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p38 and JNK induction by TRAIL Notably, Mcl‐1 knockdown cells were not responding to either the pro‐apoptotic effect of JNK inhibition or anti‐apoptotic effect of p38 inhibition, respectively (Fig. 5d). This indicates that Mcl‐1 is a mediator of the apoptosis modulatory effects of p38 and JNK. Figure 5. Mcl‐1 is a mediator of the anti‐apoptotic effect of JNK and pro‐apoptotic effect of p38 in TRAIL signaling. (a) Quantitative RT‐PCR was performed to determine mRNA expression of Mcl‐1 and Bcl‐2 after exposure to 10 µM SP600125 or 10 µM SB203580 for 24 h in H460 cells. (b) Mcl‐1 and Bcl‐ 2 expression in H460 cells treated with TRAIL (50 ng/ml) ± SP600125 (10 µM) or ± SB203580 (10 µM) for 24 h. (c) Mcl‐1 expression was silenced using siRNA in H460 cells and the expression levels were determined by Western blotting and compared with control shRNA transfected cells. (d) H460‐ shCTRL and H460‐shMcl‐1 cells were treated with TRAIL (50 ng/ml) with and without SP600125 (10 µM) or SB203580 (10 µM) and effect on apoptosis was determined. P38 and JNK activation in a panel of NSCLC cells Finally, the effect of p38 and JNK inhibition on TRAIL sensitivity was examined in an extended panel of NSCLC cells. TRAIL sensitivity was variable in these cells (Fig. 6a). H1299 and SW1573 cells showed less than 5% apoptosis when corrected for background apoptosis levels. H322 cells displayed intermediate sensitivity with approximately 10% apoptosis, whereas H1975 cells with around 25% apoptosis were most sensitive. Co‐ incubation with the JNK inhibitor SP600125 led to an increase in the amount of cell death, mainly in the H1975 and H322 cell lines, in which the increase in sub‐G1 levels was around 8% and 6%, respectively. The increase in TRAIL‐induced cell death by SP600125 was only 2% in H322 cells and 3% in SW1573 cells. The p38 inhibitor SB203850 did have little or no ‐ 51 ‐
Chapter 3 effect on TRAIL‐induced cell death in these cells. TRAIL‐induced phosphorylation of p38 and JNK was also studied in these cell lines. Cells were treated with 50 ng/ml TRAIL for 3 h and expressions of p38 and JNK and their phosphorylated forms were evaluated by Western blotting (Fig. 6b). Pleiotropic responses were observed, with clear p38 phosphorylation only in resistant H1299 cells. In these cells inhibition of p38 did not affect apoptosis (Fig. 6a) indicating that in the absence of TRAIL‐ dependent apoptotic signaling modulation of p38 activity alone has no apparent effect on apoptosis. JNK phosphorylation was detected in H1975, H322, and SW1573 cells following TRAIL treatment. In these three cell lines RIP1 cleavage by TRAIL could be seen. Taken together, inhibition of JNK has a TRAIL apoptosis stimulatory effect in NSCLC displaying already sensitivity to TRAIL‐dependent apoptosis. Figure 6. Effect of p38 and JNK inhibition on TRAIL‐induced apoptosis in a panel of NSCLC cells. (a) H1299, H1975, H322, and SW1573 cells were exposed to TRAIL (50 ng/ml) ± SP600125 (10 µM) or ± SB203580 (10 µM) for 24h. Cell death was determined as percentage of sub‐G1 cells. *p
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
Page 9 and 10: ‐ 8 ‐
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 and 50: Chapter 3 Figure 2 (continued). (e)
Page 51: Chapter 3 previously established H4
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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