High-Dimensional Analysis Identifies Mechanisms of Gilteritinib Resistance in FLT3-Mutated AML

Abstract While clinical benefit has been observed with gilteritinib in patients with FLT3 mutated relapsed/refractory acute myeloid leukemia (AML), most patients relapse through mechanisms that are incompletely understood. In this study, to investigate mechanisms of gilteritinib sensitivity and resistance, we performed targeted sequencing (21 patients) and scRNASeq analysis (8 patients) of FLT3-ITD-positive AML samples obtained before and during treatment. Before treatment, co-occurring mutations were observed in 33 genes among 21 patients. Mutations in RAS pathway genes (PTPN11, KRAS, NRAS, CBL) were the most common and observed in 57% (12/21) of patients. Seven patients pretreatment already contained RAS pathway mutations, of which 6 of these mutations were maintained over the course of treatment. During treatment, 9 patients showed emerging RAS mutations, 4 of which initially presented with a different RAS pathway mutation pre-treatment. Other mutations that arose during treatment were observed in CEBPA, IDH1, SF1 and WT1; as well as CSF3R, CUX1, PLEKHG5, and XPO1, not previously identified in gilteritinib-treated patients. Mutational clonality was generally maintained over treatment in both responders and non-responders. scRNASeq revealed global gene expression differences in myeloblast populations between gilteritinib-responsive and -unresponsive patients. Previous studies in vitro have shown that bone marrow-derived hematopoietic and inflammatory cytokines/chemokines confer resistance to FLT3 inhibitors. In the unresponsive group, we observed an increase in expression of CCL5, CXCL1, CXCL2, CXCL8, FLT3, IL6R, IL3RA, and CSF2RA during gilteritinib treatment, supporting the concept from preclinical studies that AML microenvironment-mediated factors play a critical role in drug resistance. Baseline expression of the Tec kinase BMX was significantly higher in unresponsive patients (Log2FoldChange, 6.65; adjusted P value, 0.00186), and this was maintained in the expanding myeloblast populations during treatment. Previously, upregulated BMX was shown to contribute to sorafenib resistance in patients with FLT3-ITD-positive AML, through cell-nonautonomous microenvironment hypoxia-dependent effects. Further in vitro investigation confirmed gilteritinib resistance could be reversed through genetic and pharmacological manipulation of BMX. Gene module analysis showed associations between gilteritinib responsive and upregulation of genes and pathways involved in lymphocyte differentiation and myeloid leukocyte activation, including TBX21, GATA3, CD33, and LYZ. By contrast, there was association between unresponsiveness to gilteritinib and upregulation of cell-cycle, DNA, and RNA metabolic processes, including pathways involving METTL1 and DNMT3A, as well as pre-treatment expression of pathways associated with protein translation. Together, these data provide support for microenvironment-dependent escape from targeted therapy and suggest that BMX may contribute to gilteritinib resistance. High-dimensional analysis with scRNA-seq provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity. Disclosures Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

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An in vitro investigation of pre-treatment effects before fissure sealing

International Journal of Paediatric Dentistry ◽

10.1111/ipd.12290 ◽

2017 ◽

Vol 27 (6) ◽

pp. 514-522 ◽

Cited By ~ 5

Author(s):

Mahshid Bagheri ◽

Peter Pilecki ◽

Salvatore Sauro ◽

Martyn Sherriff ◽

Timothy F. Watson ◽

...

Keyword(s):

Treatment Effects ◽

In Vitro Investigation ◽

Pre Treatment ◽

Fissure Sealing

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DUSP7 inhibits cervical cancer progression by inactivating the RAS pathway

10.21203/rs.3.rs-131949/v1 ◽

2020 ◽

Author(s):

Huimin Bai ◽

Ruili Jiao ◽

Weihua Li ◽

Jing Zhao ◽

Meizhu Xiao ◽

...

Keyword(s):

Cervical Cancer ◽

Cancer Progression ◽

Tumor Formation ◽

Differentially Expressed ◽

P Value ◽

Ras Pathway ◽

Paired Samples ◽

Siha Cells ◽

Gene Functions

Abstract Objective The present study was aimed to determine the differentially expressed proteins (DEPs) between paired samples of cervical cancer (CC) and paracancerous tissue by quantitative proteomics and to examine the effects of DUSP7 expression on tumorigenesis and progression of CC. Materials and methods Proteomic profiles of three paired samples of CC and paracancerous tissue were quantitatively analyzed to identify the DEPs. The gene functions of the DEPs were presented based on bioinformatics analysis in combining with bibliographical information. The expression of the DEPs was validated by IHC examination in the CC tissue arrays. The relationship between the DEPs expression and patients’ clinicopathological characteristics and prognosis were evaluated using the Spearman rank correlation test and Kaplan-Meier survival analysis respectively. The effects of the selected DEPs on CC progression were examined in SIHA (human CC cell line) cells. Results According to the TMT (Tandem Mass Tags) ratios (≥ 1.5 or ≤ 0.5 ), a total of 129 proteins were found to be differentially expressed in all 3 pairs of samples with a p value < 0.05. To investigate gene functions of these DEPs in tumor growth and progresssion, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed. Three DEPs, HRAS, DUSP7, and PLD1, and P-ERK1/2, associated with RAS pathway, were selected for further investigation. Western Blot and IHC staining analyses confirmed the results from quantitative proteomic analysis showing that HRAS, P-ERK1/2, and PLD1 levels were increased whereas DUSP7 level was decreased in CC tissue compared with the paired normal paracancerous tissues. The IHC results from the CC TMA analysis showed that the decreased expression of DUSP7 (P = 0.045 and 0.044, respectively) and increased expression of PLD1 (P = 0.046 and 0.028, respectively) were significantly associated with a tumor size > 2 cm and parametrial infiltration. In addition, the decreased expression of DUSP7 and increased expression of PLD1 and p-ERK1/2 were adversely related to patients’ relapse (P = 0.003, 0.040, and 0.001, respectively) and survival (P = 0.034, 0.001, and 0.006, respectively). The expression of HRAS and p-ERK1/2 was decreased in DUSP7-SIHA cells compared to NC-SIHA cells (P = 0.0003 and 0.0026, respectively). Biological functions in vitro, including invasion, migration, and proliferation, and tumor formation in vivo were decreased in the DUSP7-SIHA cells (all P < 0.05), yet increased in the siDUSP7-SIHA cells (all P < 0.05). Conclusions DUSP7 is decreased in cervical cancer tissues compared to normal tissues. Increasing or decreasing DUSP7 expression was found to significantly reduce or enhance the anchorage-independent growth of SIHA cells, respectively. The biological function of DUSP7 is possibly achieved through dephosphorylation of the ERK1/2 and inactivation of the RAS pathway. Up-regulating the expression of DUSP7 may be potential useful for the prevention or treatment of CC.

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Comparative assessment of in vitro BBB tight junction integrity following exposure to cigarette smoke and e-cigarette vapor: a quantitative evaluation of the protective effects of metformin using small-molecular-weight paracellular markers

Fluids and Barriers of the CNS ◽

10.1186/s12987-021-00261-4 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Hossam Kadry ◽

Behnam Noorani ◽

Ulrich Bickel ◽

Thomas J. Abbruscato ◽

Luca Cucullo

Keyword(s):

Tight Junction ◽

Negative Impact ◽

Electronic Cigarettes ◽

Critical Role ◽

Protective Effects ◽

Small Molecular Weight ◽

Stroke Incidence ◽

The Central Nervous System ◽

Pre Treatment

Abstract Background The blood–brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) from blood-borne agents and potentially harmful xenobiotics. Our group’s previous data has shown that tobacco smoke (TS) and electronic cigarettes (EC) affect the BBB integrity, increase stroke incidence, and are considered a risk factor for multiple CNS disorders. Metformin was also found to abrogate the adverse effects of TS and EC. Methods We used sucrose and mannitol as paracellular markers to quantitatively assess TS and EC’s impact on the BBB in-vitro. Specifically, we used a quantitative platform to determine the harmful effects of smoking on the BBB and study the protective effect of metformin. Using a transwell system and iPSCs-derived BMECs, we assessed TS and EC’s effect on sucrose and mannitol permeability with and without metformin pre-treatment at different time points. Concurrently, using immunofluorescence (IF) and Western blot (WB) techniques, we evaluated the expression and distribution of tight junction proteins, including ZO-1, occludin, and claudin-5. Results Our data showed that TS and EC negatively affect sucrose and mannitol permeability starting after 6 h and up to 24 h. The loss of barrier integrity was associated with a reduction of TEER values. While the overall expression level of ZO-1 and occludin was not significantly downregulated, the distribution of ZO-1 was altered, and discontinuation patterns were evident through IF imaging. In contrast to occludin, claudin-5 expression was significantly decreased by TS and EC, as demonstrated by WB and IF data. Conclusion In agreement with previous studies, our data showed the metformin could counteract the negative impact of TS and EC on BBB integrity, thus suggesting the possibility of repurposing this drug to afford cerebrovascular protection.

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Inhibition of histone readers bromodomain extra-terminal proteins alleviates scleroderma fibrosis

10.1101/2020.08.07.242198 ◽

2020 ◽

Author(s):

Sirapa Vichaikul ◽

Pamela J. Palisoc ◽

Mustafa Ali ◽

Phillip L. Campbell ◽

M. Asif Amin ◽

...

Keyword(s):

Therapeutic Option ◽

Critical Role ◽

Dermal Fibroblasts ◽

Skin Fibrosis ◽

P Value ◽

Bet Inhibitor ◽

Cycle Arrest ◽

Terminal Proteins

AbstractObjectivesBinding of the bromodomain and extra-terminal domain proteins (BETs) to acetylated histone residues is critical for gene transcription in many fibrotic diseases. This study sought to determine the anti-fibrotic efficacy and potential mechanisms of BET inhibition in scleroderma (SSc).MethodsDermal fibroblasts were isolated from biopsies from healthy subjects or patients with diffuse cutaneous (dc)SSc. Fibroblasts were treated with pan BET inhibitor JQ1, BRD2 inhibitor BIC1, or BRD4 inhibitors AZD5153 or ARV825. Knockdown of BETs was achieved by siRNA transfection. The in vivo anti-fibrotic efficacy of JQ1 was determined in a bleomycin-induced skin fibrosis mouse model. T-test or ANOVA were used to compare differences between groups, and a p-value of <0.05 was considered significant.ResultsBET inhibitor JQ1 dose-dependently downregulated pro-fibrotic genes in dcSSc fibroblasts, and inhibited cell migration and gel contraction. It suppressed proliferation by inducing cell cycle arrest. The anti-fibrotic effects of JQ1 were also observed in TGFβ-treated normal fibroblasts. JQ1 prevented bleomycin-induced skin fibrosis in mice. The anti-fibrotic effect of JQ1 was mediated by inhibition of BRD4, as specific blockade or knockdown of BRD4 led to downregulation of fibrotic markers and inhibition of myofibroblast properties, while inhibition or knockdown of BRD2 or BRD3 had minimal effects in dcSSc fibroblasts.ConclusionsBET inhibition showed promising anti-fibrotic effects in SSc both in vitro and in vivo. Specifically, we showed that BRD4 plays a critical role in SSc fibrosis and that targeting BRD4 might be a novel therapeutic option for this disease.Significance statementBlockade of histone readers bromodomain extra-terminal proteins (BETs) shows potent anti-fibrotic properties in SSc dermal fibroblasts and bleomycin-induced skin fibrosis model. Among the BETs, BRD4 appears to regulate essential processes involved in SSc fibrosis. Results from this study provide the scientific foundation for the use of BET or BRD4 inhibitors in treating SSc.

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Effects of Flavonoid Quercetin on Thrombo-Inflammatory Processes in Patients with Sickle Cell Disease

Blood ◽

10.1182/blood-2021-148601 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2020-2020

Author(s):

Maria A. Lizarralde ◽

Brenda Merriweather ◽

Anna Conrey ◽

Ankit Saxena ◽

Arun S Shet

Keyword(s):

Flow Cytometry ◽

Sickle Cell Disease ◽

Platelet Activation ◽

P Value ◽

Cell Disease ◽

Platelet Surface ◽

Flavonoid Quercetin ◽

Pre Treatment ◽

Stimulation Of

Abstract Introduction: Altered erythrocyte physiology in Sickle Cell Disease (SCD) results in severe vascular complications, including vaso-occlusion (VOC) along with various other manifestations, such as thrombosis and end organ damage. Moreover, platelet activation endothelial dysfunction and fibrin formation, are critical processes implicated in thrombo-inflammatory vasculopathy in SCD. Since flavonoids intake is associated with lowered adverse cardiovascular disease outcomes and cancer associated thrombosis, we are evaluating the effects of the flavonoid Isoquercetin (IQ) on thrombo-inflammatory biomarkers in patients with SCD (NCT:04514510). Here we report the mechanistic effects of Quercetin (Que), an aglycone that differs from IQ by the absence of 3-linked glucoside moiety, on markers of erythrocyte and platelet activation in patients with SCD. Methods: We studied patients with SCD (Genotype SS, mean age: 39 yrs, N=8) and ethnic matched controls (Genotype AA, mean age: 42 yrs, N=8). Erythrocyte reactive oxygen species (ROS) was measured in washed red cells by flow cytometry using the fluorescent probe 2', 7'-dichlorofluorescein diacetate (DCFDA) in the presence or absence of Quercetin (Que) (100 µM). Effects of in vitro erythrocyte ROS induction by hydrogen peroxide (H 2O 2) and ROS inhibition by N-acetyl-L-cysteine (NAC) were evaluated. Baseline and agonist-induced (ADP 20 µM) platelet P-selectin expression in vitro in the presence or absence of Que (100 µM) was assessed by flow cytometry. Results: At baseline, SS erythrocytes demonstrated elevated levels of ROS reflected by greater florescent intensity with DCFDA when compared with erythrocytes from ethnic matched controls (SS mean= 1884, SD: 508.8 vs. AA mean: 1604, SD: 554.5; p value= 0.3798). Stimulation of AA erythrocytes in vitro with H 2O 2 led to accumulation of ROS that approached levels similar to those exhibited by SS erythrocytes at baseline (AA mean: 1890, SD: 748.3). Pre-treatment of SS and AA erythrocytes in vitro with the antioxidant NAC exhibited minimal effects on ROS in SS erythrocytes (SS baseline+NAC mean: 1942, SD: 526.8, p value 0.4609 and SS H 2O 2+NAC mean: 1889, SD: 437.4, p value 0.5469) but lowered ROS levels in both baseline and H 2O 2 treated AA erythrocytes (AA baseline+NAC mean: 1442, SD: 453.9, p value 0.1563 and AA H 2O 2+NAC mean: 1581, SD: 518.7, p value 0.078). Pre-treatment of SS and AA erythrocytes in vitro with Que substantially lowered erythrocyte ROS in patients and controls at the baseline (SS baseline ROS with Que mean: 1582, SD: 542.0, p value 0.0078; AA baseline ROS with Que mean: 1341, SD: 490.9, p value 0.0313; Fig. 1A), and after H 2O 2 stimulation (SS H 2O 2 induced ROS+Que mean: 1677, SD: 572.5, p value 0.0156; AA H 2O 2 induced ROS+Que mean: 1417, SD: 512, p value 0.0078). Pre-treatment of erythrocytes with a combination of Que and NAC did not reveal synergistic anti-oxidant activity (SS NAC+Que mean: 1616, SD: 464.8; AA NAC+Que mean: 1333, SD: 524.1). We simultaneously investigated the effects of Que on platelet P-selectin expression in the same subjects. As expected, baseline platelet surface P-selectin expression was higher in SS patients compared with controls (SS N=5, mean: 1.48%, Max: 4.9%, Min: 0.03%; AA N=4, mean: 0.89%, Max: 2.3%, Min: 0%; p value 0.55). Stimulation of platelet rich plasma (PRP) with ADP increased surface P-selectin expression in comparison with baseline, in both patients and controls (SS N=5, mean: 18.04%, Max: 40.4%, Min: 5.8%, p value 0.0047; AA N=4, mean: 20.60%, Max: 43.8%, Min: 5.4%; p value 0.0140). Pre-treatment of PRP with Que prior to ADP stimulation revealed a trend toward decreased platelet surface P-selectin expression in both patients and controls (SS N=5, mean: 10.49%, Max: 24.8%, Min: 3.1%; AA N=4, mean: 12.09%, Max: 21.9%, Min: 3.4%; Fig 1B) although this did not return to baseline. Conclusion: The flavonoid Quercetin appears to have a beneficial effect in lowering erythrocyte ROS and platelet surface P-selectin and warrant further evaluation as a strategy to ameliorate thrombo-inflammatory pathophysiology in sickle hemoglobinopathies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Dynamic pneumococcal genetic adaptations support bacterial growth and inflammation during coinfection with influenza

Infection and Immunity ◽

10.1128/iai.00023-21 ◽

2021 ◽

Author(s):

Amanda P. Smith ◽

Lindey C. Lane ◽

Tim van Opijnen ◽

Stacie Woolard ◽

Robert Carter ◽

...

Keyword(s):

Single Gene ◽

Critical Role ◽

Protein Translation ◽

Bacterial Metabolism ◽

Virus Infections ◽

Nucleotide Biosynthesis ◽

Cytokines And Chemokines ◽

Bacterial Genes

Streptococcus pneumoniae (pneumococcus) is one of the primary bacterial pathogens that complicates influenza virus infections. These bacterial coinfections increase influenza-associated morbidity and mortality through a number of immunological and viral-mediated mechanisms, but the specific bacterial genes that contribute to post-influenza pathogenicity are not known. Here, we used genome-wide transposon mutagenesis (Tn-Seq) to reveal bacterial genes that confer improved fitness in influenza-infected hosts. The majority of the 32 identified genes are involved in bacterial metabolism, including nucleotide biosynthesis, amino acid biosynthesis, protein translation, and membrane transport. We generated single-gene deletion (SGD) mutants of five identified genes: SPD1414, SPD2047 (cbiO1), SPD0058 (purD), SPD1098, and SPD0822 (proB), to investigate their effect on in vivo fitness, disease severity, and host immune responses. Growth of SGD mutants was slightly attenuated in vitro and in vivo, but each still grew to high titers in the lungs of mock- and influenza-infected hosts. Despite high bacterial loads, mortality was significantly reduced or delayed with all SGD mutants. Time-dependent reductions in pulmonary neutrophils, inflammatory macrophages, and select proinflammatory cytokines and chemokines were also observed. Immunohistochemical staining further revealed altered neutrophil distribution with reduced degeneration in the lungs of influenza-SGD coinfected animals. These studies demonstrate a critical role for specific bacterial genes and for bacterial metabolism in driving virulence and modulating immune function during influenza-associated bacterial pneumonia.

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In vitro investigation of Varizella zoster virus as an oncolytic virus in glioblastoma therapy

Aktuelle Neurologie ◽

10.1055/s-0028-1086549 ◽

2008 ◽

Vol 35 (S 01) ◽

Author(s):

H Leske ◽

A Baiker ◽

C Schichor ◽

J.C Tonn ◽

R Goldbrunner ◽

...

Keyword(s):

Oncolytic Virus ◽

In Vitro Investigation ◽

Varizella Zoster Virus

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Fibrin Polymerization Defect in HIV-infected Patients-Evidence for a Critical Role of Albumin in the Prolongation of Thrombin and Reptilase Clotting Times

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653779 ◽

1995 ◽

Vol 73 (03) ◽

pp. 349-355 ◽

Cited By ~ 4

Author(s):

Pierre Toulon ◽

Elyane Frere ◽

Claude Bachmeyer ◽

Nathalie Candia ◽

Philippe Blanche ◽

...

Keyword(s):

Critical Role ◽

Human Albumin ◽

Albumin Level ◽

Final Concentration ◽

Albumin Concentration ◽

Clotting Time ◽

Fibrin Polymerization ◽

Small Series ◽

Group 1

SummaryThrombin clotting time (TCT) and reptilase clotting time (RCT) were found significantly prolonged in a series of 72 HIV-infected patients drawn for routine coagulation testing. Both TCT and RCT were highly significantly correlated with albumin (r = -0.64, and r = -0.73 respectively, p<0.0001). TCT and RCT were significantly higher (p<0.0001) in a series of 30 other HIV-infected patients selected on their albumin level below 30.0 g/l (group l) than in 30 HIV-infected patients with albumin level above 40.0 g/l or in 30 HIV-negative controls; the two latter groups were not different. In vitro supplementation of plasma from group 1 patients with purified human albumin up to 45.0 g/l (final concentration) lead to a dramatic shortening effect on both TCT and RCT, which reached normal values. The TCT and RCT of the purified fibrinogen solutions (2.0 g/l final concentration) were not different in the three groups, and normal polymerization curves were obtained in all cases. This further ruled out the presence of any dysfibrinogenemia in the plasma from group 1 patients. Using purified proteins, highly significant correlations were demonstrated between the albumin concentration and the prolongations of both TCT and RCT, which were of the same magnitude order than those found in the patients plasma. These results suggest that hypo-albuminemia is responsible for the acquired fibrin polymerization defect reported in HIV-infected patients. The pathophysiological implication of the low albumin levels was suggested by the finding of decreased albumin levels (associated with prolonged TCT and RCT) in a small series of the eight HIV-infected patients who developed thrombotic complications.

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INHIBITION OF THYROGLOBULIN BIOSYNTHESIS AND DEGRADATION BY EXCESS IODIDE. SYNERGISM WITH LITHIUM

Acta Endocrinologica ◽

10.1530/acta.0.0810495 ◽

1976 ◽

Vol 81 (2) ◽

pp. 495-506 ◽

Cited By ~ 9

Author(s):

A. Radvila ◽

R. Roost ◽

H. Bürgi ◽

H. Kohler ◽

H. Studer

Keyword(s):

Protein Synthesis ◽

Thyroid Gland ◽

Inhibitory Action ◽

Inhibit Protein Synthesis ◽

Thyrotrophic Hormone ◽

Thyroid Glands ◽

Pre Treatment ◽

Excess Iodide ◽

Kidney Liver

ABSTRACT Lithium and excess iodide inhibit the release of thyroid hormone from preformed stores. We thus tested the hypothesis that this was due to an inhibition of thyroglobulin breakdown. Rats were pre-treated with propylthiouracil (PTU) for 3 weeks in order to deplete their thyroids of thyroglobulin. While the PTU was continued, lithium chloride (0.25 mEq./100 g weight) or potassium iodide (3 mg per rat) were injected every 12 h for 3 days. Thereafter the thyroglobulin content in thyroid gland homogenates was measured. PTU pre-treatment lowered the thyroglobulin content from 4.21 to 0.22 mg/100 mg gland. Lithium caused a marked re-accumulation of thyroglobulin to 0.60 mg/100 mg within 3 days. While iodide alone had only a borderline effect, it markedly potentiated the action of lithium and a combination of the two drugs increased the thyroglobulin content to 1.04 mg/100 mg. Thyroxine was injected into similarly pre-treated animals to suppress secretion of thyrotrophic hormone. This markedly inhibited the proteolysis of thyroglobulin and 1.3 mg/100 mg gland accumulated after 3 days. Excess iodide, given in addition to thyroxine, decreased the amount of thyroglobulin accumulated to 0.75 mg/100 mg gland. To study whether this could be explained by an inhibitory action of iodide on thyroglobulin biosynthesis, thyroid glands from animals treated with excess iodide were incubated in vitro in the presence of 0.2 mm iodide for 3 h. Iodide decreased the incorporation of radioactive leucine into total thyroidal protein and into thyroglobulin by 25 and 35 % respectively. Iodide did not inhibit protein synthesis in the kidney, liver or muscle tissue. Thus, large doses of iodide selectively inhibit thyroglobulin biosynthesis.

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Rhenium-188 labeled recombinant human plasminogen kringle5 (rhk5) and preliminary biodistribution

Nuklearmedizin ◽

10.3413/nukmed-0349-10-09 ◽

2011 ◽

Vol 50 (06) ◽

pp. 234-239 ◽

Cited By ~ 3

Author(s):

R. Guo ◽

Y. Ma ◽

R. Zhang ◽

S. Liang ◽

H. Shen ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Human Serum ◽

Critical Role ◽

Angiogenesis Inhibitor ◽

Simple Method ◽

Tumour Formation ◽

Human Plasminogen ◽

Specificity Of Binding ◽

A549 Lung

Summary Aim: Angiogenesis plays a critical role in tumour formation and metastasis. Suitable radiolabeled angiogenesis inhibitor can be used for noninvasive imaging of angiogenesis and radionuclide therapy. Here we prepare rhenium-188 labeled recombinant human plasminogen kringle5 (188Re-rhk5) in a convenient manner than evaluate its properties in A549 lung adenocarcinoma. Methods: 188Rerhk5 was obtained by conjugating His group at the C end of rhk5 with fac- [188Re(H2O)3(CO)3]+. Chelating efficiency of fac-[188Re(H2O)3(CO)3]+ and radiolabeling efficiency of 188Re-rhk5 were measured by radio thin-layer chromatography (RTLC). In vitro stability of 188Re-rhk5 was determined in human serum at 37°C and analyzed by RTLC. Competition test was also performed to verify the specificity of binding. A biodistribution study was carried out in nude mice bearing A549 lung adenocarcinoma. Results: 188Rerhk5 was obtained with a radiolabel efficiency of 66.1%, the radiochemical purity (RCP) can marreach 95.2% after purification. 188Re-rhk5 showed high stability in human serum, the RCP was more than 80% even 12 h after incubation. Competition test showed a high binding specificity. Furthermore, this radio-complex was excreted mainly through kidneys and showed specific tumour uptake in mice bearing A549 tumours. Conclusion: 188Re-rhk5 was prepared by a simple method. Preliminary biodistribution results showed its potential as an agent for possible tumour imaging, therapy and encouraged further investigation.

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