Ovarian cancer is the most fatal of all gynecological malignancies. Epithelial ovarian cancer (EOC) accounts for about 90% of malignant ovarian tumours and is thought to originate mostly from ovarian surface epithelium (OSE) cells. Epidemiological data suggest that hormone replacement treatment (HRT) users have a higher risk of ovarian cancer, which is related to the use of oestrogen-only HRT. In addition, EOC is oestrogen responsive. This thesis reveals the capacity for production and metabolism of oestrogen in normal OSE and malignant primary EOC cells, and describes the action of oestrogen in the development of EOC at three levels. First, the expression of the genes encoding oestrogen production and metabolism and oestrogen receptor (ER) was investigated in OSE and EOC cells at RNA and protein levels. Immunohistochemistry revealed that steroid sulphatase (STS), oestrogen sulfotransferase (EST), 17βhydroxysteroid dehydrogenase (17βHSD) 2 and 17βHSD5 proteins were present in pre-menopausal, post-menopausal and inclusion cystic OSE as well as EOC cells. Taqman qRT-PCR revealed STS, EST, 17βHSD1, 17βHSD2, 17βHSD5, ERα, ERβ and oestrone sulphate (E1S) transporters such as organic anion transporting polypeptide (OATP)-B, OATP-D and OATP-E mRNAs were expressed in pre-menopausal OSE and EOC at different levels. When basal mRNA levels were compared among untreated samples of pre-menopausal OSE and EOC, EST mRNA expression was significantly higher in the OSE compared to EOC cells (P<0.05) while OATP-B mRNA level was the opposite (P<0.05). Radiometric enzyme activity assays demonstrated different metabolism patterns of E1S and oestrone (E1) between normal and malignant cells, indicating overall activities of STS and 17βHSD1 or 17βHSD5 to be higher than the overall activities of EST and 17βHSD2 in cancer cells while enzyme activities in OSE cells were opposite to this. Second, the impact of inflammation on oestrogen production, metabolism and action was compared in OSE and EOC cells by testing the response of target genes to a panel of pro-inflammatory cytokines. The data revealed that in OSE cells, EST (P<0.01) and 17βHSD2 (P<0.001) mRNAs were decreased while ERα mRNA (P<0.001) was increased by IL-1α. In addition, EST mRNA was inhibited by IL-4 (P<0.05). In SKOV-3 (EOC cell line) cells, IL-1α stimulated STS mRNA (P<0.001)and enzyme activity (P<0.05). Moreover, IL-4 inhibited (P<0.05) while IL-8 and IL- 10 enhanced (P<0.01) ERα mRNA levels. Finally, the effect of oestrogenic components of HRT medication (equilin and equilin-sulphate) on the expression of cancer-associated genes was compared to that of 17β-oestradiol (E2) in PEO-1 (an oestrogen-responsive EOC cell line) cells. Expression of the oestrogen-responsive genes FN1 and IGFBP3 mRNA expression was similarly inhibited by E2 and equilin (P<0.05) as well as E1S and sodium equilin-sulphate (P<0.05). In conclusion, this thesis presents evidence that intracrine oestrogen formation and metabolism differs between OSE and EOC cells, such that E2 formation is inhibited in normal OSE but is promoted in EOC. Inflammatory cytokines also influence the local production of E2 by regulating genes encoding oestrogen production and metabolism and receptors. Finally, local HRT metabolites can regulate cancer-associated gene expression in EOC. Together, these data suggest a role for local oestrogen production and action in inflammation-associated development of EOC. Conversely, differential regulation of the same parameters in OSE cells from premenopausal women minimizes oestrogen formation and ‘protects’ against the promotion of EOC.

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In Canada, ovarian cancer kills about 67% of diagnosed patients, largely due to difficulties in early diagnosis. Current treatment consists of debulking surgery and intermittent chemotherapy every three weeks. This approach leads to insufficient drug concentrations at disease sites, and long treatment-free intervals cause accelerated tumor proliferation and drug resistance, resulting in a 5-year survival rate of only 25-35%. Drug resistance development is the ultimate cause of the majority of patient deaths. Improvements yielding more effective treatment are fundamental for successful management of this disease. This thesis investigated a continuous chemotherapy strategy devoid of treatment-free intervals for ovarian cancer treatment. A biocompatible, biodegradable polymer-lipid injectable formulation PoLigel, was used for continuous DTX delivery. The formulation was well tolerated; no alterations in body weight, behaviour, histology of peritoneal tissues, or interleukin-6 levels were seen in CD-1 mice treated with the PoLigel. Continuous DTX therapy via the PoLigel was considerably more efficacious than intermittent therapy, resulting in significantly less tumor burden and ascites fluid in models of human and murine ovarian cancer. Continuous therapy resulted in less tumor cell proliferation and angiogenesis, and more tumor cell death than intermittent DTX. The presence and length of treatment-free intervals was shown to contribute to the development of drug resistance. Eliminating these intervals by continuous dosing resulted in superior antitumor efficacy in both chemosensitive and chemoresistant xenograft models of human ovarian cancer, and prevented drug resistance increase after a 21-day treatment period. Survival studies revealed that intermittent dosing led to a mild survival prolongation of 36% and 10% in chemosensitive and chemoresistant models, respectively, whereas continuous DTX prolonged survival by a striking 114% and 95%. Although long-term continuous chemotherapy substantially improved survival, increased drug resistance mechanisms were found at the endpoint. Overall, results presented here encourage the clinical implementation of continuous chemotherapy due to greater achievable therapeutic advantages.

In Canada, ovarian cancer kills about 67% of diagnosed patients, largely due to difficulties in early diagnosis. Current treatment consists of debulking surgery and intermittent chemotherapy every three weeks. This approach leads to insufficient drug concentrations at disease sites, and long treatment-free intervals cause accelerated tumor proliferation and drug resistance, resulting in a 5-year survival rate of only 25-35%. Drug resistance development is the ultimate cause of the majority of patient deaths. Improvements yielding more effective treatment are fundamental for successful management of this disease. This thesis investigated a continuous chemotherapy strategy devoid of treatment-free intervals for ovarian cancer treatment. A biocompatible, biodegradable polymer-lipid injectable formulation PoLigel, was used for continuous DTX delivery. The formulation was well tolerated; no alterations in body weight, behaviour, histology of peritoneal tissues, or interleukin-6 levels were seen in CD-1 mice treated with the PoLigel. Continuous DTX therapy via the PoLigel was considerably more efficacious than intermittent therapy, resulting in significantly less tumor burden and ascites fluid in models of human and murine ovarian cancer. Continuous therapy resulted in less tumor cell proliferation and angiogenesis, and more tumor cell death than intermittent DTX. The presence and length of treatment-free intervals was shown to contribute to the development of drug resistance. Eliminating these intervals by continuous dosing resulted in superior antitumor efficacy in both chemosensitive and chemoresistant xenograft models of human ovarian cancer, and prevented drug resistance increase after a 21-day treatment period. Survival studies revealed that intermittent dosing led to a mild survival prolongation of 36% and 10% in chemosensitive and chemoresistant models, respectively, whereas continuous DTX prolonged survival by a striking 114% and 95%. Although long-term continuous chemotherapy substantially improved survival, increased drug resistance mechanisms were found at the endpoint. Overall, results presented here encourage the clinical implementation of continuous chemotherapy due to greater achievable therapeutic advantages.

Clinical information was collected from 283 randomly chosen ovarian cancer cases from at the Saskatoon Cancer Centre between the years 1983-1995. The data was evaluated for its significance in predicting survival and relapse free survival (RFS) using univariate and multivariate analysis. Several clinical prognostic factors were identified by univariate analysis. Additionally, using Cox's regression model the independent markers of survival and RFS were FIGO stage, and residual disease in 173 and 178 patients respectively. Data on CA 125 serum level, (available in 89 patients) was a marker of prognostic significance in the patients treated with platinum based chemotherapy. CA 125 and CEA antigen expression were also evaluated in seventy one cases. It was found that mucinous neoplasms exclusively expressed CEA antigen. This study indicates that the evaluation of serum level CEA may be a complementary tool for patients with cancers not expressing CA 125. In this retrospective study, DNA from paraffin embedded tissue (PET) in patients with ovarian carcinoma was examined to identify gene abnormalities in p53, p16INK4A, RB-1, p21WAF1/CIP1, Cyclin D1, Erb-B2, and MSH2. Adverse outcome was also examined in addition to survival and RFS, to identify novel molecular prognostic markers. P53 overexpression in 44 of 112 (39%) was associated with reduced survival and RFS (' p' = '0.04' and 'p' = '0.008 '). Aneuploid DNA content, found in 34 of 112 (30%) cases, was associated with shorter survival and RFS ('p' = '0.03' and 'p' = '0.01'). Dot blot hybridization of G1-S control genes (p16INK4A, Cyclin D1, RB-1, and CDK4) did not identify amplification or deletion events to be associated with adverse outcome. A number of gene alterations in 59 of 63 (94%) ovarian cancer cases were detected by dot blot hybridization; the lack of association with clinical outcome indicated that there may be some other genes in addition to those examined that are of prognostic significance. For eighteen cases, microsatellite instability (MSI) was evaluated by using fluorescently labeled primers at nine loci. LOH was a common event in ovarian carcinoma but MSI was infrequent. Molecular and clinical marker multivariate analysis indicated: (a) residual disease for survival, (b) stage and residual disease for RFS, were independent markers of prognosis.

Ovarian cancer is the leading cause of death among all gynecological disorders. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in this malignancy. Several sialyltransferase genes have been shown to be up-regulated at both mRNA and and protein levels in a number of cancers, including that of the ovary. In the present study, we have analyzed the glycosylation patterns of kallikrein 6 in the context of ovarian cancer. We have discovered that the carbohydrate structures found at the single N-glycosylation site of kallikrein 6 derived from ovarian cancer cells found in the ascites fluid of ovarian cancer patients is enriched in sialic acid moieties and has an increased branching pattern when compared to controls. We have also developed a reliable anion-exchange HPLC-based methodology capable of quantifying different glycoform subpopulations of kallikrein 6 in serum and other biological fluids, which was capable of differentiating between samples from ovarian cancer patients and healthy controls. A variety of classic molecular biology and mass spectrometry based techniques were utilized in these experiments. Based on the results of the analysis of kallikrein 6 glycosylation and other literature reports showing upregulated sialylation of proteins in ovarian cancer, we have also identified sialylated glycoproteins from ovarian cancer proximal fluids and conditioned media of ovarian cancer cell lines. Sialylated proteins were enriched utilizing lectin affinity or hydrazide chemistry. In total, 333 sialylated glycoproteins and 579 glycosylation sites were identified. A list of 21 potential candidate ovarian cancer biomarkers was produced from proteins that were identified solely in ovarian cancer proximal fluids, which could form the basis for any future studies.

Ovarian cancer is the leading cause of death among all gynecological disorders. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in this malignancy. Several sialyltransferase genes have been shown to be up-regulated at both mRNA and and protein levels in a number of cancers, including that of the ovary. In the present study, we have analyzed the glycosylation patterns of kallikrein 6 in the context of ovarian cancer. We have discovered that the carbohydrate structures found at the single N-glycosylation site of kallikrein 6 derived from ovarian cancer cells found in the ascites fluid of ovarian cancer patients is enriched in sialic acid moieties and has an increased branching pattern when compared to controls. We have also developed a reliable anion-exchange HPLC-based methodology capable of quantifying different glycoform subpopulations of kallikrein 6 in serum and other biological fluids, which was capable of differentiating between samples from ovarian cancer patients and healthy controls. A variety of classic molecular biology and mass spectrometry based techniques were utilized in these experiments. Based on the results of the analysis of kallikrein 6 glycosylation and other literature reports showing upregulated sialylation of proteins in ovarian cancer, we have also identified sialylated glycoproteins from ovarian cancer proximal fluids and conditioned media of ovarian cancer cell lines. Sialylated proteins were enriched utilizing lectin affinity or hydrazide chemistry. In total, 333 sialylated glycoproteins and 579 glycosylation sites were identified. A list of 21 potential candidate ovarian cancer biomarkers was produced from proteins that were identified solely in ovarian cancer proximal fluids, which could form the basis for any future studies.