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The Antitumor Activity of a Novel Diterpene Ester Maria-Anna

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The Antitumor Activity of a Novel Diterpene Ester Maria-Anna The Antitumor Activity of a Novel Diterpene Ester Maria-Anna Marjorie Agnela D’Souza Master of Science (MSc) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 School of Medicine Abstract ________________________________________________________________________________ Abstract Previous work in this laboratory has shown that topical application of a protein kinase C (PKC) activator (the ingenol ester PEP005) cured subcutaneous tumors in mice with a favourable cosmetic outcome. Efficacy involved a neutrophil-dependent host inflammatory response; characteristic of diterpene esters that activate PKC. The aim of this study was to explore the antitumor efficacy and mechanism of action of EBC-46, a novel diterpene ester of the tigliane (phorbol) class, isolated by QBiotics Pty Ltd from a native rainforest plant in North Queensland, Australia, when given by intratumoral injection rather than by topical application. Preliminary work with EBC-46 in vivo resulted in successful treatment of tumors via intratumoral injection, showing outcomes similar to topical application of PEP005. Intralesional therapy bypasses the barrier zone and establishes a sub-epidermal depot, and, depending on the mode of action, may minimize the circulating level of the drug. EBC-46 activated protein kinase C (PKC), as shown by the translocation of PKC isoforms to the plasma membrane of HeLa. Here, PKC can phosphorylate other proteins involved in signal transduction of cellular activities such as growth, differentiation, migration, proliferation and inflammatory responses. TPA, a prototype PKC activator, was used for in vitro and in vivo experiments as a positive control. A single intratumoral injection of EBC-46 resulted in a local inflammatory response and ablation of tumors in human xenograft (melanoma, HNSCC) and mouse tumors (B16-F0, MC38) in immunocompromised (BALB/c Foxn1nu) and immunocompetent (C57BL6/J) mice respectively. The cure rate was > 75% and there were no recurrences over a period of up to 12 months. Compared with the sub-microgram per ml IC50 levels of current anticancer drugs, the cytotoxic effect of EBC-46 was minimal in most tumor lines in vitro, requiring doses of 100-300 µg/ml to stop growth. IC50 levels in the region of 100 ng/ml however could be achieved in the same cell lines by over-expression of PKC-α and -δ. EBC-46 had minimal effect on the cell cycle, but caused phosphorylation of ERK 1/2. It was inferred that the mechanism of action was co-dependent on the host response to the drug, thus further studies focused on the host. Tumors exhibited an inflamed appearance within 1-2 hr of injection, changing to greenish black color by 24 hr, typical of a severe bruise. Indurations were formed at the now-flattened site, and ________________________________________________________________________________ pg. - 1 - Abstract ________________________________________________________________________________ resolved over the next 2 weeks with sloughing of the scab, resulting in cure and excellent healing of the skin. A key finding in this project, emerging from the novel approach of ex vivo culture of treated tumor sites, was that no clonogenic tumor cells remained by 4 hr after injection of EBC-46; and significant loss of such cells even within 1 hr. This established a short time window for drug action and cell kill, and focused subsequent studies of mechanism. Immunohistochemical analysis confirmed the clinical observations, revealing loss of capillaries and the endothelial cell marker PECAM, tumor cell necrosis, and presence of neutrophils. EBC-46 induced the production of the pro-inflammatory cytokines IL-1β, TNF-α and IL-8 in tumor cells, dependent on activation of PKC isoforms. Global gene expression of treated tumor sites showed rapidly changing profiles but no dominant cell signaling pathway, with no RNA recoverable after 8 hr. The target tumor cells, analyzed with human-specific chips, showed .>10-fold elevation of 3 SNORD RNA sequences at the early time points, non-coding RNA sequences involved in regulation of gene expression. The antitumor role of this striking finding, if any, is yet to be determined but may reflect a novel, possibly EBC-46-specific cellular response to hemorrhagic necrosis. Such changes were not found using TPA, and numerous other differences in gene expression caused by the two drugs in vivo were observed. The host response at tumor sites, analyzed with murine- specific chips, also showed no single significant signaling pathway but induction of some proinflammatory markers was found. A more controlled in vitro expression profiling of human lymphocytes also revealed induction of proinflammatory markers by EBC-46, some but not all of which were paralleled by release of cytokine proteins. EBC-46 was therefore distinguished from most current anticancer agents by triggering a rapid and tumor-destructive host response at and around the tumor site, with no discernible adverse effects. Tumor ablation was associated with local inflammation, hemorrhagic necrosis, and cellular effects consistent with PKC activation in both tumor and host cells. Intratumoral injection of EBC-46 has been successfully trialed by QBiotics in a range of cutaneous and subcutaneous tumors in more than 250 companion animals (dogs, cats and horses). The macroscopic changes at the tumor site were similar to those found in mouse tumors in this study. The evidence overall suggests that EBC-46 may find use as a palliative treatment, local disease control or adjuvant therapy preceding surgery or radiation. Ongoing advances in drug delivery and imaging technologies may facilitate intratumoral administration to less accessible sites. ________________________________________________________________________________ pg. - 2 - Declaration by Author ________________________________________________________________________________ Declaration by Author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. ________________________________________________________________________________ pg. - 3 - List of Publications ________________________________________________________________________________ Publications during candidature Boyle, G.M*., D’Souza, M.M.A*., Pierce, C.J., Adams, R.A., Cantor, A.S., Johns, J.P., Maslovskaya, L., Gordon, V.A., Reddell, P.W. and Parsons, P.G. (2014) “Intra-lesional injection of the novel PKC activator EBC-46 rapidly ablates tumors in mouse models.” PLoS One. DOI:10.1371/journal.pone.0108887 *equal contribution Publications included in this thesis: No publications. ________________________________________________________________________________ pg. - 4 - Contributions to the thesis ________________________________________________________________________________ Contributions by others to this thesis Dr. Glen Boyle provided the melanoma cell lines over-expressing PKC isoforms –α and –δ and empty vector control lacZ used in Chapters Three and Four and gave guidance on microarray analysis in Chapter Six. Carly Chapman transiently transfected HeLa cells with EGFP-tagged PKC isoforms for PKC isoforms translocation studies in Chapter Three. Jenny Johns and Lidia Maslovskaya isolated and purified EBC-46 from the kernel of the Blushwood plant. Statement of parts of the thesis submitted to qualify for the award of another degree None ________________________________________________________________________________ pg. - 5 - ________________________________________________________________________________ ________________________________________________________________________________ pg. - 6 - Acknowledgements ________________________________________________________________________________ Acknowledgements Over the period of my PhD research years, there have been several people who have immensely contributed to the completion of my thesis. In the first place, I owe a debt of gratitude to my principle supervisor Professor Peter Parsons for his scholarly guidance, his diligent supervision and his constant support, without which this thesis would have not materialized. My heartfelt thanks to my associate supervisor
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