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TRAIL and Hsp90 inhibition Figure 5. The effect of 17‐AAG on Hsp90 client proteins possibly involved in TRAIL sensitization. (A) A549 and H460 cells were treated with TRAIL, 17‐AAG or the combination with the indicated concentrations for different time‐points and the expressions of RIP1, p‐Akt/ Akt, p‐ERK/ ERK, p‐IκBα/ IκBα, Survivin and XIAP were determined by Western blotting. Representative blots of two independent experiments are shown. β‐Actin was taken as loading control. (B) H460 and A549 cells were treated with 50 ng/ml TRAIL for 24 h with or without the Akt inhibitor, LY294002 (10 µM) or (C) the ERK inhibitor, PD098059 (10 µM). Cells were stained with PI, analyzed by flow cytometry, and sub‐G1 levels were determined. Each point represents the mean ± SEM of three independent experiments. * p
Chapter 5 DISCUSSION In the present study, we found synergistic activity between 17‐AAG and TRAIL in TRAIL‐ sensitive H460 and TRAIL‐resistant A549 cells. Hsp90 is amongst others a chaperone of proteins regulating cell cycle progression [25]. Cell cycle arrest in the G2‐M phase by Hsp90 inhibitors has been related to Plk1 [26]. In Hodgkin’s lymphoma cells, 17‐AAG decreased the expressions of Plk1 and cyclin B1, which regulates the transition of G2‐M phase to mitosis and is activated by Plk1 [27]. Although 17‐AAG affected cell cycle progression differently in A549 and H460 cells, inducing a G2/M‐arrest versus a G1 arrest, respectively, synergistic activity was the result of enhanced apoptosis activation. 17‐AAG enhanced TRAIL‐induced cell death in H460 cells, and sensitized resistant A549 cells for TRAIL. The synergistic cytotoxicity was caspase‐dependent; 17‐AAG stimulated caspase‐8 activation in both cell lines. In H460 cells, enhanced caspase‐9 activation was also involved in increased apoptosis activation. In order to obtain insight in the underlying mechanism of apoptosis sensitization by 17‐AAG we have examined several Hsp90 chaperones that are known to regulate apoptosis. RIP1 and IκB are two essential components of the NF‐κB pathway and are client proteins of Hsp90 [28]. RIP1 expression was reported to decrease upon Hsp90 inhibition leading to sensitization for TRAIL‐induced apoptosis in breast and lung cancer cells [18;19]. Inhibition of NF‐κB as a molecular mechanism of 17‐AAG‐dependent TRAIL sensitization has been described in colon and lung cancer cells [18;24]. In the present study, 17‐AAG resulted in a decrease in RIP expression in A549 cells that was accompanied by an increase in RIP cleavage. This was not observed in H460 cells that already demonstrate RIP1 cleavage following TRAIL treatment. Since RIP1 silencing in A549 cells does not lead to sensitization for TRAIL [21], RIP1 is unlikely to be the primary mediator of the effect of 17‐AAG on TRAIL sensitivity in our study. TRAIL‐dependent phosphorylation of IκBα, leading to degradation of this inhibitory protein and activation of the NF‐κB pathway was observed only in TRAIL resistant A549 cell line. Several studies have shown that the activation of NF‐ κB confers resistance to TRAIL‐induced apoptosis, including NSCLC, and inhibition of NF‐κB can lead to TRAIL‐sensitization [11;29;30]. However, 17‐AAG did not attenuate the phosphorylation of IκBα induced by TRAIL, making this pathway an unlikely effector of 17‐ AAG activity in our study. IAP family members, in particularly XIAP and survivin have also been linked to resistance to TRAIL‐induced apoptosis [31]. In resistant glioma cells, 17‐AAG sensitized for TRAIL by reducing survivin levels through enhanced proteasomal degradation [23]. Furthermore, the 17‐AAG/TRAIL combination has been reported to cause down‐regulation of XIAP, leading to enhanced caspase‐3 activation in colon cancer cells [24]. In the present study, we found no differences in survivin and XIAP expression in NSCLC cell lines between single and combined treatment with 17‐AAG and TRAIL. Examination of Akt revealed an inhibitory effect of 17‐AAG on Akt phosphorylation only in ‐ 94 ‐
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TRAIL-induced kinases activation an
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TRAIL‐induced kinases activation
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“In order to be irreplaceable one
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‐ 8 ‐
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Chapter 1 GENERAL INTRODUCTION Canc
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Chapter 1 OUTLINE OF THE THESIS As
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Chapter 1 Reference List 1. Jemal A
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Chapter 2 ABSTRACT Tumor necrosis f
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Chapter 2 INTRODUCTION The death li
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Chapter 2 In preclinical studies, a
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Chapter 2 Table 1| Summary of the k
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Chapter 2 proliferation could be pr
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Chapter 2 Figure 2. Canonical and n
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Chapter 2 associated with TRAIL res
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Chapter 2 adhesion molecules [109].
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Chapter 2 Reference List 1. Ashkena
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Chapter 2 37. Tolcher AW, Mita M, M
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Chapter 2 melanoma cells from TRAIL
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Chapter 2 and ERK pathways. Circula
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Chapter 3 ABSTRACT Tumor necrosis f
Page 43 and 44: Chapter 3 inhibitors and TRAIL rece
Page 45 and 46: Chapter 3 of amplification of the t
Page 47 and 48: Chapter 3 P38 and JNK have opposing
Page 49 and 50: Chapter 3 Figure 2 (continued). (e)
Page 51 and 52: Chapter 3 previously established H4
Page 53 and 54: Chapter 3 effect on TRAIL‐induced
Page 55 and 56: Chapter 3 induced apoptosis in H460
Page 57 and 58: Chapter 3 Reference List 1. Jemal A
Page 59 and 60: Chapter 3 40. Domina AM, Vrana JA,
Page 61 and 62: Chapter 4 ABSTRACT Tumor necrosis f
Page 63 and 64: Chapter 4 role in the activation of
Page 65 and 66: Chapter 4 the tip with a diameter o
Page 67 and 68: Chapter 4 RESULTS TRAIL induces a s
Page 69 and 70: Chapter 4 vehicle control or with 5
Page 71 and 72: Chapter 4 Non‐canonical TRAIL res
Page 73 and 74: Chapter 4 A549‐shRIP1 cells clear
Page 75 and 76: Chapter 4 Figure 7. Inhibition of S
Page 77 and 78: Chapter 4 DISCUSSION The TRAIL rece
Page 79 and 80: Chapter 4 Src‐independent mechani
Page 81 and 82: Chapter 4 variants. Cell Death Dis
Page 83 and 84: Chapter 4 ‐ 82 ‐
Page 85 and 86: Chapter 5 ABSTRACT TRAIL is an inte
Page 87 and 88: Chapter 5 targets and subsequent pr
Page 89 and 90: Chapter 5 RESULTS Synergistic activ
Page 91 and 92: Chapter 5 ng/ml TRAIL (Fig. 3B). Ap
Page 93: Chapter 5 by sub‐G1 levels in the
Page 97 and 98: Chapter 5 Reference List 1. Jemal A
Page 101 and 102: Chapter 6 ABSTRACT TRAIL is a tumor
Page 103 and 104: Chapter 6 various stress stimuli [1
Page 105 and 106: Chapter 6 Subsequently, the membran
Page 107 and 108: Chapter 6 B H460 A549 Figure 1. Rep
Page 109 and 110: Chapter 6 TRAIL‐dependent cell de
Page 111 and 112: Chapter 6 B H460 A549 C Figure 4 (c
Page 113 and 114: Chapter 6 Figure 5 (continued). Mec
Page 115 and 116: Chapter 6 regulation and checkpoint
Page 117 and 118: Chapter 6 mechanism of immune evasi
Page 119 and 120: Chapter 7 ABSTRACT Thymidine phosph
Page 121 and 122: Chapter 7 Figure 1. Schematic overv
Page 123 and 124: Chapter 7 that was measured before
Page 125 and 126: Chapter 7 RESULTS TdR conversion to
Page 127 and 128: Chapter 7 dR is secreted from the c
Page 129 and 130: Chapter 7 Figure 4. Accumulation of
Page 131 and 132: Chapter 7 angiogenic properties. Ho
Page 133 and 134: Chapter 7 activity of enzymes. Natu
Page 137 and 138: Chapter 8 SUMMARIZING DISCUSSION AN
Page 139 and 140: Chapter 8 Recently, various differe
Page 141 and 142: Chapter 8 in phase II clinical tria
Page 143 and 144: Chapter 8 Reference List 1. Herbst
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Chapter 8 ‐ 144 ‐
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Chapter 9 NEDERLANDSE SAMENVATTING
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Chapter 9 waargenomen. Het gebruik
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Chapter 9 CONCLUSIE Het activeren v
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zelfs na werktijden (lees 23:45) ko
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Verder wil ik ook dr. Eric Ronken b
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