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

More documents

Recommendations

Info

TRAIL‐induced migration and invasion Non‐canonical TRAIL‐dependent kinase activities are reflected in altered levels of target protein phosphorylation The kinome profiling assay measures kinase activities, but within a cell kinase activities are counterbalanced by the enzymatic activity of phosphatases. Hence it is important to establish if the differences in kinase activities as determined by kinome profiling are reflected in altered levels of phosphorylation of target proteins. In accordance with the PepChip results, Western blot experiments revealed that already within 5 min after TRAIL treatment Src and ERK were phosphorylated, whereas Akt, STAT3 and FAK phoshorylation were detectable at 10 to 15 min post‐treatment in A549 cells (Fig. 3A). However, not all identified possible targets or upstream kinases could be corroborated. For example, TRAIL‐induced VAV2 and JAK2 phosphorylation was not observed by Western Blotting, while we did not find evidence of activation of ROCK (not shown). Basal and TRAIL‐induced phosphorylation of a number of kinases, including Src, Akt, ERK, FAK and STAT3, was variable in the other cell lines (Fig. 3B). In TRAIL resistant SW1573 cells increases in phosphorylation of Src ERK, and FAK by TRAIL were seen. In H460 no detectable effects were observed and in intermediate TRAIL sensitive H322 cells, ERK phosphorylation was slightly elevated after TRAIL exposure (Fig. 3B). Thus the non‐ canonical TRAIL signaling pathway identified by kinome profiling is reflected in meaningful changes in target protein phosphorylation. Figure 3. TRAIL‐induced kinase activation in NSCLC cells; confirming kinases identified by PepChip kinase arrays. (A) the indicated kinases were evaluated for TRAIL‐induced phosphorylation during time by Western blotting in A549 cells using antibodies detecting phosphorylated and total kinase. Numbers represent the quantified bands as ratio of the intensity of bands for the phosphorylated form: band of the total protein. The untreated control was set at 1.0. (B) kinase activation in H460, H322, SW1573, and A549 cells for 15 min after treatment with 50 ng/ml TRAIL determined by Western blotting. ‐ 69 ‐
Chapter 4 Non‐canonical TRAIL responses are mediated through the TRAIL‐R2 Earlier we generated TRAIL variants that selectively engage either TRAIL‐R1 or TRAIL‐R2, named 4C7 and DHER respectively [21;22]. We found these variants to be more potent than wild‐type TRAIL in triggering apoptosis in a tumor cell line‐dependent fashion. We now employed these variants to determine the TRAIL receptor mainly responsible for the induction of non‐canonical pro‐invasive properties of TRAIL. A549 cells express both TRAIL‐R1 and –R2, whereas decoy receptors are hardly detectable on the cell surface (Fig. 4A), in agreement with our previous findings [23]. As shown in Fig. 4B and C, migration and invasion of A549 cells is enhanced when stimulated with the TRAIL‐R2 selective ligand (DHER), and not by TRAIL‐R1 selective 4C7. Thus, the non‐canonical migratory and invasive features of TRAIL in resistant NSCLC cells are predominantly mediated by TRAIL‐ R2. Furthermore, comparing DHER‐ with TRAIL‐treated A549 cells showed a similar kinase phosphorylation profile (Fig. 4D). Figure 4. TRAIL induces migration and invasion in A549 cells mainly via TRAIL‐R2. (A) TRAIL receptor surface expression in A549 cells determined by FACS analysis. (B) effect of TRAIL‐R1 (4C7) and TRAIL‐R2 (DHER) specific TRAIL variants on migration, and (C) invasion are shown. Levels of untreated cells were set at 100%. The averages of experimental triplicates (±s.d.) are shown. *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 41 and 42: 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: Chapter 4 vehicle control or with 5
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
Page 99 and 100: Chapter 5 ‐ 98 ‐
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 135 and 136:
Chapter 7 ‐ 134 ‐
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
Page 145 and 146:
Chapter 8 ‐ 144 ‐
Page 147 and 148:
Chapter 9 NEDERLANDSE SAMENVATTING
Page 149 and 150:
Chapter 9 waargenomen. Het gebruik
Page 151 and 152:
Chapter 9 CONCLUSIE Het activeren v
Page 153 and 154:
zelfs na werktijden (lees 23:45) ko
Page 155 and 156:
Verder wil ik ook dr. Eric Ronken b
show all

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

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?