- Title
- Sensitising human melanoma cells to TRAIL-induced apoptosis
- Creator
- Tseng, Hsin-Yi
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2012
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Melanoma is a skin cancer that remains a major public health problem in Australia because of its high incidence and the high morbidity and high mortality associated with the disease. Melanoma has proven largely resistant to many chemotherapeutic and biological agents. The introduction of a member of Tumour Necrosis Factor (TNF) family named TNF-Related Apoptosis Inducing Ligand (TRAIL) seemed to be a promising candidate due to its differential sensitivity to cancer and normal cells. Although many studies have reported approaches for sensitising cancer cells to TRAILinduced apoptosis via up-regulation of its death receptors, TRAIL-R1 and TRAIL-R2, little was known about the regulation of these receptors. Previously, reports from our laboratory have shown that the sensitivity of melanoma cells to TRAIL-induced apoptosis is in general correlated with the levels of the cell surface expression of TRAIL-R2. Therefore, the general aim of this thesis was to understand the underlying mechanism by which TRAIL-R2 is regulated and to provide more information in identifying new therapeutic approaches for increasing the sensitivity of melanoma cells to apoptosis by TRAIL. In Chapter Three, we identified the Murine Double Minute 2 (MDM2) antagonist, Nutlin-3, could enhance TRAIL-induced apoptosis as a result of p53-mediated upregulation of TRAIL-R2. Unexpectedly, Nutlin-3 up-regulated Myeloid-Cell Leukaemia Sequence 1 (Mcl-1) and inhibited apoptosis induced by the microtubule-targeting drug docetaxel. The contrasting effects of Nutlin-3 on TRAIL- and docetaxel-induced apoptosis demonstrated that Nutlin-3 may be a useful agent in improving the therapeutic efficacy of TRAIL in melanoma but could have unexpected adverse effects in combination with other chemotherapeutic drugs such as docetaxel. The MAGE proteins have been demonstrated to impinge on cell survival, proliferation and apoptosis in cancer. Studies in Chapter Four demonstrated that one of the MAGE proteins, MAGE-D2, plays an important role in protecting melanoma cells from TRAIL-induced apoptosis by suppressing TRAIL-R2 expression. We determined that MAGE-D2 is generally expressed at high levels in melanoma cells compared to melanocytes. Although its inhibition by small interfering RNA (siRNA) did not cause XX cell death, it rendered melanoma cells more sensitive to TRAIL-induced apoptosis which was associated with enhanced formation of Death-Inducing Signalling Complexes (DISC) and up-regulation of TRAIL-R2. Regulation of TRAIL-R2 by Melanoma-associated Antigen D2 (MAGE-D2) also appeared to be mediated by p53. We have shown that MAGE-D2 plays a role in repressing p53 expression in melanoma cells, as knockdown of MAGE-D2 resulted in up-regulation of p53 activity which in turn leads to the up-regulation of TRAIL-R2 protein expression. This up-regulation is not observed in p53-null or mutant p53 melanoma cells with MAGE-D2 knocked down, suggesting the dependency of p53 in regulating TRAIL-R2. Altogether, this suggests that targeting MAGE-D2 may be a useful strategy in improving the therapeutic efficacy of TRAIL in melanoma. Although it is well-known that TRAIL-R2 can be up-regulated by p53, the study in Chapter Five showed that up-regulation of TRAIL-R2 by 2-Deoxy-D-Glucose (2-DG) was independent of p53. Instead, X-box Binding Protein 1 (XBP1) in the endoplasmic reticulum (ER) stress pathway was responsible for this up-regulation. Results in this chapter demonstrated that p53-null and mutant p53 melanoma cells displayed increased levels of TRAIL-R2 expression upon 2-DG treatment and that inhibiting p53 expression in p53 wild-type melanoma cell lines did not impact on the up-regulation of TRAIL-R2 by 2-DG. In Chapter Six, we further demonstrated that conditional induction of p53 expression did not regulate TRAIL-R2 protein expression in melanoma cells and that other mechanisms may be involved. In particular, enhancing p53 levels in p53-inducible cell lines did not impact on the level of TRAIL-R2 expression or sensitise melanoma cells to TRAIL-induced apoptosis. Interestingly, we determined that Cisplatin (CDDP), a DNAdamaging drug that activates p53, could up-regulate TRAIL-R2 mRNA but did not upregulate TRAIL-R2 protein levels. This evidence pointed to regulation by translational mechanisms. The results were further supported by studies in TRAIL-selected cells where it was found that exposure to TRAIL for a prolonged period of time resulted in the down-regulation of cell surface TRAIL-R2 but not its mRNA. The precise mechanism of translational control remains to be defined but appears to involve capindependent mechanisms.
- Subject
- melanoma; TRAIL; TRAIL-R2; p53; MAGE; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/938533
- Identifier
- uon:12634
- Rights
- Copyright 2012 Hsin-Yi Tseng
- Language
- eng
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| View Details Download | ATTACHMENT02 | Thesis | 9 MB | Adobe Acrobat PDF | View Details Download |