scholarly journals Involvement of Na+/H+ exchanger in desmopressin-induced platelet procoagulant response.

2004 ◽  
Vol 51 (3) ◽  
pp. 773-788 ◽  
Author(s):  
Maria M Tomasiak ◽  
Halina Stelmach ◽  
Anna Bodzenta-Łukaszyk ◽  
Marian Tomasiak
Keyword(s):  
Flow Cytometry ◽  
Thrombin Generation ◽  
Annexin V ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Platelet Volume ◽  
Cell Sizing ◽  
Dose Dependent ◽  
In Vitro Treatment

Desmopressin (DDAVP) action on platelets is associated with the development of procoagulant response but the underlying mechanism of this phenomenon is not known. We investigated whether this effect of DDAVP might be due to activation of plasma membrane Na+/H+ exchanger. The DDAVP-induced platelet procoagulant response, measured as phospholipid-dependent thrombin generation, was dose dependent and significantly weaker than that produced by collagen or monensin (mimics Na+/H+ antiport). Both the DDAVP- and collagen-produced procoagulant responses were less pronounced in the presence of EIPA, an Na+/H+ exchanger inhibitor. Flow cytometry studies revealed that in vitro treatment of platelets with DDAVP or collagen was associated with the appearance of both degranulated (and fragmented) and swollen cells. The DDAVP-evoked rise in size and granularity heterogeneity was similar to that produced by collagen or monensin and was not observed in the presence of EIPA. Using flow cytometry and annexin V-FITC as a probe for phosphatidylserine (PS) we demonstrated increased and uniform binding of this marker to all subsets of DDAVP-treated platelet population. The DDAVP-evoked PS expression was dose dependent, strongly reduced by EIPA and weaker than that caused by monensin or collagen. As judged by optical swelling assay, DDAVP in a dose dependent manner produced a rise in platelet volume. The swelling was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Electronic cell-sizing measurements showed an increase in mean platelet volume and a decrease in platelet count and platelet crit upon treatment with DDAVP. DDAVP elicited a slow (much slower than collagen) alkalinization of platelet cytosol. Altogether the data indicate an involvement of Na+/H+ exchanger in the generation of procoagulant activity in DDAVP-treated platelets.

scholarly journals The Olive Leaves Extract Has Anti-Tumor Effects against Neuroblastoma through Inhibition of Cell Proliferation and Induction of Apoptosis

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2178
Author(s):  
Fabio Morandi ◽  
Veronica Bensa ◽  
Enzo Calarco ◽  
Fabio Pastorino ◽  
Patrizia Perri ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Cycle Progression ◽  
Treatment Strategies ◽  
Annexin V ◽  
Dependent Manner ◽  
Induction Of Apoptosis ◽  
Dose Dependent

Neuroblastoma (NB) is the most common extra-cranial solid tumor of pediatric age. The prognosis for high-risk NB patients remains poor, and new treatment strategies are desirable. The olive leaf extract (OLE) is constituted by phenolic compounds, whose health beneficial effects were reported. Here, the anti-tumor effects of OLE were investigated in vitro on a panel of NB cell lines in terms of (i) reduction of cell viability; (ii) inhibition of cell proliferation through cell cycle arrest; (iii) induction of apoptosis; and (iv) inhibition of cell migration. Furthermore, cytotoxicity experiments, by combining OLE with the chemotherapeutic topotecan, were also performed. OLE reduced the cell viability of NB cells in a time- and dose-dependent manner in 2D and 3D models. NB cells exposed to OLE underwent inhibition of cell proliferation, which was characterized by an arrest of the cell cycle progression in G0/G1 phase and by the accumulation of cells in the sub-G0 phase, which is peculiar of apoptotic death. This was confirmed by a dose-dependent increase of Annexin V+ cells (peculiar of apoptosis) and upregulation of caspases 3 and 7 protein levels. Moreover, OLE inhibited the migration of NB cells. Finally, the anti-tumor efficacy of the chemotherapeutic topotecan, in terms of cell viability reduction, was greatly enhanced by its combination with OLE. In conclusion, OLE has anti-tumor activity against NB by inhibiting cell proliferation and migration and by inducing apoptosis.

scholarly journals Ascorbic acid attenuates activation and cytokine production in sepsis-like monocytes

2021 ◽  
Author(s):  
Tobias Schmidt ◽  
Robin Kahn ◽  
Fredrik Kahn
Keyword(s):  
Flow Cytometry ◽  
Ascorbic Acid ◽  
Cytokine Production ◽  
In Vitro Study ◽  
Surface Expression ◽  
High Dose ◽  
Functional Assay ◽  
Dependent Manner ◽  
Dose Dependent

Objective To investigate the effects of high dose ascorbic acid (AA) on monocyte polarization and cytokine production in vitro Design Experimental in vitro study of cells from healthy subjects and patients with sepsis Setting University research laboratory and academic hospital Subjects Six healthy controls and three patients with sepsis Interventions Monocytes were isolated from whole blood of healthy donors (n=6) and polarized in vitro for 48hrs using LPS or LTA. Polarization was confirmed by surface marker expression using flow cytometry. As a comparison, monocytes were also isolated from septic patients (n=3) and analyzed for polarization markers. The effect of AA on monocyte polarization was evaluated. As a functional assay, AA-treated monocytes were analyzed for cytokine production of TNF and IL-8 by intracellular staining and flow cytometry following activation with LPS or LTA. Measurements and Main Results Both LPS and LTA induced polarization in healthy monocytes in vitro, with increased expression of both pro- (CD40 and PDL1, p<0.05) and anti-inflammatory (CD16 and CD163, p<0.05) polarization markers, with non-significant effects on CD86 and CD206. This pattern resembled, at least partly, that of monocytes from septic patients. Treatment with AA significantly inhibited the upregulation of surface expression of CD16 and CD163 (p<0.05) in a dose dependent manner, but not CD40 or PDL-1. Finally, AA attenuated LPS or LTA-induced cytokine production of IL-8 and TNF in a dose-dependent manner (both p<0.05). Conclusions AA inhibits upregulation of anti-, but not pro-inflammatory related markers in LPS or LTA polarized monocytes. Additionally, AA attenuates cytokine production from in vitro polarized monocytes, displaying functional involvement. This study provides important insight into the immunological effects of high dose AA on monocytes, and potential implications in sepsis.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

scholarly journals Brain-Derived Microparticles Induce Systemic Coagulation Associated with Traumatic Brain Injury

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1497-1497
Author(s):  
Jing-fei Dong ◽  
Ye Tian ◽  
Breia Salsbery ◽  
Hengjie Yuan ◽  
Min Wang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Traumatic Brain Injury ◽  
Flow Cytometry ◽  
Coagulation Factors ◽  
Endothelial Barrier ◽  
Dependent Manner ◽  
Activated Platelets ◽  
Dose Dependent ◽  
Made In

Abstract Uncontrolled hemorrhage is a leading cause of the preventable deaths that occur in patients with trauma. The cause of trauma-associated coagulopathy is multifactorial, including blood loss, consumption of coagulation factors and platelets, the dilution of coagulation factors and platelets due to fluid resuscitation, and hypothermia. Traumatic brain injury (TBI) lacks two key causal factors for coagulopathy: heavy blood loss and a large volume of fluid resuscitation, but is associated with a significantly higher incidence of coagulopathy. The pathogenesis of this TBI-associated coagulopathy remains poorly understood. We tested the hypothesis that brain-derived microparticles (BDMPs) released from an injured brain play a causal role in developing systemic coagulopathy after TBI. Here, we report that mice subjected to fluid percussion injury (1.9±0.1 atm) developed a BDMP-dependent hypercoagulable state, with a peak level of plasma glial cell and neuronal microparticles, reaching 17,496 ± 4,833/µl and 18,388 ± 3,657/µl 3 hrs after TBI. BDMPs were measured by flow cytometry using triple gating based on particle size and the expression of neural cell markers and phosphatidylserine (PS). To exclude contributions to the coagulopathy of non-neural cell microparticles released during trauma stress, BDMPs were made from normal brain by freeze-thawing and mechanical injury. BDMPs thus made had below detection levels of microparticles from leukocytes (CD45), endothelial cells (CD144), erythrocytes (CD235a), and platelets (CD42b). Uninjured mice injected with BDMPs made in vitro developed a hyper-turn-hypo-coagulable state in a dose-dependent manner as measured by the rates of clot formation and fibrinogen depletion, resulting in microvascular fibrin deposition in the lungs, kidney and heart. BDMPs measured 50 – 500 nm with relatively intact membranes under transmission electron microscopy and expressed neuronal or glial cell markers and procoagulant PS and tissue factor (TF). BDMPs promoted clot formation in a PS-dependent assay at a maximal activity of ~1 x 105 BDMPs/µl, equivalent to 1.6 µg/µl of purified brain PS. They were equally active in promoting thrombin generation in a PS-and TF-dependent manner, BDMPs at 2.5 x 104 /µl yielding an activity equivalent to 1 pM of soluble TF. The procoagulant activity of BDMPs was significantly stronger than microparticles generated from collagen-stimulated platelets and was blocked by the PS-binding lactadherin in a dose-dependent manner. Consistent with observations made in the mouse models, fetal hippocampal cells in culture produced microparticles upon injury. These microparticles transmigrated through the disrupted endothelial barrier in the presence of live, but not lyophilized platelets. BDMP-bound platelets were detected by flow cytometry and scan electron microscopy. They activated platelets as measured by increases in calcium influx and CD62p expression, but did not induce platelet aggregation directly or in the presence of low doses of collagen. In summary, we have studied acute changes in coagulation associated with TBI using a mouse FPI model combined with in vitro experiments. Focusing on the first 6 hrs post-TBI minimizes confounding changes induced by secondary events, such as ischemic injury. The results define a causal role for BDMPs in the TBI-associated systemic coagulation. We also show that BDMPs activated platelets. Activated platelets may facilitate the transmigration of BDMPs through the disrupted endothelial barrier by releasing pro-inflammatory mediators to promote local inflammation at a site of vascular injury. This notion is supported by the finding that live, but not lyophilized platelets and, to lesser degree, plasma from activated platelets promoted BDMP transmigration through a monolayer of endothelial cells. Finally, the PS binding lactadherin blocked the BDMP-dependent procoagulant activity, raising two interesting perspectives. First, PS scavengers and neutralizing molecules may reduce or prevent coagulopathy associated with TBI. Second, an intrinsic or acquired deficiency in the PS-dependent clearance of microparticles may predispose an individual to consumptive coagulopathy associated with TBI and other conditions. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tanshinone IIA suppresses the progression of lung adenocarcinoma through regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ziheng Li ◽  
Ying Zhang ◽  
Yuan Zhou ◽  
Fuqian Wang ◽  
Chao Yin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Lung Adenocarcinoma ◽  
Annexin V ◽  
Tanshinone Iia ◽  
Dependent Manner ◽  
Information Analysis ◽  
Cdk2 Complex ◽  
Dual Staining

AbstractLung adenocarcinoma (LUAD) belongs to a subgroup of non-small cell lung cancer (NSCLC) with an increasing incidence all over the world. Tanshinone IIA (TSA), an active compound of Salvia miltiorrhiza Bunge., has been found to have anti-tumor effects on many tumors, but its anti-LUAD effect and its mechanism have not been reported yet. In this study, bio-information analysis was applied to characterize the potential mechanism of TSA on LUA, biological experiments were used to verify the mechanisms involved. TCGA, Pubchem, SwissTargetPrediction, Venny2.1.0, STRING, DAVID, Cytoscape 3.7.2, Omicshare, GEPIA, RSCBPDB, Chem Draw, AutoDockTools, and PyMOL were utilized for analysis in the bio-information analysis and network pharmacology. Our experiments in vitro focused on the anti-LUAD effects and mechanisms of TSA on LUAD cells (A549 and NCI-H1975 cells) via MTT, plate cloning, Annexin V-FITC and PI dual staining, flow cytometry, and western blot assays. A total of 64 differentially expressed genes (DEGs) of TSA for treatment of LUAD were screened out. Gene ontology and pathway analysis revealed characteristic of the DEGs network. After GEPIA-based DEGs confirmation, 46 genes were considered having significant differences. Further, 10 key DEGs (BTK, HSD11B1, ADAM33, TNNC1, THRA, CCNA2, AURKA, MIF, PLK1, and SORD) were identified as the most likely relevant genes from overall survival analysis. Molecular Docking results showed that CCNA2, CDK2 and PLK1 had the lowest docking energy. MTT and plate cloning assays results showed that TSA inhibited the proliferation of LUAD cells in a concentration-dependent manner. Annexin V-FITC and PI dual staining and flow cytometry assays results told that TSA promoted the apoptosis of the two LUAD cells in different degrees, and induced cycle arrest in the G1/S phase. Western blot results showed that TSA significantly down-regulated the expression of CCNA2, CDK2, AURKA, PLK1, and p-ERK. In summary, TSA could suppress the progression of LUAD by inducing cell apoptosis and arresting cell cycle, and these were done by regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway. These findings are the first to demonstrate the molecular mechanism of TSA in treatment of LUAD combination of network bio-information analysis and biological experiments in vitro.

scholarly journals The in Vitro Effect of Polyvinylpyrrolidone and Citrate Coated Silver Nanoparticles on Erythrocytic Oxidative Damage and Eryptosis

2018 ◽  
Vol 49 (4) ◽  
pp. 1577-1588 ◽  
Author(s):  
Zannatul Ferdous ◽  
Sumaya Beegam ◽  
Saeed Tariq ◽  
Badreldin H Ali ◽  
Abderrahim Nemmar
Keyword(s):  
Oxidative Stress ◽  
Silver Nanoparticles ◽  
Antimicrobial Agents ◽  
Annexin V ◽  
Drug Carriers ◽  
Cellular Protein ◽  
Dependent Manner ◽  
Vitro Effect ◽  
Dose Dependent

Background/Aims: Silver nanoparticles (AgNPs) are increasingly used as antimicrobial agents and drug carriers in various biomedical fields. AgNPs can encounter erythrocytes either directly following intravenous injection, or indirectly via translocation from the site of administration. However, information regarding the pathophysiological effects and possible mechanism of action of AgNPs on the erythrocytes are still inadequately studied. Thus, the aim of our study was to investigate the mechanism underlying the effect of coating and concentration of AgNPs on mouse erythrocytes in vitro. Methods: We studied the interaction of polyvinylpyrrolidone (PVP) and citrate (CT) coated AgNPs (10 nm) at various concentrations (2.5, 10, 40 µg/ml) with mouse erythrocytes in vitro using various techniques including transmission electron microscopy (TEM), hemolysis, and colorimetric measurement of markers of oxidative stress comprising malondialdehyde (MDA), reduced glutathione (GSH), and catalase (CAT). Intracellular calcium (Ca2+) was determined using Fura 2AM fluorescence. Annexin V was quantified using ELISA and the caspase 3 was determined both flurometrically and by western blot technique. Results: Following incubation of the erythrocytes with AgNPs, both PVP- and CT- AgNPs induced significant and dose - dependent increase in hemolysis. TEM revealed that both PVP- and CT- AgNPs were taken up by erythrocytes. The erythrocyte susceptibility to lipid peroxidation measured by MDA was significantly increased in both PVP-and CT- AgNPs. The concentration of GSH and CAT activity were significantly decreased by both types of AgNPs. Additionally, PVP- and CT- AgNPs significantly increased intracellular Ca2+ in a dose -dependent manner. Likewise, the concentration of the cellular protein annexin V was significantly and dose - dependently enhanced by both types of AgNPs. Furthermore, PVP- and CT- AgNPs induced significant increase in calpain activity in incubated erythrocytes. Conclusion: We conclude that both PVP- and CT- AgNPs causes hemolysis, and are taken up by erythrocytes. Moreover, we demonstrated that AgNPs induces oxidative stress and eryptosis. These findings provide evidence for the potential pathophysiological effect of PVP-and CT- AgNPs on erythrocyte physiology.

Influence of Newly Developed Resin and MMA Resin on Mouse Fibroblasts Cellular Viability

2006 ◽  
Vol 309-311 ◽  
pp. 817-820
Author(s):  
S. Jinno ◽  
T. Suzuki ◽  
A. Ishikawa ◽  
T. Hayashi ◽  
M. Deguchi ◽  
...  
Keyword(s):  
Phase Contrast ◽  
High Performance ◽  
Annexin V ◽  
Dependent Manner ◽  
Cellular Viability ◽  
Mouse Fibroblasts ◽  
Propidium Iodide Staining ◽  
Ic50 Value ◽  
Dose Dependent

The aim of this study is evaluate to the cellular viability of elution from the newly developed resin and Osteobond® in vitro. The basis of the newly developed resin are methacryloyloxyethyl methyl succinate and 1,6-Hexanediol dimethacrylate. The basis of Osteobond is methyl methacrylate. The concentrations of basis in each elution were determined by high-performance liquid chromatography (HPLC). Cellular viabilities of L-929 mouse fibroblasts were evaluated by direct cells counting, and then, each IC50 value was calculated. Moreover, patterns of cell death were analyzed using annexin V/propidium iodide staining with the phase-contrast microscope and flow cytometry. The concentration of Osteobond elution was 2.16 mM of MMA, and the newly developed resin elution was 1.02 mM of TA and 1.87 x 10-2 mM of HX. Until 72 hours of incubation, treatment with each elution impaired the viability of L-929 cells in a dose-dependent manner. IC50 value of Osteobond was 6.48 x 10-4 mM of MMA. However, IC50 of the newly developed resin was not calculated. Treatment with Osteobond elution showed more necrotic cells than with the newly developed resin elution. In conclusion, the results demonstrated much more excellent cellular viability of the newly developed resin than that of MMA resin. Thus, it is suggested that the newly developed resin will be more useful as an implantation material for dentistry and orthopaedics.

The Novel Proteasome Inhibitor MLN2238 (Ixazomib) Induces Death In CLL Cells In Vitro and Potentiates Lethality Of Fludarabine and The BCL2 Inhibitor At-101

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4189-4189
Author(s):  
Kasyapa S. Chitta ◽  
Aneel Paulus ◽  
Sharoon Akhtar ◽  
Maja Kuranz ◽  
Kena Miller ◽  
...  
Keyword(s):  
Cell Death ◽  
Annexin V ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Dose Effect ◽  
Response To Treatment ◽  
Dependent Manner ◽  
Proteasomal Activity ◽  
Dose Dependent

Abstract Background Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells in the peripheral blood, bone marrow, lymph nodes, and spleen. This is due to a combined effect of deferred apoptosis with slow, but persistent proliferation of malignant cells. In CLL, tumor-sustaining homeostasis is critically maintained by the ubiquitin proteasome system. The proteasome mediates degradation of various transcription factors such as TP53 as well as upholding a balance between the anti and pro apoptotic proteins of the BCL2 family. Previous studies have demonstrated that the clinical course of the disease is negatively associated with malfunctioning apoptotic pathways that result in increased levels of BCL2. Thus, identification and correction of defects that affect programmed cell death offer therapeutic vantage to reset and engage cell death pathways in CLL. Aim Examination of the anti-CLL properties of the investigational agent MLN2238 (Millennium Pharmaceuticals, Inc., Cambridge, MA) and its ability to inhibit the proteasomal machinery; induce CLL cell death and downregulate BCL2. MLN2238 activity was also investigated in conjunction with anti-CLL therapies such as fludarabine and dexamethasone along with the BH3 mimetic BCL2 inhibitor, AT-101 (Ascenta Pharmaceuticals, Malvern, PA). Methods CLL cells with >90% CD19+ tumor population were obtained from 28 patients with a confirmed diagnosis of CLL. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Apoptosis was measured by annexin-v/PI staining, and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiles were ascertained by western blot. Results MLN2238 inhibited the chymotrypsin-like proteasomal activity by more than 90% (p<0.005) in all patient samples without altering PSMB5 protein levels. Moderate to minimal inhibitory effect on caspase-like and trypsin-like proteasomal activities, respectively, was also noted. CLL cells showed a concentration dependent decrease in viability in response to treatment with MLN2238 at an IC50 of 50 nM. MLN2238 treated cells underwent apoptosis in a dose dependent manner with a median dose effect (cell death) observed in 42% of cells at 25 nM (range 10% - 54%) and 60% of cells at a 50 nM concentration (range, 25% - 73%). PARP-1 and caspase-3 cleavage along with an increase in MOMP was also noted after CLL cells were treated with MLN2238; however, apoptosis was only partially blocked by the pan-caspase inhibitor z-VAD.fmk. BCL2 downregulation was dose-dependent and was observed as early as 12 hours. We sought to determine whether directly disrupting BCL2 function with AT-101 could enhance the anti-CLL effects of MLN2238. When used at sub-IC50 concentrations, AT1-10 synergized with MLN2238 to induce CLL cell death. Synergy was also observed when MLN2238 was paired with the cytotoxic agent fludarabine, whereas the combination of MLN2238 and dexamethasone resulted in additive anti-CLL activity. Conclusion While PI have made an important impact in various B cell cancers, their role in CLL has not been well established. We investigated preclinically, a novel PI and noted that targeting the proteasome with MLN2238 resulted in lethal events in CLL cells, which were further enhanced by disruption of the BCL2 prosurvival pathway. Moreover, proteasome disruption sensitized CLL cells to the cytotoxic effect of fludarabine, an important therapeutic in CLL. These data provides the mechanistic basis for evaluation of MLN2238 in CLL through rationale design of drug combination strategies based on CLL biology. We would like to acknowledge the Leukemia and Lymphoma Society (A.C.-K. is a Leukemia and Lymphoma Scholar in Clinical Research) for their ongoing support. We are also grateful to Mary Ella Mahoney Davidson (Millennium Pharmaceuticals) for providing logistical support. Disclosures: Foran: Celgene: Research Funding.

scholarly journals Viperin inhibits hepatitis C virus replication by interfering with binding of NS5A to host protein hVAP-33

2012 ◽  
Vol 93 (1) ◽  
pp. 83-92 ◽  
Author(s):  
Shanshan Wang ◽  
Xianfang Wu ◽  
Tingting Pan ◽  
Wuhui Song ◽  
Yaohui Wang ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Microscopic Analysis ◽  
Underlying Mechanism ◽  
Host Protein ◽  
Type I ◽  
Dependent Manner ◽  
C Terminus ◽  
Dose Dependent

Viperin is a type-I and -II interferon-inducible intracytoplasmic protein that mediates antiviral activity against several viruses. A previous study has reported that viperin could limit hepatitis C virus (HCV) replication in vitro. However, the underlying mechanism remains elusive. In the present study, we found that overexpression of viperin could inhibit HCV replication in a dose-dependent manner in both the replicon and HCVcc systems. Furthermore, through co-immunoprecipitation and laser confocal microscopic analysis, viperin was found to interact with the host protein hVAP-33. Mutagenesis analysis demonstrated that the anti-HCV activity of viperin was located to its C terminus, which was required for the interaction with the C-terminal domain of hVAP-33. Competitive co-immunoprecipitation analysis showed that viperin could interact competitively with hVAP-33, and could therefore interfere with its interactions with HCV NS5A. In summary, these findings suggest a novel mechanism by which viperin inhibits HCV replication, possibly through binding to host protein hVAP-33 and interfering with its interaction with NS5A.

Ternary Copper (II) Complex Induced Apoptosis and Cell Cycle Arrest in Colorectal Cancer Cells

2021 ◽  
Vol 21 ◽  
Author(s):  
Sathiavani Arikrishnan ◽  
Jian Sheng Loh ◽  
Xian Wei Teo ◽  
Faris bin Norizan ◽  
May Lee Low ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Annexin V ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Colorectal Cancer Cells ◽  
Dose Dependent ◽  
Parp 1 ◽  
Ht 29

Background: The lack of specificity, severe side effects, and development of drug resistance have largely limited the use of platinum-based compounds in cancer treatment. Therefore, copper complexes have emerged as potential alternatives to platinum-based compounds. Objective: Ternary copper (II) complex incorporated with 1-10-phenanthroline and L-tyrosine was investigated for its anti-cancer effects in HT-29 colorectal cancer cells. Methods: Cytotoxic effects of ternary copper (II) complex in HT-29 cells were evaluated using MTT assay, Real-Time Cell Analysis (RTCA), and lactate dehydrogenase (LDH) assay. Cell cycle analysis was performed using flow cytometry. Apoptosis induction was studied by Annexin V-FITC/propidium iodide (PI) staining and mitochondrial membrane potential analysis (JC-10 staining) using flow cytometry. Intracellular reactive oxygen species (ROS) were detected by DCFH-DA assay. The expression of proteins involved in the apoptotic signalling pathway (p53, caspases, and PARP-1) was evaluated by western blot analysis. Results: Ternary copper (II) complex reduced the cell viability of HT-29 cells in a time- and dose-dependent manner, with IC50 of 2.4 ± 0.4 and 0.8 ± 0.04 µM at 24 and 48 hours, respectively. Cell cycle analysis demonstrated induction of S-phase cell cycle arrest. Morphological evaluation and Annexin V-FITC/PI flow cytometry analysis confirmed induction of apoptosis that was further supported by cleavage and activation of caspase-8, caspase-9, caspase-3, and PARP-1. Mutant p53 was also downregulated in a dose-dependent manner. No LDH release, mitochondrial membrane potential disruption, and ROS production were observed. Conclusion: Ternary copper (II) complex holds great potential to be developed for colorectal cancer treatment.

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