Eltrombopag Decreases Proliferation of Ovarian, Lung and Breast Tumor Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2409-2409
Author(s):  
Connie L. Erickson-Miller ◽  
Jennifer Kirchner ◽  
Kodandaram Pillarisetti ◽  
Lone Ottesen ◽  
Yasser Mostafa Kamel ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Cell Lines ◽  
Mrna Levels ◽  
Tumor Cell Lines ◽  
Employment Equity ◽  
Qrt Pcr ◽  
Thrombopoietin Receptor ◽  
Equity Ownership ◽  
Low Levels

Abstract Abstract 2409 Poster Board II-386 Background: Eltrombopag (Promacta®) is a novel, oral thrombopoietin receptor (TpoR) agonist that interacts with the TpoR on bone marrow progenitors to stimulate megakaryocyte production, thus increasing platelet counts in thrombocytopenic patients. The effects of eltrombopag on the proliferation of solid tumor cell lines and the expression of thrombopoietin receptor (MPL, TpoR) on patient tumors is of interest given that chemotherapy can cause thrombocytopenia. Materials and methods: Proliferation was measured by Cell Titer Glo assay on 3 ovarian (OVCAR3, OVCAR4, SKOV3), 4 lung (A549, NCI-H226, NCI-H510, NCI-H460) and 3 breast (BT-474, MCF7, HCC1937) cancer cell lines from the ATCC treated with 0.01 – 100 ug/mL eltrombopag. Quantitative RT-PCR (qRT-PCR) for MPL expression was performed on the tumor cell lines and on 40 tumor samples, each from subjects with ovarian, lung or breast cancer. Microarray analysis for MPL mRNA expression was examined from 118 subjects with breast cancer and 29 with non-small cell lung cancer (NSCLC). Microarray data was normalized using robust multiarray average (RMA) and relative mRNA expression was determined. To determine expression of TpoR protein, western blot analyses was performed on some of the tumor cell lines. Results: Eltrombopag induced an inhibition of proliferation on all of the ovarian, lung and breast solid tumor cell lines tested. The IC50 ranged from 3.7 to 49.7 ug/mL (see table below). The Cmax of ITP patients treated with 75 mg eltrombopag is 11.4 ug/mL, demonstrating that these concentrations are clinically achievable. There was no enhancement of proliferation at any concentration of eltrombopag, consistent with the very low or undetectable level of MPL expression on samples of tumors from patients with these diseases. MPL was expressed at very low or undetectable levels in these tumor cell lines with the exception of the lung cancer line, NCI-H510. However, western blot analyses showed no detectable TpoR protein expression regardless of the higher levels of MPL mRNA in NCI-H510 cells. Erythropoietin receptor (EPOR) mRNA was expressed at low-to-moderate levels, while ERBB2 and IGF1R were expressed at higher levels in these cell lines. Microarray analysis showed undetectable MPL mRNA levels in all 118 samples from patients with breast cancer and 52% of the NSCLC samples, the remaining NSCLC samples expressed low levels of MPL. In contrast, EPOR was expressed in 75–100% of the breast cancer, and NSCLC samples. ERBB2 was expressed in 97–100% of the samples and IGF1R was expressed in 54–100% of the samples. When 40 other tumor samples each from subjects with ovarian, lung and breast cancer were examined by qRT-PCR, MPL mRNA levels were also very low or undetectable. EPOR, ERBB2, and IGF1R expression levels varied according to tumor type, but were greater than MPL levels. Conclusions: In summary, similar to its effects on leukemia and lymphoma cell lines, all of the nine lung, ovarian, breast or prostate tumor cell lines demonstrated decreased proliferation in response to eltrombopag. The undetectable or very low levels of expression of MPL mRNA in tumors of patients with lung, ovarian, breast or prostate cancer supports the proliferation results. Disclosures: Erickson-Miller: GlaxoSmithKline: Employment, Equity Ownership, Patents & Royalties, Research Funding. Kirchner:GlaxoSmithKline: Employment. Pillarisetti:GSK: Employment, Equity Ownership, Patents & Royalties. Ottesen:GSK: Employment, Equity Ownership. Mostafa Kamel:GSK: Employment, Equity Ownership. Liu:GSK: Employment, Equity Ownership. Martin:GSK: Employment, Equity Ownership. Messam:GSK: Employment, Equity Ownership.

Low or Undetectable Levels of Thrombopoietin Receptor (Tpo-R; c-mpl) Expression on Tumor Cell Lines and Primary Tumors Compared with Epo-R, ErbB2, and IGF-1R

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5394-5394 ◽  
Author(s):  
Connie L Erickson-Miller ◽  
Antony Chadderton ◽  
Anna Gibbard ◽  
Jennifer Kirchner ◽  
Lone Ottesen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Mrna Expression ◽  
Cell Lines ◽  
American Association ◽  
Breast Tumors ◽  
Tumor Cell Lines ◽  
Qrt Pcr ◽  
Thrombopoietin Receptor ◽  
Low Levels

Abstract INTRODUCTION: Recent discovery of thrombopoietin receptor (Tpo-R; c-mpl) agonists and thrombopoietin mimetics has warranted a better understanding of their effects on solid and liquid tumors. These agents bind to different components of the Tpo-R and therefore signal differently. Previously published data on Tpo-R agonists have shown antiproliferative effects on leukemia and lymphoma cells in vitro (Kalota A, Gewirtz AM. American Association for Cancer Research. 2008. Abstract 2392; Erickson-Miller CL, et al. American Association for Cancer Research. 2008. Abstract 5691). Although the expression of Tpo-R is well documented on cells of the megakaryocyte lineage, there is little quantitative information available on the expression of Tpo-R on tumors. METHODS & RESULTS: To better define Tpo-R expression, we first performed quantitative reverse-transcriptase PCR (qRT-PCR) on 378 tumor cell lines available from the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DSMZ). Tpo-R was consistently expressed at low levels, with a mean normalized abundance of 1,447 and a mode of 621. Only 3 cell lines expressed Tpo-R mRNA below the limits of reliable quantitation (SAOS-2, SF-539, and WIDR; bone, brain, and colon tumor cell lines, respectively). In comparison, the erythropoietin receptor (Epo-R) was expressed in low-to-moderate levels (mean, 12,587; mode, 7,811) and ErbB2 was expressed at higher levels (mean, 280,190; mode, 40,828), with expression, as expected, much higher among the breast tumor cell lines. IGF-1R was also expressed at higher levels (mean, 78,977; mode, 56,624). Three cell lines had greater than 9,500 normalized abundance: HEL 92.1.7, KG-1 (2 erythroleukemia cell lines), and NCI-H510 (lung cancer cell line). To determine if these trends also occurred in patient tumor samples, microarray data were examined from 118 breast cancer, 29 non-small cell lung cancer (NSCLC), and 151 renal cell carcinoma (RCC) samples collected prior to treatment in GlaxoSmithKline clinical trials. Robust multiarray average (RMA) analysis was used to determine relative mRNA expression levels. Tpo-R mRNA levels were too low for accurate measurement in all breast cancer and RCC samples, but were detectable at low levels in 14 (48%) NSCLC samples (Table 1). In contrast, Epo-R was expressed in 75% breast cancer samples, in all NSCLC samples, and in 87% RCC samples. ErbB2 was expressed in all breast samples, 97% of the NSCLC, and in 81% RCC samples. IGF-1R was expressed in 86% breast cancer samples, all NSCLC samples, and 54% RCC samples. For breast tumors, the levels of Tpo-R message expression rank as follows: Tpo-R<Epo-R<IGF-1R<ErbB2. To determine the relationship between Tpo-R message expression and Tpo-R protein expression, Western blot analyses were performed on several of the tumor cell lines, including 2 with the highest Tpo-R mRNA expression as determined by qRT-PCR (HEL 92.1.7 and NCI-H510) and 2 with undetectable Tpo-R mRNA expression (ML-2 and NCI-H360). Western blots demonstrated that Tpo-R protein was detectable in the lysates of HEL 92.1.7 cells and normal human platelets, which were used as a positive control. However, Tpo-R protein was not detected in NCI-H510, ML-2, or NCI-H360 cells. Thus, even the high levels of Tpo-R mRNA in NCI-H510 cells did not correlate to detectable Tpo-R protein expression. CONCLUSIONS: In summary, low or undetectable levels of Tpo-R mRNA expression were observed in tumor cell lines and in limited samples of patient tumors, compared with Epo-R, ErbB2, and IGF-1R. In the tumor cells tested, Tpo-R protein was not always detectable, even when Tpo-R mRNA was expressed. Table 1. Number (%) of primary tumor samples with accurately detectable Tpo-R mRNA expression from RMA analysis of microarray data. Breast tumors N = 118 NSCLC N = 29 RCC N = 151 Tpo-R (c-mpl), n (%) 0 14 (48) 0 Epo-R, n (%) 89 (75) 29 (100) 132 (87) ErbB2, n (%) 118 (100) 28 (97) 122 (81) IGF-1R, n (%) 102 (86) 29 (100) 81 (54)

scholarly journals Comparative TKI Profiling Analyses to Explore Potential Mechanisms of Ponatinib-Associated Arterial Thrombotic Events

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1783-1783 ◽  
Author(s):  
Victor M. Rivera ◽  
Justin R. Pritchard ◽  
Francois Gonzalvez ◽  
Theresa Baker ◽  
Joseph M. Gozgit ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tumor Cell ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Umbilical Vein ◽  
Effective Concentration ◽  
Tumor Cell Lines ◽  
Employment Equity ◽  
Equity Ownership

Abstract Background: Ponatinib is a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) indicated for patients with T315I positive or treatment-refractory CML and Ph+ ALL. To develop hypotheses regarding molecular and cellular targets of ponatinib that could contribute to arterial thrombotic events observed in some patients we are undertaking a broad comparative profiling analysis of ponatinib and other TKIs. Methods: Cellular activities of ponatinib and additional BCR-ABL (imatinib, nilotinib, dasatinib, bosutinib) and VEGFR2/multi-targeted (sunitinib, regorafenib) TKIs were examined in panels of Ba/F3 cell lines expressing activated kinase variants (N=61), tumor cell lines (N=246), and primary lines derived from human vasculature (aortic smooth muscle cells [ASMC] and umbilical vein [HUVEC], aortic [HAEC] and pulmonary artery [HPAEC] endothelial cells). Human steady-state Cave concentrations of 45 mg ponatinib (101 nM) were corrected for the functional effects of protein binding (3.6-fold) to derive the clinically-effective concentration. Results: Ponatinib inhibits the in vitro activity of multiple kinases with IC50s within 10-fold of ABL, including members of the VEGFR, PDGFR, FGFR, EPH receptor and SRC families of kinases, KIT, RET, TIE2, and FLT3. This profile was largely recapitulated in cellular assays using engineered Ba/F3 cells, with ponatinib demonstrating substantially greater potency against VEGFRs, FGFRs, TIE2, RET and FLT3 than other ABL TKIs. Across a broad panel of tumor cell lines, ponatinib inhibited viability with a median IC50 of 598 nM. Ponatinib only inhibited 16 cell lines (6.5%) with IC50s below its clinically effective concentration (28 nM) with the 5 most sensitive lines (IC50 <1 nM) all being BCR-ABL positive. Within the vasculature-derived cell panel, ponatinib inhibited viability of HUVECs grown in full serum with an IC50 of 261 nM, with all of the other ABL and non-ABL TKIs tested having IC50s >2000 nM. Effects of ponatinib on HAECs, HPAECs and ASMCs were more modest (IC50s 1533, 490 and 750 nM, respectively). Finally, ponatinib (IC50 20 nM) and other VEGFR2 inhibitors potently inhibited survival of HUVECs grown in VEGF-dependent conditions, while other BCR-ABL inhibitors, except dasatinib (IC50 14 nM), did not. Conclusions: Ponatinib is a potent BCR-ABL inhibitor that also inhibits VEGFR2 and other kinases at clinically achievable concentrations in vitro. Modest effects of ponatinib on endothelial cells have been observed that warrant further exploration in vivo. Developing a precise understanding of the mechanism by which ponatinib contributes to arterial thrombotic events should facilitate development of strategies to optimize its benefit/risk in patients. Disclosures Rivera: ARIAD Pharmaceuticals Inc: Employment, Equity Ownership. Pritchard:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Gonzalvez:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Baker:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Gozgit:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Hodgson:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership.

scholarly journals Proteomic Profiling and Mechanistic Investigating of a Novel Anti-Cancer Small Molecule Inhibitor of Sec61

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2076-2076
Author(s):  
Yu Qian ◽  
Henry W.B. Johnson ◽  
Christopher J. Kirk ◽  
Eric Lowe ◽  
Dustin McMinn ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Er Stress ◽  
Cell Lines ◽  
Life Sciences ◽  
Tumor Cell Lines ◽  
Proteomic Profiling ◽  
Employment Equity ◽  
Multiple Tumor ◽  
Equity Ownership

Secreted and transmembrane (TM) proteins play key roles in malignant transformation and tumor growth, including autocrine growth factor expression, receptor oncogene signal transduction pathways, metastasis, and immune system evasion. During translation, the majority of such proteins require translocation through the Sec61 translocon into the Endoplasmic Reticulum (ER) for further processing. This process is negotiated by unique signal sequences of the translating protein. Therefore, Sec61 represents a novel therapeutic target for cancer treatment through selective blockade of protein secretion. We generated Sec61 inhibitors and assessed their potential against target proteins using HEK293 cell lines stably expressing secreted or TM proteins of interest fused to a luciferase reporter. Additionally, anti-tumor activity was determined across both solid and liquid tumor cell lines in vitro and in mouse models. KZR-8834, a lead candidate identified through a medicinal chemistry campaign, induced cell death in multiple tumor cell lines in vitro, including multiple myeloma (MM), and was effective in xenograft models at doses that did not induce significant body weight loss or clinical signs of toxicity. We utilized quantitative proteomic methods to study KZR-8834 for inhibition of protein secretion and global modulation of protein homeostasis in sensitive and resistant tumor cell lines. Multiple tumor cell types were tested at various doses and time courses followed by subcellular fractionation of cytosolic and membrane/ER proteomes. Subsequent proteomic profiling was performed with Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) and/or Tandem Mass Tag 6-plex (TMT-sixplex). Sensitive targets from both proteomes were further verified using downstream biochemical methods. Sec61 client proteins showed both time- and dose-dependent inhibition upon compound treatment and proteomic results were verified via western blot analysis. Approximately 20% of the total Sec61 clientome and 25% of total proteins detected in a sensitive multiple myeloma (MM) cell line, H929, were significantly down-regulated in response to KZR-8834 treatment at concentrations leading to cell death. IPA pathway analysis suggested that activation of the ER stress response gene ATF4 was induced by KZR-8834 treatment in H929 cells. In a resistant MM cell line, U266, only 13% of the total Sec61 clientome and 5% of total protein detected were significantly down-regulated in response to the same compound treatment. A distinct profile of down-regulated Sec61 clientome was noted with overlap in only 11 of 394 commonly expressed proteins across those two cell lines. Interestingly, in compound treated cells, 39 down-regulated Sec61 client proteins in H929 were either unchanged or upregulated in U266 cells. Conversely, 38 upregulated H929 Sec61 clients were either unchanged or down-regulated in U266 cells. We further explored the ER stress response induced by KZR-8834 via comparative proteomic analysis in H929 cells treated with known ER stress inducers, Tunicamycin and Thapsigargin. These agents, which exert ER stress upon inhibition of N-linked glycosylation and blockade of ER Ca2+ flux, respectively, showed distinct cytosolic proteomic profiles in H929 cells relative to KZR-8834 treatment. These data suggest that KZR-8834-induced blockade of Sec61 results in a unique form of proteotoxic stress in sensitive MM cells. Collectively our results highlight quantitative proteomic profiling as a valuable tool toward elucidating the mechanism of pleiotropic acting molecules like KZR-8834. These studies constitute important first steps toward clarifying the anti-tumor mechanism inhibiting Sec61, a novel pathway agent, for the potential treatment of hematologic tumors. Disclosures Qian: Kezar Life Sciences: Employment, Equity Ownership. Johnson:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Lowe:Kezar Life Sciences: Employment, Equity Ownership. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Millare:Kezar Life Sciences: Employment, Equity Ownership. Muchamuel:Kezar Life Sciences: Employment, Equity Ownership. Wang:Kezar Life Sciences: Employment, Equity Ownership.

scholarly journals CC-92480 Is a Novel Cereblon E3 Ligase Modulator with Enhanced Tumoricidal and Immunomodulatory Activity Against Sensitive and Resistant Multiple Myeloma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1812-1812 ◽  
Author(s):  
Antonia Lopez-Girona ◽  
Courtney G. Havens ◽  
Gang Lu ◽  
Emily Rychak ◽  
Derek Mendy ◽  
...  
Keyword(s):  
Cell Lines ◽  
Acquired Resistance ◽  
E3 Ligase ◽  
Immunomodulatory Activity ◽  
Employment Equity ◽  
Ic50 Value ◽  
Ic50 Values ◽  
Equity Ownership ◽  
Induction Of Apoptosis ◽  
Low Levels

Lenalidomide- and pomalidomide-based therapies are effective drugs in the treatment of patients with multiple myeloma (MM), however most patients with MM eventually relapse or become resistant. CC-92480, a novel cereblon (CRBN) E3 ligase modulator (CELMoD) with multiple activities including potent immunomodulation and single-agent antiproliferative effects, is being investigated in a phase 1 clinical trial (CC-92480-MM-001; NCT03374085) for patients with relapsed/refractory MM (RRMM). The present study investigates the preclinical data and mechanism of action of CC-92480 in MM models. CELMoD agents bound to CRBN confer differentiated substrate-degradation specificity on the CRL4CRBN E3 ubiquitin ligase. CRBN-modulator agents mediate destruction of Ikaros and Aiolos, transcription factors that contribute to myeloma cell survival. CC-92480 was found to produce rapid, deep, and sustained degradation of Ikaros and Aiolos, with superior antimyeloma activity. Accordingly, in a CRBN protein competitive binding assay, CC-92480 displaced a Cy-5-labeled CELMoD analog from CRBN with a 50% inhibitory concentration (IC50) value of 0.03 μM, whereas lenalidomide competed with an IC50 value of 1.27 μM in the same assay, demonstrating a higher binding affinity of CC-92480 for CRBN. Additionally, CC-92480 promoted the recruitment of Ikaros to the CRBN E3 ligase complex more effectively than pomalidomide in 2 orthogonal CRBN/Ikaros binding assays; it also triggered a more extensive cellular ubiquitination of Ikaros, and a faster, more efficient depletion of cellular Ikaros and Aiolos than pomalidomide. In various MM cell lines, including those with acquired resistance to lenalidomide or pomalidomide and low levels of CRBN, CC-92480 produced robust degradation of Ikaros and Aiolos followed by strong reduction of 2 additional and highly critical transcription factors, c-Myc and interferon regulatory factor 4, which are linked to the induction of apoptosis as measured by cleaved caspase-3. The tumoricidal activity of CC-92480 was shown to be CRBN dependent, since the effect was prevented by complete loss of CRBN or by the stabilization of Ikaros and Aiolos. CC-92480 displayed broad and potent antiproliferative activity across a panel of 20 MM cell lines that are either sensitive, have acquired resistance, or are refractory to lenalidomide or pomalidomide; the cell lines also contained diverse chromosomal translocations and oncogenic drivers typically found in MM patients. Approximately half of the MM cell lines evaluated were highly sensitive to CC-92480, with IC50 values for antiproliferative activity ranging from 0.04 to 5 nM; only 2 cell lines had IC50 values > 100 nM. CC-92480 inhibits cell proliferation and induces apoptosis in MM cell lines that are not sensitive to lenalidomide or pomalidomide. This panel of cell lines includes both refractory cell lines and resistant cell lines generated through continuous exposure to lenalidomide and pomalidomide that acquired low levels of CRBN protein or mutations in the CRBN gene. CC-92480 also induced deep destruction of Ikaros and Aiolos in cultures of peripheral blood mononuclear cells (PBMCs), which led to the activation of T cells and increased production of the cytokines interleukin-2 and interferon gamma. These responses occurred at the range of CC-92480 concentrations that show potent tumoricidal effect against MM cells. The T cell activation and enhanced cytokine production by CC-92480 led to the potent and effective immune-mediated killing of MM cells in co-cultures with PBMCs. CC-92480 is a potent antiproliferative and proapoptotic novel CELMoD with enhanced autonomous cell-killing activity in MM cells that are either sensitive, resistant, or have acquired resistance to lenalidomide and pomalidomide. CC-92480 has a unique and rapid degradation profile stemming from the enhanced efficiency to drive the formation of a protein-protein interaction between Ikaros and Aiolos and CRBN, inducing cytotoxic effects in a CRL4CRBN-dependent fashion that leads ultimately to the induction of apoptosis, even in the context of low or mutated CRBN protein. Additionally, similar to lenalidomide, CC-92480 conserves immunomodulatory activity against MM cells. These data support the clinical investigation of CC-92480 in patients with RRMM. Disclosures Lopez-Girona: Celgene Corporation: Employment. Havens:Pfizer: Employment, Equity Ownership; Celgene: Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Gaffney:Celgene: Employment. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Matyskiela:Celgene corporation: Employment. Khambatta:Celgene: Employment. Wong:Celgene Corporation: Employment, Equity Ownership. Hansen:Celgene Corporation: Employment. Pierce:Celgene Corporation: Employment, Equity Ownership. Cathers:Global Blood Therapeutics (GBT): Employment; Celgene Corporation: Equity Ownership. Carmichael:Celgene plc: Employment, Equity Ownership.

scholarly journals A Nuclear-Directed Ribonuclease Variant Targets Cancer Stem Cells and Inhibits Migration and Invasion of Breast Cancer Cells

Cancers ◽  
2021 ◽  
Vol 13 (17) ◽  
pp. 4350
Author(s):  
Jessica Castro ◽  
Giusy Tornillo ◽  
Gerardo Ceada ◽  
Beatriz Ramos-Neble ◽  
Marlon Bravo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Tumor Cell ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Breast Cancer Cells ◽  
Migration And Invasion ◽  
Tumor Cell Lines

Despite the significant advances in cancer research made in recent years, this disease remains one of the leading causes of death worldwide. In part, this is due to the fact that after therapy, a subpopulation of self-renewing tumor cells can survive and promote cancer relapse, resistance to therapies and metastasis. Targeting these cancer stem cells (CSCs) is therefore essential to improve the clinical outcome of cancer patients. In this sense, multi-targeted drugs may be promising agents targeting CSC-associated multifocal effects. We have previously constructed different human pancreatic ribonuclease (RNase) variants that are cytotoxic for tumor cells due to a non-classical nuclear localization signal introduced in their sequence. These cytotoxic RNases affect the expression of multiple genes involved in deregulated metabolic and signaling pathways in cancer cells and are highly cytotoxic for multidrug-resistant tumor cell lines. Here, we show that these cytotoxic nuclear-directed RNases are highly selective for tumor cell lines grown in 3D, inhibit CSCs’ development and diminish the self-renewal capacity of the CSCs population. Moreover, these human RNase variants reduce the migration and invasiveness of highly invasive breast cancer cells and downregulate N-cadherin expression.

scholarly journals CSIG-18. NERVE/GLIAL ANTIGEN (NG)2 EXPRESSION IN GLIOBLASTOMA IS REGULATED BY miR-29b-3p

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi47-vi48
Author(s):  
Beate Schmitt ◽  
Anne Boewe ◽  
Yuan Gu ◽  
Christoph Sippl ◽  
Steffi Urbschat ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Therapeutic Strategy ◽  
Mrna Levels ◽  
Glioblastoma Cell ◽  
Glioblastoma Cells ◽  
Qrt Pcr ◽  
Glioblastoma Patient ◽  
Low Levels ◽  
Glioblastoma Cell Lines

Abstract Overexpression of NG2 in human glioblastoma cells is associated with an elevated drug resistance and thereby worsens clinical outcome. However, the regulatory mechanisms of NG2 expression are largely unknown. In this study, we identified miR-29b-3p as a posttranscriptional factor of NG2 expression. The basal mRNA levels of miR-29b-3p and NG2 were detected in the NG2-positive glioblastoma cell lines A1207 and U87 by qRT-PCR. The cells were transfected with miR-29b-3p-mimic or scrambled-miR (control) and the expression of NG2 was analyzed by qRT-PCR, flow cytometry and Western blot. Reporter gene analyses of the NG2 promotor region and 3’UTR were performed to study the effect of miR-29b-3p on NG2 expression. Finally, we analyzed the mRNA levels of NG2 and miR-29b-3p in samples from glioblastoma patients. We found that the two NG2-positive glioblastoma cell lines A1207 and U87 are positive for miR-29b-3p. Transfection with miR-29b-3p-mimic reduced NG2 mRNA levels in A1207 (29%±9.9; Mean±SD) and U87 (6%±2.8), resulting in a significantly decreased NG2 protein expression in A1207 (67%±6.4) and U87 (75%±4) when compared to controls. The analysis of the 3’UTR revealed that miR-29b-3p is a posttranscriptional regulator of NG2 expression. Moreover, miR-29b-3p affects the pretranscriptional NG2 expression by diminishing SP-1-dependent NG2 promotor activity. These results were confirmed by the analysis of glioblastoma patient-derived samples, demonstrating that a high NG2 expression is associated with low levels of miR-29b-3p. In conclusion, we identified miR-29b-3p as a crucial regulator of NG2 expression in glioblastoma. Hence, targeting NG2 expression by miR-29b-3p may provide a novel therapeutic strategy to overcome drug resistance in NG2-positive glioblastoma cells.

scholarly journals Synthesis of a tubugi-1-toxin conjugate by a modulizable disulfide linker system with a neuropeptide Y analogue showing selectivity for hY1R-overexpressing tumor cells

2019 ◽  
Vol 15 ◽  
pp. 96-105 ◽  
Author(s):  
Rainer Kufka ◽  
Robert Rennert ◽  
Goran N Kaluđerović ◽  
Lutz Weber ◽  
Wolfgang Richter ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Neuropeptide Y ◽  
Cell Lines ◽  
Normal Breast ◽  
Receptor Subtype ◽  
Tumor Cell Lines ◽  
Peptide Toxin ◽  
Targeting Peptide

Tubugi-1 is a small cytotoxic peptide with picomolar cytotoxicity. To improve its cancer cell targeting, it was conjugated using a universal, modular disulfide derivative. This allowed conjugation to a neuropeptide-Y (NPY)-inspired peptide [K4(C-βA-),F7,L17,P34]-hNPY, acting as NPY Y1 receptor (hY1R)-targeting peptide, to form a tubugi-1–SS–NPY disulfide-linked conjugate. The cytotoxic impacts of the novel tubugi-1–NPY peptide–toxin conjugate, as well as of free tubugi-1, and tubugi-1 bearing the thiol spacer (liberated from tubugi-1–NPY conjugate), and native tubulysin A as reference were investigated by in vitro cell viability and proliferation screenings. The tumor cell lines HT-29, Colo320 (both colon cancer), PC-3 (prostate cancer), and in conjunction with RT-qPCR analyses of the hY1R expression, the cell lines SK-N-MC (Ewing`s sarcoma), MDA-MB-468, MDA-MB-231 (both breast cancer) and 184B5 (normal breast; chemically transformed) were investigated. As hoped, the toxicity of tubugi-1 was masked, with IC50 values decreased by ca. 1,000-fold compared to the free toxin. Due to intracellular linker cleavage, the cytotoxic potency of the liberated tubugi-1 that, however, still bears the thiol spacer (tubugi-1-SH) was restored and up to 10-fold higher compared to the entire peptide–toxin conjugate. The conjugate shows toxic selectivity to tumor cell lines overexpressing the hY1R receptor subtype like, e.g., the hard to treat triple-negative breast cancer MDA-MB-468 cells.

Expression and secretion of VEGF in solid tumor cells is mediated by the mammalian target of rapamycin (mTOR)

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 14123-14123
Author(s):  
E. M. Lackner ◽  
M. T. Krauth ◽  
R. Kondo ◽  
L. Rebuzzi ◽  
K. Eigenberger ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Tumor Cell Lines ◽  
Neoplastic Cells ◽  
Malignant Effusions ◽  
Vegf Protein

14123 Background: Tumor progression and metastasis formation are often associated with enhanced angiogenesis and with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and a mediator of vascular permeability. We here describe that VEGF is produced and secreted by neoplastic cells in various solid tumors and its production mediated through mTOR. Methods and Results: As assessed by ELISA, the VEGF protein was detected in supernatants of cell lines derived from breast cancer (MDA-MB231), pancreatic carcinoma (BxPC-3), lung cancer (A-427), colon carcinoma (HCT8), and cholangiocellular carcinoma (EGI-1). In addition, VEGF was detected in supernatants of primary tumor cells obtained from malignant effusions in various malignancies (breast cancer, n=4; pancreatic cancer, n=1; ovarial cancer, n=1; parotic carcinoma, n=1; oesophageal carcinoma, n=1). In each case, VEGF protein was detectable in neoplastic cells by immunocytochemistry, and was found to accumulate in supernatants of cultured tumor cells over time, suggesting constant production and secretion. Correspondingly, as assessed by RT-PCR, primary tumor cells as well as the cell lines tested were found to express VEGF mRNA in a constitutive manner. Since mTOR is a well known regulator of VEGF synthesis, we applied rapamycin on primary neoplastic cells and on tumor cell lines. Rapamycin (20–200 nM) was found to counteract the production and secretion of VEGF in all tumor cells tested (VEGF in supernatants in cultures supplemented with rapamycin at 100 nM compared to control=100% on day 6: MDA-MB231: 11.8±0.2%; BxPC-3: 23.6±18.8%; A-427: 30.1±3.4%; HCT8 17.2±0.5%; EGI-1 28.4±1.1%; p<0.05). By contrast, neither rapamycin nor VEGF were found to modulate growth of primary tumor cells or the growth of the tumor cell lines tested. Conclusions: Various human tumor cells express and secrete VEGF. VEGF production is mediated through mTOR. These observations may have implications for the design of new treatment approaches attempting to counteract VEGF production/secretion and thus VEGF-dependent angiogenesis and effusion- formation in solid tumors. No significant financial relationships to disclose.

scholarly journals High periostin expression correlates with aggressiveness in papillary thyroid carcinomas

2008 ◽  
Vol 197 (2) ◽  
pp. 401-408 ◽  
Author(s):  
Cinzia Puppin ◽  
Dora Fabbro ◽  
Mariavittoria Dima ◽  
Carla Di Loreto ◽  
Efisio Puxeddu ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Thyroid Tumor ◽  
Specific Gene ◽  
Mrna Levels ◽  
Tumor Cell Lines ◽  
Papillary Thyroid ◽  
Osteoblastic Cell Line ◽  
Thyroid Carcinomas ◽  
Normal Tissues

Periostin is a mesenchyme-specific gene product, which acts as an adhesion molecule during bone formation and supports osteoblastic cell line attachment and spreading. However, periostin expression is activated in a large variety of epithelial human tumors and correlates with their aggressiveness. Knowledge of expression of periostin in thyroid tumors is still scanty. The aim of the present work was to investigate periostin expression in differentiated neoplasms of the thyroid and to correlate it with several clinical and molecular features of these tumors. Periostin expression was evaluated by quantitative PCR and immunohistochemistry in normal thyroid tissues, papillary thyroid carcinomas (PTCs), follicular thyroid carcinomas (FTCs), and follicular adenomas (FAs). Periostin mRNA levels were also evaluated in several thyroid tumor cell lines. PTCs show mean periostin mRNA levels significantly higher than corresponding normal tissues. In five PTCs, periostin mRNA values were at least 30-fold higher than corresponding normal tissues. Conversely, mean periostin mRNA levels of FTCs and FAs were similar to those of normal tissues. Consistent with mRNA studies, periostin was detectable by immunohistochemistry in cancerous epithelial cells only in several cases of PTCs but not in normal tissue, FTCs, and FAs. In PTCs, periostin mRNA levels positively correlate with extrathyroidal invasion, distant metastasis, and higher grade staging. A negative correlation between periostin and expression of some markers of the thyroid-differentiated phenotype (thyroglobulin, thyrotropin receptor) was also present in the PTCs. These results indicate that an increase in periostin gene expression is present in several PTCs, in which it appears as a marker of aggressiveness. Experiments in thyroid tumor cell lines indicate that high levels of periostin mRNA are due, at least in part, to the increase in periostin promoter activity.

scholarly journals Anticancer evaluation of di- and trifunctional substituted 1,3-thiazoles

2020 ◽  
Vol 15 (1) ◽  
pp. 2-11
Author(s):  
Kostyantyn Turov
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Cell Lines ◽  
Human Tumor ◽  
Tumor Cell Lines ◽  
Scientific Program ◽  
Epithelial Cancer ◽  
Human Tumor Cell ◽  
Human Tumor Cell Lines

Anticancer activity of a series of polyfunctional substituted 1,3-thiazoles has been studied within the international scientific program “NCI-60 Human Tumor Cell Lines Screen”. Screening was performed in vitro on 60 cell lines of lungs, kidneys, CNS, ovaries, prostate, and breast cancer, epithelial cancer, leukemia, and melanoma. The most effective compounds were those with a piperazine substituent at C2 of the 1,3-thiazole cycle: 1-(4-((4-methylphenyl)-sulfonyl)-2-phenyl-1,3-thiazol-5-yl)piperazine (average lg GI50 = -5.87, lg TGI = -5.54, lg LC50 = -5.21), 1-(2-(3,5-dimethyl-1H-pyrazol-1-yl)-4-((4-methylpheyl)sulfonyl)-1,3-thiazol-5-yl)piperazine (average lg GI50 = -5.66, lg TGI = -5.26, lg LC50 = -4.83), and 1-(2,4-bis((4-methylphenyl)sulfonyl)-1,3-thiazol-5-yl)piperazine (average lg GI50 = -5.67, lg TGI = -5.21, lg LC5050 = -4.67).

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