sirolimus and chelerythrine

The mammalian target of rapamycin (mTOR) inhibitor temsirolimus is utilized for the treatment of malignancy. Temsirolimus is at least in part effective by triggering suicidal tumor cell death. The most common side effect of temsirolimus treatment is anemia. At least in theory, the anemia following temsirolimus treatment could result from stimulation of eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the orchestration of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of staurosporine and chelerythrine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The purpose of the present study was to test whether temsirolimus influences eryptosis and, if so, to shed light on the signaling involved.. Flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was determined from hemoglobin concentration in the supernatant.. A 48 hours exposure of human erythrocytes to temsirolimus (5 - 20 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Temsirolimus significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance at the erythrocyte surface. The effect of temsirolimus on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+ and by addition of staurosporine (1 µM) or chelerythrine (10 µM) but not significantly modified by addition of SB203580 (2 µM), D4476 (10 µM), or zVAD (10 µM). Chelerythrine (10 µM) further significantly blunted the effect of temsirolimus on DCFDA fluorescence but not ceramide formation. Removal of extracellular Ca2+ had no effect on temsirolimus induced ROS formation or ceramide abundance.. Temsirolimus triggers eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and activation of staurosporine/Chelerythrine sensitive kinase(s).

Topics: Aniline Compounds; Annexin A5; Antineoplastic Agents; Benzamides; Benzophenanthridines; Calcium; Casein Kinase I; Caspases; Ceramides; Eryptosis; Erythrocytes; Fluoresceins; Gene Expression Regulation; Hemolysis; Humans; Imidazoles; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Phosphatidylserines; Primary Cell Culture; Protein Kinase Inhibitors; Pyridines; Reactive Oxygen Species; Signal Transduction; Sirolimus; Staurosporine; TOR Serine-Threonine Kinases; Xanthenes

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells, we identified a series of molecules antagonized by rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine or GSH cotreatment protected TSC2-null cells from chelerythrine's effects, indicating that chelerythrine-induced cell death is ROS dependent. Induction of heme-oxygenase-1 (HMOX1/HO-1) with hemin also blocked chelerythrine-induced cell death. In vivo, chelerythrine inhibited the growth of TSC2-null xenograft tumors with no evidence of systemic toxicity with daily treatment over an extended period of time. This study reports the results of a bioactive compound screen and the identification of a potential lead candidate that acts via a novel oxidative stress-dependent mechanism to selectively induce necroptosis in TSC2-deficient tumors.. This study demonstrates that TSC2-deficient tumor cells are hypersensitive to oxidative stress-dependent cell death, and provide critical proof of concept that TSC2-deficient cells can be therapeutically targeted without the use of a rapalog to induce a cell death response.

Topics: Benzophenanthridines; Cell Death; Cell Line, Tumor; Drug Screening Assays, Antitumor; Glutathione; Heme Oxygenase-1; Humans; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Oxidative Stress; Reactive Oxygen Species; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD's pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin-chelerythrine-acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.

Topics: Acetylcholinesterase; Amyloid beta-Peptides; Benzophenanthridines; Binding Sites; Butyrylcholinesterase; Catalytic Domain; Cholinesterase Inhibitors; Humans; Isoquinolines; Kinetics; Molecular Docking Simulation; Structure-Activity Relationship

We have recently demonstrated that in quiescent fibroblasts protein kinase C (PKC) epsilon(95) is phosphorylated at Ser(729), Ser(703), and Thr(566) and that upon passage of quiescent cells phosphorylation at Ser(729) is lost, giving rise to PKCepsilon(87). Ser(729) may be rephosphorylated later, suggesting cycling between PKCepsilon(87) and PKCepsilon(95). Here we show that the dephosphorylation at Ser(729) is insensitive to okadaic acid, calyculin, ascomycin C, and cyclosporin A, suggesting that dephosphorylation at this site is not mediated through protein phosphatases 1, 2A or 2B. We demonstrate that this dephosphorylation at Ser(729) requires serum and cell readhesion and is sensitive to rapamycin, PD98059, chelerythrine, and Ro-31-8220. These results suggest that the phosphorylation status of Ser(729) in the hydrophobic domain at Ser(729) is regulated independently of the phosphorylation status of other sites in PKCepsilon, by a mTOR-sensitive phosphatase. The mitogen-activated protein kinase pathway and PKC are also implicated in regulating the dephosphorylation at Ser(729).

Topics: 3T3 Cells; Alkaloids; Animals; Anti-Bacterial Agents; Benzophenanthridines; Blotting, Western; Calcineurin; Cell Adhesion; Cell Line; Culture Media; Cyclosporine; Down-Regulation; Enzyme Inhibitors; Flavonoids; Indoles; Isoenzymes; MAP Kinase Signaling System; Marine Toxins; Methionine; Mice; Models, Biological; Okadaic Acid; Oxazoles; Phenanthridines; Phosphoprotein Phosphatases; Phosphorylation; Plasmids; Precipitin Tests; Protein Binding; Protein Kinase C; Protein Kinase C-epsilon; Protein Structure, Tertiary; Serine; Signal Transduction; Sirolimus; Tacrolimus; Transfection

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Alkaloids; Amides; Animals; Benzamides; Benzophenanthridines; Benzopyrans; Butadienes; Cell Line; Enzyme Inhibitors; Flavonoids; Humans; Imidazoles; Indoles; Inhibitory Concentration 50; Isoquinolines; Lithium; Magnesium; Nitriles; Phenanthridines; Phosphorylation; Potassium Chloride; Protein Kinase Inhibitors; Protein Kinases; Pyridines; Sirolimus; Substrate Specificity; Sulfonamides