Acquired Resistance to Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand (TRAIL): Histone Deacetylase Inhibitors Resensitize TRAIL-Resistant Jurkat Acute Lymphocytic Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5032-5032
Author(s):  
Pavel Klener ◽  
Jan Molinsky ◽  
Tereza Simonova ◽  
Emanuel Necas ◽  
Ladislav Andera ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Fas Ligand ◽  
Death Receptor ◽  
Lymphocytic Leukemia ◽  
Jurkat Cells ◽  
Tnf Alpha ◽  
Apoptotic Pathway ◽  
Induced Apoptosis ◽  
Disc Formation

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a death-ligand from the TNF family. TRAIL induces programmed cell death by the cell-extrinsic p53-independent apoptotic pathway. A potential of TRAIL as cancer-specific therapeutic agent has been proposed and is preclinically and clinically tested. Development of TRAIL-resistant clones in the TRAIL-sensitive tumor cells may be a serious complication of TRAIL based cancer therapy. Jurkat acute lymphocytic leukemia cells are sensitive to TRAIL-induced apoptosis, as well as other apoptosis inducing ligands from TNF family, Fas and TNF-alpha. Jurkat cells express only one of the four receptors for TRAIL, death receptor 5 (DR5). Prolonged exposure of TRAIL-sensitive Jurkat cells to recombinant soluble TRAIL (1000 ng/mL) resulted in the establishment of three TRAIL-resistant (TR) Jurkat cell subclones, Jurkat TR1, TR2, and TR3. The Jurkat TR subclones were also resistant to TNF-alpha and Fas ligand, suggesting disruption of the extrinsic apoptotic pathway. TRAIL-resistant subclone TR1, but not TR2 and TR3, demonstrated decreased susceptibility to undergo apoptosis in response to histone-deacetylase inhibitors, valproic acid (VA), sodium butyrate (SB) and suberoylanilide hydroxamic acid (SAHA) and was resistant to fludarabine. Flow cytometry analysis showed Jurkat TR subclones had unchanged expression of cell surface death receptor DR5, Fas, and receptors for TNF-alpha, TNF-R1 and TNF-R2, compared to TRAIL-sensitive Jurkat cells. Analysis of death-inducing signaling complex (DISC) formation by immunoprecipitation (anti-TRAIL, anti-DR5) and subsequent western blotting (anti-caspase 8, anti-FADD) clearly demonstrated that the DISC formation in response to TRAIL binding to DR5 was significantly decreased in subclones TR2 and TR3, but remained unchanged in subclone TR1 compared to TRAIL-sensitive Jurkat cells. To gain further insight into potential molecular aletarations associated with acquired TRAIL resistance of Jurkat subclones, we measured gene expression of several key apoptotic regulators, including receptors for TRAIL, cFLIP, BCL2 family, IAP family, HSP family members in TRAIL-resistant and TRAIL-sensitive Jurkat cells and did not detect any significant (>2-fold) change. These results suggest acquired TRAIL resistance of Jurkat cells might be mediated by changes on the protein rather than mRNA level. We analyzed whether the TRAIL-resistant Jurkat cells could be resensitized to TRAIL-induced apoptosis by pretreatment with diverse inhibitors of important prosurvival pathways, including inhibitors of proteosynthesis (cycloheximid), inhibitors of transcription (actinomycin D), NFkB inhibitors (bortezomib, SN-50), PI3K-Akt-mTOR inhibitors (rapamycine, LY294002, Hsp90 inhibitor (17-AAG), cyclin-dependent kinase inhibitors (roscovitine), casein kinase II inhibitors (DRB), or histone deacetylase inhibitors (HDACi: SAHA, VA, SB). Pretreatment with HDAC inhibitors for 12 hour was able to resensitize all three TRAIL-resistant Jurkat subclones to TRAIL-induced apoptosis. The percentage of apoptotic cells of HDACi-pretreated subclones was 70–95% 24 h after the exposure to TRAIL compared to 5–15% apoptosis for HDACi-untreated TRAIL-exposed controls, and to 10–15% apoptosis for HDACi-treated TRAIL unexposed controls. We established TRAIL-resistant subclones from the original TRAIL-sensitive Jurkat cells. Acquired resistance to TRAIL was not mediated by downregulation of TRAIL death receptor DR5 and was associated with (cross)resistance to TNFa and Fas ligand, suggesting disruption of cell-extrinsic apoptotic pathway. We assume diverse molecular mechanisms were involved in the development of TRAIL-resistant subclones upon exposure to TRAIL, as exemplified by disrupted formation of DISC in case of subclones TR2 and TR3 and normal DISC formation and fludarabine resistance in subclone TR1, suggesting deregulated apoptotic pathway downstream of DISC. Finally, we observed that HDACi resensitized the TRAIL-resistant subclones to TRAIL. The results provide substantiation for combinatorial approaches in the potential TRAIL-based therapies of hematological malignancies.

Involvement of Fas Signalling in 7β-Hydroxycholesterol-and Cholesterol-5β,6β-Epoxide-Induced Apoptosis

2008 ◽  
Vol 27 (3) ◽  
pp. 279-285 ◽  
Author(s):  
Sinéad Lordan ◽  
John J. Mackrill ◽  
Nora M. O’Brien
Keyword(s):  
Arterial Wall ◽  
Caspase 3 ◽  
Fas Ligand ◽  
Specific Interaction ◽  
Death Receptor ◽  
Critical Event ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

The induction of apoptosis in cells of the arterial wall is a critical event in the development of atheroma. 7 β-Hydroxycholesterol (7 β-OH) and cholesterol-5 β,6 β-epoxide ( β-epoxide) are components of oxLDL and have previously been shown to be potent inducers of apoptosis. However, the exact mechanisms through which these oxysterols induce apoptosis remains to be fully elucidated. The specific interaction of the Fas death receptor with Fas ligand (FasL) initiates a caspase cascade culminating in apoptosis. The purpose of the present study was to determine the involvement of Fas signalling in 7 β-OH- and β-epoxide-induced apoptosis. To this end we employed the Fas/FasL antagonist, Kp7-6, and examined the effect of Fas inhibition on oxysterol-induced cell death in U937 cells. Fas levels were increased following 24 h exposure to 30 μM 7 β-OH while treatment with 30 μM β-epoxide had no effect. Kp7-6 reduced the Fas content of 7 β-OH-treated cells to control levels and partially protected against 7 β-OH-induced apoptosis. This coincided with a decrease in cytochrome c release along with a reduction in caspase-3 and caspase-8 activity. Our data implicate Fas signalling in the apoptotic pathway induced by 7 β-OH and also highlight differences between apoptosis induced by 7 β-OH and β-epoxide.

The Role of Histone Acetylation and Death Receptor 5 (DR5) Expression in the Treatment of Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5017-5017
Author(s):  
Bonnie A. Graham ◽  
Brenda Kuschak ◽  
James B. Johnston ◽  
Spencer B. Gibson
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Histone Acetylation ◽  
Hdac Inhibitors ◽  
Death Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Apoptotic Response ◽  
Apoptotic Pathway ◽  
Histone Deacetylase 1 ◽  
Induced Apoptosis

Abstract Chronic lymphocytic leukemia (CLL) is a disease in which two-thirds of patients will require therapy, usually with the alkylating agent, chlorambucil (CLB) or the nucleoside analog, fludarabine (FLU). TRAIL is a death receptor ligand that has shown selective cytotoxicity towards a variety of malignancies. CLL cells, however, are relatively resistant to this agent. We have previously demonstrated that CLB- and Flu-induced apoptosis is partially mediated through activation of the TRAIL apoptotic pathway and this is related to the up-regulation of the TRAIL death receptors, DR4 and DR5 (Oncogene, 22:8356–8369, 2003). Combining CLB or Flu with TRAIL produced a synergistic apoptotic response in CLL cells. In contrast, the upregulation of DR5 and the synergistic apoptotic response was not observed in normal lymphocytes. We have subsequently demonstrated that the up-regulation of DR5 is mediated by transcription factors, nuclear factor κB (NFκB) and p53, and histone acetylation. Using chromatin immunoprecipitation assays, we have found that the p65 subunit of NFκB and p53 are bound to the DR5 gene in CLL cells following either CLB or Flu treatment. In addition, histone 3 is acetylated following CLB and Flu treatment corresponding to histone acetylase p300 binding to the DR5 gene. Histone deacetylase 1 (HDAC1) also binds to the DR5 gene following CLB or Flu treatment, but generally at later time points. Overall histone acetylation was also found to be increased in CLL cells, as compared to normal lymphocytes. Treatment with HDAC inhibitors, which are being evaluated in clinical trials, resulted in increased binding of p53 and NFκB, but not HDAC1, to the DR5 gene and increased DR5 mRNA levels in cells. Furthermore, enhanced apoptosis was also observed in CLL cells treated with combinations of SAHA (another HDAC inhibitor) and TRAIL. These findings suggest that histone acetylation is important in regulating DR5 expression and that HDAC inhibitors increase DR5 expression mediated by p53 and NFκB. This could provide a mechanism to sensitize CLL cells to TRAIL-induced apoptosis.

The Induction of Apoptosis by Histone Deacetylase Inhibition in Chronic Lymphocytic Leukemia (CLL) Cells Is Mediated through the TNF-Related Apoptosis-Inducing Ligand (TRAIL) Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2982-2982
Author(s):  
David Szwajcer ◽  
Spencer B. Gibson ◽  
James B. Johnston
Keyword(s):  
Protein Expression ◽  
Cell Viability ◽  
Acridine Orange ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Drug Exposure ◽  
Apoptotic Pathway ◽  
Acridine Orange Staining ◽  
Trail Receptors ◽  
Induced Apoptosis

Abstract Background and Objectives: Histone Deacetylase Inhibitors (HDIs) have been shown to induce cell cycle arrest, differentiation and apoptosis in a variety of myeloid and lymphoid malignancies while being relatively nontoxic to normal cells. HDIs have also been shown to sensitize CLL cells to TRAIL-induced apoptosis. In this study we investigated the effects of two HDIs, trichostatin (TSA) and valproic acid (VPA), alone and in combination with fludarabine (FLU) on apoptosis in the BJAB lymphoblastoma B-cell line and in primary CLL cells. Materials and Methods: Cell viability was assessed by the MTT (3-(3,5-dimethylthiazol-2yl)- 2,5 diphenyl tetrazolium bromide) colorimetric assay, following 4 days of continuous drug treatment. Apoptosis was assessed by staining cell suspensions with acridine orange and quantitating apoptotic cell populations using fluorescence microscopy. Protein expression for the TRAIL receptors, DR4 and DR5, was determined by western blotting. Results: As determined by the MTT assay, the mean concentrations of TSA and VPA to reduce cell viability by 50% (IC50) in CLL cells were 0.23 uM and 650uM, respectively. TSA at 0.5uM and VPA at 2mM induced 60% apoptosis following 48 hours of drug exposure in both BJAB and CLL cells, as measured by acridine orange staining. Addition of DR4:FC, which sequesters TRAIL away from its receptors, decreased TSA- and VPA-induced apoptosis in BJAB and CLL cells by 9 and 20% respectively, indicating activation of the TRAIL apoptotic pathway by these HDIs. In addition, exposure to TSA at a concentration of 1uM for 24 hours, up regulated the expression of DR5 in BJAB cells, but this was not observed with VPA at a concentration of 1.5mM, after up to 48 hours of treatment. Neither HDI influenced DR4 protein expression in these cells. Preliminary results show that VPA potentiated FLU-induced apoptosis by 20% in both BJAB and CLL cells. Conclusions: Both TSA and VPA induce apoptosis in BJAB and CLL cells by activation of the TRAIL apoptotic pathway. For TSA, this effect may be potentiated by up regulation of DR5 expression. VPA potentiates FLU-induced apoptosis in BJAB and CLL cells, suggesting a therapeutic role for the combination of HDIs and nucleoside analogues in the management of CLL. Ongoing studies are determining whether this effect is specific for CLL cells, as compared to normal lymphocytes, and the optimum scheduling for synergy.

scholarly journals Histone Deacetylase Inhibitors Down-Regulate bcl-2 Expression and Induce Apoptosis in t(14;18) Lymphomas

2005 ◽  
Vol 25 (5) ◽  
pp. 1608-1619 ◽  
Author(s):  
Hong Duan ◽  
Caroline A. Heckman ◽  
Linda M. Boxer
Keyword(s):  
Cell Cycle ◽  
Histone Deacetylase ◽  
Chromatin Immunoprecipitation ◽  
Histone Deacetylase Inhibitors ◽  
Antitumor Agents ◽  
Trichostatin A ◽  
Hdac Inhibitors ◽  
Transcriptional Level ◽  
Cycle Arrest ◽  
Induced Apoptosis

ABSTRACT Histone deacetylase (HDAC) inhibitors are promising antitumor agents, but they have not been extensively explored in B-cell lymphomas. Many of these lymphomas have the t(14;18) translocation, which results in increased bcl-2 expression and resistance to apoptosis. In this study, we examined the effects of two structurally different HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), on the cell cycle, apoptosis, and bcl-2 expression in t(14;18) lymphoma cells. We found that in addition to potent cell cycle arrest, TSA and NaB also dramatically induced apoptosis and down-regulated bcl-2 expression, and overexpression of bcl-2 inhibited TSA-induced apoptosis. The repression of bcl-2 by TSA occurred at the transcriptional level. Western blot analysis and quantitative chromatin immunoprecipitation (ChIP) assay showed that even though HDAC inhibitors increased overall acetylation of histones, localized histone H3 deacetylation occurred at both bcl-2 promoters. TSA treatment increased the acetylation of the transcription factors Sp1 and C/EBPα and decreased their binding as well as the binding of CBP and HDAC2 to the bcl-2 promoters. Mutation of Sp1 and C/EBPα binding sites reduced the TSA-induced repression of bcl-2 promoter activity. This study provides a mechanistic rationale for the use of HDAC inhibitors in the treatment of human t(14;18) lymphomas.

Insights into histone deacetylase inhibitors-induced apoptosis in cancer cell lines

2008 ◽  
Vol 6 (9) ◽  
pp. 28
Author(s):  
P. Ruiz-Rico ◽  
M.P. Menéndez-Gutiérrez ◽  
E. Carrasco-García ◽  
R. Martínez-Mira ◽  
L. Rocamora-Reverte ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Cell Lines ◽  
Cancer Cell ◽  
Histone Deacetylase Inhibitors ◽  
Cancer Cell Lines ◽  
Induced Apoptosis

scholarly journals Histone Deacetylase Inhibitors Sensitize TRAIL-Induced Apoptosis in Colon Cancer Cells

Cancers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 645 ◽  
Author(s):  
Baojie Zhang ◽  
Bin Liu ◽  
Deng Chen ◽  
Rita Setroikromo ◽  
Hidde J. Haisma ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Combined Treatment ◽  
Specific Inhibitor ◽  
Normal Mucosa ◽  
Class I ◽  
Colon Cancer Cells ◽  
Induced Apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as a promising anti-cancer therapeutic. However, many cancers have been found to be or to become inherently resistant to TRAIL. A combination of epigenetic modifiers, such as histone deacetylase inhibitors (HDACi’s), with TRAIL was effective to overcome TRAIL resistance in some cancers. Broad spectrum HDACi’s, however, show considerable toxicity constraining clinical use. Since overexpression of class I histone deacetylase (HDAC) has been found in colon tumors relative to normal mucosa, we have focused on small spectrum HDACi’s. We have now tested agonistic receptor-specific TRAIL variants rhTRAIL 4C7 and DHER in combination with several class I specific HDACi’s on TRAIL-resistant colon cancer cells DLD-1 and WiDr. Our data show that TRAIL-mediated apoptosis is largely improved in WiDr cells by pre-incubation with Entinostat-a HDAC1, 2, and 3 inhibitor- and in DLD-1 cells by RGFP966-a HDAC3-specific inhibitor- or PCI34051-a HDAC8-specific inhibitor. We are the first to report that using RGFP966 or PCI34051 in combination with rhTRAIL 4C7 or DHER represents an effective cancer therapy. The intricate relation of HDACs and TRAIL-induced apoptosis was confirmed in cells by knockdown of HDAC1, 2, or 3 gene expression, which showed more early apoptotic cells upon adding rhTRAIL 4C7 or DHER. We observed that RGFP966 and PCI34051 increased DR4 expression after incubation on DLD-1 cells, while RGFP966 induced more DR5 expression on WiDr cells, indicating a different role for DR4 or DR5 in these combinations. At last, we show that combined treatment of RGFP966 with TRAIL variants (rhTRAIL 4C7/DHER) increases apoptosis on 3D tumor spheroid models.

Inhibition of Bortezomib-Induced Apoptosis in Chronic Lymphocytic Leukemia by Red Blood Cell Uptake.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5027-5027
Author(s):  
Luise M.C. Wheat ◽  
Susan L. Kohlhaas ◽  
Johan Monbaliu ◽  
Roland De Coster ◽  
Aneela Majid ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Cell Uptake ◽  
Reversible Inhibitor ◽  
Apoptotic Pathway ◽  
Mutational Status ◽  
Induced Apoptosis

Abstract Bortezomib (PS-341/Velcade™) is a reversible inhibitor of the proteasome that has shown promising activity in clinical trials in several malignancies including multiple myeloma, mantle cell lymphoma and follicular lymphoma, including those with refractory disease. However, results have been less encouraging in chronic lymphocytic leukemia (CLL) and we have, therefore, sought to determine the barriers to effective therapy with bortezomib in this disease. Patients with CLL were eligible but were required to have received no therapy in the six months prior to the study. In a panel of 26 patients with CLL, both purified mononuclear cells and whole blood were tested for their apoptotic response to bortezomib (1–100 nM) up to 24 h by flow cytometry and western blotting. In all cases, purified CLL cells were sensitive to bortezomib-induced apoptosis in a concentration and time-dependent fashion, irrespective of stage of disease, resistance to prior therapy, IGHV mutational status or the presence of TP53 mutations. Apoptosis was induced at low (>10 nM) nanomolar concentrations of bortezomib by activation of the intrinsic apoptotic pathway. Bortezomib-induced apoptosis correlated with levels of ubiquitination, Bax activation, and caspase cleavage. Apoptosis of CLL cells was obtained at drug levels readily obtained in vivo using currently-used dosing protocols. However, in vitro, it was necessary to maintain these concentrations for 16–24 hours to obtain maximal apoptosis. Apoptosis measured in a whole blood apoptosis assay was markedly less than in isolated lymphocytes at comparable time points and concentrations. Activity of bortezomib in purified cells was not diminished by addition of exogenous plasma but was abrogated by addition of autologous red blood cells (RBC), suggesting preferential active uptake of the drug by these cells. These data were confirmed in animal models showing preferential distribution of bortezomib to the RBC fraction. RBC uptake may therefore account for the low serum levels of bortezomib attained in vivo during terminal half-life and thus the lack of activity against cells in the peripheral blood. Together with pharmacokinetic and in vivo data, these studies suggest that different dosing schedules of bortezomib other than bolus injections may be more effective in patients with CLL.

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