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p38 and JNK induction by TRAIL enhanced phosphorylation of both p38 and JNK were seen upon RIP1 knockdown (Fig. 3c), suggesting that RIP1 to some extent can suppress the activation of these kinases by TRAIL. Similarly, treatment with the selective RIP1 kinase inhibitor necrostatin‐1 [27] also enhanced TRAIL‐induced phosphorylation of JNK and p38 (Fig. 3d). However, as found, activation of p38 under RIP1 knockdown conditions does not contribute to apoptosis activation, contrasting its pro‐apoptotic activity in the presence of RIP1. Figure 3. RIP1 knockdown enhances TRAIL‐induced apoptosis and affects p38 and JNK phosphorylation in H460 cells. (a) H460‐shCTRL and H460‐shRIP1 cells were treated with TRAIL (50 ng/ ml) in absence or presence of 10 µM SP600125 or 10 µM SB203580. *p
Chapter 3 previously established H460‐derived cell line, H460‐CrmA [28]. In this cell line, the cowpox virus‐encoded anti‐apoptotic protein cytokine response modifier A (CrmA), an inhibitor of caspase‐8, is stably overexpressed. Apoptosis by TRAIL was efficiently prevented in CrmA expressing cells and inhibition of p38 or JNK under these conditions had no effect, as depicted in Fig. 4a. As expected, TRAIL‐induced caspase‐8 and RIP1 cleavage was not observed in the H460‐CrmA cells as shown in Fig. 4b. Furthermore, inhibition of caspase‐8 activation did not affect p38 phosphorylation after TRAIL exposure (Fig. 4c). However, TRAIL‐induced JNK phosphorylation was not detected in H460‐CrmA cells, indicating that caspase‐8 activity is important for JNK activation by TRAIL. Figure 4. TRAIL‐induced caspase‐8 activation is required for JNK phosphorylation. (a) H460 cells overexpressing the caspase‐8 inhibiter CrmA (H460‐CrmA) were treated with and without TRAIL alone, or in combination with SP600125 (10 µM) or SB203580 (10 µM) for 24 h. Cell death was determined by FACS analysis of the sub‐G1 fraction. Means of triplicate experiments (± s.d.) are shown. (b) Caspase‐8 and RIP1 cleavage by TRAIL in H460 and H460‐CrmA cells. (c) Western blots displaying p38 and JNK and phosphorylated variants. The cells were treated with 50 ng/ml TRAIL for 3 h. Mcl‐1 is a mediator of pro‐apoptotic and anti‐apoptotic effects of p38 and JNK, respectively. Anti‐apoptotic Mcl‐1 and Bcl‐2 are known transcriptional targets of both p38 and JNK signaling [23;29;30]. Therefore, we studied the effect of p38 and JNK inhibition on Mcl‐1 and Bcl‐2 mRNA levels using qPCR in H460 cells. As shown in Fig. 5a, SP600125 treatment decreased Mcl‐1 expression, whereas p38 inhibition by SB203580 enhanced transcription. Bcl‐2 transcription was not affected by these treatments. Examination of protein levels by Western blotting revealed a reduction in Mcl‐1 expression upon JNK inhibition, while p38 inhibition clearly enhanced Mcl‐1 expression (Fig. 5b). Similar results were obtained when silencing JNK and p38 expressions with specific siRNAs (not shown). It thus appears that normally JNK activity enhances Mcl‐1 expression, while in contrast p38 activity reduces Mcl‐1 levels. To further explore the role of Mcl‐1 in this context its expression was silenced in H460 cells using a specific siRNA (Fig. 5c). In these Mcl‐1 knockdown cells TRAIL‐induced cell death was enhanced by approximately 100% when compared to control H460 cells. ‐ 50 ‐
Page 2 and 3: TRAIL-induced kinases activation an
Page 4 and 5: TRAIL‐induced kinases activation
Page 6: “In order to be irreplaceable one
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Page 11 and 12: Chapter 1 GENERAL INTRODUCTION Canc
Page 13 and 14: Chapter 1 OUTLINE OF THE THESIS As
Page 15 and 16: Chapter 1 Reference List 1. Jemal A
Page 17 and 18: Chapter 2 ABSTRACT Tumor necrosis f
Page 19 and 20: Chapter 2 INTRODUCTION The death li
Page 21 and 22: Chapter 2 In preclinical studies, a
Page 23 and 24: Chapter 2 Table 1| Summary of the k
Page 25 and 26: Chapter 2 proliferation could be pr
Page 27 and 28: Chapter 2 Figure 2. Canonical and n
Page 29 and 30: Chapter 2 associated with TRAIL res
Page 31 and 32: Chapter 2 adhesion molecules [109].
Page 33 and 34: Chapter 2 Reference List 1. Ashkena
Page 35 and 36: Chapter 2 37. Tolcher AW, Mita M, M
Page 37 and 38: Chapter 2 melanoma cells from TRAIL
Page 39 and 40: Chapter 2 and ERK pathways. Circula
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: Chapter 3 Figure 2 (continued). (e)
Page 53 and 54: Chapter 3 effect on TRAIL‐induced
Page 55 and 56: Chapter 3 induced apoptosis in H460
Page 59 and 60: Chapter 3 40. Domina AM, Vrana JA,
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 and 94: Chapter 5 by sub‐G1 levels in the
Page 95 and 96: Chapter 5 DISCUSSION In the present
Page 99 and 100: Chapter 5 ‐ 98 ‐
Page 101 and 102:
Chapter 6 ABSTRACT TRAIL is a tumor
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Chapter 6 various stress stimuli [1
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Chapter 6 Subsequently, the membran
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Chapter 6 B H460 A549 Figure 1. Rep
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Chapter 6 TRAIL‐dependent cell de
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Chapter 6 B H460 A549 C Figure 4 (c
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Chapter 6 Figure 5 (continued). Mec
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Chapter 6 regulation and checkpoint
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Chapter 6 mechanism of immune evasi
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Chapter 7 ABSTRACT Thymidine phosph
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Chapter 7 Figure 1. Schematic overv
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Chapter 7 that was measured before
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Chapter 7 RESULTS TdR conversion to
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Chapter 7 dR is secreted from the c
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Chapter 7 Figure 4. Accumulation of
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Chapter 7 angiogenic properties. Ho
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Chapter 7 activity of enzymes. Natu
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Chapter 7 ‐ 134 ‐
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Chapter 8 SUMMARIZING DISCUSSION AN
Page 139 and 140:
Chapter 8 Recently, various differe
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Chapter 8 in phase II clinical tria
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Chapter 8 Reference List 1. Herbst
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Chapter 8 ‐ 144 ‐
Page 147 and 148:
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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