by Kenneth Kwing-chin Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references. / ACKNOWLEDGEMENT / ABSTRACT / Chapter CHAPTER I : --- INTRODUCTION AND SOME DISPOSITION PRINCIPLES --- p.1-16 / Chapter CHAPTER II: --- DETERMINATION OF EPIRUBICIN IN BIOLOGICAL FLUIDS --- p.17-26 / Chapter CHAPTER III : --- DISPOSITION OF EPIRUBICIN IN PATIENTS WITH HEPATIC CARCINOMA --- p.27-44 / Chapter CHAPTER IV : --- DESIGN OF THE EMULSION FOR INJECTION --- p.45-85 / Chapter CHAPTER V : --- STUDIES ON THE ACUTE TOXICITY OF THE FORMULATED EMULSION --- p.86-113 / Chapter CHAPTER VI : --- PHARMACOKINETIC STUDIES OF THE FORMULATED EMULSION IN RABBITS --- p.114-123 / REFERENCES --- p.124-130 / APPENDICES

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 5 uppsatser.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

Hung Sau Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 131-137). / Table of Contents --- p.1 / Acknowledgements --- p.V / Abbreviations --- p.VI / Aim of investigation --- p.IX / Abstract --- p.XI / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Cancer Chemotherapy --- p.2 / Chapter 1.2 --- Plants as sources of useful drugs --- p.4 / Chapter 1.3 --- Potent antitumor compounds found in Goniothalamus giganteus --- p.7 / Chapter 1.4 --- Brief introduction of GONIOTRIOL --- p.8 / Chapter 1.5 --- The study on the antitumor activities of the antitumor compounds --- p.9 / Chapter 1.6 --- Biochemistry study of the anticancer agents --- p.10 / Chapter Chapter 2 --- Materials and Methods --- p.18 / Chapter 2.1 --- Materials --- p.19 / Chapter 2.1.1 --- Animals --- p.19 / Chapter 2.1.2 --- "Buffers, Culture Media and Chemicals" --- p.19 / Chapter 2.1.3 --- Cell lines --- p.20 / Chapter 2.1.4 --- Dye solutions --- p.21 / Chapter 2.1.5 --- Reagents and buffers for Agarose gel --- p.21 / Chapter 2.1.6 --- Synthetic goniotriol and its derivatives --- p.21 / Chapter 2.2 --- Methods --- p.23 / Chapter 2.2.1 --- Radioactive Precursor Incorporation Assays --- p.23 / Chapter 2.2.2 --- MTT assay --- p.24 / Chapter 2.2.3 --- Neutral Red assay --- p.24 / Chapter 2.2.4 --- Isolation and preparation of cells --- p.25 / Chapter 2.2.5 --- Assay for the solvent effect --- p.25 / Chapter 2.2.6 --- Assay for the in vitro antitumor activity THC88 on different cell lines --- p.27 / Chapter 2.2.7 --- Assay of the effect of THC86 on solid sarcoma Scl80 in vivo --- p.28 / Chapter 2.2.8 --- Assay of the effect of THC86 on peritoneal Scl80 in vivo --- p.28 / Chapter 2.2.9 --- Assay of the effect of THC89 on peritoneal EAT in vivo --- p.28 / Chapter 2.2.10 --- Assay of synthetic compound (THC89 and THC87) on the mitogenic activity of spleen lymphocytes --- p.29 / Chapter 2.2.11 --- Assay of synthetic compound (THC87) on the proliferation of murine bone marrow cells from compound- treated mice --- p.30 / Chapter 2.2.12 --- "Assay of synthetic compounds (Ml, P51 and P1) on nonmalignant cell-line" --- p.31 / Chapter 2.2.13 --- Assay of antitumor activity of synthetic compound (THC86)on PU5-1.8 --- p.31 / Chapter 2.2.14 --- Assay of the cytocidal effect of THC86 --- p.32 / Chapter 2.2.15 --- "Assay on the effect of THC86 on the synthesis of DNA, RNA and protein" --- p.32 / Chapter 2.2.16 --- Direct DNA cleavage by THC86 --- p.33 / Chapter 2.2.17 --- DNA fragmentation assay / Chapter 2.2.18 --- Assay of the effect of the synthetic compound (THC86) on different growth fraction of the cells / Chapter 2.2.19 --- Mitosis Study / Chapter 2.2.20 --- Assay for the stability of the synthetic compounds / Chapter Chapter 3 --- Structure / activity relationship of the synthetic compounds --- p.36 / Chapter 3.1 --- Results --- p.37 / Chapter 3.1.1 --- In vitro antitumor activity of the synthetic compounds --- p.37 / Chapter 3.2 --- Discussion --- p.45 / Chapter Chapter 4 --- Antitumor activities of the synthetic compounds --- p.63 / Chapter 4.1 --- Results --- p.64 / Chapter 4.1.1 --- Solvent effect in the screening process --- p.64 / Chapter 4.1.2 --- The effect of the synthetic compound (THC88) on different cell lines --- p.69 / Chapter 4.1.3 --- In vivo anti-tumor activities of the synthetic compounds --- p.71 / Chapter 4.1.3a --- Effect of THC86 on solid sarcoma Sc180 in vivo --- p.71 / Chapter 4.1.3b --- Effect of THC86 on peritoneal Scl80 in vivo --- p.71 / Chapter 4.1.3c --- Effect of THC89 on peritoneal EAT in vivo --- p.72 / Chapter 4.1.4 --- Cytotoxic effect of the tested compounds on normal cells --- p.77 / Chapter 4.1.4a --- Cytotoxic effect of THC89 on normal splenocytes in vitro --- p.77 / Chapter 4.1.4b --- Effect of THC87 on the proliferation of splenocytes --- p.77 / Chapter 4.1.4c --- Effect of THC87 on the proliferation of murine bone marrow cells --- p.78 / Chapter 4.1.4d --- Cytotoxic effect on non-malignant cell-line BALB/c 3T3/A31 --- p.78 / Chapter 4.2 --- Discussion --- p.85 / Chapter Chapter 5 --- The study on the antiproliferative mechanisms of the synthetic compounds --- p.88 / Chapter 5.1 --- Results --- p.89 / Chapter 5.1.1 --- "Effect of the synthetic compounds on Cell Growth, DNA, RNA and Protein" --- p.89 / Chapter 5.1.1a --- Effect of THC86 on PU5-1.8 (macrophage-like tumor) --- p.89 / Chapter 5.1.1b --- Cytocidal effect of THC86 on EAT --- p.89 / Chapter 5.1.1c --- "Effect of the synthetic compounds on synthesis of DNA, RNA and protein" --- p.90 / Chapter 5.1.2 --- Study of the synthetic compounds on the interactions of DNA --- p.101 / Chapter 5.1.2a --- DNA cleavage assay --- p.101 / Chapter 5.1.2b --- DNA fragmentation assay --- p.101 / Chapter 5.1.3 --- Effect of the synthetic compounds on different growth fraction of the cells --- p.104 / Chapter 5.1.4 --- Mitosis study of the synthetic compounds --- p.106 / Chapter 5.1.5 --- Investigation of the stability of the synthetic compounds in culture medium --- p.112 / Chapter 5.2 --- Discussion --- p.117 / Chapter Chapter 6 --- General Discussion --- p.122 / References --- p.131

Cheung, Ka Yan. / "Aug 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 157-167). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xi / Abbreviations --- p.xii / Chapter Chapter1 --- General Introduction --- p.1 / Chapter 1.1 --- Cancer epidemiology and managements --- p.2 / Chapter 1.2 --- Photodynamic therapy (PDT) as cancer treatment --- p.7 / Chapter 1.3 --- Pheophorbide a (Pa) as a photosensitizer for PDT --- p.13 / Chapter 1.4 --- Aim of study --- p.15 / Chapter Chapter2 --- The anti-proliferative effect of pheophorbide a- mediated photodynamic therapy on human breast adenocarcinoma cell line MCF-7 --- p.17 / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Cell cycle regulation --- p.18 / Chapter 2.1.2 --- Growth arrest and DNA damage inducible (GADD) genes as cell cycle regulators --- p.22 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Materials / Chapter 2.2.1.1 --- Cell line --- p.29 / Chapter 2.2.1.2 --- "Cell culture medium, supplements and other reagents" --- p.29 / Chapter 2.2.1.3 --- Gene expression assay reagents --- p.30 / Chapter 2.2.1.4 --- Reagents and buffers for Western blotting --- p.32 / Chapter 2.2.1.5 --- Cell cycle analysis reagents --- p.35 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Cell line propagation and subculture --- p.36 / Chapter 2.2.2.2 --- Whole-transcript expression micro array analysis --- p.37 / Chapter 2.2.2.3 --- GADD genes expression assay- RT-PCR --- p.37 / Chapter 2.2.2.4 --- Cell cycle analysis --- p.40 / Chapter 2.2.2.5 --- Western Blotting --- p.41 / Chapter 2.2.2.6 --- Statistical analysis --- p.43 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Effect of Pa-PDT on GADD genes expression by whole-transcript expression microarray analysis --- p.44 / Chapter 2.3.2 --- Effect of Pa-PDT on GADD genes expression by RT-PCR --- p.46 / Chapter 2.3.3 --- Temporal change in the cell cycle profile after Pa-PDT --- p.48 / Chapter 2.3.4 --- Effect of Pa-PDT on cell cycle associated proteins --- p.65 / Chapter 2.4 --- Discussion --- p.67 / Chapter Chapter3 --- Development of drug resistance in human breast adenocarcinoma cell line MDA and the circumvention by pheophorbide a-mediated photodynamic therapy --- p.77 / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Clinical Importance of multidrug resistance (MDR) --- p.78 / Chapter 3.1.2 --- Mechanisms of MDR --- p.78 / Chapter 3.1.3 --- Development of MDR cell lines --- p.82 / Chapter 3.1.4 --- Reversal of MDR by P-glycoprotein modulators --- p.83 / Chapter 3.1.5 --- Therapeutic potential of Pa-PDT in treating MDR cancers --- p.83 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Materials / Chapter 3.2.1.1 --- Cell line --- p.85 / Chapter 3.2.1.2 --- "Cell culture medium, supplements and other reagents" --- p.85 / Chapter 3.2.1.3 --- Cell viability assay reagents --- p.85 / Chapter 3.2.1.4 --- Gene expression assay reagents --- p.86 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell line propagation and subculture --- p.87 / Chapter 3.2.2.2 --- Drug-resistance development --- p.88 / Chapter 3.2.2.3 --- Measurement of cell viability - MTT reduction assay --- p.88 / Chapter 3.2.2.4 --- ABCB1 expression assay- RT-PCR --- p.89 / Chapter 3.2.2.5 --- Doxorubicin uptake assay --- p.91 / Chapter 3.2.2.6 --- Pheophorbide a uptake assay --- p.91 / Chapter 3.2.2.7 --- Statistical analysis --- p.92 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Cytotoxicity of doxorubicin on MDA and MDA-R cells --- p.93 / Chapter 3.3.2 --- mRNA expression of ABCB1 (P-glycoprotein) in MDA and MDA-R cells --- p.96 / Chapter 3.3.3 --- Doxorubicin uptake by MDA and MDA-R cells --- p.98 / Chapter 3.3.4 --- Circumvention of drug resistance in MDA-R cells by Pa-PDT --- p.102 / Chapter 3.3.5 --- Pheophorbide a uptake by MDA and MDA-R cells --- p.104 / Chapter 3.4 --- Discussion --- p.106 / Chapter Chapter4 --- Synergistic anti-proliferation of pheophorbide a-mediated photodynamic therapy and doxorubicin on multidrug resistant uterine sarcoma cell line Dx5 --- p.113 / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- Clinical limitations of doxorubicin as chemotherapeutic drug --- p.114 / Chapter 4.1.2 --- Clinical limitations of photodynamic therapy --- p.115 / Chapter 4.1.3 --- Combination therapy with Dox and Pa-PDT --- p.117 / Chapter 4.1.4 --- Uterine sarcoma cell line Dx5 as in vitro model for combination therapy --- p.118 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Materials / Chapter 4.2.1.1 --- Cell line --- p.120 / Chapter 4.2.1.2 --- "Cell culture medium, supplements and other reagents" --- p.120 / Chapter 4.2.1.3 --- Anti-cancer drugs --- p.121 / Chapter 4.2.1.4 --- "ROS inhibitor, α-tocopherol" --- p.121 / Chapter 4.2.1.5 --- Cell viability assay reagents --- p.122 / Chapter 4.2.1.6 --- P-glycoprotein activity assay reagents --- p.122 / Chapter 4.2.2 --- Methods - / Chapter 4.2.2.1 --- Cell line propagation and subculture --- p.123 / Chapter 4.2.2.2 --- Cell viability assay --- p.123 / Chapter 4.2.2.3 --- P-glycoprotein activity assay --- p.124 / Chapter 4.2.2.4 --- Statistical analysis --- p.125 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Combination therapy of Pa-PDT and doxorubicin in Dx5 cells --- p.126 / Chapter 4.3.2 --- Effect of α-tocopherol on the synergism between Pa-PDT and doxorubicin in Dx5 cells --- p.129 / Chapter 4.3.3 --- Effect of Pa-PDT on P-glycoprotein activity in Dx5 cells --- p.132 / Chapter 4.3.4 --- Combination therapy of Pa-PDT and doxorubicin in SA cells --- p.138 / Chapter 4.4 --- Discussion --- p.141 / Chapter Chapter5 --- General Discussion --- p.148 / Chapter 5.1 --- Pa-PDT induced growth arrest and DNA fragmentation in breast cancer MCF-7 cells --- p.149 / Chapter 5.2 --- Circumvention of doxorubicin resistance by Pa-PDT in breast cancer MDA cells --- p.151 / Chapter 5.3 --- Synergistic anti-proliferation of Pa-PDT and doxorubicin on uterine sarcoma cell line Dx5 --- p.151 / Chapter 5.4 --- Clinical implication --- p.153 / Chapter 5.5 --- Conclusions and future perspectives --- p.153 / References --- p.157

Interactions between the retinoic acid receptor alpha (RARalpha) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In acute promyelocytic leukemia (APL), RARalpha is fused with the promyelocytic leukemia (PML) gene, resulting in the expression of the fusion protein PML/RARalpha. Here, I report that topoisomerase II beta (topoIIbeta) associates with and negatively modulates PML/RARalpha and RARalpha transcriptional activity, and increased levels and association of topoIIbeta cause resistance to retinoic acid (RA) in APL cell lines. Knock down of topoIIbeta was able to overcome resistance by permitting RA-induced differentiation and increased RA-gene expression. Overexpression of topoIIbeta, in clones from an RA-sensitive cell line, conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicate that topoIIbeta is bound to an RA-response element, and inhibition of topoIIbeta causes hyper-acetylation of histone 3 at lysine 9 and activation of transcription. These results identify a novel mechanism of resistance in APL and provide further insights to the role of topoIIbeta in gene regulation and differentiation. / Studies to determine the mechanism by which topoIIbeta protein is regulated found that levels of protein kinase C delta (PKCdelta) correlated with topoIIbeta protein expression. Moreover, activation of PKCdelta, by RA or PMA, led to an increase of topoIIbeta protein levels. Most notably, in NB4-MR2 cells, we observed increased phosphorylation levels of threonine 505 on PKCdelta, a marker of activation. Inhibition of PKCdelta was able to overcome the topoIIbeta repressive effects on RA-target genes. In addition, the combination of RA and PKCdelta inhibition led to increased expression of the granulocytic marker, CD11c, in NB4 and NB4-MR2 cells. These results suggest that PKCdelta regulates topoIIbeta expression, and a constitutively active PKCdelta in the NB4-MR2 cell line leads to overexpression of topoIIbeta. / In conclusion, these studies demonstrate that topoIIbeta associates with RARalpha, binds to RAREs and plays a critical role in RA dependent transcriptional regulation and granulocytic differentiation. In addition, I show that topoIIbeta overexpression leads to RA resistance and provide evidence that topoIIbeta protein levels are regulated via a mechanism involving the PKCdelta pathway. This work has contributed to an enhanced understanding of the role of topoIIbeta in gene regulation and brings novel perspectives in the treatment of RA-resistance in APL.

The altered protein expression and activity of receptor tyrosine kinases (TK) are implicated in the progression of various types of cancers. One such dysfunction is the overexpression of the epidermal growth factor receptor (EGFR) that correlates with aggressive tumor progression and poor prognosis. On the other hand, c-Src non-receptor tyrosine kinase is overexpressed and activated in a large number of human malignancies and has been strongly linked to progression to distant metastases. c-Src-induced phosphorylation of EGFR is required for EGF-mediated mitogenesis, tumorigenesis and tumour invasiveness. Thus we surmised that molecules termed "combi-molecules" designed to block both EGFR and c-Src should not only possess significant growth inhibitory potency but also strong anti-invasive properties. In this thesis, we utilized molecular modeling to design molecules containing two moieties: one that straddles the structure of the known Src inhibitor PP2 and the other that mimics the backbone of Iressa, a potent EGFR inhibitor. Of all the molecules synthesized, only SB163 containing the longest spacer between the two moieties was capable of inducing a dose dependent inhibition of both Src and EGFR. More importantly, SB163 blocked cell motility in the wound healing assay and showed significantly greater anti-invasive activity than a PP2+Iressa combination. The observation that SB163 was a less potent EGFR or Src inhibitor than Iressa and PP2 suggests that its superior potency when compared with the PP2+ Iressa combination may be at least partially attributed to mechanisms other than EGFR or Src blockade. This was also corroborated by the fact that SB163, despite its significant bulkiness (>700) could induce dose dependent inhibition of other kinase such PDFGR and Abl. The results in toto suggest that conferring multiple kinase targeting properties to single molecules can lead to highly anti-proliferative and anti-invasive agents. Traditionally, multi-kinase targeted molecules were discovered serendipitously through multi-kinase testing. Here we initiated a more rational approach to the design of single multi-targeted molecules. Cancer being a complex disease driven by tumours characterized by multiple disordered signaling pathways, this approach may well represent a novel avenue in the therapy of refractory malignancies.

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), is a key calcium (Ca++) regulatory hormone. It is also associated with functions unrelated to Ca++ homeostasis. Here, special attention is paid towards the anticancer properties of 1,25D. 1,25D strongly inhibits the growth of well-differentiated head and neck squamous cell carcinoma (HNSCC) derived cell lines. However, advanced, less differentiated, HNSCC cell lines (e.g. SCC4) are partially resistant to 1,25D. Resistance to nuclear receptor (NR) agonists is a common event that occurs in other NR-related treatments. For example, some leukemias develop resistance to the usually effective retinoic acid (RA) treatment. However, treating RA-resistant cells with HDAC inhibitors (HDACi) sensitizes them to RA. Thus, this study aims to investigate how treatment with TSA, an HDACi, would affect the response of SCC4 cell lines to 1,25D. We found that TSA had a variety of effects on 1,25D-regulated gene expression. Combined treatment with 1,25D and TSA increased the expression of cell-cycle regulating proteins, but also enhanced the downregulation of key target genes. Given the potential of the 1,25D/HDACi combination in combating cancers, two chimeric compounds, each containing parts of 1,25D and an HDACi, were synthesized in collaboration with Dr. James Gleason (Dept. of Chemistry, McGill). These 1,25D analogs have the HDACi-like structure replacing the 1,25D side chain. Both compounds proved to be agonists of the vitamin D receptor. Moreover, the TSA-substituted compound, called triciferol, effectively induced a-tubulin as well as histones acetylation. This study underlines the potential of combining 1,25D and TSA in cancer treatment, and reveals that bi-functional 1,25D analogs can be produced with potentially enhanced therapeutic activity.

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