperature of DSPC from 55 °C to 51 °C. Furthermore, using DSC, we also observed another transition, a sharp exothermic peak above 275 °C in the interaction of resveratrol with DSPC. We performed computational simulations of the DSPC membrane at different temperatures with and without resveratrol. The simulation indicates that resveratrol affects the transition temperature of the DSPC, which is in agreement with our DSC data. In conclusion, our data indicate that resveratrol abolishes the transition of DLPC and acts as a plasticizer for phospholipids with longer fatty acyl chains.

Transcriptional profiling of radiation damage and preventive treatments in a 3-dimensional (3D) human cell culture model of oral mucositis

Cancer patients who receive radiation are often afflicted by oral mucositis, a debilitating disease, characterized by mouth sores and difficulty in swallowing. Oftentimes, cancer patients afflicted with mucositis must stop life-saving therapies. Thus it is very important to prevent mucositis before it develops. Using a validated organotypic model of human oral mucosa, a 3-dimensional cell culture model of human oral keratinocytes, it has been shown that a mixture (NAC–QYD) of N-acetyl cysteine (NAC) and a traditional Chinese medicine, Qingre Liyan decoction (QYD), prevented radiation damage (Lambros et al., 2014). Here we provide detailed methods and analysis of microarray data for non-irradiated and irradiated human oral mucosal tissue with and without pretreatment with NAC, QYD and NAC-QYD. The microarray data been deposited in Gene Expression Omnibus (GEO): GSE62397. These data can be used to further elucidate the mechanisms of irradiation damage in oral mucosa and its prevention.