Targeting xIAP Is More Important Than Bcl-Xl in Lymphomas with Constitutive Activation of NF-kB.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2390-2390
Author(s):  
Yanjuan He ◽  
Joan Cain ◽  
Lee Ratner ◽  
Leon Bernal-Mizrachi
Keyword(s):  
Cell Lines ◽  
Annexin V ◽  
Fold Increase ◽  
Daudi Cell ◽  
Xiap Expression ◽  
Apoptotic Effect ◽  
Effective Doses ◽  
Induction Of Apoptosis ◽  
Apoptotic Pathways

Abstract Pathways resulting in resistance to apoptosis are essential to the process of lymphomagenesis. One such pathway, the nuclear factor-kB (NFkB), has been shown to be a key element in coordinating the anti-apoptotic effect of these malignancies. However the mechanisms used by which NFkB prevents apoptosis are not well understood. It has been suggested that NFkB inhibits activation of the intrinsic, extrinsic and common apoptotic pathways. Previous work in our lab using two different virally mediated lymphoma models (Tax/HTLV1 and LMP1/EBV driven tumors) has identified two candidates that could explain these results: X chromosome-linked inhibitor of apoptosis (xIAP) and BCL-xL. Although the current literature extensively demonstrates the role of BCL-xL in lymphomas, little is known about the importance of xIAP in these malignancies. To answer this question we tested the apoptotic effect of etoposide or tumor necrosis factor (TNF) after knocking down bcl-xL and xIAP expression in our lymphoma models (SC and Daudi cell lines) using a lentivirus expressing siRNAs. After 24 hours of treatment with etoposide and TNF, we measured apoptosis by flow cytometry using double staining with Annexin V-Alexa Fluorescense and propidium iodide. Interestingly, xIAP siRNA-expressing cell lines demonstrated 2–4 fold increase in the induction of apoptosis after treatment with etoposide as compared to a nearly 2 fold increase in those expressing Bcl-xL siRNA (see Table below). No synergism was seen after treatment with TNF. Based on this finding, we then tested a novel small molecule, homolog smac, (SHC, kindly provided by Dr. PG Harren) to determine the possible therapeutic effect of xIAP inhibitors. After titration, the two most effective doses were selected (25 μM and 50 μM) to treat Daudi cell lines for 24hrs, with either etoposide or TNF. At doses of 25 μM , we observed a 2 fold increase in the induction of apoptosis produced by etoposide compared to that seen in control (DMSO + etoposide) or SHC alone and no synergism with TNF confirming the siRNA data. More importantly, at doses of 50 μM, SHC alone demonstrated activity with a 5 fold increase in apoptosis and a nearly 10 fold increase as compared to control (DMSO) when etoposide was added. Overall, we have demonstrated that xIAP and bcl-xL are important in mediating NFkB-resistance to apoptosis. However, our findings suggested that xIAP is a more potent anti-apoptotic signal and opens the door for further drug development aimed at testing xIAP-inhibitors in lymphomas. Induction of Apoptosis in xIAP or Bcl-xL siRNA expressing cell lines siRNA/Compound Etoposide TNF Untreated xIAP 43.1 ± 17.6 17.04 ± 1.4 14.3 ± 2 SC Bcl-xL 18.39± 3.7 9.4 ± 0.22 12.5 ± 2.7 Luc/DMSO 14.9 ± 1.8 14.4 ± 5.6 14.03 ± 1.25 xIAP 9.2 ± 3.2 4.7 ± 0.48 4.6 ± 0.44 Bcl-xL 8.9 ± 0.5 5.3 ± 1.7 4.16 ± 0.4 Daudi Luc/DMSO 5.49 ± 1.71 4.28 ± 0.5 6.2 ± 0.9 SHC 25 μM 20.07 ± 4.8 12.8 ± 3.9 12.1 ± 3.2 SHC 50 μM 47.7 ± 14.55 38.3 ± 0.99 32.7 ± 8.99

Caffeic Acid Phenethyl Ester Induces Apoptosis in Acute Myeloid Leukemia Cells - Possible Role of Chemokine/G-Protein Signaling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4802-4802
Author(s):  
Priscila S. Scheucher ◽  
Guilherme Augusto Silva dos Santos ◽  
Hamilton Luiz G Teixeira ◽  
Roberto P. Falcao ◽  
Eduardo Magalhaes Rego
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Cell Lines ◽  
Annexin V ◽  
Caffeic Acid Phenethyl Ester ◽  
Nb4 Cells ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Abstract Abstract 4802 Caffeic acid phenethyl ester (CAPE) is an active phenolic compound present in propolis obtained from honeybee hives. It is reported to present a spectrum of biological activities including antioxidant, anti-inflammatory and antitumoral. The antitumoral activity of CAPE as evaluated by several studies in vitro and in vivo seems to be related to distinct effects like inhibition of angiogenesis, invasion and metastasis and induction of apoptosis or differentiation of cancer cells. In the scenario of AML the demonstration of CAPE-induced apoptosis or cellular differentiation is restricted to the HL-60 cell line. Our aim was to evaluate the effects of CAPE treatment on primary AML samples as well as APL cell lines NB4 and NB4-R2 (a cell resistant to ATRA-induced differentiation) and on AML cell line Kasumi-1 (representative of core binding factor leukemia with AML1-ETO rearrangement). Proliferation and viability was evaluated by cell count with tripan blue in Neubauer chamber at fixed time intervals. Differentiation was evaluated by flow cytometer determination of CD11b expression. Apoptotic cells were defined as sub-G0 fraction and were evaluated by flow cytometer determination of propidium iodide- DNA fluorescence. Also apoptosis was detected by the annexin-V method. Leishman stained cytospins were used to confirm apoptosis or differentiation. CAPE did not induce differentiation in the cell lines NB4, NB4-R2 or Kasumi-1 and did not alter the differentiation induced by ATRA in NB4 cells. CAPE inhibited the proliferation of AML cell lines in a time and dose dependent fashion. The ED50 in 24h treatment for NB4 cell line (tripan blue) was 32.1 mcg/ml. ED50 (at 24h) for induction of apoptosis in the more sensitive assay using annexin-V in NB4 cells after 24h was 7.5mcg/ml and for Kasumi-1 was 10.2mcg/ml. CAPE (32 mcg/ml) significantly induced apoptosis after 24h in cells from AML patients (n=10), mean (IC95%) of 40.5% (29.26 – 51.76) versus control treated cells 18.16% (12.27 – 24.05); p=0.0004 In order to evaluate the mechanisms of CAPE-induced apoptosis in NB4 cells we performed a microarray analysis after 12 hours treatment (32mcg/ml). The majority of downregulated genes fall into two categories: positive cell cycle regulators and ribosomal genesis / protein traduction. In the other hand, upregulated genes fall into several categories, we point out chemokines and G- protein signalization genes. (Table 1 and 2) The role of IL-8 and Gro chemokines, that signaling by G-protein coupled receptors, has been determined in tumor progression and invasiveness. We are currently investigating the possibility that CAPE exerts an inhibitory effect in chemokine signaling in APL. In conclusion, CAPE-induced apoptosis in AML is associated with the regulation of specific genes. These properties are interesting and need further investigation. Disclosures: No relevant conflicts of interest to declare.

Accumulation of NFkB1 (p105) and NFkB2 (p100) Is Essential for Apoptosis Induced by Proteasome Inhibition in a Lymphoma Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2607-2607
Author(s):  
Leon Bernal-Mizrachi ◽  
Sarah K. Edwards ◽  
Lee Ratner
Keyword(s):  
Transcription Factors ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Nuclear Translocation ◽  
Annexin V ◽  
Proteasome Inhibition ◽  
Luciferase Reporter ◽  
Apoptotic Effect ◽  
Induction Of Apoptosis ◽  
Novel Target

Abstract The proteasome is a novel target in the therapeutic approach against different subtypes of lymphomas due to its role in the degradation of many proteins involved in cell cycle progression and apoptosis. This multi-enzyme complex controls gene expression by degrading transcription factors such as NF-kB, p53, c-Jun, c-Myc, HIFla, and MATa2. Among these, the best characterized is the NFkB family of transcription factors. Mammals express five NFkB proteins including REL-A (p65), c-REL, REL-B, p50 and p52. All NFkB proteins contain a highly conserved REL-homology domain (RHD), which is responsible for DNA binding, dimerization, nuclear translocation, and interaction with the IkB proteins. Active NFkB is present in the nucleus as heterodimers of p65 and p50 or p52 and REL-B subunits. In contrast, inactive NFkB dimers are sequestered in the cytoplasm due to their interaction with inhibitory proteins such as the IkB’s or the large NFkB subunits p105 and p100. Activation of the NFkB pathway is mediated by proteasome processing of p105 and p100 to produce p50 and p52, respectively, and proteasome degradation of the IkB proteins. Therefore, the proteasome plays a central role in the equilibrium between anti-apoptotic signals derived from p50 and p52, and the NFkB inhibitory signals of p105 and p100. However, it is still undetermined whether modulation of the inhibitory signals or the anti-apoptotic signals by proteasome inhibition is more important for the induction of apoptosis in lymphomas. To address this question we tested the apoptotic effect of the proteasome inhibitor PS341 in two lymphoma cell lines (Daudi and SC) after knocking down p105 and/or p100, using lentivirus expressing siRNA’s. After 24 hours of treatment with titration doses of PS341 (5, 10, 50, and 100nM), we measured apoptosis and NFkB inhibition by Annexin V-Alexa fluorescence and an NFkB luciferase reporter assay, respectively. Interestingly, co-expression of siRNA against both p100 and p105 rendered cells resistant to the induction of apoptosis at clinical doses of PS341 (5 and 10nM) compared to the controls (see table). Similar results were obtained after selective knock down of either p105 or p100. Western Blot analysis showed much lower accumulation of p105 and p100 in siRNA expressing cells than in control cells. However, IkB levels remain stable or increase during treatment in the p105 and/or p100 siRNA expressing cells, despite the observed reduction in apoptosis. These results suggest that the accumulation of p105 and p100, rather than IkB, contributes to the induction of apoptosis. In addition, the expression of NFkB anti-apoptotic signals, such as BCL-xL, increased in all cell lines, including controls. Therefore, the presence of anti-apoptotic signals has no impact on the apoptotic effect produced by PS341. In conclusion, the current study in our lymphoma model demonstrates that the accumulation of p105 and p100 is essential for the induction of apoptosis produced by proteasome inhibition. siRNA Against p100 and/or p105 Reduces PS341 Induction of Apoptosis ps314(nM) 5 10 50 100 SC Luciferase siRNA 61.2±3.2 70±3.6 72.5±3.6 79.5±5 Both siRNA’s 23.5±1.5 33±2.9 59.2±3.9 72.1±5.7 p100 siRNA 27.2±0.4 29±0.5 56±2.7 66.3±6.3 p105siRNA 32.2±7.3 45±2.4 55.4±7.7 78.4±4.1 DAUDI Luciferase siRNA 45.6±3.3 49.5±5.1 61.5±7.1 69.9±5 Both siRNA’s 21.4±4.6 29.3±1.7 34.5±2.4 55.3±7.7 p100 siRNA 17.3±4.2 24.4±9.9 44.5±3.3 56.6±4.7 p105 siRNA 19.7±1.6 34.3±3.3 31.3±2.6 46.3±3.4

scholarly journals EXPERIMENTAL MODELING OF APOPTOSIS UNDER CONDITIONS OF STABLE STRONTIUM EXPOSURE

2019 ◽  
Vol 21 (1) ◽  
pp. 137-140
Author(s):  
O. V. Dolgikh ◽  
N. V. Zaitseva ◽  
D. G. Dianova ◽  
A. V. Krivtsov ◽  
K. D. Starkova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Annexin V ◽  
Fold Increase ◽  
Apoptosis Markers ◽  
Stable Strontium ◽  
Cell Death Mechanisms ◽  
Apoptotic Pathways ◽  
And Control

Apoptosis is defined as a highly regulated form of programmed cell death with typical morphological and biochemical features. A variety of factors, including heavy metals, may influence the intensity of programmed cell death. The aim of the work was to simulate apoptosis in an in vitrosystem under the conditions of stable strontium exposure. The children’s population consuming drinking water with high strontium (Sr2+) content (n = 49) was observed. The level of lymphocyte apoptosis was determined with flow cytometry technique, by means of labeled annexin V-FITC conjugate (AnnV-FITC) and propidium iodide (PI) staining. AnnV-FITC+PI- cells were regarded as early apoptotic forms, whereas late apoptotic and/or necrotic cells were AnnV-FITC+PI+. The isolated leukocytes were incubated with Sr2+ at a concentration of 7.0 mg/l, the maximal permitted concentration (MPC) for water of aqueous objects, for 4 hours at 37 ºC. Expression of CD95 and p53 apoptosis markers was performed by flow cytometry using labeled monoclonal antibodies.In vitroexposure to strontium was associated with significantly decreased expression of apoptosisregulating factors, i.e., membrane marker CD95 and intracellular transcription protein p53, 1.56- and 1.68-fold, respectively. Meanwhile, we revealed a significantly (4.68-fold) decreased amounts of AnnV-FITC+PI--cells, as well as a statistically significant (1.35-fold) increase of the AnnV-FITC+PI+-cells. Moreover, the amounts of AnnV-FITC+ PI--lymphocytes in all samples were below the physiological ranges and control values. The number of samples with higher contents of AnnV-FITC+PI+-lymphocyte exceeding the established standards and control values, was 30.8%. Thus, it has been experimentally proven that strontium, at a concentration corresponding to MPC for water objects may significantly inhibit cell death along apoptotic pathways, with switching to necrotic cell death mechanisms, according to phosphatidylserine contents, as detected by annexin V binding test. The data have revealed an ability of strontium to have a significant effect upon the parameters of regulation and maintenance of cellular homeostasis, by influencing the apoptosis intensity, due to shifting a balance towards necrosis and reducing expression of apoptosis-regulating factors. The results of this study may be used in order to identify some marker indexes of immune disorders potentially induced by external influence of strontium upon human health under specific environmental factors.

Anti‐apoptotic effect of vinpocetine on cisplatin‐induced hepatotoxicity in mice: The role of Annexin‐V, Caspase‐3, and Bax

2020 ◽  
Vol 34 (10) ◽  
Author(s):  
Sally A. Habib ◽  
Rehab S. Abdelrahman ◽  
Mona Abdel Rahim ◽  
Ghada M. Suddek
Keyword(s):  
Caspase 3 ◽  
Annexin V ◽  
Apoptotic Effect

The role of calcium in the induction of apoptosis in human tumour cell lines

1992 ◽  
Vol 20 (1) ◽  
pp. 77S-77S ◽  
Author(s):  
SEAMUS V. LENNON ◽  
STEPHEN A. KILFEATHER ◽  
THOMAS G. COTTER
Keyword(s):  
Cell Lines ◽  
Tumour Cell ◽  
Human Tumour ◽  
Tumour Cell Lines ◽  
Induction Of Apoptosis ◽  
Human Tumour Cell Lines

scholarly journals Voruciclib, an Oral, Selective CDK9 Inhibitor, Enhances Cell Death Induced By the Bcl-2 Selective Inhibitor Venetoclax in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1361-1361 ◽  
Author(s):  
Daniel A Luedtke ◽  
Yongwei Su ◽  
Holly Edwards ◽  
Lisa Polin ◽  
Juiwanna Kushner ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Combination Index ◽  
Survival Rates ◽  
Annexin V ◽  
Antileukemic Activity ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Abstract Introduction: Patients with acute myeloid leukemia (AML) face overall 5-year survival rates of 65% and 27% for children and adults, respectively, leaving significant room for improvement. Relapse remains a major contributor to such low overall survival rates, and leukemic stem cells (LSCs) that survive treatment are believed to be responsible for AML relapse. The anti-apoptotic protein Bcl-2 is overexpressed in bulk AML cells and LSCs and is associated with poor clinical outcomes. Thus, Bcl-2 represents a promising therapeutic target for the treatment of AML. Venetoclax (ABT-199) is a selective Bcl-2 inhibitor that has shown great potential for treating a number of malignancies, including AML. Venetoclax inhibits Bcl-2, preventing it from sequestering pro-apoptotic Bcl-2 family protein Bim, leading to Bim activated Bax/Bak, resulting in apoptosis. However, Mcl-1 can also sequester Bim and prevent apoptosis. We previously showed that directly targeting Mcl-1 can enhance the antileukemic activity of venetoclax (Luedtke DA, et al. Signal Transduct Target Ther. Apr 2017). Alternatively, we proposed that indirect targeting of Mcl-1 may preserve or enhance the antileukemic activity of venetoclax, and prevent resistance resulting from Mcl-1. It has been reported that inhibition of CDK9 can downregulate cell survival genes regulated by superenhancers, including Mcl-1, MYC, and Cyclin D1. One CDK9 inhibitor in clinical development, flavopiridol (alvocidib), has progressed to phase II clinical trials in AML. However, off target effects and dose-limiting toxicities remain a concern. Voruciclib is an oral, selective CDK inhibitor differentiated by its potent inhibition of CDK9 as compared to other CDK inhibitors. This selectivity may potentially circumvent toxicities resulting from inhibition of non-CDK targets like MAK and ICK that are inhibited by flavopiridol. Voruciclib has been shown in vitro to promote apoptosis and decrease Mcl-1 expression levels in chronic lymphocytic leukemia (CLL) cells (Paiva C, et al. PLOS One. Nov 2015) and inhibit tumor growth in mouse xenograft models of diffuse large B-cell lymphoma (DLBCL) in combination with venetoclax (Dey J. et al Scientific Reports. Dec 2017). Based on these data, voruciclib may downregulate Mcl-1 in AML cells and therefore synergistically enhance the antileukemic activity of venetoclax. Methods/Results: Culturing AML cell lines (THP-1, U937, MOLM-13, MV4-11, and OCI-AML3) and primary patient samples with various concentrations of voruciclib resulted in a concentration-dependent increase in Annexin V+ cells (2 μM voruciclib induced 13.8-55.8% Annexin V+ cells) along with increased levels of cleaved caspase 3 and PARP, demonstrating that voruciclib induces apoptosis in AML cells. Next, we tested the combination of voruciclib and venetoclax in AML cell lines and primary AML patient samples at clinically achievable concentrations. Annexin V/PI staining, flow cytometry analysis, and combination index calculation (using CalcuSyn software) revealed synergistic induction of apoptosis by voruciclib and venetoclax combination (combination index values for MV4-11, U937, THP-1, and MOLM-13 cells were <0.73; treatment with 2 µM voruciclib and venetoclax for 24 h resulted in >80% apoptosis). Importantly, synergy was observed in both venetoclax sensitive and resistant cell lines. This was accompanied by increased cleavage of caspase 3 and PARP. Lentiviral shRNA knockdown of Bak and Bax partially rescued AML cells from voruciclib-induced apoptosis, showing that voruciclib induces apoptosis at least partially through the intrinsic apoptosis pathway. However, Bak and Bax knockdown had little to no effect on induction of apoptosis by the combination treatment, indicating that there might be other molecular mechanisms underlying the synergistic interaction between the two agents. Treatment with the pan-caspase inhibitor Z-VAD-FMK partially rescued cells from combination treatment induced-apoptosis. Discussion: Collectively, these results demonstrate that voruciclib and venetoclax synergistically induce apoptosis in AML cells in vitro and reverse venetoclax resistance. Further studies to determine the mechanism of action and in vivo efficacy of this promising combination in AML xenografts and PDX models are underway. Disclosures Ge: MEI Pharma: Research Funding.

Bortezomib Sensitizes Non-Hodgkin’s Lymphoma Cells to Apoptosis Induced by Antibodies to TRAIL Receptors R1 and R2.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4614-4614
Author(s):  
Mitchell Reed Smith ◽  
Fang Jin ◽  
Indira Joshi
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Annexin V ◽  
Hematologic Malignancies ◽  
Caspase Inhibitor ◽  
Apoptosis Induction ◽  
Minimal Growth ◽  
Trail Receptors ◽  
Independent Mechanism ◽  
Induction Of Apoptosis

Abstract TRAIL-R1 and -R2 signaling induces apoptosis via a pathway that activates caspase 8. The proteosome inhibitor bortezomib may act via several pathways. Agonistic antibodies to TRAIL-R1 and -R2 and bortezomib are in clinical trials in solid tumors and hematologic malignancies. To develop rational combinations for future clinical studies, we investigated the actions of these agents on non-Hodgkin s lymphoma (NHL) cell lines. The t(14;18)+, EBV- NHL cell lines DoHH2 and WSU-FSCCL were treated with agonistic monoclonal antibodies to TRAIL-R1 (HGS-ETR1) and -R2 (HGS-ETR2) (Human Genome Sciences, Rockville, MD) and/or bortezomib. While HGS-ETR 1 and HGS-ETR 2 are effective inducers of apoptosis in FSCCL, DoHH2, which expresses dim TRAIL-R1 (DR4, HGS-ETR1 target) and TRAIL-R2 (DR5, HGS-ETR2 target), shows minimal growth inhibition or apoptosis induction by HGS-ETR1 or HGS-ETR2. Bortezomib has modest effects on DoHH2 cells in growth inhibition and apoptosis assays. HGS-ETR1 and HGS-ETR2 induction of apoptosis in WSU-FSCCL is efficiently blocked by the caspase inhibitor ZVAD. In contrast, bortezomib effects are not blocked by ZVAD, indicating an independent mechanism of action. To determine if these separate pathways would provide enhanced combination activity, DoHH2 cells were pre-treated with bortezomib for 30 min, followed by incubation with HGS-ETR1 or HGS-ETR2. This led to supra-additive induction of apoptosis (annexin V staining). We conclude that bortezomib sensitizes DoHH2 cells to the action of HGS-ETR1 and HGS-ETR2. Further, bortezomib induces apoptosis in DoHH2 cells by an independent mechanism, and the combination of TRAIL-receptor signaling and bortezomib may be a useful combination to explore.

Bim Promoter Is Highly Methylated in Malignant Lymphoid Cell Lines, Leading to Downregulation of Bim Expression and Protection from Apoptosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2625-2625 ◽  
Author(s):  
Rocco G. Piazza ◽  
Vera Magistroni ◽  
Federica Andreoni ◽  
Anna Franceschino ◽  
Carlo Gambacorti
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Protein Level ◽  
Methylation Status ◽  
Annexin V ◽  
Cpg Sites ◽  
Induction Of Apoptosis ◽  
High Level ◽  
Lymphoid Cell Lines

Abstract Bim, a proapoptotic, BH3-only, Bcl-2 family member, is the major physiological antagonist of the antiapoptotic Bcl-2 proteins in B and T lymphocytes. It is essential for the induction of apoptosis of activated T cells following an immune response and for the homeostasis of B cells; it also plays a key role in the induction of apoptosis of early hematopoietic progenitors following cytokine-deprivation. We performed a CpG Islands prediction analysis on Bim promoter, identifying a putative CpG Island. Using a Bisulfite Modification-Clonal Sequencing Analysis (BMCSA), we investigated the methylation status of 19 CpG sites (from nucleotide −504 to +64 from the ATG start site) in the Bim promoter in 12 malignant hematological cell lines: 7 of lymphoid and 5 of myeloid origin. A minimum of 6 clones were analysed. An homogeneous, very high level of methylation was present in all the lymphoid cell lines (Average Level of Methylation (ALM) 93.4 ± 4.4% Standard Deviation [SD]) and a variable level of methylation in the myeloid cell lines (ALM 37.1 ± 32.4%). The lowest ALM was found in lymphocytes from healthy donors (15.5 ± 2.1%). Evidence of Bim promoter methylation was also found in frozen tumor samples from patients affected by NPM/ALK+ lymphomas. We treated the 12 cell lines with the demethylating agent 5-azacytidine (AZA). The changes in the methylation status of Bim promoter were evaluated by BMCSA and the corresponding induction of Bim by Real-Time PCR (TaqMan) and by Western Blot. The demethylation of Bim promoter led to a potent induction of Bim at the mRNA and protein level. In the lymphoid, NPM/ALK positive, SUDHL-1 cell line, in which a complete demethylation (from 100% to 0%) was achieved, the increase in the expression of Bim was 7.7-fold and this correlated with a potent induction of apoptosis, as assessed by TUNEL and Annexin V assays. Similar results were obtained using a different demethylating agent: 5-aza-2′deoxycytidine (DAC). To assess whether the methylation of Bim promoter is an active process, a wash-out experiment was performed on the SUDHL-1 (high level of methylation, 100%), on the PML/RAR alpha positive myeloid NB4 (intermediate level of methylation, 33%) and on the BCR/ABL positive LAMA-R cell lines (unmethylated) previously treated with AZA or DAC. This experiment showed that the demethylation is reversible and that, following remethylation, the expression of Bim at mRNA and protein level is reduced to the initial value. In the NB4 cell line, in which methylation is clustered on the last 6 CpG sites, remethylation occurs following the same pattern. No de novo methylation was seen in LAMA-R after the wash-out. To address the biological role for the methylation of Bim promoter, we generated a TET-ON inducible system for BimS (the most potent proapoptotic isoform of Bim) in the highly methylated NPM/ALK+ Karpas-299 cell line, showing that, following an induction of Bim expression, the cells are potently induced to apoptosis, as assessed by FACS using TUNEL and Annexin V assays. We conclude that Bim promoter is actively methylated in several leukemias/lymphomas of T and B origin and that its methylation is associated with the downregulation of Bim expression and with protection from apoptosis.

Mdm2 Inhibitor Nutlin-3 Enhances the Cytotoxic Synergism Induced by the Combination of MEK1 Inhibitor and Arsenic Trioxide (ATO) in AML Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1364-1364
Author(s):  
Paolo Lunghi ◽  
Laura Mazzera ◽  
Vittorio Rizzoli ◽  
Antonio Bonati
Keyword(s):  
Fixed Ratio ◽  
Annexin V ◽  
Fold Increase ◽  
Negative Regulator ◽  
Myelogenous Leukemia ◽  
P53 Pathway ◽  
Mdm2 Antagonist ◽  
High Doses ◽  
Apoptotic Pathways ◽  
Induced Apoptosis

Abstract We recently reported that PD184352 (PD) (Pfizer), a highly selective inhibitor of MEK1 phosphorylation and activation, sensitizes primary acute myelogenous leukemia (AML) to ATO-induced apoptosis via p73, a p53 paralogue, and Bad pro-apoptotic pathways activation (Blood107: 4549–4554, 2006). We also demonstrated that high doses of ATO (2μM) induced p53 accumulation (more than two-fold increase compared with control) in 11 out of 21 patients (52%), indicating a possible contribution of p53 pathway in apoptosis induction of dual treated blasts. TP53 mutations are quite rare in AML (5–10%) and MDM2 (murine double minute 2), its principal negative regulator, has been found to be frequently overexpressed in AML, a process that can actively enhance tumorigenic potential and resistance to apoptosis. The aim of this study was to investigate whether Nutlin-3, a potent and selective small-molecule MDM2 antagonist, can potentiate the apoptotic effect of the PD plus ATO combination in AML cells that retain a functional p53. Apoptosis was evaluated, after 24 and 48 hours of treatment, by measurement of sub-G1 DNA content, annexin V binding and mitochondrial transmembrane potential assays. We first analyzed the pharmacologic interactions between Nutlin-3, PD and ATO (0, 0.25, 0.5, 1, 2, 5, or 10 μM) using a fixed-ratio (1:1:1) experimental design in OCI-AML-3 cell line that has wild-type p53. We found that the three-drugs combination showed cytotoxic synergism stronger than PD plus ATO combination indicating that the inhibition of the p53-MDM2 interaction can positively influence the pro-apoptotic efficacy of dual-treated (PD plus ATO) cells: the averaged Combination Index (CI) values calculated from the ED50 (50% effective dose), ED75 and ED90, in Nutlin-3 plus PD plus ATO versus PD plus ATO treated cells were 0.36± 0.03 and 0.66± 0.02 respectively (CI values equal to 0.85 or less indicate synergism). In order to investigate the molecular effectors involved in Nutlin-3-PD-ATO or PD-ATO-induced apoptosis we first studied the kinetics (2h, 12h, 24h and 48h) of p53, p73 and phospho-Bad at Ser112 in OCI-AML-3. In the absence of Nutlin-3 ATO, even at high doses, did not promote a p53 accumulation whereas modulated the expression of the p73 gene by inducing the pro-apoptotic and anti-proliferative TAp73 and the anti-apoptotic and pro-proliferative ΔNp73 isoforms, thereby failing to elevate the TA/ΔNp73 ratio. Conversely, treatment with PD reduced the level of ΔNp73 and blunted the ATO-mediated up-regulation of ΔNp73 thus causing an increase in the TA/ΔNp73 ratio of dual-treated cells. In the presence of Nutlin-3, p53 accumulated and the triple combination of Nutlin-3, PD and ATO enhanced the loss of mitochondrial membrane potential occurred in PD plus ATO treatment. Furthermore, pretreatment with MEK1 inhibitor strongly increased the expression of dephosphorylated Bad and blunted the ATO-mediated phosphorylation of Bad at Ser112, thereby activating its pro-apoptotic functions. These findings suggest that the pro-apoptotic p73 and Bad pathways, both involved in PD plus ATO efficacy, can be potentiated by the rescue of p53 pathway in AML cells that possess a functional p53 pathway.

Synergistic Induction of Cell Death and Apoptosis by the Histone Deacetylase Inhibitor SAHA (Vorinostat) and the Demethylating Agent DAC in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2393-2393
Author(s):  
Edgar Jost ◽  
Claudia Schubert ◽  
Tim Bruemmendorf ◽  
Oliver Galm
Keyword(s):  
Cell Lines ◽  
Apoptotic Cell ◽  
Hdac Inhibitors ◽  
Annexin V ◽  
Rt Pcr ◽  
Advisory Committees ◽  
Dnmt Inhibitors ◽  
Induction Of Apoptosis ◽  
Rpmi 8226

Abstract Abstract 2393 Poster Board II-370 Introduction: Hypermethylation of CpG islands in the promoter region of genes is a well characterized epigenetic modification associated with transcriptional silencing of cancer related genes and plays a crucial role in carcinogenesis. In addition, acetylation of core histones is necessary for the maintenance of transcriptional activity of genes. DNA methylation and histone deacetylation are reversible and can be influenced by DNA methyltransferase (DNMT) inhibitors such as 5-aza-2`-deoxycytidine (DAC) or 5-azacytidine (AZA) and histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA), respectively. Clinical trials using a strategy based on the modification of epigenetic changes with DAC or AZA in combination with HDAC inhibitors have been promising and may help to generate new strategies in treatment of hematopoietic malignancies including multiple myeloma (MM). In MM however, only limited data are published about the possible synergistic effects between DNMT inhibitors and the highly potent pan-HDAC inhibitor SAHA. Material and Methods: To assess the in vitro effects of SAHA on the MM cell lines U266, LP-1, RPMI8226 or OPM-2 and the possible interactions with DNMT inhibitors, cells were first incubated with DAC in a final concentration of 0.1 or 0.2 mM for 72 hours. After exposure to DAC, cells were incubated for 72 or 96 hours with SAHA in a final concentration between 0.1 and 20 mM. The toxic effect of the treatment was assessed by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The drug concentration inducing a 50 % killing of the cells compared to control cell survival was calculated from the dose-response curve (IC50). Induction of apoptosis was analysed by flow cytometry with annexin V-binding. In addition, the expression of the epigenetically silenced tumor suppressor genes SFRP-2 and DAB2 was determined by real time RT-PCR before and after exposure to DAC and SAHA. Results: In LP-1 and U-266 cells, no relevant enhancement in the cytotoxic effect of SAHA was observed after previous exposure to DAC. In contrast, in OPM-2 and RPMI-8226 cells, a significant increase in cytotoxicity of SAHA was observed, when the cells were first incubated with DAC with a decrease of the IC50 from 6.5 μM to 2.43 μM and 10.37 μM to 4.5 μM, respectively. We further analysed a possible synergism between SAHA and DAC for the induction of apoptosis by flow cytometry. After sequential exposure of the cells with DAC for 72 hours and with SAHA for 72 hours, no change in the apoptotic cell fraction was observed for the cell lines OPM-2 and RPMI-8226. However, for U-266 and LP-1, a significant increase in apoptotic cells was observed after incubation with SAHA, when the cells were previously exposed to DAC with a increase in the apoptotic cell fraction of 39.5 % to 55.4 % and 2.5 % to 14.4 %, respectively. By real-time RT-PCR, corresponding transcriptional silencing for SFRP-2 and DAB2 was demonstrated in untreated cells, and exposure of cell lines to DAC and SAHA resulted in reexpression. A synergism for the induction of reexpression of these genes was observed when cells were incubated with DAC and SAHA sequentially. Discussion: After treatment with SAHA, we observed a dose-dependent induction of cell death and apoptosis as assessed by MTT and annexin V assay, respectively. In the different MM cell lines, we observed a synergism between SAHA and DAC both for cytotoxic effects and the induction of apoptosis. A synergism was also observed for the reexpression of epigenetically silenced genes after exposure to DAC and SAHA. These in vitro data can be considered as a basis for further in vitro studies and preclinical models with SAHA in combination with demethylating agents such as DAC in order to improve treatment response and survival in MM patients. Disclosures: Jost: MSD: Research Funding. Bruemmendorf:Genzyme: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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