Raf-1 Is Over Expressed in Chronic Lymphocytic Leukemia and Promotes Cell Survival by Phosphorylation of ERK and BAD

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3120-3120 ◽  
Author(s):  
Miao Wang ◽  
P.M. Kluin ◽  
Stefano Rosati ◽  
Marjan Luinge ◽  
Simon M. G. J. Daenen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Western Blot ◽  
Cell Survival ◽  
Cell Lines ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Cyclin D3 ◽  
Qrt Pcr ◽  
Mrna Ratio

Abstract Introduction: The serine threonine kinase Raf-1 plays a protective role in many cell types, but its expression and function in CLL cells has not been studied in detail. In the present study, we analyzed Raf-1 expression and tested the hypothesis that Raf-1 is critical for CLL cell survival. Materials and Methods: By using qRT-PCR and western blot, we compared the expression of Raf-1 of mRNA and protein levels in purified B cells from 45 CLL cases and CD38 negative B cells from 5 reactive tonsils. By western blot, we analyzed the activity of phospho-Raf-1 (ser338) and its downstream targets (phospho-ERK1/2, phospho-BAD) in 23 CLL cases and 4 CLL cell lines (JVM-3, MEC-1, MEC-2 and MO1043) before and after IgM stimulation. By immunoprecipitation, we analyzed if Raf-1 co-localizes with Bcl-2. We correlated the change in phosphorylation status (Raf-1, ERK and BAD) in response to IgM stimulation with the ZAP-70/SYK mRNA ratio, which was detected by qRT-PCR in purified B cells from CLL cases. After using specific inhibitors, including the Raf-1 inhibitor GW5074, the Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137, we investigated apoptosis by Annexin V flowcytometry in CLL cases and CLL cell lines as well as cell cycle changes in the 4 cell lines by flowcytometry. Results: In comparison to normal resting (CD38 negative) B cells, there was a strongoverexpression of Raf-1 in CLL cells, both at at the mRNA and protein level. Using qRT-PCR there was an almost linear correlation between Raf-1 and Bcl-2 expression. Moreover, the phosphorylation status of Raf-1 and ERK in response to IgM stimulation strongly correlated with the ZAP-70/SYK mRNA ratio. Using immunoprecipitation and confocal miscroscopy we found colocalization of Raf-1 with Bcl-2, which might account for the observed constitutive activation of BAD in CLL cells. The Raf-1 inhibitor GW5074, Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137 all led to p-Raf-1 inhibition as well as downregulation of p-ERK and p-BAD. Additionally, all three inhibitors downregulated cyclin D3 and cyclin E, which are important for G0/G1 transition. We also found that GW5074 induced apoptosis in CLL cell lines and primary cells of CLL cases. Conclusion/Discussion: In conclusion, our study identifies Raf-1 as a critical anti-apoptotic and cell cycle regulating kinase in CLL cells.

Introduction of Constitutivelly Active Akt in Primary CLL B-Cells Results in Upregulation of Mcl-1, Bcl-XL and Cyclin D3 and Promotes Leukemic Cell Survival.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2805-2805
Author(s):  
Pablo Longo ◽  
Stefania Gobessi ◽  
Luca Laurenti ◽  
Simona Sica ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Survival ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Cyclin D3 ◽  
Leukemic Cells ◽  
Interleukin 4 ◽  
Antiapoptotic Proteins ◽  
Sustained Activation ◽  
Constitutively Active

Abstract The PI3K/Akt and Raf/MEK/ERK pathways are key regulators of various cellular responses, including proliferation, survival, differentiation, migration and malignant transformation. These pathways are activated in chronic lymphocytic leukemia B-cells by a number of survival or growth stimulatory signals, such as immobilized anti-IgM antibodies, interleukin-4, phorbol-ester, CXCL12, or stimulatory CpG oligonucleotides. Moreover, enhanced activation of Akt has been implicated in the pathogenesis of the CLL-like disorder that develops in mice transgenic for the TCL1 oncogene. To further delineate the relative contribution of the PI3K/Akt and Raf/MEK/ERK pathways in regulating leukemic cell growth and survival, we introduced constitutively active Akt or constitutively active MEK2 in primary CLL B-cells by nucleofection. Expression of constitutively active Akt consistently promoted survival, as evidenced by a higher percentage of Annexin V/PI negative cells after 48 hours in culture (median 52%) compared to samples transfected with control vector (median 31%). Immunoblot analysis of several important antiapoptotic proteins revealed that enforced activation of Akt upregulates Mcl-1 and Bcl-xL, whereas no changes were observed in the levels of Bcl-2. Expression of constitutively active Akt also induced an increase in size and granularity of the leukemic cells, indicating increased metabolic activity. These changes were associated with significant induction of cyclin D3, indicating that activation of Akt is required for both leukemic cell survival and cell cycle progression. In contrast, introduction of constitutively active MEK2 induced sustained activation of ERK, but showed only a modest increase in the percentage of viable CLL B-cells (median 36%) and no significant changes in the levels of any of the investigated antiapoptotic proteins. These experiments provide direct evidence that sustained activation of Akt promotes leukemic cell survival and upregulates Mcl-1, an antiapoptotic protein that has been associated with resistance to chemotherapy in patients with CLL.

CD74 Is a Novel Survival Receptor in Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2812-2812
Author(s):  
Inbal Binsky ◽  
Michal Haran ◽  
Diana Starlets ◽  
Nurit Harpaz ◽  
Lev Shvidel ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
Cell Survival ◽  
Early Stage ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Signaling Cascade ◽  
Advanced Stage ◽  
C Cells ◽  
Actin Mrna

Abstract Scientific Background: Previous studies have shown that chronic lymphocytic leukemia (CLL) lymphocytes express relatively large amounts of CD74 mRNA compared to normal B cells. We have recently demonstrated in a murine model that CD74 stimulation with anti-CD74 antibody leads to an induction of a signaling cascade resulting in NF-κ B activation, entry of the stimulated cells into the S phase, elevation of DNA synthesis, cell division, and augmented expression of BCL-XL. These findings therefore demonstrated that surface CD74 functions as a survival receptor. In the current study we aimed to determine whether activation of cell surface CD74 in B-CLL cells leads to induction of a signaling cascade resulting in cell survival. Meterials and methods: B cells were purified from the peripheral blood of CLL patients of different stages. CD74 stimulation was achieved using anti-CD74 or MIF (a natural occurring ligand of CD74). IL-8 expression and function was determined by RT-PCR, western blot, ELISA and Annexin V staining. Results: In all CLL patients there was a significantly increased expression of cell surface CD74. Activation of cell surface CD74 initiates a signaling cascade that results in secretion of Interleukin 8 (see Fig1), which in turn regulates cell survival(see Fig 1,2). Conclussion: Our data show that over-expression of CD74 in CLL is an important survival mechanism, operational from the very early stages of the disease, and inherent in all further stages. This survival mechanism thus appears to be an early and significant event in the pathogenesis of the disease. Our findings open prospects for novel therapeutic strategies aimed at this survival pathway. Legends: Fig 1: CD74 induces IL-8 and Bcl-2 expression in CLL B cells. (A,C) Cells were incubated in the presence or absence of anti-CD74 antibody, Id2, a control antibody or MIF for 18 h. (A,C) RNA was purified and levels of IL-8, Bcl-2 and actin mRNA were analyzed. The results presented are representative of 7 early-stage and 5 advanced-stage B-CLL patients. (B) ) Cells’ conditioned medium was collected and their IL-8 levels were analyzed by ELISA. The results presented are representative of 3 independent experiments. Fig 1:. CD74 induces IL-8 and Bcl-2 expression in CLL B cells. (A,C) Cells were incubated in the presence or absence of anti-CD74 antibody, Id2, a control antibody or MIF for 18 h. (A,C) RNA was purified and levels of IL-8, Bcl-2 and actin mRNA were analyzed. The results presented are representative of 7 early-stage and 5 advanced-stage B-CLL patients. (B) ) Cells’ conditioned medium was collected and their IL-8 levels were analyzed by ELISA. The results presented are representative of 3 independent experiments. Fig 2: IL-8 secreted following CD74 stimulation regulates B-CLL cell survival.
 (A, B) B-CLL cells were incubated in the presence or absence of anti-CD74 (B), anti-IL-8 (A, B) or a control antibody (c-jun; A) for 48 h. Cells were stained with annexin V and analyzed by FACS. The results presented are representative of 3 early-stage and 4 advanced-stage B-CLL patients. (C) B-CLL cells were incubated in the presence or absence of a control antibody (c-jun), anti-CD74, anti-IL-8 or IL-8 for 48 h. Cell death was analyzed by ELISA. The graph shows the average of one experiment, representative of 4. Fig 2:. IL-8 secreted following CD74 stimulation regulates B-CLL cell survival.
 (A, B) B-CLL cells were incubated in the presence or absence of anti-CD74 (B), anti-IL-8 (A, B) or a control antibody (c-jun; A) for 48 h. Cells were stained with annexin V and analyzed by FACS. The results presented are representative of 3 early-stage and 4 advanced-stage B-CLL patients. (C) B-CLL cells were incubated in the presence or absence of a control antibody (c-jun), anti-CD74, anti-IL-8 or IL-8 for 48 h. Cell death was analyzed by ELISA. The graph shows the average of one experiment, representative of 4.

Anti-CD20 Antibody Rituximab and Anti-CD23 Antibody IDEC-152 Induce Apoptosis of Malignant B-Cells in Combination with Chemical Antagonists of XIAP.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1401-1401 ◽  
Author(s):  
Massimo Mangiola ◽  
Kate Welsh ◽  
Shinichi Kitada ◽  
Irene M. Pedersen ◽  
Nuzhat Pathan ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Lymphoma Cell ◽  
Efficient Induction ◽  
Anti Cd20

Abstract We tested the effects of Rituximab (anti-CD20) and IDEC-152 (anti-CD23) on apoptosis of B-cell malignancies, using established non-Hodgkin’s B-Cell lymphoma cell lines and freshly isolated Chronic Lymphocytic Leukemia (CLL) B-cells. We used monolayers of stably transfected CHO-cells expressing FcRγIII-A to present antibody to B-cells and promote crosslinking. Established B-cell lymphomas (n = 3) were cultured in the presence of FcRγIIIA-expressing CHO monolayer with or without MAbs and apoptosis was measured by annexin V/propidium iodide staining at various times thereafter. Both antibodies induced time-dependent apoptosis of B-cell lymphoma cell lines. After 48 hrs of treatment with either Rituximab or IDEC-152, the majority of the malignant B-cells were apoptotic (remaining viable cells = 28.7% ± 0.2137% for Rituximab and 30.87% ± 0.7332% for IDEC-152). Rituximab and IDEC-152 also induced marked increases in caspase activity in B-cell lymphoma cell lines, with fold-increases above baseline control cells of 25 ± 0.9031 and 24 ± 0.3839, respectively. In contrast, neither Rituximab nor IDEC-152 induced striking effects on primary CLL B-cells (n = 6). We therefore tested the combination of Rituximab or IDEC-152 with other agents that target anti-apoptotic proteins, exploring whether more efficient induction of apoptosis can be achieved. We cultured lymphoma cell lines and primary CLL specimens with chemical antagonists of XIAP (Schimmer, et al. Cancer Cell5: 25, 2004), an anti-apoptotic protein that inhibits effector caspases. When used at concentrations where XIAP antagonists alone were non-apoptotic (approximately 2.5 μM), a significant increase in apoptosis was achieved in cultures of lymphoma and CLL cells treated with either Rituximab or IDEC-152. These findings suggest that Rituximab or IDEC-152 may more efficiently induce apoptosis of malignant B-cells when combined with an apoptosis-sensitizing agent. (Supported by CA-81534; CA-78040; and an unrestricted grant from Genentech, Inc.).

Functional Analysis of Cyclin D1 in Mantle Cell Lymphoma (MCL) by Specific Lentiviral shRNA Mediated Knockdown.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1584-1584
Author(s):  
Margit Klier ◽  
Natasa Anastasov ◽  
Daniela Angermeier ◽  
Mark Raffeld ◽  
Falko Fend ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Infection Rates ◽  
Facs Analysis ◽  
Cyclin D2 ◽  
Qrt Pcr

Abstract Introduction: Cyclin D1 overexpression is the hallmark of MCL. However, the importance of cyclin D1 for the maintenance of MCL still remains to be defined. Therefore, the aim of this study is to elucidate the role of cyclin D1 overexpression using the siRNA technology in well-characterized MCL cell lines, as a model system. Material and Methods: A highly efficient cyclin D1-shRNA (96% knockdown) was identified using a lacZ-cyclin D1 fusion gene reporter system in HEK-293T cells. This shRNA was cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Seven MCL cell lines were analyzed (Granta 519, Jeko-1, Rec-1, Z-138, UPN-1, Hbl-2 and JVM-2), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantitate the knockdown effect. The effect of cyclin D1 knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis. Results: The infection rates varied among the different MCL cell lines. Rec-1 and Hbl-2 showed low infection rates (50%) even at high MOI’s (multiplicity of infection), whereas UPN-1 and JVM-2 had moderate infection rates (80%). Jeko-1, Granta 519 and Z-138 showed high infection rates (almost 100% of the cells). Despite the good tranfection rate, the downregulation of cyclin D1, as measured by Western Blot and qRT-PCR, was about 80% in Granta 519, and 65% in Jeko-1 and Z-138. No IFN response, as secondary effect was identified. Interestingly, no apoptosis was observed, and there was only a moderate retardation of growth (60% of control cells) with 10% shift from the S phase to G1 phase of the cell cycle when compared to the controls, suggesting that other cell cycle proteins might compensate, at least partially, for the loss of cyclin D1. Accordingly, cyclin D2 showed upregulation in Western blot analysis and qRT-PCR, whereas the phosphorylation status of retinoblastoma protein on Ser780 was reduced and the expression of the CDK inhibitor p27Kip1 increased. No changes were observed in the expression of cyclin D3, Cyclin E, CDK4 and CDK2. Conclusions: In this study, a system that enables the specific downregulation of cyclin D1 in MCL cell lines was established. Surprisingly, the downregulation of cyclin D1 in MCL cell lines resulted in only a moderate inhibition on cell growth with no apoptosis. The reasons for this might be 1) that the upregulation of cyclin D2 compensates for cyclin D1 downregulation, and/or 2) that the chromosomal translocation leading to cyclin D1 overexpression is an initiating event in MCL lymphomagenesis followed by secondary genetic events at later stages of the disease, which make cyclin D1 dispensable. This finding has important implications for MCL therapy, as strategies targeting only cyclin D1 might be hampered by the redundancy of the system, resulting in a low probability of treatment response.

Cardamonin, a Novel Blocker of STAT3 and Nuclear Factor-κB Activation, Suppresses Proliferation, Arrests Cell Cycle, and Potentiates Apoptosis In Human Multiple Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1820-1820
Author(s):  
You Qin ◽  
Chunyan Sun ◽  
Tao Guo ◽  
Yadan Wang ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Western Blot ◽  
Cell Survival ◽  
Cell Lines ◽  
Nuclear Factor Κb ◽  
Time Dependent ◽  
Nuclear Factor ◽  
Rpmi 8226 ◽  
Cell Survival And Proliferation

Abstract Abstract 1820 Background and Objective: Although there have been introductions of novel and more potent therapeutic regimens, multiple myeloma (MM) remains an incurable hematological malignant disorder. Current goals in MM treatment are focused on finding new therapies that target the deregulated signaling cascades which promote MM cell survival and proliferation, such as STAT3 and nuclear factor-κB (NF-κB) (Bharti AC et al, Blood 2004). Cardamonin (2′,4′-dihydroxy-6′-methoxychalcone) is isolated from Alpinia katsumadai (Zingiberaceae), a plant widely used in traditional Chinese medicine. Previous reports have demonstrated that cardamonin possesses diverse pharmacologic actions, such as anti-inflammation (Hatziieremia S et al, Br J Pharmacol 2006), anti-melanogenesis (Cho M et al, Biochem Biophys Res Commun 2009) and anti-platelet properties (Jantan I et al, Phytomedicine 2008). However, very little is known about anti-myeloma activity of cardamonin. Thus, in the present report we investigated the therapeutic potential of cardamonin against MM, specifically studying the capacity of cardamonin to inhibit STAT3 and NF-κB pathways. Methods and Results: Cardamonin directly inhibited the growth of MM cell lines in a dose-dependent (from 0 μM to 100 μM) and time-dependent (from 0 h to 72 h) manner by using a cell counting kit-8 assay kit. Growth inhibition of MM cell lines, including RPMI 8226, U266 and ARH-77 cells, was demonstrated with an IC50 of less than 50 μM cardamonin at 24 h. Cardamonin also induced chemosensitivity to vincristine, doxorubicin, and dexamethasone in MM cells. Flow cytometric analysis showed that cardamonin caused accumulation of MM cells in G2 phase (from 14.6% to 81.9% for RPMI 8226, 24 h at 10 μM). A more precise evaluation of cell cycle by Hochest 33342 and 5-ethynyl-2′-deoxyuridine (EdU) fluorescence staining indicated that the cells in S phase were reduced in cardamonin-treated cells. Indeed, cardamonin strongly induced cell apoptosis as shown by annexin V-fluoroisothyocyanate analysis (from 5.3% to 49.4% for RPMI 8226, 48 h at 25 μM), which was also proved by a combination of acridine orange and ethidium bromide staining assay. Furthermore, cardamonin significantly enhanced the apoptotic effects of bortezomib from 23.1% to 74.3% and of thalidomide from 20.1% to 65.8%. Because of the pivotal role of STAT3 and NF-κB in MM cell survival and proliferation, we explored whether the above effects of cardamonin were mediated by interfering with STAT3 and NF-κB pathways by western blot analysis and immunofluorescence. We found that cardamonin blocked IL-6-inducible STAT3 phosphorylation and sequent STAT3 nuclear translocation. The constitutive phosphorylation of STAT3 found in certain cells was also abrogated by treatment with cardamonin in a dose- and time-dependent manner. In addition, we discovered that NF-κB, constitutively active in all human MM cell lines examined, was downregulated by cardamonin through suppression of phosphorylation of NF-κB p65 as evaluated by western blot. This correlated with reduction of the nuclear retention of p65. Moreover, cardamonin suppressed phosphorylation of IκBα, an inhibitor of NF-κB, and phosphorylation of Akt, which has been shown to phosphorylate p65. Additionally, immunoblotting analysis indicated that the expression of STAT3 and NF-κB-regulated gene products associated with proliferation (cyclin D1, TF and COX2), antiapoptosis (Bcl-2, Bcl-xL, Survivin, XIAP and Bfl-1/A1), invasion (ICAM-1), and angiogenesis (vascular endothelial growth factor) were down-regulated by cardamonin. ELISA assay showed that IL-6 release was also suppressed by cardamonin in certain MM cells. Conclusions: Taken together, our results suggest that cardamonin is a potent in vitro inhibitor of STAT3 and NF-κB pathways, which provides the molecular basis for its anti-myeloma activities, including suppression of proliferation, arrest of cell cycle, and induction of apoptosis. Disclosures: No relevant conflicts of interest to declare.

BI 836826, a Novel Fc-Engineered Antibody in Combination with Phosphoinositide-3-Kinase Inhibitor for Treatment of High Risk Chronic Lymphocytic Leukemia and Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2767-2767
Author(s):  
Deborah M Stephens ◽  
Kyle A. Beckwith ◽  
Priscilla Do ◽  
Carolyn Cheney ◽  
Xiaokui Mo ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Viability ◽  
B Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Advisory Committees

Abstract Background Targeting new antigens in chronic lymphocytic leukemia (CLL) and lymphoma may increase flexibility in the clinic and help circumvent resistance. The tetraspanin CD37 domain mediates transduction of survival and apoptotic signals (Lapalombella et al.,Cancer Cell, 2014), and has been clinically validated by recent trials of otlertuzumab (TRU-016) in CLL and Non-Hodgkin Lymphoma . Ligation of CD37 by this reagent simultaneously induced pro-apoptotic signaling and inhibited pro-survival signaling of phosphoinositide 3-kinase δ (PI3Kδ), which introduces a unique opportunity to use combination strategies employing activation of CD37 and inhibition of PI3Kδ. A new agent BI 836826 is an Fc-engineered anti-CD37 IgG1 that displays improved effector activities as well as crosslinker-independent direct cytotoxicity. We have evaluated the efficacy of BI 836826 combined with the PI3Kδ-selective inhibitor idelalisib in diffuse large B-cell lymphoma (DLBCL) cell lines and primary human CLL B-cells in the University and then by industry to validate the synergistic finding initially reported. Methods Cell viability assays usedCellTiterGlo to measure inhibition of antibody, isotype control, idelalisib or a combination of antibody and compound over 72h in culture. The cell viability of vehicle is measured at the time of dosing (T0) and after seventy-two hours (T72). A GI reading of 0% represents no growth inhibition, GI 100% represents complete growth inhibition, and a GI 200% represents complete death of all cells in the culture well. Annexin V-FITC and propidium iodide measure by flow cytometry was used to assess enhanced killing of primary CLL cells, with incubation of BI 836826 (0.1 µg/mL) and/or idelalisib (1 µM) at 37°C for 24 hours. Trastuzumab included as a non-specific IgG1 control. Data was reported as percentage of viable cells (Annexin V negative, PI negative) normalized to untreated control. Results DLBCL cell lines were variably sensitive to single agent BI 836826. In most of the cell lines tested, the cell viability was inhibited by 40%-50% with BI 836826 in the concentration range of 1-1000 ng/mL (Figure 1A). A synergistic effect was noted in several DLBCL cell lines when BI 836826 was combined with idelalisib. When the maximal effect of BI 836826 was greater than isotype control (GI% > 12, dotted line) and the effect of idelalisib showed a GI50 < 1uM, 3/5 cell lines showed synergy in combination (red dot, Figure 1B). A shift in the EC50of idelalisib can be seen with the addition of increasing amounts of BI 836826 (Figure 1C). In primary CLL B-cell cultures, 1 µM idelalisib displayed weak single agent activity following 24-hour incubation. The cytotoxicity of BI 836826 at 0.1 µg/mL was more variable, although treatment of samples from most CLL patients resulted in 20-50% B-cell death. The combination of these 2 agents resulted in enhanced cytotoxic activity (Figure 2A), and this effect was not attenuated by the presence of del(17)(p13.1), as there was no significant difference in cytotoxicity against these cells compared to those with lower risk cytogenetics (Figure 2B,C). Additionally, the combination was beneficial in CLL B-cells isolated from patients who were refractory to ibrutinib (Figure 2D). Conclusions This collaborative industry and academic endeavor with cross validation of initial mechanistic studies of synergy between CD37 and idelalisib demonstrates that addition of idelalisib to BI 836826 augments cytotoxicity against DLBCL cell lines and primary human CLL B-cells in an additive-to-synergistic manner. In addition, it maintains efficacy against CLL B-cells with del(17)(p13.1) and those from ibrutinib-refractory patients. Further exploration of this therapeutic strategy in clinical trials is strongly warranted. Disclosures Jones: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Grosmaire:Gilead: Employment. Jones:Gilead: Employment. DiPaolo:Gilead: Employment. Tannheimer:Gilead Sciences: Employment. Heider:4Boehringer Ingelheim RCV: Employment.

scholarly journals Micromeria fruticosa Induces Cell Cycle Arrest and Apoptosis in Breast and Colorectal Cancer Cells

2020 ◽  
Vol 13 (6) ◽  
pp. 115 ◽  
Author(s):  
Waseem El-Huneidi ◽  
Naglaa G. Shehab ◽  
Khuloud Bajbouj ◽  
Arya Vinod ◽  
Ahmed El-Serafi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Expression Profiles ◽  
Annexin V ◽  
Cyclin B1 ◽  
Dependent Manner ◽  
Qrt Pcr ◽  
Hct 116 ◽  
Micromeria Fruticosa ◽  
Mcf 7

Micromeria fruticosa (L.) Druce subsp. serpyllifolia (Lamiaceae) has been used widely in folk medicine to alleviate various ailments such as abdominal pains, diarrhea, colds, eye infections, heart disorders and wounds. A few reports have confirmed different therapeutic potentialities of its extracts, including the anti-inflammatory, gastroprotective, analgesic, antiobesity and antidiabetic activities. This study aimed to investigate the mechanistic pathway of the antiproliferative activity of the ethanolic extract of M. fruticosa on two different cancer cell lines, namely human breast (mammary carcinoma F7 (MCF-7)) and human colorectal (human colon tumor cells (HCT-116)) cell lines. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) assay, Annexin V-FITC/PI, caspases 8/9 and cell cycle analyses, qRT-PCR and Western blot were used to assess the effect of M. fruticosa on cytotoxicity, apoptosis, cell cycle, cell cycle-related genes and protein expression profiles in MCF-7 and HCT-116. The extract inhibits cell proliferation in a time- and dose-dependent manner. The half-maximal inhibitory concentration (IC50) for both cell lines was found to be 100 μg/mL. Apoptosis induction was confirmed by Annexin V-FITC/PI, that was related to caspases 8 and 9 activities induction. Furthermore, the cell cycle analysis revealed arrest at G2/M phase. The underlying mechanism involved in the G2/M arrest was found to be associated with the downregulation of CDK1, cyclin B1 and survivin that was confirmed by qRT-PCR and Western blotting.

scholarly journals The Chromosome 21 Kinase DYRK1A and Its Substrate FOXO1 Constitute a Novel Therapeutic Pathway in B-ALL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 548-548
Author(s):  
Rahul S. Bhansali ◽  
Malini Rammohan ◽  
Ji Heon Paul Lee ◽  
Sebastien Malinge ◽  
Yi-Chien Tsai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
B Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Negative Regulator ◽  
Chromosome 21 ◽  
Cyclin D3 ◽  
B Lymphopoiesis

Abstract Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase 1A (DYRK1A) is a serine/threonine kinase that regulates diverse pathways such as splicing, cell cycle, differentiation, apoptosis, and transcription. DYRK1A is encoded within the Down syndrome (DS) critical region of chromosome 21, underlying its importance in DS-related pathologies, such as Alzheimer's disease. Children with DS have an increased risk of developing hematologic malignancies, namely acute megakaryoblastic leukemia (DS-AMKL) and B-cell acute lymphoblastic leukemia (DS-ALL). We previously reported that DYRK1A promotes DS-AMKL by regulating subcellular localization of its substrate NFAT. In a subsequent study, we examined its role in normal hematopoiesis and found that DYRK1A is necessary for B and T cell development through phosphorylation and destabilization of Cyclin D3. Dyrk1a-deficient large pre-B cells and double negative thymocytes are unable to enter quiescence for maturation. Despite elevated levels of Cyclin D3, however, these cells lose proliferative capacity due to a block at the G2-M transition. This observation suggests that DYRK1A inhibition may exhibit anti-tumor activity in lymphocytes by first stimulating exit from quiescence but then blocking repeated rounds of cell division. Notably, DYRK1A is overexpressed in acute leukemias, including both T-ALL and B-ALL, relative to normal hematopoietic counterparts. Moreover, overexpression of dominant-negative DYRK1A-K188R impairs proliferation in human B-ALL cell lines, suggesting that DYRK1A kinase activity is required for B-ALL growth. In order to assess the physiologic relevance of targeting DYRK1A in vivo, we generated a murine model of B-ALL with a floxed Dyrk1a allele and observed significant survival advantages with homozygous (p=0.0045) and heterozygous deletion (p=0.0015). Additionally, both B-ALL cell lines and patient samples were sensitive to EHT1610, a potent and selective DYRK1 inhibitor. Relevant to the localization of DYRK1A on chromosome 21, DS-ALL samples were especially sensitive to kinase inhibition. EHT1610 also conferred synergistic growth inhibition of B-ALL cells when combined with cytotoxic chemotherapy drugs used in traditional ALL treatment regimens, such as dexamethasone, methotrexate and cytarabine. We next aimed to elucidate the mechanism by which DYRK1A inhibition cause a failure of G2-M progression. Using global and directed phosphoproteomic studies, we identified several DYRK1A substrates in pre-B cells that are involved in cell cycle, splicing, transcriptional regulation, and RNA metabolism. In addition to Cyclin D3, a notable substrate is FOXO1, an indispensable transcription factor in B lymphopoiesis. We observed that inhibition of DYRK1A led to an accumulation of FOXO1 in the nucleus of large pre-B cells despite intact PI3K/Akt signaling, which is the predominant negative regulator of FOXO1. Treatment of pre-B cells with AS1842856, an inhibitor of FOXO1 nuclear translocation, rescued the G2-M blockade and proliferative impairment induced by EHT1610 treatment. Despite FOXO1 acting as a tumor suppressor in normal lymphocytes, B-ALL cell lines and patient samples were paradoxically sensitive to FOXO1 inhibition, suggesting a unique requirement in the survival of B-ALL cells. This may be due to regulation of DNA damage, as DYRK1A inhibition alone led to negligible changes in gamma-H2AX foci, whereas FOXO1 inhibition increased DNA damage. When DYRK1A and FOXO1 were inhibited in combination, we observed a synergistic accumulation of DNA damage along with cell death in B-ALL cell lines. Finally, as both EHT1610 and AS1842856 are potent inhibitors of B-ALL cell growth in vitro, we assessed their in vivo efficacy. Both EHT1610 and AS1842856 significantly increased survival in xenograft models of B-ALL (p=0.0002 and p=0.001, respectively). We therefore conclude that both DYRK1A and its substrate FOXO1 are therapeutic targets in B-ALL. Importantly, EHT1610 represents the first selective DYRK1A inhibitor with suitable in vivo activity. Ultimately, we have determined that the DYRK1A pathway is integral to the maintenance of normal and malignant B-lymphopoiesis, the latter which can be effectively targeted through 1) a primary proliferative impairment, 2) sensitization to cell cycle-dependent chemotherapy, and 3) downstream inhibition of DYRK1A substrates such as FOXO1. Disclosures Lee: AbbVie: Employment. Bourquin:Amgen: Other: Travel Support. Crispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

scholarly journals Down-regulation of CCNE1 expression suppresses cell proliferation and sensitizes gastric carcinoma cells to Cisplatin

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Chao Zhang ◽  
Qiang Zhu ◽  
Jianzhong Gu ◽  
Shan Chen ◽  
Qian Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Gastric Carcinoma ◽  
Western Blot ◽  
Cell Lines ◽  
Survival Curve ◽  
Lymphatic Invasion ◽  
Cell Counting ◽  
Down Regulation ◽  
Qrt Pcr

AbstractA novel oncogene CCNE1 (cyclin E) is considered to be associated with the development of various tumor types, its role in gastric carcinoma (GC) is little studied and the effect of CCNE1 on chemotherapy also remains unclear. We recruited 55 cases of GC tissues and corresponding normal tissues. Immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and Western blot analysis were performed to detect the expression of CCNE1. We also examined the expression of CCNE1 in gastric mucosal GES-1 cells and five GC cell lines. Silencing CCNE1 was used to assess its effect on proliferation and cell cycle in MGC-803 and NCI-N87 cells, as performed by Cell counting kit-8 (CCK-8) and flow cytometry assay. Meanwhile, cell cycle related genes were also detected through qRT-PCR and Western blot. The results showed CCNE1 up-regulation mainly expressed in GC tissues and GC cell lines, also was associated with tumor node metastasis (TNM) stage and lymphatic invasion. Three-year survival curve analysis showed CCNE1 with high expression had a poor prognosis. Silencing CCNE1 significantly reduced cell viability in 48 h, cultured and arrested cell cycle in G1 phase, moreover, Cyclin A, D1 and C-myc all revealed down-regulation in both MGC-803 and NCI-N87 cells. CCNE1 expression was significantly increased at low and moderate concentrations of Cisplatin. Down-regulation of CCNE1 expression would remarkably promote cell apoptosis induced by Cisplatin, and regulate the rate of Bax/Bcl-2. Down-regulation of CCNE1 expression could inhibit cell proliferation and enhance GC cells sensibility to Cisplatin, possibly involving the regulation of Bcl-2 family.

Panobinostat (LBH589) a Promising New Partner for Combination with Doxorubicin in Acute Myeloid Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1638-1638 ◽  
Author(s):  
Patricia Maiso ◽  
Enrique Colado ◽  
Enrique M Ocio ◽  
Mercedes Garayoa ◽  
Peter Atadja ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Cell Population ◽  
Mechanism Of Action ◽  
Drug Combination ◽  
Ex Vivo ◽  
Annexin V ◽  
Hel Cells ◽  
Acute Myeloid

Abstract Background and Aims: The combination of cytarabine with an anthracyclin has been the gold standard for the induction treatment of acute myeloid leukaemia (AML) for the last three decades. Nevertheless, 20–50% of patients fail to respond to this scheme and among those who achieve complete response (CR) the relapse rate come close to 50%. In the present study, we have investigated the potential value of panobinostat (LBH589) for the treatment of AML. This drug has already demonstrated antileukemic activity. Nevertheless, since there is a large body of evidence indicating that AML treatment requires drug combination, we analysed the potential synergism of panobinostat with other well established anti-AML agents. Material and Methods: The efficacy of panobinostat and of its combination with each one of three other agents (cytarabine, doxorubicin and fludarabine) was analyzed both in vitro, (in four AML cell lines: HEL, HL-60, KG-1 and MV4.11, by MTT) and ex vivo (in freshly isolated cells from 6 AML patients by flow cytometry). In addition the toxicity in normal hematopoietic cells was analyzed. The mechanism of action was investigated by Annexin V, cell cycle profile, DioC6 staining, Western Blot and gene expression profile (GEP) analysis by microarrays. Results: Panobinostat potently suppressed the viability of AML cells. IC50 values were 9 nM (HEL), 7 nM (HL-60), 17 nM (KG1), and 6 nM (MV4-11). Comparison of the IC50 of panobinostat with other drugs commonly used in AML, indicated that panobinostat was more potent than cytarabine, fludarabine, and doxorubicin (IC50 = 740 nM, 362 nM and 220 nM respectively, for HEL cells). Panobinostat increased the anti-AML effect of cytarabine and fludarabine. Nevertheless, the most significant effect was observed for the combination with doxorubicin. The CI range values were 0.05–0.41 in all cell lines, and accordingly, remaining experiments focused on this combination. This efficacy was also confirmed in ex vivo experiments. Using quadruple staining (annexin V-FITC/CD33-PE/CD45-PerCP/CD34-APC), we identify and distinguish the blast cell population (CD34−/+, CD33−/+, CD45dim) from the normal residual lymphocytes (CD45+, SSClo) and quantify the number of apoptotic cells in each cell population. In all six cases a potentiation was observed with P+D. Interestingly no effect was observed in terms of toxicity on non leukemic residual hematopoietic cells from the same patients’ samples. An important question to be asked, upon using drug combinations, is whether the genes deregulated by the combination just represent the sum of those targeted by each of the drugs or if the drug combination is inducting new targeting pathways. In order to answer this question we compared the GEP of HEL cells exposed to the P+D with those as single agents. While there were 285 genes deregulated with panobinostat and 43 with doxorubicin after 24 hours with each drug; 12 hours of treatment with P+D resulted in the deregulation of 841 genes. Accordingly, 588 genes were exclusively deregulated after P+D treatment, indicating that panobinostat and doxorubicin affect different groups of genes and pathways. The two most significantly deregulated functional categories were genes involved in the control of cell cycle and apoptosis. Treatment with P+D down-regulated Cyclin B1 (−3.04), AVEN (−2.58), Bcl-X (−2.96), TNFRSF25 (−3.48) or HSPA5 (−3.87), and up-regulated the levels of Cyclin G2 (5.49), BTG1 (5.47), or BNIP3L (2.17). c-jun was upregulated after treatment with panobinostat (3.77) and particularly with doxorubicin (26.30), and the upregulation was even higher upon treatment the AML cells with the combination of both drugs (58.38). Mechanistic experiments showed that P+D activated apoptosis (Annexin V staining and PARP- and caspases-cleavage by Western Blot) at concentrations that did not induce any cytotoxic effect when panobinostat and doxorubicin were used as single agents. P+D activated the intrinsic pathway of apoptosis with loss of mitochondrial membrane potential and subsequent release of Cytochrome C to the cytoplasm. A decrease in MCL-1 and BCL-X cleavage was also observed with the combination while not with the single agents. Interestingly, P+D also induced cell cycle arrest. Conclusion: Panobinostat + doxorubicin show a marked synergistic activity against AML cells, with unique mechanism of action, and represent a most attractive combination for clinical investigation.

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