towards improved death receptor targeted therapy for ... - TI Pharma

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RESULTS p38 and JNK induction by TRAIL TRAIL‐dependent activation of p38 and JNK MAPKs in NSCLC cells TRAIL‐induced p38 and JNK phosphorylation were studied in H460 and A549 cells representing TRAIL sensitive and resistant cells, respectively [21]. Time course experiments applying TRAIL (50 ng/ml) for up to 4 h confirmed strong caspase‐8, ‐9 and ‐3 cleavage in H460 cells already after 1 h exposure. A549 cells showed only limited caspase cleavage, which was not sufficient for triggering apoptosis (Fig. 1a). These results were in line with hardly any increase in caspase activity after TRAIL treatment in A549 cells contrasting strong activation in H460 cells (data not shown). Using the same cell lysates TRAIL‐induced phosphorylation of p38 and JNK were examined. In H460 cells, p38 and JNK were phosphorylated after 2 h and 3 h TRAIL exposure, respectively, whereas no phosphorylation was seen in A549 cells (Fig. 1b). Since RIP1 kinase has been reported to mediate MAPK activation [22], we determined RIP1 expression in the NSCLC cells. Both A549 and H460 expressed RIP1, but only H460 cells displayed clear TRAIL‐induced RIP1 cleavage that was detectable at around 30 min post‐treatment and with strong cleavage after 2 h treatment. Figure 1. Caspases activation, JNK and p38 phosphorylation and RIP expression in TRAIL‐sensitive H460 and ‐resistant A549 cells. (a) The H460 and A549 cell lines were treated with 50 ng/ml TRAIL for the indicated time points and caspase‐8, ‐9, ‐3 cleavage was assessed by Western blotting. (b) Phosphorylation of p38 (p‐p38) and phosphorylation of JNK1/2/3 (p‐JNK1/2/3), as well as RIP1 expression following TRAIL treatment (50 ng/ml) were determined by Western blotting. The blots are representative for at least three independent experiments. ‐ 45 ‐
Chapter 3 P38 and JNK have opposing effects on TRAIL‐induced apoptosis P38 and JNK have been described to mediate both pro‐ and anti‐apoptotic activities depending on the stimulus applied and the cell type studied [23]. In order to explore their role in TRAIL‐induced apoptosis in NSCLC cells we employed selective chemical JNK and p38 inhibitors. As shown in Fig. 2a, JNK inhibition with SP600125 resulted in enhanced TRAIL‐induced cell death in H460 cells, indicating anti‐apoptotic activity of JNK. P38 inhibition with SB203580, on the other hand, decreased the level of cell death, indicating that p38 has pro‐apoptotic activity in TRAIL signaling (Fig. 2b). The chemical inhibitors SP600125 and SB203580 have been generated against the ATP binding pockets of JNK and p38, respectively, and do not affect the phosphorylation status of the kinases themselves or only partially in case of autophosphorylation [24;25]. As a control and in line with this we did not observe a decrease in TRAIL‐dependent phosphorylation of JNK and p38 in the presence of these inhibitors (Fig. 2f). Furthermore, at concentrations lower than 25 µM hardly any significant cross‐inhibition of other MAPKs have been reported, which has been linked to the unique binding properties of these chemicals in the ATP‐binding sites of the enzymes [26]. Nonetheless, to further corroborate our findings we also employed specific siRNAs against JNK and p38 that upon transfection strongly reduced the expression of these kinases in H460 cells when compared to control siRNA transfected cells (Fig 2c, d). In resistant A549 cells, JNK and p38 inhibition did not significantly affect TRAIL‐dependent apoptosis (Fig. 2a, b). Next, the effect of kinases inhibition on TRAIL‐induced caspases activation was studied by Western blotting following 3 or 24 h treatment. JNK inhibition by SP600125 appeared to stimulate caspase‐8, Bid, RIP1, caspase‐9, caspase‐3 and PARP cleavage, as judged by more intense cleaved products and/ or a decrease in the full length proteins (Fig. 2e). Inhibition of p38 with SB203580 in combination with TRAIL hardly affected cleavage of caspase‐8, Bid and RIP1, whereas cleavage of caspase‐9, caspase‐3 and PARP was somewhat decreased compared to TRAIL alone. ‐ 46 ‐
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: Chapter 3 of amplification of the t
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 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 97 and 98:
Chapter 5 Reference List 1. Jemal A
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Chapter 5 ‐ 98 ‐
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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 8 SUMMARIZING DISCUSSION AN
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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 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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