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

More documents

Recommendations

Info

Combination study: TRAIL and TFT the synergistic activity of TFT and TRAIL. Chemotherapy and radiation are known to cause an enhancement of TRAIL receptor cell surface expression, mostly affecting TRAIL‐R2 expression and to a lesser extent TRAIL‐R1 [25]. We also found that TFT specifically enhances TRAIL‐R2 expression and to a lesser extent that of TRAIL‐R1. The TRAIL‐R2 gene has transcription factor binding sites for p53 that mediate cytotoxic agent‐dependent enhancement of expression [23]. In A549 cells, the up‐regulation of TRAIL‐R2 by TFT was indeed largely p53‐dependent. In H460 cells the TFT‐mediated increases in TRAIL receptor expression appeared not to be dependent on p53. Alternative mechanisms of TRAIL receptor up‐regulation have been described such as by NF‐κB [23]. Chemotherapeutics are known to affect cFLIP expression amongst others via transcriptional regulation by multiple factors and by posttranslational regulation involving rates of proteasomal degradation [8]. TFT downregulated cFLIPL, a property that was also found for the well‐studied nucleoside analogue 5‐fluorouracil in colon cancer cells [26]. Accordingly we observed stronger caspase‐8 cleavage after combined TRAIL/TFT treatment. However, Galligan and co‐workers found no effect of 5‐fluorouracil on TRAIL receptor expression independent of p53 status [26], in contrast to our observations with TFT. In another study, an increase of TRAIL receptor expression was reported for another nucleoside analogue, fludarabine, in chronic lymphocytic leukemia cells [27]. Comparable to TFT, fludarabine has been reported to decrease the expression of XIAP in chronic lymphocytic leukemia (CLL) cells [28]. Furthermore, the nucleoside analogs, fludarabine, cladribine and clofarabine promote cell death by alterations in MOMP causing cytochrome c release, as a consequence of Bax and Bak translocation to the mitochondria, in a p53‐dependent manner [29]. Together this illustrates that available nucleoside analogues have different or overlapping TRAIL sensitizing properties, depending on the tumor type and cellular context. The property of TFT to enhance TRAIL‐R2 expression may indicate that combined treatment with TRAIL‐R2 agonists may have beneficial effects. We also explored possible drug interactions at the level of cell cycle progression. TRAIL caused an accumulation of cells in the G1 phase. A small increase in phosphorylated Chk1 after TRAIL exposure may be responsible for this arrest. An increase of p‐Chk1 upon TRAIL treatment has not been reported before. However, phosphorylation of Chk2 by TRAIL has been described in colon and cervix carcinoma cells [30]. TRAIL was found to induce a DNA damage response pathway in which also DNA‐PK (DNA‐dependent protein kinase) and ATM (ataxia telangiectasia) were activated leading to Chk2 phosphorylation. In a similar way this may lead to Chk1 phosphorylation by TRAIL as we observed in this study. Alternatively, TRAIL‐induced accumulation of p21 (WAF1), may lead to cell cycle arrest as has been reported previously in human T‐cells [31]. However, we did not detect a TRAIL‐ dependent increase in p21 levels. Combined treatment resulted in similar cell cycle effects compared to TFT alone, indicating that drug interactions at the level of cell cycle ‐ 113 ‐
Chapter 6 regulation and checkpoint‐dependent apoptosis activation are not likely to underlie the synergistic effects. The mechanism of TFT‐induced cell death was also further addressed in this study. Since cell death by TFT was largely caspase‐independent we examined the possible involvement of the lysosomal protease cathepsin B in cell death activation. Previously, we found TFT as well as 5‐fluorouracil to be able to activate cathepsin B in colon cancer cell lines and a variable contribution of cathepsin B cleavage to cell death activation was found [11]. Others have reported that TRAIL can induce apoptotic cell death in oral squamous cell carcinoma cells through activation of cathepsin B [32]. In the current study we also observed cleavage of cathepsin B induced by both TFT and TRAIL, in particular in A549 cells. However, inhibition of cathepsin B by a chemical inhibitor did not affect cell death activation. Thus, the more precise way by which TFT eradicates NSCLC cells remain to be identified. Regardless of this, the property of TFT to kill NSCLC tumor cells partially via caspase‐independent mechanisms may be an advantage compared to other nucleoside analogues with respect to bypassing apoptosis‐resistance mechanisms. In conclusion, the present study demonstrates synergistic interactions between TFT and TRAIL in NSCLC cells. As TFT is also able to sensitize TRAIL‐resistant NSCLC cells, this combination of TFT and TRAIL may offer a potential novel regime for NSCLC treatment. Acknowledgements This research was performed within the framework of project T3‐112 of the Dutch Top Institute Pharma and partly supported by Taiho Pharmaceuticals, Tokushima, Japan. Conflict of interest Dr. Fukushima was employee of Taiho Pharmaceuticals. ‐ 114 ‐
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:
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:
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
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: Chapter 6 Figure 5 (continued). Mec
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
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

Create successful ePaper yourself

Delete template?

Save as template?