scholarly journals Strategic Development of an Immunotoxin for the Treatment of Glioblastoma and Other Tumours Expressing the Calcitonin Receptor

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2347
Author(s):  
Pragya Gupta ◽  
David L. Hare ◽  
Peter J. Wookey
Keyword(s):  
Cell Lines ◽  
High Grade Glioma ◽  
Endosomal Escape ◽  
Receptor Protein ◽  
Calcitonin Receptor ◽  
Strategic Development ◽  
Response Profiles ◽  
Potential Strategy ◽  
Malignant Glioma Cells ◽  
New Strategies

New strategies aimed at treatment of glioblastoma are frequently proposed to overcome poor prognosis. Recently, research has focused on glioma stem cells (GSCs), some quiescent, which drive expansion of glioblastoma and provide the complexity and heterogeneity of the tumour hierarchy. Targeting quiescent GSCs is beyond the capability of conventional drugs such as temozolomide. Here, we discuss the proposal that the calcitonin receptor (CT Receptor), expressed in 76–86% of patient biopsies, is expressed by both malignant glioma cells and GSCs. Forty-two percent (42%) of high-grade glioma (HGG; representative of GSCs) cell lines available from one source express CT Receptor protein in cell culture. The pharmacological calcitonin (CT)-response profiles of four of the HGG cell lines were reported, suggesting mutational/splicing inactivation. Alternative splicing, commonly associated with cancer cells, could result in the predominant expression of the insert-positive isoform and explain the atypical pharmacology exhibited by CT non-responders. A role for the CT Receptor as a putative tumour suppressor and/or oncoprotein is discussed. Both CT responders and non-responders were sensitive to immunotoxins based on an anti-CT Receptor antibody conjugated to ribosomal-inactivating proteins. Sensitivity was increased by several logs with the triterpene glycoside SO1861, an endosomal escape enhancer. Under these conditions, the immunotoxins were 250–300 times more potent than an equivalent antibody conjugated with monomethyl auristatin E. Further refinements for improving the penetration of solid tumours are discussed. With this knowledge, a potential strategy for effective targeting of CSCs expressing this receptor is proposed for the treatment of GBM.

scholarly journals Building the case for the calcitonin receptor as a viable target for the treatment of glioblastoma

2020 ◽  
Vol 12 ◽  
pp. 175883592097811
Author(s):  
Pragya Gupta ◽  
Sebastian G. B. Furness ◽  
Lucas Bittencourt ◽  
David L. Hare ◽  
Peter J. Wookey
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Splice Variants ◽  
High Grade Glioma ◽  
Receptor Protein ◽  
Calcitonin Receptor ◽  
Direct Role ◽  
Malignant Glioma Cells ◽  
Satellite Stem Cells ◽  
Quiescent Stem Cells

Researchers are actively seeking novel targeted therapies for the brain tumour glioblastoma (GBM) as the mean survival is less than 15 months. Here we discuss the proposal that the calcitonin receptor (CT Receptor), expressed in 76–86% of patient biopsies, is expressed by both malignant glioma cells and putative glioma stem cells (GSCs), and therefore represents a potential therapeutic target. Forty-two per cent (42%) of high-grade glioma (HGG; representative of GSCs) cell lines express CT Receptor protein. CT Receptors are widely expressed throughout the life cycle of organisms and in some instances promote apoptosis. Which of the common isoforms of the CT Receptor are predominantly expressed is currently unknown, but a functional response to cell stress of the insert-positive isoform is hypothesised. A model for resistant malignancies is one in which chemotherapy plays a direct role in activating quiescent stem cells for replacement of the tumour tissue hierarchy. The putative role that the CT Receptor plays in maintenance of quiescent cancer stem cells is discussed in view of the activation of the Notch–CT Receptor–collagen V axis in quiescent muscle (satellite) stem cells. The pharmacological CT response profiles of four of the HGG cell lines were reported. Both CT responders and non-responders were sensitive to an immunotoxin based on an anti-CT Receptor antibody. The CALCR mRNA exhibits alternative splicing commonly associated with cancer cells, which could result in the atypical pharmacology exhibited by CT non-responders and an explanation of tumour suppression. Due to the inherent instability of CALCR mRNA, analysis of CT Receptor protein in patient samples will lead to improved data for the expression of CT Receptor in GBM and other cancers, and an understanding of the role and activity of the splice variants. This knowledge will aid the effective targeting of this receptor for treatment of GBM.

scholarly journals Metabolic drug survey highlights cancer cell dependencies and vulnerabilities

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tea Pemovska ◽  
Johannes W. Bigenzahn ◽  
Ismet Srndic ◽  
Alexander Lercher ◽  
Andreas Bergthaler ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput Screening ◽  
Drug Response ◽  
Fatty Acid Synthase ◽  
Leukemia Cell ◽  
Cellular Metabolism ◽  
Acid Synthesis ◽  
Metabolic Drug ◽  
Response Profiles ◽  
Synthase Inhibitor

AbstractInterrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.

scholarly journals Proteomic analysis of human glioblastoma cell lines differently resistant to a nitric oxide releasing agent

2015 ◽  
Vol 11 (6) ◽  
pp. 1612-1621 ◽  
Author(s):  
Roberta Leone ◽  
Paola Giussani ◽  
Sara De Palma ◽  
Chiara Fania ◽  
Daniele Capitanio ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Lines ◽  
Glioma Cell ◽  
High Grade Glioma ◽  
High Grade ◽  
Glioblastoma Cell ◽  
Proteomic Profile ◽  
Glioma Cell Lines ◽  
Nitric Oxide Releasing ◽  
Glioblastoma Cell Lines

NO exposure of two human high grade glioma cell lines (CCF-STTG1 and T98G) characterized by a different proteomic profile shows differential ceramide distribution and proliferation.

scholarly journals Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines

2020 ◽  
Vol 21 (12) ◽  
pp. 4322 ◽  
Author(s):  
Jacqueline Reinhard ◽  
Natalie Wagner ◽  
Miriam M. Krämer ◽  
Marvin Jarocki ◽  
Stephanie C. Joachim ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Mrna Expression ◽  
Cell Lines ◽  
Cluster Formation ◽  
Collagen Iv ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Receptor Protein ◽  
Tenascin C ◽  
Chemotherapy Drugs ◽  
Protein Levels

Retinoblastoma (RB) represents the most common malignant childhood eye tumor worldwide. Several studies indicate that the extracellular matrix (ECM) plays a crucial role in tumor growth and metastasis. Moreover, recent studies indicate that the ECM composition might influence the development of resistance to chemotherapy drugs. The objective of this study was to evaluate possible expression differences in the ECM compartment of the parental human cell lines WERI-RB1 (retinoblastoma 1) and Y79 and their Etoposide resistant subclones via polymerase chain reaction (PCR). Western blot analyses were performed to analyze protein levels. To explore the influence of ECM molecules on RB cell proliferation, death, and cluster formation, WERI-RB1 and resistant WERI-ETOR cells were cultivated on Fibronectin, Laminin, Tenascin-C, and Collagen IV and analyzed via time-lapse video microscopy as well as immunocytochemistry. We revealed a significantly reduced mRNA expression of the proteoglycans Brevican, Neurocan, and Versican in resistant WERI-ETOR compared to sensitive WERI-RB1 cells. Also, for the glycoproteins α1-Laminin, Fibronectin, Tenascin-C, and Tenascin-R as well as Collagen IV, reduced expression levels were observed in WERI-ETOR. Furthermore, a downregulation was detected for the matrix metalloproteinases MMP2, MMP7, MMP9, the tissue-inhibitor of metalloproteinase TIMP2, the Integrin receptor subunits ITGA4, ITGA5 and ITGB1, and all receptor protein tyrosine phosphatase β/ζ isoforms. Downregulation of Brevican, Collagen IV, Tenascin-R, MMP2, TIMP2, and ITGA5 was also verified in Etoposide resistant Y79 cells compared to sensitive ones. Protein levels of Tenascin-C and MMP-2 were comparable in both WERI cell lines. Interestingly, Fibronectin displayed an apoptosis-inducing effect on WERI-RB1 cells, whereas an anti-apoptotic influence was observed for Tenascin-C. Conversely, proliferation of WERI-ETOR cells was enhanced on Tenascin-C, while an anti-proliferative effect was observed on Fibronectin. In WERI-ETOR, cluster formation was decreased on the substrates Collagen IV, Fibronectin, and Tenascin-C. Collectively, we noted a different ECM mRNA expression and behavior of Etoposide resistant compared to sensitive RB cells. These findings may indicate a key role of ECM components in chemotherapy resistance formation of RB.

scholarly journals Proteasome inhibition as a therapeutic approach in atypical teratoid/rhabdoid tumors

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Andrew Morin ◽  
Caroline Soane ◽  
Angela Pierce ◽  
Bridget Sanford ◽  
Kenneth L Jones ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
High Grade Glioma ◽  
Proteasome Inhibition ◽  
Atypical Teratoid ◽  
Rhabdoid Tumors ◽  
Approved Drugs

Abstract Background Atypical teratoid/thabdoid tumor (AT/RT) remains a difficult-to-treat tumor with a 5-year overall survival rate of 15%–45%. Proteasome inhibition has recently been opened as an avenue for cancer treatment with the FDA approval of bortezomib (BTZ) in 2003 and carfilzomib (CFZ) in 2012. The aim of this study was to identify and characterize a pre-approved targeted therapy with potential for clinical trials in AT/RT. Methods We performed a drug screen using a panel of 134 FDA-approved drugs in 3 AT/RT cell lines. Follow-on in vitro studies used 6 cell lines and patient-derived short-term cultures to characterize selected drug interactions with AT/RT. In vivo efficacy was evaluated using patient derived xenografts in an intracranial murine model. Results BTZ and CFZ are highly effective in vitro, producing some of the strongest growth-inhibition responses of the evaluated 134-drug panel. Marizomib (MRZ), a proteasome inhibitor known to pass the blood–brain barrier (BBB), also strongly inhibits AT/RT proteasomes and generates rapid cell death at clinically achievable doses in established cell lines and freshly patient-derived tumor lines. MRZ also significantly extends survival in an intracranial mouse model of AT/RT. Conclusions MRZ is a newer proteasome inhibitor that has been shown to cross the BBB and is already in phase II clinical trials for adult high-grade glioma (NCT NCT02330562 and NCT02903069). MRZ strongly inhibits AT/RT cell growth both in vitro and in vivo via a moderately well-characterized mechanism and has direct translational potential for patients with AT/RT.

Identification of the soluble granulocyte-macrophage colony stimulating factor receptor protein in vivo

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 461-469 ◽  
Author(s):  
Farzana Sayani ◽  
Felix A. Montero-Julian ◽  
Valerie Ranchin ◽  
Jay M. Prevost ◽  
Sophie Flavetta ◽  
...  
Keyword(s):  
Cell Lines ◽  
State Level ◽  
Leukemic Cell ◽  
Protein Product ◽  
Receptor Protein ◽  
Soluble Form ◽  
Human Neutrophils ◽  
Gm Csf ◽  
Leukemic Cell Lines

On the basis of the finding of alternatively spliced mRNAs, the -subunit of the receptor for GM-CSF is thought to exist in both a membrane spanning (tmGMR) and a soluble form (solGMR). However, only limited data has been available to support that the solGMR protein product exists in vivo. We hypothesized that hematopoietic cells bearing tmGMR would have the potential to also produce solGMR. To test this hypothesis we examined media conditioned by candidate cells using functional, biochemical, and immunologic means. Three human leukemic cell lines that express tmGMR (HL60, U937, THP1) were shown to secrete GM-CSF binding activity and a solGMR-specific band by Western blot, whereas a tmGMR-negative cell line (K562) did not. By the same analyses, leukapheresis products collected for autologous and allogeneic stem cell transplants and media conditioned by freshly isolated human neutrophils also contained solGMR. The solGMR protein in vivo displayed the same dissociation constant (Kd = 2-5 nmol) as that of recombinant solGMR. A human solGMR ELISA was developed that confirmed the presence of solGMR in supernatant conditioned by the tmGMR-positive leukemic cell lines, hematopoietic progenitor cells, and neutrophils. Furthermore, the ELISA demonstrated a steady state level of solGMR in normal human plasma (36 ± 17 pmol) and provided data suggesting that plasma solGMR levels can be elevated in acute myeloid leukemias.

scholarly journals Upregulation of KCNQ1OT1 promotes resistance to stereotactic body radiotherapy in lung adenocarcinoma by inducing ATG5/ATG12-mediated autophagy via miR-372-3p

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Huanyu He ◽  
Xinmao Song ◽  
Zuozhang Yang ◽  
Yuchi Mao ◽  
Kunming Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Stereotactic Body Radiotherapy ◽  
Clinical Stage ◽  
Resistant Cell ◽  
Antitumor Effects ◽  
Large Tumor ◽  
Advanced Clinical Stage ◽  
Potential Strategy

Abstract Stereotactic body radiotherapy (SBRT) has emerged as a standard treatment for non-small-cell lung cancer. However, its therapeutic advantages are limited with the development of SBRT resistance. The SBRT-resistant cell lines (A549/IR and H1975/IR) were established after exposure with hypofractionated irradiation. The differential lncRNAs were screened by microarray assay, then the expression was detected in LUAD tumor tissues and cell lines by qPCR. The influence on radiation response was assessed via in vitro and in vivo assays, and autophagy levels were evaluated by western blot and transmission electron microscopy. Bioinformatics prediction and rescue experiments were used to identify the pathways underlying SBRT resistance. High expression of KCNQ1OT1 was identified in LUAD SBRT-resistant cells and tissues, positively associated with a large tumor, advanced clinical stage, and a lower response rate to concurrent therapy. KCNQ1OT1 depletion significantly resensitized A549/IR and H1975/IR cells to radiation by inhibiting autophagy, which could be attenuated by miR-372-3p knockdown. Furthermore, autophagy-related 5 (ATG5) and autophagy-related 12 (ATG12) were confirmed as direct targets of miR-372-3p. Restoration of either ATG5 or ATG12 abrogated miR-372-3p-mediated autophagy inhibition and radiosensitivity. Our data describe that KCNQ1OT1 is responsible for SBRT resistance in LUAD through induction of ATG5- and ATG12-dependent autophagy via sponging miR-372-3p, which would be a potential strategy to enhance the antitumor effects of radiotherapy in LUAD.

1,25-Dihydroxyvitamin D3 receptor RNA: expression in hematopoietic cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1238-1247 ◽  
Author(s):  
M Kizaki ◽  
AW Norman ◽  
JE Bishop ◽  
CW Lin ◽  
A Karmakar ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
Constitutive Expression ◽  
Peripheral Blood Lymphocytes ◽  
Leukemic Cells ◽  
Receptor Protein ◽  
Dihydroxyvitamin D3 ◽  
1,25 Dihydroxyvitamin D3 ◽  
Normal Individuals

Abstract 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] induces differentiation and inhibits proliferation of myeloid leukemic cells from various lines and patients; these effects are probably mediated through the 1,25(OH)2D3 receptor. Little is known of expression of 1,25(OH)2D3 receptor RNA in hematopoietic cells. We examined the expression and modulation of expression of 1,25(OH)2D3 receptor RNA in various proliferating and nonproliferating hematopoietic cells. Constitutive expression of 1,25(OH)2D3 receptor RNA was detected in various kinds of hematopoietic cells, including macrophages and activated T lymphocytes, as well as in cell lines KG-1 (myeloblasts), HL-60 (promyelocytes), ML-3 (myelomonoblasts), U937, THP-1 (monoblasts), K562 (erythroblasts), and S-LB1 (HTLV-1-transfected T lymphocytes). Receptor transcripts were 4.6 kilobases (kb), and no variant sizes were observed. All cell lines examined in this group also expressed 1,25(OH)2D3 receptors. Most B lymphocyte lines expressed negligible levels of 1,25(OH)2D3 receptor RNA and protein; however; analysis of a lymphoid/myeloid somatic hybrid suggested that suppression of expression of 1,25(OH)2D3 receptor RNA in B lymphocytes may be a dominant characteristic. HL-60 cells were cultured with 10(-7) mol/L 1,25(OH)2D3 for 24 to 72 hours, and levels of expression of 1,25(OH)2D3 receptor and its RNA were examined. Levels of RNA coding for the receptor were not modulated by exposure to high levels of ligand. Levels of occupied 1,25(OH)2D3 receptor protein increased in these HL-60 cells; but the total number of 1,25(OH)2D3 receptors decreased about 50% at 24 hours and returned toward normal at 72 hours. Steady-state levels of 1,25(OH)2D3 receptor RNA were not affected by terminal differentiation of HL-60 toward either granulocytes or macrophages. Nondividing macrophages from normal individuals also expressed 1,25(OH)2D3 receptor RNA. In contrast, nondividing peripheral blood lymphocytes from normal individuals did not express 1,25(OH)2D3 receptor RNA; with stimulation of proliferation of these cells, accumulation of 1,25(OH)2D3 receptor RNA increased markedly. Half-life (t1/2) of 1,25(OH)2D3 receptor RNA in T lymphocytes was short (1 hour) as determined by measuring decay of the message after addition of actinomycin D. Consistent with this short t1/2, accumulation of 1,25(OH)2D3 receptor RNA increased in cells as their protein synthesis was inhibited. Further studies are required to understand the physiologic role of 1,25(OH)2D3 receptors in myeloid cells and proliferating T lymphocytes.

scholarly journals Smarcd1 Inhibits the Malignant Phenotypes of Human Glioblastoma Cells via Crosstalk with Notch1

2020 ◽  
Author(s):  
Yihao Zhu ◽  
Handong Wang ◽  
Maoxing Fei ◽  
Ting Tang ◽  
Wenhao Niu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
High Grade Glioma ◽  
Glioblastoma Cell ◽  
Glioblastoma Cells ◽  
Human Glioblastoma ◽  
Protein Levels ◽  
Enhanced Expression ◽  
Human Glioblastoma Cells ◽  
Glioblastoma Cell Lines

AbstractSmarcd1 is a component of an evolutionary conserved chromatin remodeling complex—SWI/SNF, which is involved in transcription factor recruitment, DNA replication, recombination, and repair. Suppression of the SWI/SNF complex required for cellular differentiation and gene regulation may be inducible for cell proliferation and tumorigenicity. However, the inhibitory role of Smarcd1 in human glioblastoma cells has not been well illustrated. Both U87 and U251 human glioblastoma cell lines were employed in the present study. The lentivirus-mediated gene knockdown and overexpression approach was conducted to determine the function of Smarcd1. The protein levels were tested by western blot, and the relative mRNA contents were detected by quantitative real-time PCR. Cell viability was tested by CCK-8 and colony-forming assay. Transwell assays were utilized to evaluate the motility and invasive ability. Flow cytometry was employed to analyze cell cycle and apoptosis. SPSS software was used for statistical analysis. Low expression of Smarcd1 was observed in glioblastoma cell lines and in patients with high-grade glioma. Importantly, the depletion of Smarcd1 promoted cell proliferation, invasion, and chemoresistance, whereas enhanced expression of Smarcd1 inhibited tumor-malignant phenotypes. Mechanistic research demonstrated that overexpression of Smarcd1 decreased the expression of Notch1, while knockdown of Notch1 increased the expression of Smarcd1 through Hes1 suppression. Hence, the crosstalk between Smarcd1 and Notch1, which formed a feedback loop, was crucial in regulation of glioblastoma malignant phenotypes. Furthermore, targeting Smarcd1 could be a potential strategy for human glioblastoma treatment.

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