published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Clinical Oncology / Master / Master of Philosophy

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia. While external radiotherapy is the mainstay of treatment, adjuvant chemotherapy is required in advanced disease. Current chemotherapy heavily relies on cisplatin and docetaxel. The disease relapse rate is relatively high with poor survival chance for recurrent or metastatic disease. Development of novel therapeutic strategies against the disease is clearly needed. Bortezomib and suberoylanilide hydroxamic acid are respectively classified as proteasome inhibitor and histone deacetylase inhibitor. Bortezomib and SAHA induce apoptosis in various cancers including renal cell carcinoma, hepatoma and mantle cell lymphoma. However, the effect of bortezomib and SAHA on NPC cells was not mentioned. We sought to study the molecular and cellular effects of the bortezomib and SAHA on NPC cells hoping to look for drug alternatives in NPC treatment. Since SAHA reactivates EBV in NPC cells, the combined effect of bortezomib and SAHA on EBV lytic cycle was also evaluated. NPC proliferation was assessed by MTT assay. 5 EBV-positive NPC cell lines authenticated by Short Tandem Repeats (STR) profiling were used as most NPC in Chinese contains EBV. Isobologram and combination index analysis confirmed that the anti-proliferative effect on NPC mediated by the drug combination was synergistic. 30 nM bortezomib and 5μM SAHA were chosen for further studies on apoptosis because the synergism of the drugs was maximal at these concentrations. NA and C666-1 were chosen for further studies because C666-1 was the only NPC cell line that consistently harboured native NPC and the combination index was lowest in NA among the rest of the NPC cell lines. Bortezomib led to apoptosis in NPC cells. The effect was more pronounced after the addition of SAHA as evidenced by greater TUNEL positive population and earlier cleavage of poly ADP ribose polymerase (PARP). In previous cancer studies, ROS induction was commonly suggested pathways of bortezomib and SAHA’s antiproliferative effects. Staining with dichlorofluorescein diacetate (DCFH-DA) revealed enhanced reactive oxygen species (ROS) level in cells treated with both drugs. At the same time, addition of N-acetyl cysteine, a ROS scavenger, markedly reduced their effect on cytotoxicity. SAHA is known for its effect on EBV lytic cycle induction. Yet, the addition of bortezomib diminished SAHA-induced viral load, lytic protein expression and EBV infectivity. The expression of Latent Membrane Protein 1 (LMP1) was much lower in NPC treated with both drugs than in NPC treated with SAHA alone, which would reduce NF-κB activation. This, together with reduced EBNA1 expression upon treatment with both drugs, would theoretically reduce oncogenic activity. In conclusion, bortezomib and SAHA induced ROS-driven apoptosis of NPC in a synergistic manner and bortezomib inhibited SAHA-induced EBV lytic cycle. It suggests that bortezomib and SAHA are potential drug candidates for the treatment of nasopharyngeal carcinoma. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr Virus (EBV).Early diagnosis of NPC will improve the overall survival. However, traditional EBV markers do not perform well in high-risk individuals or for early detection of NPC. Aberrant promoter hypermethylation of tumor suppressor genes (TSGs) is an important epigenetic change in early tumorigenesis. This study identified a promising panel of methylation markers for early detection of NPC and assessed the clinical usefulness of these markers using nasopharyngeal (NP) brushing and blood specimens. Methylation-sensitive high resolution melting (MS-HRM) assays were carried out to assess the methylation status of a selected panel of four TSGs (RASSF1A, WIF1, DAPK1, RAR2)in biopsies, NP brushings and cell-free plasma from NPC patients. NP brushing and blood samples from high-risk and cancer-free groups were used as controls. The DNA methylation panel showed higher sensitivity and specificity than the EBV DNA markerincell-free plasma for early stage (Iand II) NPC (sensitivity: 64.6% vs. 51.2% and specificity: 96.0% vs. 88.0%, respectively). In combination with plasma EBV DNA, testing for DNA methylation in plasma and NP brushings using the four-gene MS-HRM test significantly increased the detection rate for all stages of NPC(94.1% for stages I-II, 98.4% for stages III-IV) as well as recurrence(93.5%). Aberrant activation of the Wnt signaling pathway is a common mechanism for cell transformation and tumor development in a variety of human cancers. A high frequency of promoter hypermethylation of WIF1was observed in NPC cell lines (100%), primary tumor biopsies(89.7%), NP brushings (80.2%), and cell-free plasma (51.8%),with no significant correlation with NPC stage. Simultaneously, expression of WIF1 was completely silenced in NPC cell lines (HONE1, HK1, HNE1, SUNE1, CNE1, CNE2, and C666),but not in immortalized NP epithelial cells (NP460 and NP69). These together suggested an important role of WIF1 in NPC development. In vitro and in vivo functional assays revealed a tumor suppressive role of WIF1in NPC. Restoration of WIF1expression in NPC cells significantly suppressed anchorage-independent growth, in vivo tumorigenicity, invasion, migration, and angiogenesis of NPC cells. A number of important angiogenesis-related genes were down-regulated by WIF1expression, including IL6,IL8,VEGF165,VEGFA, PDGFB, and MCP1. There is inhibition of the Wnt/β-catenin signaling pathway, manifested as decreased β-catenin expression and TCF/LEF Wnt promoter activity. These data indicated the important regulatory role of Wnt signaling pathway in NPC tumorigenicity, invasion, migration, and angiogenesis, by interacting with the complex signaling network in NPC cells. To conclude, the MS-HRM assay on the selected gene panel in combination with the EBV DNA test, increases the sensitivity for NPC detection at an early stage and detection of recurrence and has great potential to become a non-invasive test for early diagnosis and disease monitoring after treatment. Collectively, results from this study reveal that WIF1is not only a sensitive biomarker, but also a tumor suppressor gene in NPC. Understanding the molecular regulatory role ofWIF1in NPC will facilitate the diagnosis of NPC, and development of novel NPC therapeutic strategy. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 212-237) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.