Author: Mahnaz Ramezanpour
Ramezanpour, Mahnaz, 2015 Characterisation of Anti-Cancer Properties of Bioactive Compounds in Sea Anemone Venom, Flinders University, School of Health Sciences
This electronic version is made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact email@example.com with the details.
The potential of crude venom extracts, obtained by the milking technique from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor), to kill cancer cells was tested on three human cancer cell lines (A549 lung cancer; T47D breast cancer and A431 skin cancer). The level of cytotoxicity depended on which sea anemone venom extract was used and which cancer cell line was tested. The study then focused on Heteractis magnifica as its venom extract had significant inhibitory effects on the three cancer cell lines tested in the initial study. The H. magnifica venom displayed potent cytotoxic activity against human lung cancer A549 cells, with less effect on the survival of MRC5 human non-cancer lung cell line. This was evident by higher IC50 values (i.e. 18.17 µg/ml on the MRC5 cell line compared to 11.14 µg/ml on the A549 cancer cell line). Heteractis magnifica venom down-regulated cell cycle progression and induced apoptosis through the activation of caspases and mitochondrial membrane pathways in the A549 cancer cell line. Conversely, apoptotic cell death was not observed for MRC5 cells; instead, the cell death that occurred was by necrosis. Furthermore, H. magnifica venom significantly killed human adherent breast cancer cells T47D and MCF7. In contrast, an equivalent concentration of the venom exerted a lower effect on the survival of the 184B5 human non-cancer origin breast cell line. This was evident after 24 hours’ (24h) treatment, with a higher IC50 value of 14.70 µg/ml on 184B5 (compared to values of 9.26 µg/ml on MCF7 and 5.67 µg/ml on T47D). The venom induced cell cycle arrest in T47D and MCF7 cell lines by apoptosis, through the activation of caspases and mitochondrial membrane pathways. The crude venom was purified using size exclusion chromatography and mass spectrophotometry, and the amino acid sequence was partially determined. To provide sufficient material for functional investigation, recombinant protein was produced in a prokaryotic expression system and purified by affinity column. The peptide’s cytotoxicity was evaluated by the MTT assay. The peptide decreased the survival of A549, T47D and MCF7 cancer cell lines. Conversely, an identical concentration of the peptide had significantly less effect on the survival of 184B5 cells. This was evident from higher IC50 values of 10.28 µg/ml on 184B5 (compared to 5.28 µg/ml on MCF7 and 6.97 µg/ml on T47D) after 24h treatment. The IC50 values for T47D and MCF7 (compared to values for 184B5) show support for selectivity against breast cancer cells. In addition, the IC50 value of T47D was much lower than published values for paclitaxel, the most common commercial drug currently used for breast cancer. These results clearly demonstrate that the purified recombinant peptide could be an excellent candidate for T47D breast cancer cell line if resistant to paclitaxel. The novel peptide identified has the potential for therapeutic development. It is able to decrease the survival of breast cancer cells in a dose-dependent manner, whereby the dose that targets and kills cancer cells has significantly less impact on non-cancer breast cells.
Keywords: Cancer cell lines, Sea anemone, H. magnifica, Apoptosis, Caspases, Purification crude venom
Subject: Health Sciences thesis, Medical Biochemistry thesis
Thesis type: Doctor of Philosophy
School: School of Health Sciences
Supervisor: Barbara Sanderson