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INTRODUCTION Combination study: TRAIL and TFT Lung cancer is the leading cause of cancer‐related death world‐wide [1]. Non‐small cell lung cancers (NSCLC) are epithelial tumors that represent around 80% of all lung carcinomas. In NSCLC almost half of all cases have locally advanced or widespread metastatic disease at diagnosis, with an overall 5‐year survival rate of approximately 1 to 5% [2]. Whereas surgery is the mainstay treatment for localized disease, most patients are candidates for systemic or adjuvant chemotherapy. It has become clear in recent years that a therapeutic plateau has been reached for patients with advanced stage NSCLC treated with conventional chemotherapeutic agents. Therefore, novel chemotherapeutics or targeted therapeutics are required to improve prognosis of these patients. A potential useful group of agents are the tumor necrosis factor apoptosis‐inducing ligand (TRAIL) receptor targeting agents that can activate apoptosis directly in tumor cells, while healthy cells are not affected [3]. TRAIL activates the extrinsic apoptotic pathway after binding cell surface membrane‐localized TRAIL death receptors (DRs) that include TRAIL‐ R1 (DR4), TRAIL‐R2 (DR5) and two decoy receptors (TRAIL‐R3 (Dc1) and TRAIL‐R4 (Dc2) that do not possess functional death domains [4]. After TRAIL binding to TRAIL‐R1 or TRAIL‐R2 several proteins are recruited to the intracellular receptor death domain, forming the death‐ inducing signaling complex (DISC) in which caspase‐8 is activated. Caspase‐8 on its turn can activate downstream caspases, such as caspase‐3, or cross‐ activate mitochondrial apoptosis via the cleavage of the Bcl‐2 family member Bid. The mitochondrial or intrinsic pathway involves mitochondrial outer membrane permeabilization (MOMP), which is regulated by the Bcl‐2 family of proteins [5]. Cleaved Bid can induce MOMP, leading to the release of pro‐apoptotic factors, such as cytochrome c and Smac/DIABLO into the cytosol. Cytochrome c facilitates the formation of the apoptosome in which procaspase‐9 is activated, and Smac/DIABLO can sequester X‐linked inhibiter of apoptosis (XIAP) thereby preventing XIAP‐mediated binding and inhibition of caspase‐9 and ‐3 [6;7]. At the DISC level, cellular FLICE‐inhibitory protein (cFLIP) is a potent inhibitor of procaspase‐8 activation. Two variants, cFLIPL and cFLIPS representing inactive procaspase‐8 analogs, have been found to prevent procaspase‐8 activation [8]. Currently, several TRAIL‐receptor targeting drugs are evaluated in clinical phase I/II trials alone or in combinations for the treatment of NSCLC [9]. However, approximately 50% of tumor cells are resistant to TRAIL and combination with other agents can sensitize tumor cells for TRAIL [9]. TFT is a thymidylate synthase (TS) inhibitor that interferes with thymidylate production and in its triphosphate form can be incorporated into the DNA causing DNA damage [10]. TFT has been found to induce apoptotic cell death in both colon and lung cancer cell lines. Moreover, apart from activating intrinsic and/or extrinsic apoptotic pathways also caspase‐independent modes of cell death can be induced by TFT [11]. The lysosomal protease cathepsin B is known to be involved in caspase‐independent cell death upon ‐ 101 ‐
Chapter 6 various stress stimuli [12]. Previously, we found that cathepsin B plays a role in TFT‐ mediated cell death in colorectal cancer [11]. TFT is part of the formulation TAS‐102, in which TFT is combined with the thymidine phosphorylase inhibitor (TPI). TPI inhibits thymidine phosphorylase, which inactivates TFT, increasing its in vivo activity [10]. TAS‐ 102 is active in tumor cells resistant to the anti‐metabolite 5‐fluorouracil (5‐FU) suggesting at least partially non‐overlapping mechanisms of action [13]. Currently, it is being evaluated in phase II studies for the treatment of several solid tumors [14] and it has shown activity in 5‐FU resistant colon cancer cells [15]. In this study, we examined the interaction between TFT and TRAIL in NSCLC cells as a possible novel combination treatment. The effects on cell cycle progression and cell death activation, and underlying molecular mechanisms were explored. MATERIALS & METHODS Cell lines and chemicals Human NSCLC cell lines A549, H292, H322 and H460 were obtained from the American Type Culture Collection (ATCC, Teddington, UK) and were grown as monolayers in 25 cm 2 culture flasks (Greiner Bio‐One, Frickenhansen, Germany) at 37°C in a humidified 5% CO2 atmosphere. The cells were cultured in RPMI, supplemented with 10% foetal calf serum, and 100 units/ml penicillin and streptomycin (Lonza, Verviers, Belgium). TFT (Taiho Pharmaceuticals Co., Ltd, Tokushima, Japan) was dissolved in PBS as a stock solution of 20 mM and was stored in aliquots at ‐20°C. Aliquots of TRAIL (Peprotech, Rocky Hill, NJ, USA) were stored at ‐80°C. The synthetic pan‐caspase inhibitor zVAD‐fmk was obtained from Bachem AG (Bubendorf, Switzerland), and was dissolved in DMSO (Sigma‐Alderich, Steinheim, Germany) at 10 mM stock solutions and stored at ‐20°C. Anti‐caspase‐3 (#9662), anti‐caspase‐8 (#9746), anti‐caspase‐9 (#9502), anti‐cleaved caspase‐3 (#9661), anti‐Chk1 (#2345), anti‐phosphorylated Chk1 (Ser345; #2341), anti‐Chk2 (#2662), anti‐ phosphorylated Chk2 (Thr68; #2661), anti‐Cdc25c (#4688) and anti‐phosphorylated Cdc25c (Ser216; #4901), anti‐p53 (#9282), anti‐FLIP (#3210), anti‐XIAP (#2042) antibodies were all purchased from Cell Signalling Technology (Danvers, MA, USA) and anti‐p21 (#sc‐ 756) was from Santa Cruz (Santa Cruz Biotechnology, Inc. Santa Cruz, California, USA). Anti‐cathepsin B antibody was purchased from Oncogene Research Products (Boston, MA, USA), anti‐β‐actin antibody from Sigma‐Aldrich Chemicals, Goat‐a‐mouse‐IRDye (800CW;#926‐32210 and 680;#926‐32220) and goat‐a‐rabbit‐IRDye (800CW;926‐32211 and 680;#926‐32221) were obtained from Licor (Westburg, Leusden, The Netherlands). Growth inhibition assay Drug cytotoxicity was determined by the MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide) assay [16]. Cells (2000/well) were seeded in 96‐well plates (Greiner ‐ 102 ‐
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TRAIL-induced kinases activation an
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TRAIL‐induced kinases activation
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“In order to be irreplaceable one
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‐ 8 ‐
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Chapter 1 GENERAL INTRODUCTION Canc
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Chapter 1 OUTLINE OF THE THESIS As
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Chapter 1 Reference List 1. Jemal A
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Chapter 2 ABSTRACT Tumor necrosis f
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Chapter 2 INTRODUCTION The death li
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Chapter 2 In preclinical studies, a
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Chapter 2 Table 1| Summary of the k
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Chapter 2 proliferation could be pr
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Chapter 2 Figure 2. Canonical and n
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Chapter 2 associated with TRAIL res
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Chapter 2 adhesion molecules [109].
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Chapter 2 Reference List 1. Ashkena
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Chapter 2 37. Tolcher AW, Mita M, M
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Chapter 2 melanoma cells from TRAIL
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Chapter 2 and ERK pathways. Circula
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Chapter 3 ABSTRACT Tumor necrosis f
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Chapter 3 inhibitors and TRAIL rece
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Chapter 3 of amplification of the t
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Chapter 3 P38 and JNK have opposing
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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 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: Chapter 6 ABSTRACT TRAIL is a tumor
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 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
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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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