Combination of Imatinib with Cisplatin and Nutlin-3: Functional and Molecular Effects on Bcr-Abl Positive Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2954-2954
Author(s):  
Ioanna Skorta ◽  
Heiko van der Kuip ◽  
Martin Henkes ◽  
Moshe Oren ◽  
Walter E. Aulitzky
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Cellular Response ◽  
Combined Treatment ◽  
Optimal Growth ◽  
Growth Conditions ◽  
P53 Expression ◽  
Complete Eradication ◽  
Abl Kinase ◽  
Highly Effective

Abstract Imatinib is highly effective in inducing remissions in chronic phase CML. However, complete eradication of the malignant clone by Imatinib monotherapy is rare. This prompted us to explore the efficacy of combination of Imatinib with the DNA damaging agent Cisplatin and with Nutlin-3, a compound which induces p53 accumulation by interfering with its binding to Mdm2. We used a Bcr-Abl positive cell line characterized by the loss of Imatinib sensitivity in the presence of optimal growth factor concentrations. Whereas combined treatment of Imatinib with either Cisplatin or Nutlin-3 in low and nontoxic doses induced ∼15–20% cell death, simultaneous treatment of all three compounds was highly effective (∼50% cell death) even in the presence of optimal growth conditions. Importantly, comparable effects were also seen in CFU assays performed with primary CD34 positive cells from 5 CML patients. To examine the molecular mechanisms of these effects we analyzed p53 dependent cellular response to Cisplatin in our cell line model in the presence or absence of Imatinib and/or Nutlin-3. The active Bcr-Abl kinase caused superinduction of p53 protein after DNA damage. We found both an upregulated p53 accumulation and enhanced p21 and Mdm2 RNA and protein levels upon Cisplatin treatment. This phenomenon could be reversed both by siRNA-mediated inhibition of Bcr-Abl expression and by Imatinib-induced inhibition of Bcr-Abl kinase activity: despite of optimal growth conditions inhibition of Bcr-Abl caused a significant reduction of p53 expression and activation. In the presence of Nutlin-3, p53 expression was significantly upregulated upon Cisplatin treatment both with and without Imatinib. However, Nutlin-3 was not capable to restore the Imatinib-induced blockade of the transcriptional activity of p53 on mdm2. The reduced p53 activation observed in Bcr-Abl positive cells treated with Imatinib was paralleled by a shift from cell cycle arrest to cell death both in the presence and absence of Nutlin selectively in Bcr-Abl positive cells rendering them hypersensitive to Cisplatin. In summary, combined treatment of Imatinib with low concentrations of a DNA damaging agent and a p53 activator is highly effective in vitro. Such combined treatment may prove to be clinically relevant for complete eradication of the malignant clone in CML, provided that conditions are found where it does not affect adversely normal hematopoietic cells in vivo.

scholarly journals The SGK1 Kinase Inhibitor SI113 Sensitizes Theranostic Effects of the 64CuCl2 in Human Glioblastoma Multiforme Cells

2017 ◽  
Vol 43 (1) ◽  
pp. 108-119 ◽  
Author(s):  
Giada Catalogna ◽  
Cristina Talarico ◽  
Vincenzo Dattilo ◽  
Vincenzo Gangemi ◽  
Ferdinando Calabria ◽  
...  
Keyword(s):  
Cell Death ◽  
Glioblastoma Multiforme ◽  
Cell Viability ◽  
Kinase Inhibitor ◽  
Therapeutic Potential ◽  
Synergistic Effects ◽  
Combined Treatment ◽  
Malignant Gliomas ◽  
P53 Expression ◽  
Radio Therapy

Background/Aims: The importance of copper in the metabolism of cancer cells has been widely studied in the last 20 years and a clear-cut association between copper levels and cancer deregulation has been established. Copper-64, emitting positrons and β-radiations, is indicated for the labeling of a large number of molecules suitable for radionuclide imaging as well as radionuclide therapy. Glioblastoma multiforme (GBM) is the CNS tumor with the worse prognosis, characterized by high number of recurrences and strong resistance to chemo-radio therapy, strongly affecting patients survival. We have recently discovered and studied the small molecule SI113, as inhibitor of SGK1, a serine/threonine protein kinase, that affects several neoplastic phenotypes and signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation, perturbs cell cycle progression and restores chemo-radio sensibility by modulating SGK1-related substrates. In the present paper we aim to characterize the combined effects of 64CuCl2 and SI113 on human GBM cell lines with variable p53 expression. Methods: Cell viability, cell death and stress/authopagic related pathways were then analyzed by FACS and WB-based assays, after exposure to SI113 and/or 64CuCl2. Results: We demonstrate here, that i) 64CuCl2 is able to induce a time and dose dependent modulation of cell viability (with different IC50 values) in highly malignant gliomas and that the co-treatment with SI113 leads to ii) additive/synergistic effects in terms of cell death; iii) enhancement of the effects of ionizing radiations, probably by a TRC1 modulation; iv) modulation of the autophagic response. Conclusions: Evidence reported here underlines the therapeutic potential of the combined treatment with SI113 and 64CuCl2 in GBM cells.

Growth characteristics of a cell line derived from the pig oviduct

1975 ◽  
Vol 21 (12) ◽  
pp. 2094-2097 ◽  
Author(s):  
A. M. P. Bouillant ◽  
P. Genest ◽  
A. S. Greig
Keyword(s):  
Cell Line ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Growth Characteristics ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Temperature Growth ◽  
A Cell ◽  
Maximum Population Density ◽  
Optimal Ph

Further evidence for the establishment of the pig fallopian tube (PFT) cell line as a continuous cell line was shown by an increase in the maximum population density as the number of subcultures increased. The optimal pH and temperature–growth ranges appeared to be 7.4–7.8 and 37–41 °C respectively, and the population doubling time was 20–25 h under optimal growth conditions. With progressive subculture, the serum requirements dropped from 20 to 2%. A plating efficiency of 2 to 4% was found in all serial subcultures. Colonies were observed in agar suspension culture at the 146th subculture and thereafter. Chromosomal alterations were found in the 100th subculture and thereafter.

scholarly journals Effects of Stress, Reactive Oxygen Species, and the SOS Response onDe NovoAcquisition of Antibiotic Resistance in Escherichia coli

2015 ◽  
Vol 60 (3) ◽  
pp. 1319-1327 ◽  
Author(s):  
Nadine Händel ◽  
Marloes Hoeksema ◽  
Marina Freijo Mata ◽  
Stanley Brul ◽  
Benno H. ter Kuile
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Cellular Response ◽  
De Novo ◽  
Sos Response ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Regulation Of Transcription ◽  
Ph Homeostasis ◽  
Content Type

Strategies to prevent the development of antibiotic resistance in bacteria are needed to reduce the threat of infectious diseases to human health. Thede novoacquisition of resistance due to mutations and/or phenotypic adaptation occurs rapidly as a result of interactions of gene expression and mutations (N. Handel, J. M. Schuurmans, Y. Feng, S. Brul, and B. H. Ter Kuile, Antimicrob Agents Chemother 58:4371–4379, 2014,http://dx.doi.org/10.1128/AAC.02892-14). In this study, the contribution of several individual genes to thede novoacquisition of antibiotic resistance inEscherichia coliwas investigated using mutants with deletions of genes known to be involved in antibiotic resistance. The results indicate thatrecA, vital for the SOS response, plays a crucial role in the development of antibiotic resistance. Likewise, deletion of global transcriptional regulators, such asgadEorsoxS, involved in pH homeostasis and superoxide removal, respectively, can slow the acquisition of resistance to a degree depending on the antibiotic. Deletion of the transcriptional regulatorsoxS, involved in superoxide removal, slowed the acquisition of resistance to enrofloxacin. Acquisition of resistance occurred at a lower rate in the presence of a second stress factor, such as a lowered pH or increased salt concentration, than in the presence of optimal growth conditions. The overall outcome suggests that a central cellular mechanism is crucial for the development of resistance and that genes involved in the regulation of transcription play an essential role. The actual cellular response, however, depends on the class of antibiotic in combination with environmental conditions.

scholarly journals Gilteritinib and Venetoclax Synergize to Eliminate FLT3/ITD+ Leukemia Cells through BIM

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2564-2564 ◽  
Author(s):  
Ruiqi Zhu ◽  
Li Li ◽  
Bao Nguyen ◽  
Amy S. Duffield ◽  
Donald Small
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Combined Treatment ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Advisory Committees ◽  
Activate Cell

Gilteritinib and Venetoclax synergize to eliminate FLT3/ITD+ leukemia cells through BIM Abstract Acute myeloid leukemia (AML) is characterized by a clonal proliferation of immature myeloid cells in the bone marrow or other tissues. The most commonly mutated gene in AML is FMS-like tyrosine kinase (FLT3). FLT3 downstream signaling pathways include PI3K/AKT, STAT5 and MAPK, which affect apoptosis, differentiation and cell proliferation. An internal tandem duplication mutation in FLT3 (FLT3/ITD) is identified in approximately 25% of patients with AML, and this mutation is associated with a particularly poor prognosis. A subset of AML also has a point mutation in the tyrosine kinase domain of FLT3 (FLT3/TKD); however, this mutation does not have the same pronounced impact on prognosis as the FLT3/ITD mutation. Since tyrosine kinases are an attractive drug target, tyrosine kinase inhibitors (TKIs) that target FLT3 have been developed, including recent agents that show enhanced specificity, such as Gilteritinib. Despite these advances, TKI monotherapy continues to show limited success, indicating that combination therapy is likely necessary for the effective treatment of FLT3/ITD AML. As FLT3 signaling pathway activity is known to be anti-apoptotic, in this study we investigated the combinatorial effect of a FLT3-selective TKI and the BCL-2 inhibitor Venetoclax. The BCL-2 protein plays a key role in apoptosis, with anti-apoptotic/prosurvival effects. Venetoclax is a selective BCL-2 inhibitor, and is used clinically in the treatment of chronic lymphocytic leukemia and relapsed/refractory AML. We first investigated the combinatorial effect of treatment with Gilteritinb and Venetoclax in a FLT3/ITD+ leukemia cell line (Molm14). Combined treatment with Gilteritinib (20nM) and Venetoclax (80nM) reduced cell proliferation by 83.7%, as compared to Gilteritinib (62.2%, P<0.05) or Venetoclax (37.3%, P<0.05) alone. The drug combination demonstrated synergy (CI=0.42). Combined treatment shows that these agents act synergistically to enhance apoptosis (88.2%), as compared with Gilteritinib (52.1%, P<0.05) or Venetoclax (12.1%, P<0.05) alone. Moreover, the combined treatment also significantly reduced cell proliferation in patient samples with FLT3/ITD+ and FLT3/TKD mutations. Further experiments with cell line (Molm14 cells resistant to 60nM CEP-701) showed that Venetoclax can re-sensitize FLT3 TKI-resistant cell lines to TKI treatment. Western blot analysis indicates that this effect is mediated by inhibiting MAKP pathway-whose reactivation is an important reason for TKI resistance in FLT3/ITD+ patients. These data demonstrate that combined treatment with Gilteritinb and Venetoclax reduces cell proliferation and enhances apoptosis in a FLT3/ITD leukemia cell line. We next investigated the mechanism of action of this drug combination. BCL-2 and other antiapoptotic proteins in this subfamily (i.e. MCL-1, BCL-XL) exert their pro-survival effects by sequestering BIM. BIM is a proapoptotic protein, that, when released by BCL-2, can activate cell death mediators. These cell death mediators include BAX and BAK, which perforate the mitochondrial membrane, resulting in apoptosis. Venetoclax acts by binding to BCL-2 and displacing BIM, freeing BIM to associate with and activate cell death mediators, resulting in apoptosis. We found that in Molm14 cells, Venetoclax not only dissociates BIM from BCL2, but also decreases expression of BIM and enhances the binding of BIM and MCL-1. In contrast, we found that Gilteritinib increases expression of BIM, reduces the binding of BIM with MCL-1 via a reduction of MCL1 expression, and shows enhanced binding of BIM with BCL2. Cells treated with both Venetoclax and Gilteritinib show dissociation of BIM from both BCL2 and MCL-1, though the interaction between BIM and BCL-XL is not affected. Combination treatment also showed increased binding between BIM and the cell death mediator BAX, leading to increased apoptosis. These studies provide evidence that the addition of Venetoclax may enhance TKI therapy in the treatment of FLT3/ITD leukemia. Additionally, these findings suggest that enhanced cell death in FLT3/ITD AML cells treated with combination therapy occurs because Venetoclax mitigates unintentded pro-survival effects of the TKI, including an increase in BIM expression and increased association between BIM and BCL-2. Disclosures Duffield: MedImmune: Consultancy; Boston Biomedical/Sumitomo Dainippon Pharma Co., Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Small:Pharos I, B & T: Consultancy, Research Funding; InSilico Medicine: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Combined inhibition of Bcl-2 and NFκB synergistically induces cell death in cutaneous T-cell lymphoma

Blood ◽  
2019 ◽  
Vol 134 (5) ◽  
pp. 445-455 ◽  
Author(s):  
Tabea C. Froehlich ◽  
Karin Müller-Decker ◽  
Jana D. Braun ◽  
Thomas Albrecht ◽  
Anne Schroeder ◽  
...  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Cell Line ◽  
Combination Treatment ◽  
Cell Lymphoma ◽  
Combined Treatment ◽  
B Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Specific Cell ◽  
Cutaneous T Cell Lymphoma

Abstract Therapeutic options for cutaneous T-cell lymphoma (CTCL) are limited and curative treatment regimens are not available. Thus, new targeted and well-tolerated therapeutic approaches are urgently needed. In this respect, we have recently shown that dimethyl fumerate (DMF) inhibits NF-κB acting as a survival factor in CTCL. Similarly, inhibition of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) has been shown to induce cell death in CTCL especially when combined with histone deacetylase inhibitors. Therefore, we hypothesized that inhibition of Bcl-2 should potentiate NF-κB inhibition in a novel combination treatment of CTCL. We show that, in vitro, the Bcl-2 inhibitors ABT-199 and ABT-263 induced specific cell death in primary CD4+ cells from CTCL patients as well as in the CTCL cell line SeAx, but not in T cells of healthy donors nor in the CTCL cell line HH, which lacks Bcl-2. Combined treatment with ABT-199 and DMF caused synergistic cell death specifically in CTCL cells engaging 2 independent signaling pathways. To verify these findings in vivo, we performed combined ABT-199 and DMF treatment in a xenograft mouse model for CTCL. The combined treatment effectively reduced tumor growth and increased overall survival via synergistic induction of CTCL cell death and suppression of tumor cell proliferation. Essentially, the combination treatment was superior to ABT-199 monotherapy with respect to both efficacy and tolerability. To sum up, our data provide proof of principle for the therapeutic potential of combining Bcl-2 and NF-κB inhibitors in treating CTCL. Next, this potential should be explored further in a clinical study.

The Histone Deacetylase Inhibitor, Sodium Butyrate, Augments Chlorambucil Cytotoxicity in the Promyelocytic Leukaemia Cell Line, HL-60.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4198-4198
Author(s):  
Faith Kwa ◽  
Miroslav Kapuscinski ◽  
Merrole Cole-Sinclair
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Cell Line ◽  
Sodium Butyrate ◽  
Cell Model ◽  
Combined Treatment ◽  
Cell Killing ◽  
Tetrazolium Salt ◽  
Leukaemia Cell Line ◽  
Drug Concentrations

Abstract Treatment of various haematological malignancies such as chronic lymphocytic leukaemia (CLL) with alkylating drugs (e.g. chlorambucil, CLB) fails to extinguish the leukaemic clone and often results in chemo-resistance. A possible mechanism of drug resistance in CLL B cells is p53 inactivation. The resistance of cells to standard chemotherapy may be ameliorated by agents that alter the chromatin remodelling processes, DNA methylation and histone acetylation (e.g. sodium butyrate, NaBu). We used the HL-60 cell line (which carries a non-functional p53 protein) as a cell model for resistant CLL B cells and determined the sensitivity of HL-60 cells to a range of concentrations of CLB (5μM,10μM and 20μM) and NaBu (0.5mM, 1mM and 2mM) by tetrazolium salt-based proliferation assays of cells incubated with the drugs over 4 days (days 0, 1, 2 and 3). Cells were resistant to 5μM CLB until day 3 where 56% of cells were killed. 10μM and 20μM CLB had very similar effects (p>0.05) with both treatments killing 83.2% and 87.3% respectively of cells by the end of the incubation. The cell death induced by NaBu was both time- and dose-dependent, with 2mM NaBu being the most effective treatment, while 0.5mM NaBu did not result in significant cell death (p>0.05). To investigate if the treatment of HL-60 cells with both drugs would enhance or have an additive killing effect on the cells, we tested combined treatment involving a series of lower drug concentrations of each of the two drugs (5μM/10μM CLB ± 0.2mM/0.5mM/1mM). All combined treatments using 5μM CLB were more effective in cell killing than when the cells were treated singly with CLB or NaBu (p<0.001). The most effective combined treatment was 5μMCLB+0.2mM NaBu (62% cell death) compared to only 10% and 40% cell killing caused by CLB and NaBu alone, respectively. In contrast, the effect of 10μM CLB was not augmented by NaBu in concentrations ranging between 0.2mM and 1mM (p>0.05). In conclusion, combined treatment involving NaBu increased the cytotoxic effect of low concentrations of CLB in p53-inactivated cells, implying the potential benefit of chromatin modifying agents in cytotoxic therapy. Further studies including the use of a wider range of concentrations of CLB with NaBu or other chromatin modifying agents are underway to investigate possible synergism in the killing of HL-60 cells by these agents.

Cytotoxicity and Cell Death Mechanisms Induced by a Novel Bisnaphthalimidopropyl Derivative against the NCI-H460 non-small Lung Cancer Cell Line

2013 ◽  
Vol 13 (3) ◽  
pp. 414-421 ◽  
Author(s):  
Raquel T. Lima ◽  
Gemma A. Barron ◽  
Joanna A. Grabowska ◽  
Giovanna Bermano ◽  
Simranjeet Kaur ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Death ◽  
Cell Line ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Lung Cancer Cell Line ◽  
Lung Cancer Cell ◽  
Cell Death Mechanisms

Poly-L-arginine: Enhancing Cytotoxicity and Cellular Uptake of Doxorubicin and Necrotic Cell Death

2019 ◽  
Vol 18 (10) ◽  
pp. 1448-1456 ◽  
Author(s):  
Bahareh Movafegh ◽  
Razieh Jalal ◽  
Zobeideh Mohammadi ◽  
Seyyede A. Aldaghi
Keyword(s):  
Cell Death ◽  
Cellular Uptake ◽  
Combined Treatment ◽  
Annexin V ◽  
Cell Penetrating Peptides ◽  
Short Exposure ◽  
Dependent Manner ◽  
Du145 Cells ◽  
Induced Apoptosis ◽  
Resistance To Doxorubicin

Objective: Cell resistance to doxorubicin and its toxicity to healthy tissue reduce its efficiency. The use of cell-penetrating peptides as drug delivery system along with doxorubicin is a strategy to reduce its side effects. In this study, the influence of poly-L-arginine on doxorubicin cytotoxicity, its cellular uptake and doxorubicin-induced apoptosis on human prostate cancer DU145 cells are assessed. Methods: The cytotoxicity of doxorubicin and poly-L-arginine, alone and in combination, in DU145 cells was evaluated at different exposure times using MTT assay. The influence of poly-L-arginine on doxorubicin delivery into cells was evaluated by fluorescence microscopy and ultraviolet spectroscopy. DAPI and ethidium bromide- acridine orange stainings, flow cytometry using annexin V/propidium iodide, western blot analysis with anti-p21 antibody and caspase-3 activity were used to examine the influence of poly-L-arginine on doxorubicininduced cell death. Results: Poly-L-arginine had no cytotoxicity at low concentrations and short exposure times. Poly-L-arginine increased the cytotoxic effect of doxorubicin in DU145 cells in a time-dependent manner. But no significant reduction was found in HFF cell viability. Poly-L-arginine seems to facilitate doxorubicin uptake and increase its intracellular concentration. 24h combined treatment of cells with doxorubicin (0.5 µM) and poly-L-arginine (1 µg ml-1) caused a small increase in doxorubicin-induced apoptosis and significantly elevated necrosis in DU145 cells as compared to each agent alone. Conclusion: Our results indicate that poly-L-arginine at lowest and highest concentrations act as proliferationinducing and antiproliferative agents, respectively. Between these concentrations, poly-L-arginine increases the cellular uptake of doxorubicin and its cytotoxicity through induction of necrosis.

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