ABSTRACT
Nigratine (also known as 6E11), a flavanone derivative of a plant natural product, was characterized as highly specific non-ATP competitive inhibitor of RIPK1 kinase, one of the key components of necroptotic cell death signaling. We show here that nigratine inhibited both necroptosis (induced by Tumor Necrosis Factor-α) and ferroptosis (induced by the small molecules glutamate, erastin, RSL3 or cumene hydroperoxide) with EC50 in the µM range. Taken together, our data showed that nigratine is a dual inhibitor of necroptosis and ferroptosis cell death pathways. These findings open potential new therapeutic avenues for treating complex necrosis-related diseases.
Subject(s)
Ferroptosis , Apoptosis , Cell Death/physiology , Humans , Necroptosis , Necrosis , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/ß (GSK-3 α/ß), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.
Subject(s)
Acacia/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Triterpenes/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Apoptosis/genetics , Binding Sites , Casein Kinase 1 epsilon/antagonists & inhibitors , Casein Kinase 1 epsilon/genetics , Casein Kinase 1 epsilon/metabolism , Cell Proliferation/drug effects , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Humans , Janus Kinase 3/antagonists & inhibitors , Janus Kinase 3/genetics , Janus Kinase 3/metabolism , K562 Cells , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Models, Molecular , NIMA-Related Kinases/antagonists & inhibitors , NIMA-Related Kinases/genetics , NIMA-Related Kinases/metabolism , Plant Bark/chemistry , Plant Extracts/chemistry , Protein Binding , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/isolation & purification , Proto-Oncogene Proteins c-abl/chemistry , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Signal Transduction , Triterpenes/chemistry , Triterpenes/isolation & purification , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
The marine α-pyrone macrolide neurymenolide A was previously isolated from the Fijian red macroalga, Neurymenia fraxinifolia, and characterized as an antibacterial agent against antibiotic-resistant strains that also exhibited moderate cytotoxicity in vitro against cancer cell lines. This compound was also shown to exhibit allelopathic effects on Scleractinian corals. However, to date no mechanism of action has been described in the literature. The present study showed, for the first time, the isolation of neurymenolide A from the New Caledonian Rhodophyta, Phacelocarpus neurymenioides. We confirmed the compound's moderate cytotoxicity in vitro against several human cell lines, including solid and hematological malignancies. Furthermore, we combined fluorescence microscopy and flow cytometry to demonstrate that treatment of U-2 OS osteosarcoma human cells with neurymenolide A could block cell division in prometaphase by inhibiting the correct formation of the mitotic spindle, which induced a mitotic catastrophe that led to necrosis and apoptosis. Absolute configuration of the stereogenic center C-17 of neurymenolide A was deduced by comparison of the experimental and theoretical circular dichroism spectra. Since the total synthesis of this compound has already been described, our findings open new avenues in cancer treatment for this class of marine molecules, including a new source for the natural product.
Subject(s)
Macrolides/chemistry , Macrolides/pharmacology , Pyrones/chemistry , Pyrones/pharmacology , Rhodophyta/chemistry , Spindle Apparatus/drug effects , Apoptosis/drug effects , Bone Neoplasms/drug therapy , Bone Neoplasms/pathology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Hep G2 Cells , Humans , K562 Cells , MCF-7 Cells , Microtubules/pathology , Mitosis/drug effects , Necrosis/drug therapy , Osteosarcoma/drug therapy , Osteosarcoma/pathologyABSTRACT
Half a century of biochemical and biophysical experiments has provided attractive models that may explain the diverse functions of microtubules within cells and organisms. However, the notion of functionally distinct microtubule types has not been explored with similar intensity, mostly because mechanisms for generating divergent microtubule species were not yet known. Cells generate distinct microtubule subtypes through expression of different tubulin isotypes and through post-translational modifications, such as detyrosination and further cleavage to Δ2-tubulin, acetylation, polyglutamylation and polyglycylation. The recent discovery of enzymes responsible for many tubulin post-translational modifications has enabled functional studies demonstrating that these post-translational modifications may regulate microtubule functions through an amazing range of mechanisms.
Subject(s)
Cytoskeleton/metabolism , Microtubules/metabolism , Protein Processing, Post-Translational , Acetylation , Animals , Humans , Tubulin/chemistry , Tubulin/metabolism , Tyrosine/metabolismABSTRACT
Clinically relevant mature cartilage cells (chondrocytes) present challenges for use in cartilage tissue engineering applications, given their low capacity for cell division and tissue production. Since the in situ environment of chondrocytes is hypertonic relative to standard culture medium conditions, in this study we tested the hypothesis that using culture medium of a hypertonic, more physiologic osmolarity during both two-dimensional (2D) expansion of mature bovine chondrocytes (MBCs) and their subsequent encapsulation culture in three-dimensional (3D) agarose hydrogel constructs produces improved engineered tissue construct mechanical and biochemical properties. Results demonstrate that 2D expansion of MBCs in hypertonic (NaCl) medium before encapsulation yielded improved construct mechanical properties. However, 3D encapsulation culture of cells in hypertonic (NaCl) medium yielded poorer construct mechanical properties. Osmolarity-related differences in construct biochemical content and organization may have contributed to differences in mechanical properties, as construct glycosaminoglycan content correlated moderately with construct mechanical properties, and construct collagen distribution varied between 3D osmotic culture groups. Results of this study suggest that application of hypertonic (NaCl) medium during 2D mature chondrocyte expansion, but not 3D encapsulated chondrocyte culture, may serve as a convenient and inexpensive method for improving mechanical properties of expanded cell-seeded constructs.
Subject(s)
Cartilage/drug effects , Cartilage/physiology , Cell Culture Techniques/methods , Chondrocytes/cytology , Saline Solution, Hypertonic/pharmacology , Tissue Engineering/methods , Animals , Cattle , Cell Proliferation/drug effects , Chondrocytes/drug effects , Chondrocytes/metabolism , Collagen/metabolism , DNA/metabolism , Elastic Modulus/drug effects , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Glycosaminoglycans/metabolism , Hydrogel, Polyethylene Glycol Dimethacrylate/pharmacology , Materials Testing , Osmosis/drug effectsABSTRACT
Galvanotaxis, that is, migration induced by DC electric fields, is thought to play a significant role in development and wound healing, however, the mechanisms by which extrinsic electric fields orchestrate intrinsic motility responses are unknown. Using mammalian cell lines (3T3, HeLa, and CHO cells), we tested one prevailing hypothesis, namely, that electric fields polarize charged cell surface molecules, and that these polarized molecules drive directional motility. Negatively charged sialic acids, which contribute the bulk of cell surface charge, redistribute preferentially to the surface facing the direction of motility, as measured by labeling with fluorescent wheat germ agglutinin. We treated cells with neuraminidase to remove sialic acids; as expected, this decreased total cell surface charge. We also changed cell surface charge independent of sialic acid moieties, by conjugating cationic avidin to the surface of live cells. Neuraminidase inhibited the electric field-induced directional polarization of membrane ruffling and alpha4 integrin, while avidin treatment actually reversed the directional polarization of sialic acids. Neuraminidase treatment inhibited directionality but did not alter speed of motility. Surprisingly, avidin treatment did not significantly alter either directionality or speed of motility. Thus, our results demonstrate that electric field-induced polarization of charged species indeed occurs. However, polarization of the bulk of charged cell surface proteins is neither necessary nor sufficient to cause motility, thus contradicting the second part of our hypothesis. Because neuraminidase inhibited directional motility, we also conclude that sialic acids are required constituents of some cell surface molecule(s) through which electric fields mount a polarized transmembrane response.
Subject(s)
Cell Movement/physiology , Cell Polarity/physiology , Electricity , Membrane Proteins/physiology , Animals , Avidin/chemistry , CHO Cells , Cell Migration Assays/methods , Cell Movement/drug effects , Cricetinae , Cricetulus , Electrodes , Fluorescent Antibody Technique/methods , HeLa Cells , Humans , Mice , Microscopy, Phase-Contrast/methods , NIH 3T3 Cells , Neuraminidase/chemistry , Neuraminidase/pharmacology , Sialic Acids/chemistry , Sialic Acids/physiologyABSTRACT
Applied electric fields (static and pulsing) are widely used in orthopedic practices to treat nonunions and spine fusions and have been shown to improve ligament healing in vivo. Few studies, however, have addressed the effect of electric fields (EFs) on ligament fibroblast migration and biosynthesis. In the current study, we applied static and pulsing direct current (DC) EFs to calf anterior cruciate ligament (ACL) fibroblasts. ACL fibroblasts demonstrated enhanced migration speed and perpendicular alignment to the applied EFs. The motility of ligament fibroblasts was further modulated on type I collagen. In addition, type I collagen expression increased in ACL fibroblasts after exposure to pulsing EFs. In vitro wound-healing studies showed inhibitory effects of static EFs, which were alleviated with a pulsing EF. Our results demonstrate that applied EFs augment ACL fibroblast migration and biosynthesis and provide potential mechanisms by which EFs may be used for enhancing ligament healing and repair.