Louisville, KY, United States
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Liu Y.,James Graham Brown Cancer Center | Liu Y.,University of Louisville | Dean D.C.,James Graham Brown Cancer Center | Dean D.C.,University of Louisville
Cell Cycle | Year: 2010

Many tumors have been found to contain a subset of cells referred to as cancer stem cells. As opposed to the remainder of the tumor, these cells are undifferentiated - they do not express markers of differentiation and they can re-express stem cell specification genes. The cells can divide asymmetrically to yield differentiated cells as well as cells comprising the original heterogeneous population of the tumor while maintaining their number. Because cancer stem cells display some properties of stem cells, it has been presumed that the presence of such cells in tumors reflects a stem cell origin for cancer. However, recent studies suggest that cancer can originate with outgrowth of differentiated somatic cells, and that cells with properties of cancer stem cells can be generated as tumors progress. Appearance of such cells may be linked to reprogramming of differentiated somatic cells to a stem cell-like phenotype by overexpression of transcription factors involved in epithelial-mesenchymal transition (EMT). We discuss recent studies from our group and other laboratories linking cell out-growth in tumors to inhibition of the RB1 pathway, loss of cell contact inhibition and generation of undifferentiated cancer stem-like cells from somatic cells. And, we compare this pathway to that arising with introduction of mutant Ras in cells. © 2010 Landes Bioscience.


Siow D.L.,James Graham Brown Cancer Center | Wattenberg B.W.,James Graham Brown Cancer Center | Wattenberg B.W.,University of Louisville
Journal of Biological Chemistry | Year: 2012

The mammalian ORMDL proteins are orthologues of the yeast Orm proteins (Orm1/2), which are regulators of ceramide biosynthesis. In mammalian cells, ceramide is a proapoptotic signaling sphingolipid, but it is also an obligate precursor to essential higher order sphingolipids. Therefore levels of ceramide are expected to be tightly controlled. We tested the three ORMDL isoforms for their role in homeostatically regulating ceramide biosynthesis in mammalian cells. Treatment of cells with a short chain (C6) ceramide or sphingosine resulted in a dramatic inhibition of ceramide biosynthesis. This inhibition was almost completely eliminated by ORMDL knockdown. This establishes that the ORMDL proteins mediate the feedback regulation of ceramide biosynthesis in mammalian cells. The ORMDL proteins are functionally redundant. Knockdown of all three isoforms simultaneously was required to alleviate the sphingolipid-mediated inhibition of ceramide biosynthesis. The lipid sensed by the ORMDL-mediated feedback mechanism is medium or long chain ceramide or a higher order sphingolipid. Treatment of permeabilized cells with C6-ceramide resulted in ORMDL-mediated inhibition of the rate-limiting enzyme in sphingolipid biosynthesis, serine palmitoyltransferase. This indicates that C6-ceramide inhibition requires only membrane-bound elements and does not involve diffusible proteins or small molecules.Wealso tested the atypical sphingomyelin synthase isoform, SMSr, for its role in the regulation of ceramide biosynthesis. This unusual enzyme has been reported to regulate ceramide levels in the endoplasmic reticulum. We were unable to detect a role for SMSr in regulating ceramide biosynthesis. Wesuggest that the role of SMSr may be in the regulation of downstream metabolism of ceramide. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.


Jala V.R.,James Graham Brown Cancer Center | Radde B.N.,University of Louisville | Haribabu B.,James Graham Brown Cancer Center | Klinge C.M.,University of Louisville
BMC Cancer | Year: 2012

Background: G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer.Methods: The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed.Results: Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue.Conclusion: The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression. © 2012 Jala et al.; licensee BioMed Central Ltd.


Wilkerson D.C.,University of Louisville | Sankar U.,University of Louisville | Sankar U.,James Graham Brown Cancer Center
International Journal of Biochemistry and Cell Biology | Year: 2011

Mitochondria have long been recognized as cellular energy power houses that also regulate cellular redox signaling to arbitrate cell survival. Recent studies of mitochondria in stem cells (SCs) demonstrate that they have critical roles beyond this traditional view. Embryonic (E) SCs, termed pluripotent for their ability to differentiate into all cell types within an organism, maintain a limited number of morphologically undifferentiated (electron translucent and poorly formed cristae) mitochondria. As these cells differentiate, their mitochondria undergo a tightly choreographed gain of number, mass and morphological complexity. Therefore, mechanisms that regulate mitochondrial growth, localization, division and partition must play active roles in the maintenance of pluripotency and execution of differentiation. Aberrant mitochondrial dynamics are associated with a plethora of human disorders, for which SCs hold curative potential. Hence, a comprehensive understanding of the mechanisms that regulate mitochondrial dynamics and function in SCs and their overall relationship to the maintenance of pluripotency is pivotal for the progression of therapeutic regenerative medicine. © 2011 Elsevier Ltd. All rights reserved.


Spencer W.A.,James Graham Brown Cancer Center | Jeyabalan J.,James Graham Brown Cancer Center | Kichambre S.,James Graham Brown Cancer Center | Gupta R.C.,James Graham Brown Cancer Center | Gupta R.C.,University of Louisville
Free Radical Biology and Medicine | Year: 2011

There is increasing evidence supporting a causal role for oxidatively damaged DNA in neurodegeneration during the natural aging process and in neurodegenerative diseases such as Parkinson and Alzheimer. The presence of redox-active catecholamine neurotransmitters coupled with the localization of catalytic copper to DNA suggests a plausible role for these agents in the induction of oxidatively generated DNA damage. In this study we have investigated the role of Cu(II)-catalyzed oxidation of several catecholamine neurotransmitters and related neurotoxins in inducing oxidatively generated DNA damage. Autoxidation of all catechol neurotransmitters and related congeners tested resulted in the formation of nearly a dozen oxidation DNA products resulting in a decomposition pattern that was essentially identical for all agents tested. The presence of Cu(II), and to a lesser extent Fe(III), had no effect on the decomposition pattern but substantially enhanced the DNA product levels by up to 75-fold, with dopamine producing the highest levels of unidentified oxidation DNA products (383 ± 46 adducts/106 nucleotides), nearly 3-fold greater than 8-oxo-7,8-dihydro-2′- deoxyguanosine (122 ± 19 adducts/106 nucleotides) under the same conditions. The addition of sodium azide, 2,2,6,6-tetramethyl-4-piperidone, tiron, catalase, bathocuproine, or methional to the dopamine/Cu(II) reaction mixture resulted in a substantial decrease (> 90%) in oxidation DNA product levels, indicating a role for singlet oxygen, superoxide, H2O 2, Cu(I), and Cu(I)OOH in their formation. Whereas the addition of N-tert-butyl-α-phenylnitrone significantly decreased (67%) dopamine-mediated oxidatively damaged DNA, three other hydroxyl radical scavengers, ascorbic acid, sodium benzoate, and mannitol, had little to no effect on these oxidation DNA product levels, suggesting that free hydroxyl radicals may have limited involvement in this dopamine/Cu(II)-mediated oxidatively generated DNA damage. These studies suggest a possible contributory role of oxidatively generated DNA damage by dopamine and related catechol neurotransmitters/neurotoxins in neurodegeneration and cell death. We also found that a naturally occurring broad-spectrum antioxidant, ellagic acid, was substantially effective (nearly 50% inhibition) at low doses (1 μM) at preventing this dopamine/Cu(II)-mediated oxidatively generated DNA damage. Because dietary ellagic acid has been found to reduce oxidative stress in rat brains, a neuroprotective role of this polyphenol is plausible. © 2010 Elsevier Inc. All rights reserved.


Siow D.,James Graham Brown Cancer Center | Wattenberg B.,James Graham Brown Cancer Center | Wattenberg B.,University of Louisville
Biochemical and Biophysical Research Communications | Year: 2014

Histone deacetylase 6 (HDAC6) controls acetylation of a number of cytosolic proteins, most prominently tubulin. Tubacin is a small molecule inhibitor of HDAC6 selected for its selective inhibition of HDAC6 relative to other histone deacetylases. For this reason it has become a useful pharmacological tool to discern the biological functions of HDAC6 in numerous cellular processes. The interest of this laboratory is in the function and regulation of sphingolipids, a family of lipids based on the sphingosine backbone. Sphingolipid biosynthesis is initiated by the rate limiting enzyme serine palmitoyltransferase (SPT). Sphingolipids have critical and diverse functions in cell survival, apoptosis, intra- and intercellular signaling, and in membrane structure. In the course of examining the role of HDAC6 in the regulation of sphingolipid biosynthesis we observed that tubacin strongly inhibited de novo synthesis whereas HDAC6 knockdown very moderately stimulated synthesis. We resolved these seemingly contradictory results by demonstrating that, surprisingly, tubacin is a direct inhibitor of SPT activity in permeabilized cells. Furthermore tubacin inhibits de novo sphingolipid synthesis in intact cells at doses commonly used to test HDAC6 function and does so in an HDAC6-independent manner. Niltubacin is a chemical analog of tubacin which lacks tubacin's HDAC6 activity, and so is often used as a control for off-target effects of tubacin. We find that niltubacin is inactive in the inhibition of sphingolipid biosynthesis, and so does not serve to distinguish the inhibitory effects of tubacin on HDAC6 from those on sphingolipid biosynthesis. These results indicate that caution should be used in the use of tubacin to study the role of HDAC6. © 2014 Elsevier Inc. All rights reserved.


Huang H.,Rosalind Franklin University of Medicine and Science | Shah K.,Rosalind Franklin University of Medicine and Science | Bradbury N.A.,Rosalind Franklin University of Medicine and Science | Li C.,James Graham Brown Cancer Center | And 2 more authors.
Cell Death and Disease | Year: 2014

Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltagedependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca2+ uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca2+-dependent ROS production. © 2014 Macmillan Publishers Limited.


Todd L.R.,James Graham Brown Cancer Center | Damin M.N.,James Graham Brown Cancer Center | Gomathinayagam R.,James Graham Brown Cancer Center | Horn S.R.,Duke University | And 3 more authors.
Molecular Biology of the Cell | Year: 2010

The relationship of mitochondrial dynamics and function to pluripotency are rather poorly understood aspects of stem cell biology. Here we show that growth factor erv1-like (Gfer) is involved in preserving mouse embryonic stem cell (ESC) mitochondrial morphology and function. Knockdown (KD) of Gfer in ESCs leads to decreased pluripotency marker expression, embryoid body (EB) formation, cell survival, and loss of mitochondrial function. Mitochondria in Gfer-KD ESCs undergo excessive fragmentation and mitophagy, whereas those in ESCs overexpressing Gfer appear elongated. Levels of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) are highly elevated in Gfer-KD ESCs and decreased in Gfer-overexpressing cells. Treatment with a specific inhibitor of Drp1 rescues mitochondrial function and apoptosis, whereas expression of Drp1-dominant negative resulted in the restoration of pluripotency marker expression in Gfer-KD ESCs. Altogether, our data reveal a novel prosurvival role for Gfer in maintaining mitochondrial fission-fusion dynamics in pluripotent ESCs. © 2010 by The American Society for Cell Biology.


Fan T.W.-M.,University of Louisville | Fan T.W.-M.,James Graham Brown Cancer Center | Lane A.N.,University of Louisville | Lane A.N.,James Graham Brown Cancer Center | And 2 more authors.
Metabolomics | Year: 2011

We have determined the time course of [U-13C]-glucose utilization and transformations in SCID mice via bolus injection of the tracer in the tail vein. Incorporation of 13C into metabolites extracted from mouse blood plasma and several tissues (lung, heart, brain, liver, kidney, and skeletal muscle) were profiled by NMR and GC-MS, which helped ascertain optimal sampling times for different target tissues. We found that the time for overall optimal 13C incorporation into tissue was 15-20 min but with substantial differences in 13C labeling patterns of various organs that reflected their specific metabolism. Using this stable isotope resolved metabolomics (SIRM) approach, we have compared the 13C metabolite profile of the lungs in the same mouse with or without an orthotopic lung tumor xenograft established from human PC14PE6 lung adenocarcinoma cells. The 13C metabolite profile shows considerable differences in [U-13C]-glucose transformations between the two lung tissues, demonstrating the feasibility of applying SIRM to investigate metabolic networks of human cancer xenograft in the mouse model. © 2010 Springer Science+Business Media, LLC.


Chagpar A.B.,University of Louisville | Chagpar A.B.,James Graham Brown Cancer Center
Surgical Oncology Clinics of North America | Year: 2010

Since its introduction in the mid-1990s, sentinel lymph node (SLN) biopsy has revolutionized the management of breast cancer patients, allowing for a minimally invasive method to accurately stage the axilla without the need for axillary lymph node dissection and its concomitant morbidity in node-negative patients. The ability to identify the lymph nodes most likely to harbor metastases has allowed for increased scrutiny of these lymph nodes, often finding minimal disease in the SLNs. The relevance of such minute disease has been controversial, and there has been considerable debate as to how best to manage such patients. © 2010 Elsevier Inc.

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