The role of cancer procoagulant on the MTOR pathway
- Authors: Chiuswa, Chengetanai
- Date: 2016
- Subjects: Cancer -- Research , Neovascularization , Biochemistry , Blood coagulation factors -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/7303 , vital:21316
- Description: Cancer procoagulant (CP) is a cysteine protease found in tumour cells and amnion chorion membranes. The main function of CP is not yet known, but it has potential roles in tumour growth and metastasis. Initially, CP was believed to increase coagulation in cancer patients; however, research has shown that increase in CP concentration does not correlate with an increase in coagulation. The location of CP in amnion chorion membranes and tumour cells only led to the hypothesis that CP might be involved in inducing blood vessel and or lymph vessel formation. CP was shown to induce lymphangiogenesis (lymph vessel formation) in human telomerase reverse transcriptase-human dermal endothelial cells (hTERT-HDLEC) (Tshaka, 2011). CP-induced tube formation was inhibited by rapamycin; indicating that CP may be signalling via the mammalian target of rapamycin (mTOR) pathway. The aim of this study was to investigate the effect of CP on the mTOR signalling pathway using human umbilical endothelial vein cells (HUVECs) as a model. CP was isolated from amnion chorion membranes and purified using two anion exchange chromatography steps. Purified CP (2 μg/ml) was used to induce tube formation in endothelial cells (HUVECs) seeded on growth factor reduced (GFR) Matrigel. In addition, the 2 μg/ml CP was used to treat cultured HUVECs. Sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) and western blotting were used to determine phosphorylation levels of protein kinase B (Akt) and ribosomal protein S6 kinase (S6K). CP was successfully isolated and purified using anion exchange chromatography. The effect of CP on tube formation was not significant relative to the control in the HUVEC cell line. The role of CP on Akt and S6K phosphorylation still needs to be verified by using sensitive methods of quantification such as enhanced chemiluminescence (ECL) and enzyme linked immunosorbent assay (ELISA). The levels of CP activity were shown to be higher in early tumour growth than in advanced cancer suggesting that certain physiological factors could be increasing CP activity during early tumour growth. This study investigated the effect of cobalt chloride on CP activity in breast cancer cell lines. Cobalt chloride reduced CP activity in MCF-7 and promoted CP activity in MDA-MB-231.
- Full Text:
- Date Issued: 2016
- Authors: Chiuswa, Chengetanai
- Date: 2016
- Subjects: Cancer -- Research , Neovascularization , Biochemistry , Blood coagulation factors -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/7303 , vital:21316
- Description: Cancer procoagulant (CP) is a cysteine protease found in tumour cells and amnion chorion membranes. The main function of CP is not yet known, but it has potential roles in tumour growth and metastasis. Initially, CP was believed to increase coagulation in cancer patients; however, research has shown that increase in CP concentration does not correlate with an increase in coagulation. The location of CP in amnion chorion membranes and tumour cells only led to the hypothesis that CP might be involved in inducing blood vessel and or lymph vessel formation. CP was shown to induce lymphangiogenesis (lymph vessel formation) in human telomerase reverse transcriptase-human dermal endothelial cells (hTERT-HDLEC) (Tshaka, 2011). CP-induced tube formation was inhibited by rapamycin; indicating that CP may be signalling via the mammalian target of rapamycin (mTOR) pathway. The aim of this study was to investigate the effect of CP on the mTOR signalling pathway using human umbilical endothelial vein cells (HUVECs) as a model. CP was isolated from amnion chorion membranes and purified using two anion exchange chromatography steps. Purified CP (2 μg/ml) was used to induce tube formation in endothelial cells (HUVECs) seeded on growth factor reduced (GFR) Matrigel. In addition, the 2 μg/ml CP was used to treat cultured HUVECs. Sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) and western blotting were used to determine phosphorylation levels of protein kinase B (Akt) and ribosomal protein S6 kinase (S6K). CP was successfully isolated and purified using anion exchange chromatography. The effect of CP on tube formation was not significant relative to the control in the HUVEC cell line. The role of CP on Akt and S6K phosphorylation still needs to be verified by using sensitive methods of quantification such as enhanced chemiluminescence (ECL) and enzyme linked immunosorbent assay (ELISA). The levels of CP activity were shown to be higher in early tumour growth than in advanced cancer suggesting that certain physiological factors could be increasing CP activity during early tumour growth. This study investigated the effect of cobalt chloride on CP activity in breast cancer cell lines. Cobalt chloride reduced CP activity in MCF-7 and promoted CP activity in MDA-MB-231.
- Full Text:
- Date Issued: 2016
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
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