published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Glioblastoma multiforme (GBM), a grade IV malignant astrocytic tumor according to WHO classification, is one of the most common and malignant brain tumor. Temozolomide (TMZ) is the current standard treatment for GBM. Nevertheless, resistance to chemotherapy in GBM is common and therefore a major obstacle to successful treatment. Adducin 3 (ADD3), a cytoskeletal protein, has been found to be associated with chemoresistance in osteosarcoma, but its potential role in glioblastoma is unclear. A TMZ-resistant model was established by chronically exposing the glioma cells (D54 cell line) to an increasing dose of TMZ. A resistant subclone (D54-R) was successfully generated. ADD3 expression level was found to be upregulated in the D54-R when compared to the parental D54 cells (D54-C). CD133 is a putative cancer stem cell marker. Its expression level was found also to be higher in D54-R when compared to D54-C cells. Among the D54-R cells, a subgroup of cells was found to express ADD3 intensely. The proportion of these spherical cells was higher in D54-R than D54-C. Moreover, these cells were spherical in morphology and expressed putative cancer stem cell markers: CD133, NANOG and OCT-3/-4. Therefore, ADD3 is associated with cancer stem cells in human glioma. The upregulation of ADD3 expression is associated with TMZ-resistance in GBM. / published_or_final_version / Surgery / Master / Master of Research in Medicine

Ovarian carcinoma is one of the most leading causes of cancer death among all gynaecologic malignancies worldwide. Although there are advances in cancer treatment for the last decades, the curative rate of this disease is just modestly improved. Chemoresistance is the major obstacle in clinical management of ovarian cancer nowadays. Thus, it is an urgent need for exploring effective alternative therapeutic strategies for ovarian cancer patients with advanced or recurrent disease. Emerging evidence has suggested that targeting cancer cell metabolism is the most promising molecular therapeutic approach in combating human cancers. Recently, the application of pharmaceutical AMPK activators is a plausible approach in selectively and specifically killing cancer cells without hampering normal cells. However, these pharmaceutical AMPK activators have many side-effects. Therefore, searching for replaceable reagents from nutraceuticals is a “new vista”. Bitter melon and its bioactive components are proposed to be natural activator of AMPK not only to reduce triglycerides levels in hyperlipidemic diabetic or insulin-resistant rodents but also to suppress human cancer cell growth specifically without toxicity to normal cells. In this study, the anti-cancer effect and molecular mechanism of bitter melon extract (BME) and one of its bioactive components, MAP30, on ovarian cancer cells were examined. Upon treatment of BME and MAP30, ovarian cancer cells showed a drastic reduction in cell proliferation and an increase of cell apoptosis in a dose dependent manner. Intriguingly, co-treatment of BME or MAP30 could enhance cisplatin-induced cell cytotoxicity in ovarian cancer cells. On the other hand, tumor microenvironement has been known as a key factor promoting cancer progression and chemoresistance. Results herein showed that BME or MAP30 could inhibit cell growth, cell migration and invasion of ovarian cancer cells mediated by omentum conditioned medium (OCM), as well as enhanced cisplatin-mediated cell cytotoxicity in a xenograft mouse tumour model. Mechanistic studies revealed that the inhibitory effect of BME and MAP30 was concomitantly associated with up-regulated AMPK activity but reduced expression of phospho-AKT, phospho-ERK and FOXM1. Such effects were similar to the functions of common AMPK activators e.g. AICAR, A23187, metformin or hypoxic stress, indicating that BME and MAP30 functions as natural AMPK activators in suppressing cancer cells growth through activating AMPK activity and inhibiting AKT/ERK/FOXM1 signaling cascade. Importantly, this study demonstrated that BME and MAP30 induced AMPK activation through an AMP-independent manner using a pair of isogenic HEK293 cells with overexpression of either the wild-type (WT) or R531G mutant isoform of AMPK2 subunit, implying the significance that BME and MAP30 may not affect the mitochondrial respiration and thus may be more tolerated by patients when used as anti-cancer medications. Taken together, the findings in this study suggest that the non-toxic BME and MAP30 function as natural AMPK activator in impairing ovarian cancer cell growth and enforcing cisplatin-mediated cell cytotoxicity in ovarian cancer cells through targeting cancer cell metabolism. Thus, BME or MAP30 may be used as a supplement for synergistically enhancing the efficacy of current chemotherapy regimes. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Human mesenchymal stromal cells (hMSCs) are part of bone marrow micro-environment that supports hematopoiesis. However, hMSCs also enhance tumor progression and survival when they become part of the cancer micro-environment. I aimed to investigate the interaction between hMSCs and cancer cells during chemotherapy. Firstly, I studied the interaction between hMSCs and T-lineage acute lymphoblastic leukemia (T-ALL) cells under pegylated arginase I (BCT-100) treatment. Three T-ALL cell lines were sensitive to BCT-100 but not hMSCs. Conversely, hMSCs could partly protect all T-ALL cell lines from BCT-100 induced cell death under transwell co-culture condition. Concerning the possible mechanism, the intermediate metabolite L-ornithine could not rescue most T-ALL cells from BCT-100 treatment. But the downstream L-arginine precursor, L-citrulline could partly rescue all T-ALL cells from BCT-100 treatment. Ornithine transcarbamylase (OTC) converts L-ornithine into L-citrulline. OTC expression level in hMSCs remained relatively high during BCT-100 treatment but OTC expressions in T-ALL cell lines declined drastically. It suggested that hMSCs may protect T-ALL cells against BCT-100 treatment by having sustained OTC expression. Suppression of hMSCs by vincristine (VCR) disrupted the protective effect of hMSCs to most T-ALL cells during BCT-100 treatment. This suggests that by transiently suppressing hMSCs, we may abolish the protective effect of hMSCs to T-ALL cells during BCT-100 treatment. Then I studied the interaction between hMSCs and neuroblastoma under cisplatin treatment. Two neuroblastoma cell lines were used for both of them are cisplatin sensitive while hMSCs are cisplatin resistant. hMSCs could partly protect neuroblastoma cells from cisplatin induced cytotoxicity. On the other hand, exogenous IL-6 but not IL-8 could also partly rescue them from cisplatin induced cytotoxicity. IL-6 activated STAT3 phosphorylation dose-dependently and enhanced expression of detoxifying enzyme (glutathione S-transferase π, GST-π) in neuroblastoma. Such effect could be counteracted by anti-IL-6R neutralizing antibody tocilizumab (TCZ). However, TCZ failed to suppress hMSCs’ protection to neuroblastoma during cisplatin treatment. This suggests involvement of multiple factors. Up-regulation of serum GST-πin some hTertMSCs/neuroblastoma co-engrafted SCID mice compared to neuroblastoma engrafted mice provided a clue that GST-π might be a possible stromal-protection factor. Caffeic acid phenethyl ester (CAPE) is a known GST inhibitor after tyrosinase activation. Neuroblastoma cells expressed tyrosinase and CAPE enhanced cisplatin cytotoxicity on them, with or without hMSCs. Paradoxically, CAPE enhanced GST-πexpression with or without cisplatin treatment in neuroblastoma suggesting possible negative feedback to GST-π inhibition. However, such additive effect of CAPE to cisplatin cytotoxicity was not observed in vivo. Further delineation of the in vivo study design may help to verify the additive effect of CAPE to cisplatin cytotoxicity in vivo. Finally, I studied the effect of apoptotic cancer cells (AC) on the immune function of hMSCs. hMSCs could phagocytose apoptotic neuroblastoma cells with respective up-regulation of many immune-mediators including two highly-expressed cytokines IL-6 and IL-8. Up-regulation of these immune-mediators may enhance immune cells chemotaxis. Further detailed investigation on the effect of AC-engulfed hMSCs to other immune cells will help us to understand the dynamic interaction between cancer cells and stromal cells during chemotherapy. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Gliomas are the commonest type of primary malignant brain tumours of the central nervous system (CNS). The highly aggressive and infiltrative characteristics of gliomas render them one of the most lethal cancers. Amongst all, the most malignant form of glioma is glioblastoma multiforme (GBM), a World Health Organization (WHO) grade IV astrocytoma. Despite well-developed multimodal treatment including surgery, radiotherapy, and chemotherapy, the prognosis of GBM patients remains poor with median survival of just over one year. This high mortality rate is commonly the result of relentless tumour recurrence secondary to the tumour’s intrinsic resistance towards its standard chemotherapeutic agent temozolomide (TMZ). Prolyl 4-hydroxylase, beta subunit (P4HB) is an endoplasmic reticulum stress response (ERSR) chaperone protein that was previously found to be overexpressed in the chemoresistant glioma cell lines D54-MG and U87-MG. Differential expressions of numerous microRNAs (miRNAs) were also found between chemosensitive and chemoresistant glioma cell lines. As such, we surmised that the dysregulation of a P4HB-regulating miRNA may contribute to P4HB upregulation and therefore chemoresistance in glioma. MiR-210, a commonly dysregulated miRNA in various cancers, is one of the most highly downregulated miRNAs in chemoresistant glioma cells (compared to chemosensitive glioma cells), and, based on bioinformatics findings, may also regulate P4HB expression. MiR-210 was therefore selected for further investigations regarding its potential roles in glioma chemoresistance. The regulatory relationship between P4HB and miR-210 was subjected for verifications. With the use of quantitative real-time polymerase chain reaction (qPCR) and western blotting, the intrinsic expressions of P4HB and miR-210 were studied. The upregulation of P4HB in D54 and U87 chemoresistant glioma (compared to the parental) cell lines were found to correlate reciprocally with the downregulation of miR-210 in the same chemoresistant glioma cells. To delineate the potential regulatory role of miR-210, a gain of function approach was adopted. Transfection of a miR-210 mimic was performed into the D54 and U87 parental chemosensitive (D54-S and U87-S) and chemoresistant (D54-R and U87-R) cells, along with a negative control. The transfection efficiency of miR-210 as well as the subsequent P4HB expressions was verified. It was found that P4HB expression was downregulated as a result of miR-210 upregulation both at the mRNA and protein levels in glioma cells. Furthermore, the effects of miR-210 overexpression on chemoresistance in the glioma cells were tested by performing cell proliferation assay. Decrease in the half maximal inhibitory concentration (IC50) of TMZ were found in all cell lines overexpressing miR-210, suggesting that miR-210 upregulation may lead to P4HB inhibition, which would at least partially mediate an alleviation of glioma cells’ resistance towards its chemotherapeutic agent TMZ. In summary, miR-210 is downregulated in chemoresistant glioma cells in vitro. It plays a potential role in regulating P4HB expression, hence chemoresistance in GBM cells. Future investigations may focus on its mechanism of action and potentiality for therapeutic intervention. / published_or_final_version / Surgery / Master / Master of Medical Sciences

abstract / Biological Sciences / Master / Master of Philosophy

abstract / Pathology / Master / Master of Philosophy

Appendix included in back. / Errata posted on back end cover. / Bibliography: leaves 236-268. / xxii, 268, [7] leaves, [41] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A contribution to the knowledge of multidrug resistance and its role in acute leukaemia. / Thesis (Ph.D.)--University of Adelaide, Dept. of Cytogenetics and molecular genetics, 1997

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.