ABT-737 Is a Useful Component of Combinatory Chemotherapies for Chronic Myelogenous Leukemias with Diverse Drug Resistance Mechanisms.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 808-808 ◽  
Author(s):  
Junya Kuroda ◽  
Shinya Kimura ◽  
Michael Andreeff ◽  
Eishi Ashihara ◽  
Yuri Kamitsuji ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Resistance Mechanisms ◽  
Cell Killing ◽  
Leukemic Cells ◽  
Killing Effect ◽  
Cell Killing Effect

Abstract Chronic myelogenous leukemia (CML) is characterized by its refractoriness to various apoptotic insults by Bcr-Abl tyrosine kinase (TK)-mediated signalling. Although imatinib mesylate (IM), a Bcr-Abl TK inhibitor, has markedly improved the therapeutic outcomes of CML, additional or alternative molecular targeting strategies are still needed. Since the interplay of anti-apoptotic Bcl-2 proteins and BH3-only proteins, such as Bim and Bad, is crucial for regulating the cellular fate of Bcr-Abl+ leukemic cells (Kuroda J et al, PNAS , 2006; Cell Death Differ, 2007), the direct targeting of anti-apoptotic Bcl-2 proteins by the use of a BH3-only protein mimetic is an attractive approach for treating CML. We here investigated the activity of ABT-737, a mimic of BH3-only proteins that inhibit anti-apoptotic Bcl-2, Bcl-XL and Bcl-w, but not Mcl-1 or A1, against CML. The Annexin-V-staining study, the assay for mitochondrial outer membrane potential and the internucleosomal fragmentation assay revealed that ABT-737 potently induced apoptosis in CML cell lines (BV173, K562, KCL22, KT-1, MEG-01 and MYL) with the IC50 for induction of cell death by 48 h of treatment ranging from 0.04 to 4.06 μM. ABT-737 was also effective in killing primary CML samples in vitro. ABT-737 prolonged the survival of mice xenografted with a CML cell line, BV173, demonstrating its in vivo bioactivity. Higher expression of Bcl-2, Bcl-XL, or Mcl-1 reduced cell killing by ABT-737 in each cell line, but we found no correlation between the sensitivities to ABT-737 and the specific expression patterns of Bcl-2 family proteins among different cell lines. The levels of Bcr-Abl and Lyn, a member of Src kinase family associated with apoptosis resistance in CML, also varied among the cell lines, and we found no consistent relationship between the sensitivity to ABT-737 and the expression levels of these proteins Thus, the cell killing effect of ABT-737 in CML must be determined in part by other drug resistance mechanisms, such as high expression of Bcr-Abl, overexpression of P-glycoprotein (P-gp), a drug-efflux pump, and/or their combination. Importantly, ABT-737 augmented the cell killing effect of IM in CML cell lines with high levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL, or even Mcl-1), Bcr-Abl, P-gp, or Lyn, unless leukemic cells harboured IM-insensitive Abl kinase domain (KD) mutations. Moreover, the combination of ABT-737 with homoharringtonine, an herbal-derived anti-CML therapeutic that potently reduces Mcl-1 within 6 hours in vitro, dramatically enhanced the killing by ABT-737 in CML cells with diverse drug resistance mechanisms, including IM-insensitive Abl KD mutations, such as T315I mutation. These results suggest that ABT-737 could be a useful component of combinatory chemotherapies for CML.

A Novel Pan-PI3K/Akt Inhibitor, SF1126, Inhibits In Vitro Growth of Multiple Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4806-4806
Author(s):  
Jeannine Silberman ◽  
Kimberly Dalbey ◽  
Claire Torre ◽  
Ebenezer David ◽  
Leif Bergsagel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Mtt Assay ◽  
Immunoblot Analysis ◽  
Akt Inhibitor ◽  
Downstream Targets

Abstract Backround: Dysregulation of the PI3K/Akt signal transduction pathway has been implicated in the development of a number of malignancies, including multiple myeloma (MM). This cellular signaling mechanism and its downstream targets (eg mTOR) regulate cell growth, proliferation and apoptosis. SF1126 (Semafore) is a water soluble prodrug of the pan-PI3K inhibitor, LY294002, whose anti-proliferative and pro-apoptotic activity has been well described in the literature. Preclinical studies using SF1126 in a variety of malignancies including glioma, prostate, non-small cell lung cancer, and breast cancer appear promising and have demonstrated profound antiangiogenic effects mediated through VEGF inhibition. Aim: To demonstrate in vitro anti-myeloma activity of SF1126, alone and in combination with dexamethasone, bortezomib, and melphalan and evaluate their effects on downstream targets of PI3K/Akt. Methods: MM cell lines (MM.1R, MM.1S, RPMI 8226) were treated with SF1126 (1–100uM), dexamethasone (5uM), bortezomib (5nM), melphalan (10uM) alone, and in combination. Growth inhibition following treatment was measured by MTT assay at 24 and 48 hours. Apoptosis was assessed by annexin-V binding assay using flow cytometry. Immunoblot analysis was performed to measure downstream targets of Akt including: p-PDK1 and mTOR (4E-BP1). Results: A clear dose response was established with an IC50 of 8.75uM in the MM.1R and 7.5uM in the MM.1S cell lines at 48 hours. At 24 and 48 hours, 5uM SF1126 alone resulted in 80% and 64% cell viability by MTT assay, respectively, in the MM.1R cell line. The combination of 5uM SF1126 with conventional agents was then tested in the MM.1R cell line. Combination with 5uM dexamethasone enhanced the efficacy of 5uM SF1126 by 26% at 48 hours. Combination with 10uM melphalan enhanced the efficacy of 5uM SF1126 by 20% at 24 hours. The combination with 5nM bortezomib enhanced the efficacy of 5uM SF1126 by 23% at 48 hours. Given prior experience demonstrating that short exposure to bortezomib activates Akt, we tested sequential administration of bortezomib and SF1126 in the MM.1R cell line. Optimal cell death was induced with bortezomib prior to SF1126, followed by concurrent administration. Immunoblot analysis of p-PDK1, downstream mTOR target (4E-BP1) were performed on the MM.1S cell line treated with 5, 10, 20, and 50uM SF1126 at 12 and 24 hours. At the 12 hour time point, p-PDK-1 appeared to increase, but was significantly reduced by 48 hours. A similar pattern of initial upregulation followed by reduction by 24 hours was seen with the mTOR protein 4E-BP1. Conclusion: SF1126 has dose dependent, in vitro activity in several multiple myeloma cell lines both as a single agent and in combination with dexamethasone, bortezomib, and melphalan. The addition of SF1126 to dexamethasone in a dexamethasone resistant cell line results in increased cell death, possibly by overcoming resistance mechanisms. The addition of SF1126 to bortezomib and melphalan also resulted in increased growth inhibition over either agent alone. These results warrant further study of this promising new pan-PI3K/Akt inhibitor.

scholarly journals Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 447-455 ◽  
Author(s):  
D Zipori ◽  
J Toledo ◽  
K von der Mark
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cell ◽  
Leukemic Cells ◽  
Matrix Composition ◽  
Mouse Bone Marrow ◽  
Type I

Abstract Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis

1993 ◽  
Vol 104 (2) ◽  
pp. 307-315 ◽  
Author(s):  
A.C. Bayly ◽  
N.J. French ◽  
C. Dive ◽  
R.A. Roberts
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferators ◽  
Hepatoma Cell Lines

A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of establishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcinogens. In view of speculation that peroxisome proliferators suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apoptotic cell death. Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO. This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1. This response was found to display intercellular heterogeneity by immunocytochemistry. Thus, the FaO cell line maintained characteristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible markers. However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium. The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA. Cells detaching from confluent FaO cultures exhibited chromatin condensation and DNA fragmentation patterns characteristic of cels undergoing apoptotic death.Interestingly, no apoptosis was seen in monolayer cells, suggesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Bim Is the Critical, and Bad and Bmf Are the Ancillary BH3-Only Proteins for Imatinib Mesylate-Induced Apoptosis of Bcr/Abl-Positive Leukemias.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2876-2876
Author(s):  
Junya Kuroda ◽  
Hamsa Puthalakath ◽  
Philippe Bouillet ◽  
Mark S. Cragg ◽  
Priscilla N. Kelly ◽  
...  
Keyword(s):  
Cell Death ◽  
Imatinib Mesylate ◽  
Cell Lines ◽  
Cell Killing ◽  
Leukemic Cells ◽  
Imatinib Treatment ◽  
Killing Activity ◽  
Induced Apoptosis ◽  
Transformed Cell Lines

Abstract Imatinib mesylate (imatinib) exerts the anti-Philadelphia-positive (Ph1+) leukemia activity both by the inhibition of cell proliferation and by the induction of apoptosis. Recent studies demonstrate that the induction of cell death is essential for eradication of Ph1+ leukemic clones in imatinib treatment; however, the molecular mechanisms have not yet been clearly described. By examining the effect of imatinib on parental K562 and subclones overexpressing either Bcl-2, Bcl-XL or a dominant interfering mutant of FADD/MORT1, which blocks death receptor apoptosis signalling, we found that imatinib triggers apoptosis exclusively via the Bcl-2 family-regulated intrinsic apoptotic pathway. We investigated the involvement of BH3-only proteins as apoptotic initiators in imatinib-induced cell death, because the cell life-or-death decision is arbitrated by the balance between pro-apoptotic BH3 only-proteins and anti-apoptotic Bcl-2 proteins. We found that imatinib treatment upregulated Bim in Ph1+ leukemic cell lines and bcr-c-abl transformed murine fetal liver cells (FLCs)-derived cell lines both by transcriptional and post-translational mechanisms. Imatinib also activated Bad through dephosphorylation and upregulated Bmf transcriptionally. To examine the role of Bim in imatinib-induced apoptosis, we examined the cell killing activity of imatinib in subclones of K562 and BV173 Ph1+ cells expressing abnormally reduced levels of Bim using stable RNA interference system. This revealed that the cell killing activity of imatinib largely dependent on Bim expression levels in these cell lines, although significant apoptosis was still evident. To further define the role of Bim, Bad and Bmf in imatinib-induced cell death, we examined the effect of imatinib on retrovirally bcr-c-abl transformed cell lines derived from FLCs from wild type C57BL/6, Bim-/-, Bad-/-, Bim-/-Bad-/- double KO and Bcl-2 transgenic fetuses. The bim-/-bcr-c-abl+ FLCs were shown to be more resistant to imatinib-induced cell death than wt.bcr-c-abl+ FLCs, however, bim-/-bcr-c-abl+ FLCs were eventually induced into cell death, indicating that Bim is not the only initiator of apoptosis. The bad-/-bcr-c-abl+ FLCs were also partially resistant to imatinib-induced cell death. Intriguingly, like in vav.bcl-2.bcr-c-abl+ FLCs, the cell death induction by imatinib (~5.0μM) was largely abrogated in bim-/-bad-/-bcr-c-abl+ FLCs, indicating that Bim collaborates with Bad for the apoptotic induction by imatinib. Importantly, we found that Bim was inducible by ex vivo imatinib treatment in primary Ph1+ leukemic cells only from clinically good responders but not from patients refractory to imatinib treatment. Collectively, these results demonstrate that Bim is the critical but not the only initiator required for imatinib-induced apoptosis of Bcr/Abl-positive hematopoietic cells; Bad and Bmf may be the ancillary BH3-only proteins in this process. Our results provide evidence for the therapeutic significance of regulation of BH3-only proteins, particularly Bim, for the eradication of Ph1+ leukemic cells.

Characterization of Gene Expression Associated with Cyclophosphamide Resistance in Chronic Myeloid Leukemia (CML).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1532-1532
Author(s):  
Fei Bao ◽  
Mary L. Nordberg ◽  
Paula Polk ◽  
Amanda Sun ◽  
David Murray ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Resistant Cell ◽  
Parental Line ◽  
Resistant Line ◽  
Rt Pcr

Abstract Cyclophosphamide (CP) is one of the alkylating agents collectively referred to as oxazaphosphorines that are used to treat many types of cancers including myeloid leukemia. Tumor cell drug resistance is an important factor for clinical treatment failure. The mechanisms of drug resistance are multifactorial and incompletely understood. KBM-7 human CML cell line was established from blast cells from a patient in the terminal phase of CML. In the CP resistance model, the B5-180 sub-line was isolated following exposure to the in vitro active CP analog 4HC. B5-180 cells were cross-resistant to busulfan and γ-radiation. Total RNA was extracted and hybridized to Affymetrix Genechip HG-U95Av2 arrays. Each array contains 12,386 probes corresponding to approximately 9000 known human genes. Each cell line was arrayed in triplicate. Quantitative RT-PCR, Fluorescence In-Situ Hybridization (FISH) and cytogenetic analysis were performed in both cell lines. Both the KBM-7/B5 parental line and B5-180 resistant sub-line expressed high-levels of BCR-ABL transcripts by real-time RT-PCR. FISH and cytogenetic analysis revealed multiple copies of t(9;22) translocation and other additional chromosomal abnormalities such as trisomy 8, and abnormalities of chromosome 18 in both cell lines. Gene array identified 794 gene transcripts that were more than twofold (range from 2-fold to 2675-fold) over-expressed or under-expressed in the resistant line relative to the parental line. ALDH1A1 (aldehyde dehydrogenase 1 family) showed the most differential expression between sensitive and resistant cell lines, ALDH1A1 was upregulated more than 2000-fold in the resistant sub-line. ALDH-2 (aldehyde dehydrogenase 2 family mitochondrial) was also expressed substantially higher in the resistant line. This finding is consistent with the established fact that elevated ALDH activity is an important factor in the resistance of B5-180 cells to 4HC. The remaining differentially expressed genes encode proteins with a wide variety of biochemical functions, which include 44 apoptosis and 7 anti-apoptosis-related genes, 56 genes related to cell cycle and cell growth, 6 DNA repair genes, 13 genes involved in hemopoiesis and B-cell activation. We also tested the expression of the hematopietic transcription factor PU-1 and PUB, a novel PU-1 binding factor. Interestingly, the expression of PU-1 was decreased and PUB increased in the resistant clone. In conclusion, we have identified a large number of differentially expressed genes in a CP resistant cell line derived from CML blast crisis by microarray technology. Our results suggest that CP resistance is a complex phenotype that involves multiple genes and a variety of mechanisms. Real-time RT-PCR analysis and further characterization of selected genes associated with CP resistance as well as the response in vitro to tyrosine kinase inhibitors are currently under investigation.

A COMBINED CELL KILLING EFFECT IN VITRO OF PHOTORADIATION AND HYPERTHERMIA

1985 ◽  
Vol 76 (7) ◽  
pp. 1059-1065 ◽  
Author(s):  
Kazuyoshi Nakajima ◽  
Haruo Hisazumi ◽  
Norio Miyoshi
Keyword(s):  
Cell Killing ◽  
Killing Effect ◽  
Cell Killing Effect

scholarly journals Annona cherimola Seed Extract Activates Extrinsic and Intrinsic Apoptotic Pathways in Leukemic Cells

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 506 ◽  
Author(s):  
Tony Haykal ◽  
Peter Nasr ◽  
Mohammad H. Hodroj ◽  
Robin I. Taleb ◽  
Rita Sarkis ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Seed Extract ◽  
Leukemic Cells ◽  
Western Blots ◽  
Toxic Activity ◽  
Annona Cherimola

Annona cherimola Mill is a large green fruit with black seeds widely known to possess toxic properties due to the presence of Annonaceous acetogenins. The present study investigates the anti-cancer properties of an Annona cherimola Mill ethanolic seed extract on Acute Myeloid Leukemia (AML) cell lines in vitro and elucidates the underlying cellular mechanism. The anti-proliferative effects of the extract on various AML cell lines and normal mesenchymal cells (MSCs) were assessed using WST-1 viability reagent. The pro-apoptotic effect of the extract was evaluated using Annexin V/PI staining and Cell Death ELISA. The underlying mechanism was deciphered by analyzing the expression of various proteins using western blots. Treatment with an A. cherimola seed ethanolic extract promotes a dose- and time-dependent inhibition of the proliferation of various AML cell lines, but not MSCs. Positive Annexin V staining, as well as DNA fragmentation, confirm an increase in apoptotic cell death by upregulating the expression of pro-apoptotic proteins which control both intrinsic and extrinsic pathways of apoptosis. GC/MS analysis revealed the presence of phytosterols, in addition to other bioactive compounds. In conclusion, Annona cherimola Mill seed extract, previously known to possess a potent toxic activity, induces apoptosis in AML cell lines by the activation of both the extrinsic and the intrinsic pathways.

scholarly journals In vitro study of enhanced photodynamic cancer cell killing effect by nanometer-thick gold nanosheets

Nano Research ◽  
2020 ◽  
Vol 13 (12) ◽  
pp. 3217-3223 ◽  
Author(s):  
Ziyi Zhang ◽  
Dalong Ni ◽  
Fei Wang ◽  
Xin Yin ◽  
Shreya Goel ◽  
...  
Keyword(s):  
Cancer Cell ◽  
In Vitro Study ◽  
Cell Killing ◽  
Vitro Study ◽  
Killing Effect ◽  
Thick Gold ◽  
Cell Killing Effect

scholarly journals Visfatin Is Actively Secreted In Vitro From U-937 Macrophages, but Only Passively Released From 3T3-L1 Adipocytes and HepG2 Hepatocytes

2017 ◽  
pp. 709-714 ◽  
Author(s):  
P. SVOBODA ◽  
E. KŘÍŽOVÁ ◽  
K. ČEŇKOVÁ ◽  
K. VÁPENKOVÁ ◽  
J. ZÍDKOVÁ ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
In Vitro Study ◽  
Active Secretion ◽  
Intracellular Enzyme ◽  
Cell Lysate ◽  
Passive Release ◽  
Monocytes And Macrophages

Visfatin is a multi-functional molecule that can act intracellularly and extracellularly as an adipokine, cytokine and enzyme. One of the main questions concerning visfatin is the mechanism of its secretion; whether, how and from which cells visfatin is released. The objective of this in vitro study was to observe the active secretion of visfatin from 3T3-L1 preadipocytes and adipocytes, HepG2 hepatocytes, U-937, THP-1 and HL-60 monocytes and macrophages. The amount of visfatin in media and cell lysate was always related to the intracellular enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), to exclude the passive release of visfatin. Visfatin was not found in media of 3T3-L1 preadipocytes. In media of 3T3-L1 adipocytes and HepG2 hepatocytes, the ratio of visfatin to the amount of GAPDH was identical to cell lysates. Hence, it is likely that these cells do not actively secrete visfatin in a significant manner. However, we found that significant producers of visfatin are differentiated macrophages and that the amount of secreted visfatin depends on used cell line and it is affected by the mode of differentiation. Results show that 3T3-L1 adipocytes and HepG2 hepatocytes released visfatin only passively during the cell death. U-937 macrophages secrete visfatin in the greatest level from all of the tested cell lines.

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