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1.
J Invest Dermatol ; 138(12): 2635-2643, 2018 12.
Article in English | MEDLINE | ID: mdl-29908149

ABSTRACT

Overexpression of hexokinase 2, and its binding to VDAC1 on the outer mitochondrial membrane of cancer cells, is key to their metabolic reprogramming to aerobic glycolysis, which enables them to proliferate. We describe Comp-1, an allosteric small molecule that selectively detaches hexokinase 2 from the mitochondria. Detachment of hexokinase 2 reduces glycolysis and triggers apoptosis in cancer cells, without affecting hexokinase 1-expressing normal cells. The anti-cancer activity of Comp-1 was demonstrated in the UVB-damaged skin model in SKH-1 mice. Topical treatment with Comp-1 led to 70% reduction in lesion number and area. This in vivo efficacy was obtained without local skin reactions or other safety findings. Mechanism-related pharmacodynamic markers, including hexokinase 2 and cleaved caspase 3 levels, are affected by Comp-1 treatment in vivo. Good Laboratory Practice toxicology studies in minipigs for 28 days and 13 weeks established no systemic toxicities and minimal dermal reaction for once-daily application of up to 20% and 15% ointment strengths, respectively. Thus, Comp-1 may address a significant unmet medical need for a non-irritating efficacious topical actinic keratosis treatment.


Subject(s)
Acetates/therapeutic use , Antineoplastic Agents/therapeutic use , Cyclopentanes/therapeutic use , Keratosis, Actinic/drug therapy , Neoplasms, Squamous Cell/drug therapy , Oxylipins/therapeutic use , Skin Neoplasms/drug therapy , Skin/pathology , Ultraviolet Rays/adverse effects , Acetates/chemical synthesis , Acetates/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line , Cyclopentanes/chemical synthesis , Cyclopentanes/pharmacology , Female , Glycolysis , Hexokinase/metabolism , Humans , Mice , Mice, Mutant Strains , Mitochondria/metabolism , Models, Animal , Oxylipins/chemical synthesis , Oxylipins/pharmacology , Skin/drug effects , Swine , Swine, Miniature , Voltage-Dependent Anion Channel 1/metabolism , Xenograft Model Antitumor Assays
2.
Bioorg Med Chem Lett ; 22(20): 6460-8, 2012 Oct 15.
Article in English | MEDLINE | ID: mdl-22963766

ABSTRACT

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.


Subject(s)
Carrier Proteins/agonists , Enzyme Activation/drug effects , Indoles/chemistry , Indoles/pharmacology , Membrane Proteins/agonists , Neoplasm Proteins/agonists , Neoplasms/enzymology , Thyroid Hormones/agonists , Caco-2 Cells , Carrier Proteins/metabolism , Humans , Membrane Proteins/metabolism , Molecular Docking Simulation , Neoplasm Proteins/metabolism , Neoplasms/drug therapy , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Multimerization/drug effects , Pyruvate Kinase/metabolism , Sulfinic Acids/chemistry , Sulfinic Acids/pharmacology , Thyroid Hormones/metabolism , Thyroid Hormone-Binding Proteins
3.
Exp Toxicol Pathol ; 57(4): 291-7, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16413765

ABSTRACT

Development of effective therapies for multiple sclerosis (MS) is dependent on the advancement of improved tools for evaluation of progression of this disease in animal models. We present a novel technique utilizing scanning electron microscopy (SEM) for imaging wet biological specimens thus enabling rapid and high-resolution imaging of myelin in mouse spinal cord (SC). We demonstrate the advantages of using the wet SEM technique to image myelin in a murine model of MS, experimental autoimmune encephalomyelitis (EAE) induced in the Biozzi (antibody-high) mouse, by sensitization with spinal cord homogenate (SCH) in adjuvant. Our studies show that the methodology allows easy identification of normal and pathological components with great clarity, which is then correlated with light microscopy (LM) and validated thereby. Furthermore, we demonstrate gold immunolabeling of specific epitopes. We conclude that the new technique provides a quick, accurate, and detailed structural evaluation of the SC that can be applied to advance the research of MS.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/pathology , Microscopy, Electron, Scanning/methods , Multiple Sclerosis/pathology , Myelin Sheath/ultrastructure , Spinal Cord/ultrastructure , Animals , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/immunology , Female , Mice , Mice, Inbred Strains , Reproducibility of Results
4.
Diabetes ; 54 Suppl 2: S137-44, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16306331

ABSTRACT

Type 2 diabetes is characterized by insulin resistance and progressive beta-cell failure. Deficient insulin secretion, with increased proportions of insulin precursor molecules, is a common feature of type 2 diabetes; this could result from inappropriate beta-cell function and/or reduced beta-cell mass. Most studies using tissues from diabetic patients are retrospective, providing only limited information on the relative contribution of beta-cell dysfunction versus decreased beta-cell mass to the "beta-cell failure" of type 2 diabetes. The gerbil Psammomys obesus is a good model to address questions related to the role of insulin resistance and beta-cell failure in nutritionally induced diabetes. Upon a change from its natural low-calorie diet to the calorie-rich laboratory food, P. obesus develops moderate obesity associated with postprandial hyperglycemia. Continued dietary load, superimposed on its innate insulin resistance, results in depletion of pancreatic insulin stores, with increased proportions of insulin precursor molecules in the pancreas and the blood. Inadequate response of the preproinsulin gene to the increased insulin needs is an important cause of diabetes progression. Changes in beta-cell mass do not correlate with pancreatic insulin stores and are unlikely to play a role in disease initiation and progression. The major culprit is the inappropriate insulin production with depletion of insulin stores as a consequence. Similar mechanisms could operate during the evolution of type 2 diabetes in humans.


Subject(s)
Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/genetics , Animal Feed , Animals , Diet , Disease Models, Animal , Environment , Gerbillinae , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/metabolism
5.
Toxicol Pathol ; 32(3): 357-63, 2004.
Article in English | MEDLINE | ID: mdl-15204979

ABSTRACT

In this report we introduce wet-tissue scanning electron microscopy, a novel technique for direct imaging of wet tissue samples using backscattered electrons. Samples placed in sealed capsules are imaged through a resilient, electron-transparent membrane. The contrast of the imaged samples may be enhanced by chemical staining. The samples several millimeters thick and imaged without sectioning, makes this technique suitable for rapid analysis of tissue specimens. We applied this technique to D-limonene-induced nephropathy where accumulation of hyaline protein droplets is induced in proximal and distal convoluted tubules of the kidney. Images obtained by scanning electron microscopy of hydrated kidney specimens exhibited superior resolution, contrast, and magnification compared with those obtained by conventional light microscopy of paraffin sections. The electron micrographs can be obtained within an hour of tissue removal, whereas preparation for light microscopy requires at least 1 day. These advantages of the wet scanning electron microscopy technique indicate its potential utility in a wide range of applications in histopathology and toxicology.


Subject(s)
Histological Techniques , Kidney Diseases/pathology , Kidney/pathology , Kidney/ultrastructure , Microscopy, Electron, Scanning/methods , Animals , Carcinogens/toxicity , Cyclohexenes , Histological Techniques/methods , Kidney Diseases/chemically induced , Limonene , Male , Microscopy, Electron, Scanning/instrumentation , Paraffin Embedding , Rats , Rats, Sprague-Dawley , Terpenes/toxicity , Time Factors
6.
Ultrastruct Pathol ; 28(1): 29-31, 2004.
Article in English | MEDLINE | ID: mdl-14967596

ABSTRACT

Progress in the processing of wet tissues, without the need of fixation and complex preparation procedures, may facilitate the microscopic examination of tissues and cells. Microscopic examination of tissues is a central tool in clinical diagnosis as well as in diverse areas of research. The authors present the application of Wet SEM, a technology for imaging fully hydrated samples at atmospheric pressure in a scanning electron microscope (SEM). The technique is based on 2 principles. First, samples are imaged in sealed specimen capsules and are separated from the evacuated interior of the electron microscope by a thin, electron-transparent partition membrane that is strong enough to sustain a 1-atm pressure difference. Second, imaging is done in a SEM, based on detection of backscattered electrons, which penetrate a few microns into the specimen and thus give information on the cellular level.


Subject(s)
Microscopy, Electron, Scanning/instrumentation , Microscopy, Electron, Scanning/methods , Specimen Handling/instrumentation , Specimen Handling/methods , Colitis/pathology , Colon/ultrastructure , Crohn Disease/pathology , Humans , Inflammatory Bowel Diseases/pathology
7.
Proc Natl Acad Sci U S A ; 101(10): 3346-51, 2004 Mar 09.
Article in English | MEDLINE | ID: mdl-14988502

ABSTRACT

A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is approximately 100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers.


Subject(s)
Cytological Techniques/methods , Histological Techniques/methods , Microscopy, Electron, Scanning/methods , Animals , CHO Cells , Cricetinae , Electrons , Gold , HeLa Cells , Humans , Mice , Scattering, Radiation , Staining and Labeling/methods , Trypanosoma brucei brucei/ultrastructure , Water
8.
Ultrastruct Pathol ; 28(4): 255-60, 2004.
Article in English | MEDLINE | ID: mdl-15693637

ABSTRACT

The authors present the application of wet SEM for histopathological assessment, a technology for imaging fully hydrated samples at atmospheric pressure in a scanning electron microscope (SEM). Both transmission and scanning electron microscopy techniques usually require long and complex sample preparation of the tissues. In marked contrast, a rapid preparation of tissues is described for evaluation by SEM imaging. The wet SEM technology successfully demonstrated both histological and ultrastructural features of several CNS tumors: Rosette formation and intracytoplasmic lumens were observed in ependymoma; numerous fibrillary processes in fibrillary astrocytoma; and focal rosette formation with no intracytoplasmic lumens in medulloblastoma. Application of this method simultaneously with frozen section may improve rapid intraoperative diagnosis of these intracranial tumors.


Subject(s)
Brain Neoplasms/pathology , Brain Neoplasms/ultrastructure , Microscopy, Electron, Scanning/instrumentation , Microscopy, Electron, Scanning/methods , Astrocytoma/pathology , Astrocytoma/ultrastructure , Ependymoma/pathology , Ependymoma/ultrastructure , Humans , Medulloblastoma/pathology , Medulloblastoma/ultrastructure , Microscopy, Electron, Transmission
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