The Synergistic Inhibitory Effect of Combining MK-2206 and AZD 6244 in MARIMO Cells Harboring a Calreticulin Gene Mutation

Chemotherapy ◽  
2021 ◽  
pp. 1-10
Author(s):  
Chunqing Wang ◽  
Xueting Hu ◽  
Yan Wan ◽  
Shujin Wang ◽  
Kunming Qi ◽  
...  
Keyword(s):  
Cell Growth ◽  
Myeloproliferative Neoplasm ◽  
Inhibitory Effect ◽  
Janus Kinase ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Akt Activation ◽  
Phosphorylated Akt ◽  
Primary Mouse ◽  
Synergistic Inhibitory Effect

<b><i>Introduction:</i></b> Somatic mutations in the calreticulin (CALR) gene occur in most myeloproliferative neoplasm (MPN) patients who lack Janus kinase 2 or thrombopoietin receptor (MPL) mutations, but the molecular pathogenesis of MPN with mutated CALR is unclear, which limited the further treatment for CALR gene mutant patients. <b><i>Objectives:</i></b> Previous studies showed that CALR mutations not only activated serine/threonine protein kinase (AKT) in primary mouse bone marrow cells but also mitogen-activated protein kinases (MAPKs) in MARIMO cells harboring a heterozygous 61-bp deletion in CALR exon 9, which were responsible for mutant CALR cell survival, respectively. Hence, we aimed to initially explore the mechanism of AKT activation and observe the synergistic inhibitory effect of combining AKT (MK-2206) and MAPK kinase (AZD 6244) inhibitors in MARIMO cells. <b><i>Methods:</i></b> We detected the expression of phosphorylated AKT in MARIMO cells treated with inhibitors for 24 or 48 h by western blotting and analyzed cell proliferation, cell cycle, and apoptosis by flow cytometry. We further examined the synergistic inhibitory effect of combining MK-2206 and AZD 6244 in MARIMO cells using the median effect principle of Chou and Talalay. <b><i>Results:</i></b> We found that the AKT was activated in MARIMO cells, and blocking its activity significantly inhibited MARIMO cell growth with downregulation of cyclin D and E, and accelerated cell apoptosis by decreasing Bcl-2 but increasing Bax and cleaved caspase-3 levels in a dose-dependent manner. Further analysis showed that AKT activation was dependent on mammalian target of rapamycin but not on the JAK signaling pathway in MARIMO cells, displaying that inhibition of JAK activity by ruxolitinib (RUX) did not decrease the AKT phosphorylation. Furthermore, the combination of MK-2206 and AZD 6244 produced a significantly synergistic inhibitory effect on MARIMO cells. <b><i>Conclusions:</i></b> AKT activation is a feature of MARIMO cells and co-targeting of AKT and MAPKs signaling pathways synergistically inhibits MARIMO cell growth.

Interactions between TGF-beta and adrenocorticotropin in growth regulation of human adrenal fetal zone cells

1994 ◽  
Vol 266 (3) ◽  
pp. E495-E500
Author(s):  
A. K. Stankovic ◽  
W. E. Grizzle ◽  
C. R. Stockard ◽  
C. R. Parker
Keyword(s):  
Cell Growth ◽  
Transforming Growth Factor Beta ◽  
Growth Regulation ◽  
Transforming Growth Factor ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Tgf Beta ◽  
Intrauterine Development ◽  
Sigma Chemical ◽  
And Function

The factors that regulate growth and function of the human adrenal gland during intrauterine development and thereafter are ill defined. Whereas others have reported that adrenocorticotropic hormone (ACTH) augments the inhibitory effect of transforming growth factor-beta (TGF-beta) on growth of fetal zone (FZ) cells of the human fetal adrenal, we recently found that ACTH interferes with TGF-beta's inhibition of growth of fetal adrenal neocortex cells. In this study we sought to assess independently the effects of TGF-beta in the absence and presence of ACTH on growth of FZ cells. TGF-beta, in a time- and dose-dependent manner, inhibited growth (i.e., [3H]thymidine incorporation) of FZ cells. ACTH (Cortrosyn), at 90 pM to 90 nM, was found to interfere with the TGF-beta inhibition of FZ growth. ACTH 1-24 and human ACTH 1-39, both from Sigma Chemical, also were found to blunt the response of FZ cells to TGF-beta. Growth inhibition due to TGF-beta action and the reversal by ACTH of TGF-beta effects on FZ cell growth were confirmed by the results of immunohistochemical analyses of 5'-bromo-2'-deoxyuridine incorporation into nuclei of FZ cells and by indirect evaluations of cell numbers. Both forskolin (10 microM) and dibutyryl adenosine 3',5'-cyclic monophosphate (1 mM), but not phorbol 12-myristate 13-acetate (1 or 100 mM), were able to mimic ACTH actions in blunting the inhibitory effects of TGF-beta on DNA synthesis. We conclude that ACTH, possibly via activation of adenylate cyclase, interferes with, rather than augments, the growth-inhibitory effect of TGF-beta on FZ cell growth.

Neuromedin U Peptide Activates STAT5 and S6 in a JAK-2 Dependent Manner and Promotes Erythroid Cell Growth in Primary Erythroid Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1241-1241
Author(s):  
Rebecca Lenzo ◽  
Martha Dua-Awereh ◽  
Martin Carroll ◽  
Susan E. Shetzline
Keyword(s):  
Cell Growth ◽  
Progenitor Cells ◽  
Surrogate Marker ◽  
Janus Kinase ◽  
Erythroid Cell ◽  
Dependent Manner ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Erythroid Progenitor Cells ◽  
Pi3k Activation

Abstract Abstract 1241 Erythropoiesis is a multi-step process during which hematopoietic stem cells terminally differentiate into red blood cells (RBCs). Erythropoietin (EPO) is the only known cytokine regulator of terminal erythroid differentiation. Previously, we reported that the neuropeptide, neuromedin U (NmU), which interacts with NmU receptor type 1 (NMUR1), functions as a novel extracellular cofactor with EPO to promote the expansion of early erythroblasts, which are CD34−, CD71+, glycophorin A (GlyA)dim(Gambone et al, Blood. 2011). Here, we describe studies to understand the mechanism whereby NmU augments EPO effects on erythroid cell growth. EPO triggers Janus kinase (Jak)-2 dependent activation of signal transducer and activator of transcription (STAT) 5 and phosphatidylinositol 3-kinase (PI3K) to promote the proliferation and/or survival of erythroid progenitor cells. We hypothesized that NmU peptide would cooperate with EPO to promote the proliferation of early erythroblasts through STAT5 and/or PI3K activation. To address this hypothesis, we cultured primary human CD34+ cells in 2-stage liquid culture with IL-3, IL-6, and stem cell factor (SCF) from day 0 to day 6. On day 6, 2U/mL of EPO was added, and the cells were cultured for an additional 5 days to expand erythroid progenitors. On day 11, cells were briefly serum starved and then stimulated with EPO and/or NmU in the absence or presence of a Jak-1/2 inhibitor. Activation of STAT5 and S6, a surrogate marker for PI3K activation, were assessed by phospho-flow in ERY3 (CD34−, CD71+, GlyA+) and ERY4 (CD34−, CD71dim, GlyA+) cells. As expected, EPO alone activated STAT5 and S6 in ERY3 cells only, and the presence of a Jak-1/2 inhibitor diminished STAT5 activation. Interestingly, STAT5 and S6 were activated by NmU peptide alone in ERY3 and ERY4. Surprisingly, in the presence of a Jak-1/2 inhibitor, NmU peptide, which binds to NMUR1 a G-protein coupled receptor, did not activate STAT5 or S6 in ERY3 or 4 cells, suggesting that NmU functions through a JAK kinase in erythroid cells. No additive or synergistic activation of STAT5 and S6 is observed in the presence of both EPO and NmU peptide when EPO was used at a dose of 2 U/mL. The mechanism whereby NmU activates a JAK dependent signaling pathway is under investigation. Preliminary evidence suggests that EPO induces the physical association of NMUR1 with EPO receptor (EPOR). Taken together, we propose that NmU is a neuropeptide expressed in bone marrow cells that cooperates to regulate erythroid expansion during early erythropoiesis through the activation of cytokine receptor like signaling pathways and perhaps through direct interaction with EPOR. NmU may be useful in the clinical management of anemia in patients unresponsive to EPO or other erythroid-stimulating agents. Disclosures: No relevant conflicts of interest to declare.

Abstract 323: A Peptide Inhibitor of PTEN Improves Mouse Survival After Cardiac Arrest

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_2) ◽  
Author(s):  
Xiangdong Zhu ◽  
Jing Li ◽  
Huashan Wang ◽  
Chunpei Lee ◽  
Zhiyi Zhu ◽  
...  
Keyword(s):  
Cardiac Arrest ◽  
Mouse Model ◽  
Glucose Utilization ◽  
Polyol Pathway ◽  
Treated Group ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Akt Phosphorylation ◽  
Akt Activation

Introduction: Prior works from our laboratory found that cooling protection after cardiac arrest is mediated by enhanced Akt activation and in cardiomyocyte the cooling protection can be reproduced using PTEN chemical inhibitor. The current study extend these works by designing a cell-permeable peptide, TAT-PTEN9c, which is more specific for PTEN. Hypothesis: We hypothesized that TAT-PTEN9c interferes with endogenous PTEN binding to cell membrane adaptor resulting in increased Akt activation, enhanced glucose utilization and improved mouse survival after cardiac arrest. Methods: Mouse cardiomyocytes were isolated from 1-3 day old mouse pups. Western blot was used to determine the efficacy of TAT-PTEN9c for Akt activation. The effect of TAT-PTEN9c on mouse survival after cardiac arrest was determined in a mouse model. TAT-PTEN9c (7.5 mg/kg) was given intravenously (IV) after CPR. As a measure of impaired glucose utilization, sorbitol content in heart and brain was determined by a fluorescence assay of NADH formation using sorbitol dehydrogenase and NAD + . Results: TAT-PTEN9c peptide enhanced Akt activation in mouse cardiomyocytes in a concentration-dependent manner. Akt phosphorylation was observed at 1 μM and further increased with 10 μM TAT-PTEN9c. TAT-PTEN9c blocked the binding of endogenous PTEN to MAGI2 in a co-immunoprecipitation assay, while TAT-PTEN3a control had no inhibitory effect. In a mouse model of cardiac arrest, survival was significantly increased in the TAT-PTEN9c treated group compared to saline controls at 4 h (10/15, 67% vs. 6/15, 40%, P < 0.05) after CPR. TAT-PTEN9c improved MAP at both R30 min and R2h. The treated mice had increased Akt phosphorylation at R15 min in both heart and brain tissues with significantly decreased sorbitol content and reduced release of taurine and glutamate into blood, suggesting improved metabolic recovery and glucose utilization. Conclusion: TAT-PTEN9c can be used after CPR in a mouse SCA model to rapidly enhance Akt activation and decrease glucose shunting to the polyol pathway in critical organs, preventing osmotic injury and early cardiovascular collapse and death.

scholarly journals Low-Dose Albendazole Inhibits Epithelial-Mesenchymal Transition of Melanoma Cells by Enhancing Phosphorylated GSK-3β/Tyr216 Accumulation

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Zhiqiang He ◽  
Shun Lei ◽  
Fucheng Liang ◽  
Liuchang Tan ◽  
Weinan Zhang ◽  
...  
Keyword(s):  
Low Dose ◽  
Epithelial Mesenchymal Transition ◽  
Inhibitory Effect ◽  
Melanoma Cells ◽  
Melanoma Metastasis ◽  
Dependent Manner ◽  
Antitumor Effects ◽  
Mesenchymal Transition ◽  
Phosphorylated Akt ◽  
Gsk 3Β

Albendazole (ABZ) is an effective broad-spectrum anthelmintic agent that has been widely used for humans and animals. Previous studies have reported that ABZ exhibits antitumor effects against melanoma and other different cancer types; however, it is unknown whether ABZ exerts the inhibitory effect against melanoma metastasis. In this study, we aimed to investigate the inhibitory effect of ABZ on melanoma cells. Through in vitro studies, we discovered that low-dose ABZ treatment significantly inhibited the migration and invasion, but not the proliferation, of A375 and B16-F10 cells in a dose-dependent manner. Further analysis revealed that ABZ treatment reduced the expression level of snail family transcriptional repressor 1 (Snail) in the cytoplasm and nucleus by decreasing the levels of phosphorylated AKT (pAKT) Ser473/GSK-3β (pGSK-3β) Ser9 and increasing pGSK-3β/Tyr216, resulting in a significant upregulation of E-cadherin and downregulation of N-cadherin and ultimately reversing the epithelial-mesenchymal transition (EMT) process of melanoma cells. In contrast, the continuous activation of AKT via transfected plasmids elevated the protein levels of pAKT Ser473/pGSK-3β Ser9 and Snail and antagonized the inhibitory action of ABZ. We also confirmed that ABZ treatment effectively inhibited the lung metastasis of melanoma in nude mice in vivo. Subsequent immunohistochemical analysis verified the decreased pAKT Ser473/pGSK-3β Ser9 and increased pGSK-3β/Tyr216 levels in ABZ-treated subcutaneous tumors. Therefore, our findings demonstrate that ABZ treatment can suppress the EMT progress of melanoma by increasing the pGSK-3β/Tyr216-mediated degradation of Snail, which may be used as a potential treatment strategy for metastatic melanoma.

scholarly journals The influence of LTS-4, a saponoside from Lysimachia thyrsiflora L., on human skin fibroblasts and human melanoma cells

2008 ◽  
Vol 13 (4) ◽  
Author(s):  
Agnieszka Galanty ◽  
Marta Michalik ◽  
Łukasz Sędek ◽  
Irma Podolak
Keyword(s):  
Cell Growth ◽  
Cell Motility ◽  
Human Skin ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Human Melanoma ◽  
Skin Fibroblasts ◽  
Dependent Manner ◽  
Human Skin Fibroblasts ◽  
Dose Dependent

AbstractWe investigated the effect of a triterpene saponoside from Lysimachia thyrsiflora L. upon the viability, proliferation, morphology and cell motility of human melanoma HTB-140 cells and human skin fibroblasts (HSFs). The compound, denoted LTS-4, decreased the viability and rate of cell growth of both cell types in a time-and dose-dependent manner, and proved cytotoxic against cancer cells at significantly lower concentrations than for fibroblasts. LTS-4 also affected the morphology of the examined cells, causing vacuolisation and actin cytoskeleton disintegration, and had an inhibitory effect on the tumour cell motility.

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

1988 ◽  
Vol 119 (1) ◽  
pp. 145-151 ◽  
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.

Probenecid Inhibits Platelet Responses to Aggregating Agents in Vitro and Has a Synergistic Inhibitory Effect with Penicillin G

1996 ◽  
Vol 76 (02) ◽  
pp. 239-244 ◽  
Author(s):  
M A Packham ◽  
M L Rand ◽  
D W Perry ◽  
D H Ruben ◽  
R L Kinlough-Rathbone
Keyword(s):  
Platelet Activating Factor ◽  
Anion Channel ◽  
Inhibitory Effect ◽  
Penicillin G ◽  
Dependent Manner ◽  
Fura 2 ◽  
Synergistic Inhibitory Effect ◽  
Dose Dependent

SummaryProbenecid is an anion channel blocker and uricosuric agent, originally developed to slow the rate of excretion of penicillin. It is now also administered with many other drugs to reduce their required dosages. Recently, probenecid (2.5 mM) has been used to prevent leakage of fura-2 or fluo-3 when these indicators of cytosolic Ca2+ levels have been introduced into cells. However, we found that probenecid markedly inhibited the increases in cytosolic Ca2+ caused by ADP, thrombin, the thrombin receptor-activating peptide (SFLLRN, TRAP), ADP, sodium arachidonate, the thromboxane A2 (TXA2) mimetic U46619, and platelet-activating factor (PAF). This finding precluded the use of probenecid with platelets in measurements of cytosolic Ca2+ with indicators such as fura-2. We then investigated the effects of probenecid on aggregation and release of 14C-serotonin from prelabeled platelets. Responses to all the agonists were inhibited by 2.5 mM probenecid, but concentrations as low as 0.25-0.5 mM inhibited responses to agonists that act largely via TXA2 (collagen, sodium arachidonate and U46619). Collagen-induced TXA2 formation was inhibited in a dose-dependent manner. Responses of aspirin-pretreated platelets to thrombin, SFLLRN, U46619 and PAF were also inhibited by probenecid, indicating that prevention of TXA2 formation does not account for all the inhibitory effects. The combination of probenecid with penicillin G produced additive or synergistic inhibition of platelet responses; responses dependent on TXA2 were synergistically inhibited by concentrations of the drugs that are reached in vivo. The synergistic inhibitory effect of probenecid on platelet functions could further impair hemostasis if it has already been partially compromised by the administration of other drugs.

scholarly journals Synergistic inhibitory effect of apomine and lovastatin on osteosarcoma cell growth

Cancer ◽  
2011 ◽  
Vol 118 (3) ◽  
pp. 750-760 ◽  
Author(s):  
Gatien Moriceau ◽  
Anke J. Roelofs ◽  
Régis Brion ◽  
Françoise Redini ◽  
Frank H. Ebetion ◽  
...  
Keyword(s):  
Cell Growth ◽  
Inhibitory Effect ◽  
Osteosarcoma Cell ◽  
Synergistic Inhibitory Effect
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