Isolation, purification and partial characterisation of cancer procoagulant from placental amnion-chorion membranes and its role in angiogenesis inflammation and metastasis
- Authors: Krause, Jason
- Date: 2014
- Subjects: Coagulation , Amnion , Chorion , Metastasis , Inflammation , Neovascularization
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10350 , http://hdl.handle.net/10948/d1020897
- Description: Cancer procoagulant (EC 3.4.22.26) is an enzyme that is derived from tumour and foetal tissue, but not normal tissue. It is a direct activator of factor X and has been isolated from amnion-chorion membranes as well as from extracts and cells from human melanoma. The presence of cancer procoagulant has been associated with the malignant phenotype, as well as having a particularly high activity in metastatic cells. Cancer procoagulant activity is elevated in the serum of early stage breast cancer patients and decreased to normal in the advanced stages of the disease. In this study, cancer procoagulant was successfully isolated from amnion-chorion membranes and purified to homogeneity. The molecular weight of cancer procoagulant was determined using SDS-PAGE and was found to be 68 kDa. Cancer procoagulant was delipidated and it was shown that its activity was increased by the presence of lipids in a dose-dependent manner. Recovery of cancer procoagulant after delipidation is poor, consequently, a larger mass of sample is required to obtain sufficient amounts of delipidated material for N-terminal amino acid analysis. The optimum pH of cancer procoagulant was determined to be pH 8 and its optimal temperature was found to be 50°C. Novel synthetic substrates were designed to assay for cancer procoagulant activity. Currently, 2 potential candidates have been identified, namely, PQVR-AMC and AVSQSKP-AMC. Cancer procoagulant-induced expression of cytokines is differently modulated in the less aggressive MCF-7 cell line as compared to the metastatic and more aggressive MDA-MB-231 cell line. There are marked similarities in the inflammatory response produced by cancer procoagulant in hTERT-HDLEC and MDA-MB-231 cells, which are both associated with migratory capacity. Furthermore, cancer procoagulant-induced PDGF-β expression in hTERT-HDLEC and MDA-MB-231 cells could point to involvement of cancer procoagulant in wound healing and metastatic spread, respectively. Cancer procoagulant induced the motility of MDA-MB-231, MCF-7 and hTERT- cells in vitro in a time- and dose-dependent manner. Together, these results suggest that cancer procoagulant plays a role in the migration of breast cancer cells as well as the migration of endothelial cells.
- Full Text:
- Date Issued: 2014
- Authors: Krause, Jason
- Date: 2014
- Subjects: Coagulation , Amnion , Chorion , Metastasis , Inflammation , Neovascularization
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10350 , http://hdl.handle.net/10948/d1020897
- Description: Cancer procoagulant (EC 3.4.22.26) is an enzyme that is derived from tumour and foetal tissue, but not normal tissue. It is a direct activator of factor X and has been isolated from amnion-chorion membranes as well as from extracts and cells from human melanoma. The presence of cancer procoagulant has been associated with the malignant phenotype, as well as having a particularly high activity in metastatic cells. Cancer procoagulant activity is elevated in the serum of early stage breast cancer patients and decreased to normal in the advanced stages of the disease. In this study, cancer procoagulant was successfully isolated from amnion-chorion membranes and purified to homogeneity. The molecular weight of cancer procoagulant was determined using SDS-PAGE and was found to be 68 kDa. Cancer procoagulant was delipidated and it was shown that its activity was increased by the presence of lipids in a dose-dependent manner. Recovery of cancer procoagulant after delipidation is poor, consequently, a larger mass of sample is required to obtain sufficient amounts of delipidated material for N-terminal amino acid analysis. The optimum pH of cancer procoagulant was determined to be pH 8 and its optimal temperature was found to be 50°C. Novel synthetic substrates were designed to assay for cancer procoagulant activity. Currently, 2 potential candidates have been identified, namely, PQVR-AMC and AVSQSKP-AMC. Cancer procoagulant-induced expression of cytokines is differently modulated in the less aggressive MCF-7 cell line as compared to the metastatic and more aggressive MDA-MB-231 cell line. There are marked similarities in the inflammatory response produced by cancer procoagulant in hTERT-HDLEC and MDA-MB-231 cells, which are both associated with migratory capacity. Furthermore, cancer procoagulant-induced PDGF-β expression in hTERT-HDLEC and MDA-MB-231 cells could point to involvement of cancer procoagulant in wound healing and metastatic spread, respectively. Cancer procoagulant induced the motility of MDA-MB-231, MCF-7 and hTERT- cells in vitro in a time- and dose-dependent manner. Together, these results suggest that cancer procoagulant plays a role in the migration of breast cancer cells as well as the migration of endothelial cells.
- Full Text:
- Date Issued: 2014
The effects of extracellular and intracellular Hop on cell migration processes
- Authors: Contu, Lara
- Date: 2014
- Subjects: Heat shock proteins , Metastasis , Cancer Chemotherapy , Molecular chaperones , Cell migration
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193961 , vital:45410
- Description: The Hsp70/Hsp90-organising protein (Hop) is a 60 kDa co-chaperone that acts as an adaptor molecule, facilitating the transfer of client proteins between the Hsp70 and Hsp90 chaperone systems. Hop functions both intracellularly and extracellularly and has been implicated in many processes involved in cancer progression, including cell migration and invasion. Little is known about the mechanisms or domains by which extracellular Hop functions. In addition, little is known about the effects of Hop on signalling molecules involved in cell migration and invasion through regulation of actin dynamics. It was hypothesised that both extracellular and intracellular pools of Hop would regulate distinct cell migration processes by activation of cell signalling pathways or direct interactions with signalling intermediates. HS578T cells were treated with recombinant full length and truncated murine Hop proteins (overexpressed and purified in this study) to determine the effects of extracellular Hop and the independent domains on cell migration processes. Additionally, RNA interference (RNAi) techniques were used to determine the effect of Hop knockdown on cell migration related signalling intermediates and cell morphologies. A short hairpin RNA (shRNA) system for the stable knockdown of Hop was developed and used for a number of these studies. Treatment of HS578T cells with the TPR2A2B and TPR1 domains of Hop resulted in a significant decrease in cell migration and caused changes in the actin cytoskeleton and extracellular matrix proteins, gelatin and fibronectin. RhoC immunoprecipitated in a common complex with Hop and Hsp90. Hop knockdown reduced levels of actin and total RhoC, as well as active RhoC. In addition, knockdown of Hop resulted in a reduced migratory phenotype. We interpreted these data to indicate that intracellular Hop played a role in cell migration through regulation of RhoC activity, either through a direct interaction between Hop and RhoC, or an indirect interaction of RhoC with the Hsp90 multichaperone heterocomplex. Taken together, the data suggested that extracellular and intracellular Hop played distinct roles in extracellular and intracellular processes that lead to actin dynamics and cell migration. Understanding the mechanistic role of Hop in these processes is essential as it would aid in assessing the viability of Hop as a potential drug target for the treatment of metastatic cancers. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2014
- Full Text:
- Date Issued: 2014
- Authors: Contu, Lara
- Date: 2014
- Subjects: Heat shock proteins , Metastasis , Cancer Chemotherapy , Molecular chaperones , Cell migration
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193961 , vital:45410
- Description: The Hsp70/Hsp90-organising protein (Hop) is a 60 kDa co-chaperone that acts as an adaptor molecule, facilitating the transfer of client proteins between the Hsp70 and Hsp90 chaperone systems. Hop functions both intracellularly and extracellularly and has been implicated in many processes involved in cancer progression, including cell migration and invasion. Little is known about the mechanisms or domains by which extracellular Hop functions. In addition, little is known about the effects of Hop on signalling molecules involved in cell migration and invasion through regulation of actin dynamics. It was hypothesised that both extracellular and intracellular pools of Hop would regulate distinct cell migration processes by activation of cell signalling pathways or direct interactions with signalling intermediates. HS578T cells were treated with recombinant full length and truncated murine Hop proteins (overexpressed and purified in this study) to determine the effects of extracellular Hop and the independent domains on cell migration processes. Additionally, RNA interference (RNAi) techniques were used to determine the effect of Hop knockdown on cell migration related signalling intermediates and cell morphologies. A short hairpin RNA (shRNA) system for the stable knockdown of Hop was developed and used for a number of these studies. Treatment of HS578T cells with the TPR2A2B and TPR1 domains of Hop resulted in a significant decrease in cell migration and caused changes in the actin cytoskeleton and extracellular matrix proteins, gelatin and fibronectin. RhoC immunoprecipitated in a common complex with Hop and Hsp90. Hop knockdown reduced levels of actin and total RhoC, as well as active RhoC. In addition, knockdown of Hop resulted in a reduced migratory phenotype. We interpreted these data to indicate that intracellular Hop played a role in cell migration through regulation of RhoC activity, either through a direct interaction between Hop and RhoC, or an indirect interaction of RhoC with the Hsp90 multichaperone heterocomplex. Taken together, the data suggested that extracellular and intracellular Hop played distinct roles in extracellular and intracellular processes that lead to actin dynamics and cell migration. Understanding the mechanistic role of Hop in these processes is essential as it would aid in assessing the viability of Hop as a potential drug target for the treatment of metastatic cancers. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2014
- Full Text:
- Date Issued: 2014
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