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 Blocking Protein Secretion with Novel Small Molecule Inhibitors of Sec61 Represents a Potential Treatment Strategy Against Hematologic Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 408-408
Author(s):  
Eric Lowe ◽  
Andrea R Fan ◽  
Jing Jiang ◽  
Henry W. B. Johnson ◽  
Christopher J. Kirk ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Cell Lines ◽  
Protein Secretion ◽  
Small Molecule ◽  
Life Sciences ◽  
Hematologic Malignancies ◽  
Employment Equity ◽  
Equity Ownership

Secreted and membrane proteins play key roles in cancer development, including as autocrine growth factors, receptors in oncogenic signaling cascades, and checkpoints in immune system evasion. The biogenesis of most secreted and transmembrane proteins involves cotranslational translocation of nascent polypeptides ("clients") into the endoplasmic reticulum (ER) through the Sec61 translocon. Multiple inhibitors of protein secretion have been described that target Sec61 and have antitumor activity but lack adequate pharmaceutical properties or tolerability to be studied clinically. Here we describe novel small molecule inhibitors of Sec61 which exhibit activity against hematologic tumor cells in vitro and in vivo. KZR-8834 (8834) was identified through a screening campaign for novel inhibitors of Sec61-dependent protein secretion, where it exhibited nanomolar potency against multiple Sec61 client proteins of therapeutic value, including several immune checkpoint proteins. The broad Sec61 client inhibition profile of 8834 led to its in vitro assessment of anti-cancer activity against a panel of 346 human cancer cell lines. 8834 displayed broad cytotoxic activity against both solid and hematologic tumor types with high potency for hematologic malignancies including acute lymphoid leukemia (mean IC50=317nM, n=8 cell lines), acute myeloid leukemia (IC50=359nM, n=14), lymphoma (IC50=250nM, n=15) and multiple myeloma (IC50=352nM, n=11). In mouse xenograft models of multiple myeloma (H929) and mantle cell lymphoma (Mino), once weekly administration of 8834 or KZR-9261 (8834 analog) resulted in >90% tumor growth inhibition without significant clinical toxicity. Two multiple myeloma cell lines, H929 and U266, were chosen to assess cellular response due to their sensitivity and resistance to 8834, respectively. In H929 cells, 250nM 8834 induced activation of caspase 3/7 (>15 fold) within 8 hours of exposure which corresponded with a cell viability IC50 of 98nM at 24 hours. In contrast, no caspase 3/7 activation was noted in U266 cells through 24 hours of exposure, which corresponded with minimal effects on cell viability. Gene expression profiling by RNAseq and quantitative proteomic profiling by mass spectrometry was performed on these cell lines to elucidate mechanisms of sensitivity and resistance to Sec61 inhibition. Both methods revealed rapid upregulation of ER stress response genes/proteins and activation of the unfolded protein response, which was greater in H929 cells and confirmed by immunoblot and QPCR. Gene set enrichment analysis revealed significantly higher basal levels of ER stress-related genes in H929 vs U266 cells, suggesting ER stress response capacity as a possible predictive biomarker. In summary, blockade of Sec61-dependent translocation of secreted and membrane proteins with novel small molecule inhibitors exhibits a broad antitumor profile in vitro, potentially in part through activation of proteotoxic stress. These effects translate into therapeutic activity in multiple mouse xenograft models, demonstrating a potential novel treatment for hematologic malignancies. Disclosures Lowe: Kezar Life Sciences: Employment, Equity Ownership. Fan:Kezar Life Sciences: Employment, Equity Ownership. Jiang:Kezar Life Sciences: Employment, Equity Ownership. Johnson:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Muchamuel:Kezar Life Sciences: Employment, Equity Ownership. Qian:Kezar Life Sciences: Employment, Equity Ownership. Tuch:Kezar Life Sciences: Consultancy.

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.

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.

Synthesis and Cytotoxicity of New Mannich Bases of 6-[3-(3,4,5-Trimetoxyphenyl)-2- propenoyl]-2(3H)-Benzoxazolone

2020 ◽  
Vol 17 (4) ◽  
pp. 512-517
Author(s):  
Ognyan Ivanov Petrov ◽  
Yordanka Borisova Ivanova ◽  
Mariana Stefanova Gerova ◽  
Georgi Tsvetanov Momekov
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Biological Activities ◽  
Human Tumor ◽  
Mannich Bases ◽  
In Vitro Cytotoxicity ◽  
Tumor Cell Lines ◽  
Human Tumor Cell ◽  
Human Tumor Cell Lines

Background: Chemotherapy is one of the mainstays of cancer treatment, despite the serious side effects of the clinically available anticancer drugs. In recent years increasing attention has been directed towards novel agents with improved efficacy and selectivity. Compounds with chalcone backbone have been reported to possess various biological activities such as anticancer, antimicrobial, anti-inflammatory, analgesic, antioxidant, etc. It was reported that aminomethylation of hydroxy chalcones to the corresponding Mannich bases increased their cytotoxicity. In this context, our interest has been focused on the design and synthesis of the so-called multi-target molecules, containing two or more pharmacophore fragments. Methods: A series of Mannich bases were synthesized by the reaction between 6-[3-(3,4,5- trimethoxyphenyl)-2-propenoyl]-2(3Н)-benzoxazolone, formaldehyde, and a secondary amine. The structures of the compounds were confirmed by elemental analysis, IR and NMR spectra. The new Mannich bases were evaluated for their in vitro cytotoxicity against a panel of human tumor cell lines, including BV-173, SKW-3, K-562, HL-60, HD-MY-Z and MDA-MB-231. The effects of selected compounds on the cellular levels of glutathione (GSH) were determined. Results: The new compounds 4a-e exhibited concentration-dependent cytotoxic effects at micromolar concentrations in MTT-dye reduction assay against a panel of human tumor cell lines, similar to those of starting chalcone 3. The tested agents led to concentration - dependent depletion of cellular GSH levels, whereby the effects of the chalcone prototype 3 and its Mannich base-derivatives were comparable. Conclusion: The highest chemosensitivity to the tested compounds was observed in BV- 173followed by SKW-3 and HL-60 cell lines.

Cytotoxic Effect of Brassica oleracea Extract on two Tumor Cell Lines in vitro

2013 ◽  
Vol 16 (1) ◽  
pp. 137-142
Author(s):  
Farooq I. Mohammed ◽  
◽  
Farah T. Abdullah ◽  
Shaimaa Y. Abdulfttah ◽  
◽  
...  
Keyword(s):  
Tumor Cell ◽  
Brassica Oleracea ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Tumor Cell Lines

scholarly journals Pharmacoinformatics and Preclinical Studies of NSC765690 and NSC765599, Potential STAT3/CDK2/4/6 Inhibitors with Antitumor Activities against NCI60 Human Tumor Cell Lines

Biomedicines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 92
Author(s):  
Bashir Lawal ◽  
Yen-Lin Liu ◽  
Ntlotlang Mokgautsi ◽  
Harshita Khedkar ◽  
Maryam Rachmawati Sumitra ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Tumor ◽  
Cancer Cell Lines ◽  
Tumor Cell Lines ◽  
Human Tumor Cell ◽  
Human Tumor Cell Lines ◽  
Anti Cancer

Signal transducer and activator of transcription 3 (STAT3) is a transcriptional regulator of a number of biological processes including cell differentiation, proliferation, survival, and angiogenesis, while cyclin-dependent kinases (CDKs) are a critical regulator of cell cycle progression. These proteins appear to play central roles in angiogenesis and cell survival and are widely implicated in tumor progression. In this study, we used the well-characterized US National Cancer Institute 60 (NCI60) human tumor cell lines to screen the in vitro anti-cancer activities of our novel small molecule derivatives (NSC765690 and NSC765599) of salicylanilide. Furthermore, we used the DTP-COMPARE algorithm and in silico drug target prediction to identify the potential molecular targets, and finally, we used molecular docking to assess the interaction between the compounds and prominent potential targets. We found that NSC765690 and NSC765599 exhibited an anti-proliferative effect against the 60 panels of NCI human cancer cell lines, and dose-dependent cytotoxic preference for NSCLC, melanoma, renal, and breast cancer cell lines. Protein–ligand interactions studies revealed that NSC765690 and NSC765599 were favored ligands for STAT3/CDK2/4/6. Moreover, cyclization of the salicylanilide core scaffold of NSC765690 mediated its higher anti-cancer activities and had greater potential to interact with STAT3/CDK2/4/6 than did NSC765599 with an open-ring structure. NSC765690 and NSC765599 met the required safety and criteria of a good drug candidate, and are thus worthy of further in-vitro and in-vivo investigations in tumor-bearing mice to assess their full therapeutic efficacy.

Evaluation of ponatinib in vitro effect in three canine mast cell tumor cell lines expressing FGFR-1, PDGFR-α, and VEGFR-2

2021 ◽  
Vol 269 ◽  
pp. 105621
Author(s):  
C.J. Fisher ◽  
A.T. Lejeune ◽  
M.J. Dark ◽  
O.M. Hernandez ◽  
K. Shiomitsu
Keyword(s):  
Mast Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
Cell Tumor ◽  
Tumor Cell Lines ◽  
Mast Cell Tumor ◽  
Canine Mast Cell Tumor ◽  
Vitro Effect

Synthesis and Characterization of New Palladium(II) Complexes with Ligands Derived from Furan-2-carbaldehyde and Benzaldehyde Thiosemicarbazone and their in vitro Cytotoxic Activities against Various Human Tumor Cell Lines

2010 ◽  
Vol 65 (10) ◽  
pp. 1271-1278 ◽  
Author(s):  
Wilfredo Hernández ◽  
Juan Paz ◽  
Fernando Carrasco ◽  
Abraham Vaisberg ◽  
Jorge Manzur ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Human Tumor ◽  
Myelogenous Leukemia ◽  
Cytotoxic Activities ◽  
Spectroscopic Techniques ◽  
Tumor Cell Lines ◽  
Human Tumor Cell ◽  
Human Tumor Cell Lines

With the ligands 4-phenyl-1-(furan-2-carbaldehyde)thiosemicarbazone, HTSC1, (1), 4-phenyl-1- (5´-phenyl-furan-2-carbaldehyde)thiosemicarbazone, HTSC2 (2), o-methoxy-benzaldehydethiosemicarbazone, HTSC3 (3), and o-cyano-benzaldehydethiosemicarbazone, HTSC4 (4), the corresponding palladium(II) complexes, Pd(TSC1)2 (5), Pd(TSC2)2 (6), Pd(TSC3)2 (7), and Pd(TSC4)2 (8) were synthesized and characterized by elemental analysis and spectroscopic techniques. The crystal structure of Pd(TSC3)2 (7) was determined by single-crystal X-ray diffraction. Complex 7 shows a squareplanar geometry, where two deprotonated ligands are coordinated to the PdII center through the nitrogen and sulfur atoms in a trans arrangement. In vitro antitumor studies against different human tumor cell lines have revealed that the palladium(II) complexes 5- 8 are more cytotoxic (IC50 values in the range of 0.21 - 3.79 μM) than their corresponding ligands (1 - 4) (> 60 μM). These results indicate that the antiproliferative activity is enhanced when thiosemicarbazone ligands are coordinated to the metal. Among the studied palladium(II) complexes, 8 exhibits high antitumor activity on K562 chronic myelogenous leukemia cells with a low value of the inhibitory concentration (IC50 = 0.21 μM).

Activation of the heat shock transcription factor by hypoxia in normal and tumor cell lines in vivo and in vitro

1992 ◽  
Vol 23 (4) ◽  
pp. 891-897 ◽  
Author(s):  
Amato J. Giaccia ◽  
Elizabeth A. Auger ◽  
Albert Koong ◽  
David J. Terris ◽  
Andrew I. Minchinton ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Tumor Cell ◽  
Cell Lines ◽  
Heat Shock Transcription Factor ◽  
Tumor Cell Lines

scholarly journals Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis

2006 ◽  
Vol 11 (8) ◽  
pp. 922-932 ◽  
Author(s):  
Andrea Ivascu ◽  
Manfred Kubbies
Keyword(s):  
Tumor Cell ◽  
Suspension Culture ◽  
Cell Lines ◽  
Cell Function ◽  
Tumor Cell Lines ◽  
Proliferating Cells ◽  
Basement Membrane Extract ◽  
Rapid Generation ◽  
Size Heterogeneity

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.

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