Centipede (Scolopendra subspinipes mutilans) venom toxin peptide exhibits cytotoxic and cell growth effects in a concentration-dependent manner

Abstract Background The rise in resistance to existing antimicrobials has prompted a need for the development of novel antibiotics. Microbion has identified a novel compound, pravibismane, with potent broad spectrum anti-infective and anti-biofilm activity. Methods Here we used a variety of assays, including Bacterial Cytological Profiling (BCP), to analyze pravibismane in E.coli to gain insight into its likely mechanism of action (MOA). The BCP profile of pravibismane suggested it rapidly shut down cell growth, potentially by turning off cellular gene or protein expression. This was confirmed using a plasmid based GFP induction assay in E.coli tolC that showed pravibismane strongly reduced expression of GFP. The kinetics, reversibility and MOA of pravibismane was further characterized by using time-lapse microscopy, wash out experiments and measurements of both membrane potential and relative intracellular ATP levels. Results We found that pravibismane acts rapidly (within 30 mins) to completely halt cell growth rather than causing immediate cell lysis such as that observed with non-specific cell damaging agents bleach or detergent. Inhibitor wash out experiments in which cells were exposed to pravibismane for 2 hours, washed to remove the compound, and then observed using time-lapse microscopy revealed that the effect of pravibismane is reversible and that cells recovered 8-12 hrs after removing the compound. Wash out experiments with an E.coli tolC strain carrying a plasmid with an IPTG inducible GFP demonstrated that transcription and translation ultimately resumed in most cells after washout. The bioenergetics of the membrane was measured using DiBAC 4(5), a membrane potential sensitive dye which can enter depolarized cells, which revealed that pravibismane caused depolarization of the membrane within 30 mins of exposure in a concentration dependent manner. Finally, a luciferase assay determined pravibismane reduced ATP levels (resulting in decreased luminescence) within 15 mins of exposure in a concentration dependent manner unlike antibiotic controls that had modest or no effect on luminescence. Conclusion Our results suggest that pravibismane acts rapidly to disrupt cellular bioenergetics, resulting in the immediate cessation of cell growth and protein expression. Disclosures Brett Baker, M.Sc., D.C., Microbion Corporation (Board Member, Employee)

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Anticancer effects of mifepristone on human uveal melanoma cells

Cancer Cell International ◽

10.1186/s12935-021-02306-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Prisca Bustamante Alvarez ◽

Alexander Laskaris ◽

Alicia A. Goyeneche ◽

Yunxi Chen ◽

Carlos M. Telleria ◽

...

Keyword(s):

Cell Death ◽

Cell Growth ◽

Progesterone Receptor ◽

Dna Fragmentation ◽

Cell Lines ◽

Caspase 3 ◽

Annexin V ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Free Dna

Abstract Background Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds. Methods The effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR. Results MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. Conclusion This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

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Na regulates growth of kidney epithelial cells induced by lowering extracellular K concentration

AJP Cell Physiology ◽

10.1152/ajpcell.1984.247.5.c321 ◽

1984 ◽

Vol 247 (5) ◽

pp. C321-C326 ◽

Cited By ~ 37

Author(s):

M. M. Walsh-Reitz ◽

H. N. Aithal ◽

F. G. Toback

Keyword(s):

Cell Growth ◽

Epithelial Cells ◽

Dependent Manner ◽

Deficient Diet ◽

Critical Determinant ◽

Concentration Dependent Manner ◽

Kidney Epithelial Cells ◽

K Concentration ◽

Low K ◽

Stimulation Of

Accelerated kidney growth and increased tissue Na content have been observed in rats fed a K-deficient diet. These observations suggest that enhanced Na influx could mediate renal growth, a hypothesis that was tested in cultures of kidney epithelial cells of the BSC-1 line. Reduction of the K concentration in the culture medium from 5.4 to 3.2 mM augmented cell growth and induced a transient increase in the cellular content of Na and a decrease in that of K. That low-K-induced growth was Na dependent was shown by decreasing the medium Na concentration from 155 to 150 mM, which abolished the increases in both growth and cell Na content in a concentration-dependent manner. The stimulation of glyceraldehyde-3-phosphate dehydrogenase (G3PD) activity that occurs in cells exposed to low-K medium for 1 h was similarly prevented by decreasing the medium Na concentration. Thus decreased availability of extracellular Na prevented the increase in cell Na content, stimulation of G3PD activity, and accelerated growth induced by low-K medium. The hypothesis was also tested by adding vasopressin to cultures of BSC-1 cells exposed to low-K medium; the hormone prevented the increments in cell Na content, G3PD activity, and growth to the same extent as did decreased availability of extracellular Na. These results are consistent with the interpretation that transient accumulation of Na is a critical determinant of the initiation of kidney epithelial cell growth.

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Anticancer Effects of Mifepristone on Human Uveal Melanoma Cells

10.21203/rs.3.rs-794697/v1 ◽

2021 ◽

Author(s):

Alexander Laskaris ◽

Prisca Bustamante ◽

Alicia A. Goyeneche ◽

Carlos M. Telleria ◽

Julia V Burnier

Keyword(s):

Cell Death ◽

Cell Growth ◽

Progesterone Receptor ◽

Dna Fragmentation ◽

Cell Lines ◽

Uveal Melanoma ◽

Annexin V ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Free Dna

Abstract Background: Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant cancer. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytostatic and cytotoxic effects of MF in human UM cell lines of different genetic backgrounds.Methods: The effects of incremental concentrations of MF (0, 5, 10, 20, 30 or 40 mM) on a panel of human UM primary (MP46, 92.1, MP41, MEL270) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 hours before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or propidium iodide (PI), caspases 3/7 activities, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by ddPCR, while the expression of progesterone and glucocorticoid receptors was determined by qPCR. Results: MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI-double positive cells, caspase 3/7 activities, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. Conclusion: This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

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Biological Effects of Osteoblast-Like Cells on Nanohydroxyapatite Particles at a Low Concentration Range

Journal of Nanomaterials ◽

10.1155/2011/104747 ◽

2011 ◽

Vol 2011 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Xiaochen Liu ◽

Jie Wei ◽

Shicheng Wei

Keyword(s):

Cell Growth ◽

Actin Cytoskeleton ◽

Protein Content ◽

Biological Effects ◽

Dependent Manner ◽

Concentration Increase ◽

Intracellular Protein ◽

Concentration Dependent Manner ◽

Low Concentration ◽

Significant Difference

The biological effects of osteoblast-like MG-63 cells on nanohydroxyapatite (n-HA) at the low concentration range (5–25 g/mL) for 5 days was investigated. The results showed the viability and actin cytoskeleton of the cells descended with the increase of the concentration of n-HA, and the actin cytoskeleton of cells was depolymerised and became more disordered. Apoptotic rate of cells (1.85%, 1.99%, and 2.29%) increased with the increase of n-HA concentration (5, 15, and 25 g/mL) and become significantly higher than the control. Total intracellular protein content decreased with n-HA concentration increase, showing significant difference between 25 g/mL and the control, and no significant change of ALP activity was observed at the 5th day. The results revealed that the cell growth was inhibited by n-HA in a concentration-dependent manner, and the obvious biological effects of MG-63 cells on n-HA existed at the low concentration range from 5 to 25 g/mL.

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The inhibitory effect of iodide on growth of rat thyroid (FRTL-5) cells

Acta Endocrinologica ◽

10.1530/acta.0.1190145 ◽

1988 ◽

Vol 119 (1) ◽

pp. 145-151 ◽

Cited By ~ 16

Author(s):

Motoyasu Saji ◽

Osamu Isozaki ◽

Toshio Tsushima ◽

Mariko Arai ◽

Megumi Miyakawa ◽

...

Keyword(s):

Cell Growth ◽

Inhibitory Effect ◽

Dependent Manner ◽

Camp Production ◽

Iodide Uptake ◽

Concentration Dependent Manner ◽

Thyroid Cells ◽

Camp Generation ◽

Rat Thyroid ◽

Sites Of Action

Abstract. The effect of iodide on growth of rat thyroid cells (FRTL-5) was studied. TSH-stimulated cell growth was inhibited by iodide in a concentration-dependent manner, and an effect of iodide was detected at 10−6 mol/l. KClO4 or 1-methylimidazole-2-thiol blocked the effect of iodide, suggesting that iodide uptake and its organification are required to produce the inhibitory effect of iodide on cell growth. Iodide not only decreased TSH-stimulated cAMP production in FRTL-5 cells but also cell growth induced by cAMP. These observations suggest that iodide inhibits TSH-stimulated growth of the cells by attenuating cAMP production and also by acting on the step(s) distal to cAMP generation. The inhibitory effect of iodide was also seen in growth stimulated by insulin, insulin-like growth factor-I or 12-O-tetradecanoyl phorbol 13-acetate, suggesting multiple sites of action of iodide in the process of growth of FRTL-5 cells.

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A chalcone inhibits the growth and metastasis of KYSE-4 esophageal cancer cells

Journal of International Medical Research ◽

10.1177/0300060520928831 ◽

2020 ◽

Vol 48 (6) ◽

pp. 030006052092883

Author(s):

Jie Chen ◽

Chun-Yan Kang ◽

Zhao-Xia Niu ◽

Hui-Cong Zhou ◽

Hong-Mei Yang

Keyword(s):

Esophageal Cancer ◽

Cell Growth ◽

Cancer Cells ◽

Migration And Invasion ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Anticancer Effects ◽

Esophageal Cancer Cells

Objective To investigate the in vitro and in vivo anticancer effects of a chalcone against KYSE-4 esophageal cancer cells. Methods A chalcone was synthesized via the molecular hybridization strategy based on the anticancer activity of chalcone and dithiocarbamate scaffolds. The anticancer effects of different concentrations of the chalcone derivative were compared in esophageal cancer cells. Results This chalcone displayed strong inhibitory effects on esophageal cancer cell growth with an IC50 of 1.06 μM in KYSE-4 cells. Analysis of the mechanism revealed that the derivative obviously inhibited KYSE-4 cell growth, migration, and invasion in a concentration-dependent manner. Furthermore, the compound regulated migration-related biomarkers (E-cadherin, N-cadherin, and Slug) and inhibited the Wnt/β-catenin pathway. According to western blotting, this chalcone suppressed the expression of proline-rich protein 11 (PRR11) in a concentration- and time-dependent manner. Conclusions This chalcone might be a leading candidate for suppressing the growth and metastasis of esophageal cancer by downregulating PRR11 expression and inhibiting Wnt/β-catenin signaling.

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The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000195 ◽

2014 ◽

Vol 84 (1-2) ◽

pp. 79-91 ◽

Cited By ~ 7

Author(s):

Amin F. Majdalawieh ◽

Hyo-Sung Ro

Keyword(s):

Cholesterol Efflux ◽

Transcriptional Activity ◽

Molecular Mechanisms ◽

Foam Cell ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Foam Cell Formation ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamins anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

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Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646319 ◽

1992 ◽

Vol 68 (05) ◽

pp. 570-576 ◽

Cited By ~ 20

Author(s):

Mary A Selak

Keyword(s):

Neutrophil Elastase ◽

Cell Activation ◽

Thromboxane A2 ◽

Creatine Phosphate ◽

Dense Granule ◽

Cathepsin G ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Low Concentrations ◽

High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

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Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651597 ◽

1993 ◽

Vol 69 (03) ◽

pp. 286-292 ◽

Cited By ~ 13

Author(s):

Che-Ming Teng ◽

Feng-Nien Ko ◽

Inn-Ho Tsai ◽

Man-Ling Hung ◽

Tur-Fu Huang

Keyword(s):

Platelet Aggregation ◽

Snake Venom ◽

Inositol Phosphate ◽

Shape Change ◽

Platelet Activating Factor ◽

Atp Release ◽

Platelet Membrane ◽

Thromboxane B2 ◽

Dependent Manner ◽

Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

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