Ovarian cancers are the second most common female cancer in the UK. In the majority of patients there are no observable symptoms of ovarian cancer at early stage and no effective screening strategy to detect cancer before metastasis. Moreover, chemo-resistance is another problem which hinders the successful treatment of ovarian cancer. Therefore, discovery of novel anti-ovarian cancer drugs remains critically important. Snake venoms contain different proteins, peptides, enzymes and low molecular weight components that have been investigated for development of new therapies to treat many diseases including cancer. The present study was carried out to evaluate the effect of Crotalus ruber ruber and Agkistrodon piscivorus leucostoma snake venoms both from the Viperidae family on dissemination and processes of metastasis of an ovarian cancer cell line (A2870). The first part of the project involved examination of changes to cell morphology, cytotoxicity, migration, invasion and the effect of the crude venom on levels of the main integrins involved in cell adhesion. Cell morphology differences were observed with both venoms; Crotalus ruber ruber crude venom caused the cells to round up so that they were poorly spread at 1.5 and 3.1μg/ml, while, Agkistrodon piscivorus leucostoma venom caused the cells to detach from the growing surface at 0.7, 1.5, and 3.1μg/ml. An AlamarBlue® Cell Viability Assay and SYTOX®Green (nucleic acid stain) were used to test the viability of the cells following treatment and to demonstrate that the morphological alterations were not due to a cytotoxic effect, but to anti-adhesive activity. Moreover, an integrin-independent substratum (poly-Llysine)was used to determine if the venom is specific for the integrin family of adhesion molecules or not. As a result, the effect of Crotalus ruber ruber venom on adhesion was found to be specific for the integrin family of adhesion receptors, while the effect of Agkistrodon piscivorus leucostoma venom on adhesion was not specific for the integrin family. Both venoms had an inhibitory effect on migration and invasion of the cells by inhibiting α5, β1integrins (fibronectin receptor) for Crotalus ruber ruber venom and α5, β1integrins and αv integrins (fibronectin and vitronectin receptor) for Agkistrodon piscivorus leucostoma venom. The next part of the project was to semi-purify both venoms. This was achieved using gel chromatography to obtain faction 3 (F3) from Crotalus ruber ruber venom and F6 from Agkistrodon piscivorus leucostoma venom. The assays carried out with the crude venoms were repeated using the fractions instead. F3 of Crotalus ruber ruber venom and F6 of Agkistrodon piscivorus leucostoma venom were found to be the fractions that contained components responsible for the activity of the venoms. F3 had an inhibitory effect on the level of expression of α5, β1 integrins while F6 had an inhibitory effect on α5, β1 and on αv integrins. Moreover, it was proposed that F6 had hydrolytic activity against the extracellular matrix. Finally preliminary analysis was carried out on F3 and F6 using SDS-PAGE, Nanoflow HPLC Electrospray Tandem Mass Spectrometry, liquid chromatography mass spectrometry and metalloproteinase inhibitor studies to elucidate the peptides as rubelysin for F3 and leucostoma peptidase A for F6. These results suggest that rubelysin and leucostoma peptidase A have antimetastatic activity against ovarian cancer.
|Date of Award||2 Jun 2017|
- University Of Strathclyde
|Supervisor||Valerie Ferro (Supervisor) & Edward Rowan (Supervisor)|