Elucidating the Modes of Action of the Antitumour New Zealand Marine Natural Product Pterocellin A
Wang, A. T. (2015). Elucidating the Modes of Action of the Antitumour New Zealand Marine Natural Product Pterocellin A (Thesis, Master of Science (Research) (MSc(Research))). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/9620
Permanent Research Commons link: http://hdl.handle.net/10289/9620
Pterocellin A is a novel bioactive alkaloid isolated from the New Zealand marine bryozoan Pterocella vesiculosa. It exhibits potent antitumour activity towards the P388 (murine leukaemia) cell line in vitro with an IC₅₀ value of 477 ng/mL. Further screening at the NCI indicated that it was selectively sensitive towards certain non-small cell lung, melanoma, and breast cancer cell lines, however, the biological mode of action of pterocellin A is unknown. This project investigated and elucidated the cytotoxicity of pterocellin A using the human cervical cancer cell line HeLa. Cellular toxicity can be manifested as either necrotic or apoptotic cell death. Cancer cells are prone to resisting apoptosis, and one of the key mechanisms recognised in anticancer agents is the induction of apoptosis. Pterocellin A exhibited similar cytotoxicity against the HeLa cell line compared to P388 cells with an IC₅₀ value of 886 ng/mL. In this study, HeLa cells were incubated with various concentrations (up to 2000 ng/mL) of pure pterocellin A, and characteristic markers of apoptotic and necrotic cell death were investigated using several in vitro biochemistry techniques. Assessments were made on the morphological and biochemical changes in HeLa cells after treatment with pterocellin A. Time-course MTT and LDH assays were carried out and the results showed that only a low level of cytosolic LDH was detected in the supernatant after all the cells had died from pterocellin A treatment at 2000 ng/mL. This indicated the cells maintained membrane integrity upon death which suggested apoptosis. Additionally, morphological changes were observed under the microscope after six hours of treatment. Cell shrinkage and nucleus condensation were observed, as well as apparent membrane blebbing, a key feature of apoptosis. The MTT data were also indicative of mitochondria impairment which could suggest that pterocellin A targets the mitochondria. This idea was supported by the observed changes in the morphology and location of the mitochondria after exposure to pterocellin A. Furthermore, the level of activated caspase-3 in HeLa cells increased after treatment with pterocellin A; activated caspase-3 can only be detected after a series of signalling events following the induction of apoptosis. These data support the notion that pterocellin A is an inducer of apoptosis in HeLa cells. This project also utilised the biochemical assays to develop a systematic bioassay screening system at the University of Waikato, and further investigated the bioactive metabolites of P. vesiculosa. The same approach taken to study pure pterocellin A in HeLa cells was successfully applied to the bioassay development. The bioactive fractions separated from the crude extract were analysed by LC-MS and several unknown metabolites were observed, however due to time constraints associated with the project, no further investigation was carried out.
University of Waikato
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