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The spirooxepinisoxazoline alkaloid psammaplysin F (1) was selected as a scaffold for the generation of a unique screening library for both drug discovery and chemical biology research. Large-scale extraction and isolation chemistry was performed on a marine sponge (Hyattella sp.) collected from the Great Barrier Reef in order to acquire >200 mg of the desired bromotyrosine-derived alkaloidal scaffold. Parallel solution-phase semisynthesis was employed to generate a series of psammaplysin-based urea (2–9) and amide analogues (10–11) in low to moderate yields. The chemical structures of all analogues were characterized using NMR and MS data. The absolute configuration of psammaplysin F and all semisynthetic analogues was determined as 6R, 7R by comparison of ECD data with literature values. All compounds (1–11) were evaluated for their effect on cell cycle distribution and changes to cancer metabolism in LNCaP prostate cancer cells using a multiparametric quantitative single-cell imaging approach. These investigations identified that in LNCaP cells psammaplysin F and some urea analogues caused loss of mitochondrial membrane potential, fragmentation of the mitochondrial tubular network, chromosome misalignment, and cell cycle arrest in mitosis.

The naturally occurring pentacyclic diterpenoid gibberellic acid (1) was used in the generation of a drug-like amide library using parallel-solution-phase synthesis. Prior to the synthesis, a virtual library was generated and prioritized based on drug-like physicochemical parameters such as log P, hydrogen bond donor/acceptor counts, and molecular weight. The structures of the synthesized analogues (2–13) were elucidated following analysis of the NMR, MS, UV, and IR data. Compound 12 afforded crystalline material, and its structure was confirmed by X-ray crystallographic analysis. All compounds were evaluated in vitro for cytotoxicity and deregulation of lipid metabolism in LNCaP prostate cancer cells. While no cytotoxic activity was identified at the concentrations tested, synthesized analogues 3, 5, 7, 10, and 11 substantially reduced cellular uptake of free cholesterol in prostate cancer cells, suggesting a novel role of gibberellic acid derivatives in deregulating cholesterol metabolism.

We report for the first time the mechanism of action of the natural product thalicthuberine (TH) in prostate and cervical cancer cells. TH induced a strong accumulation of LNCaP cells in mitosis, severe mitotic spindle defects and asymmetric cell divisions, ultimately leading to mitotic catastrophe accompanied by cell death through apoptosis. However, unlike microtubule-binding drugs (vinblastine and paclitaxel), TH did not directly inhibit tubulin polymerization when tested in a cell-free system, whereas it reduced cellular microtubule polymer mass in LNCaP cells. This suggests that TH indirectly targets microtubule dynamics through inhibition of a critical regulator or tubulin-associated protein. Furthermore, TH is not a major substrate for P-glycoprotein (Pgp), which is responsible for multidrug resistance in numerous cancers, providing a rationale to further study TH in cancers with Pgp-mediated treatment resistance. The identification of TH's molecular target in future studies will be of great value to the development of TH as potential treatment of multidrug resistant tumors.

The molecular chaperone Hsp90 is overexpressed in prostate cancer (PCa) and is responsible for the folding, stabilization and maturation of multiple oncoproteins, which are implicated in PCa progression. Compared to first-in-class Hsp90 inhibitors such as 17-allylamino-demethoxygeldanamycin (17-AAG) that were clinically ineffective, second generation inhibitor AUY922 has greater solubility and efficacy. Here, transcriptomic and proteomic analyses of patient-derived PCa explants identified cytoskeletal organization as highly enriched with AUY922 treatment. Validation in PCa cell lines revealed that AUY922 caused marked alterations to cell morphology, and suppressed cell motility and invasion compared to vehicle or 17-AAG, concomitant with dysregulation of key extracellular matrix proteins such as fibronectin (FN1). Interestingly, while the expression of FN1 was increased by AUY922, FN1 secretion was significantly decreased. This resulted in cytosolic accumulation of FN1 protein within late endosomes, suggesting that AUY922 disrupts vesicular secretory trafficking pathways. Depletion of FN1 by siRNA knockdown markedly reduced the invasive capacity of PCa cells, phenocopying AUY922. These results highlight a novel mechanism of action for AUY922 beyond its established effects on cellular mitosis and survival and, furthermore, identifies extracellular matrix cargo delivery as a potential therapeutic target for the treatment of aggressive PCa.

Treatments for advanced prostate cancer (PCa) continue to target androgen signalling leading to prolonged androgen deprivation. A major side-effect of androgen deprivation is development of insulin resistance resulting in hyperinsulinaemia, which is specifically associated with more rapid progression and increased cancer mortality, but is not clinically addressed. Metformin is used widely in the treatment of diabetes and insulin resistance but its effects in cancer cells has been shown to be dependent on cancer type and context. We examined the effect of metformin, in androgen-deprived PCa cells, utilising the androgen-dependent LNCaP and DUCaP cell lines, and the novel castrate-resistant cell line, MSK3. Microarray of LNCaP cells +/-10nM DHT, +/- 5mM metformin followed by Ingenuity pathway analysis identified apoptosis and autophagy pathways modulated by metformin. QRT-PCR revealed Bcl2, Bcl-XL and ATG12, upregulated by androgens, were dose-dependently downregulated by metformin in all cell lines. Increased rates of apoptosis with metformin treatment, measured by FACS accompanied decreases in autophagy, using CYTO-ID stain. Androgen deprivation was associated with a 15% reduction in maximal mitochondrial respiration, which was restored with 10nM DHT. Metformin dose-dependently reduced mitochondrial activity, which was accompanied by an overall decreased glycolytic capacity. Our results suggest metformin counteracts survival pathways in androgen-deprived PCa cells. These results suggest a susceptibility of PCa cells to metformin, irrespective of androgen dependence. In a parallel randomised, placebo-controlled clinical trial, we have observed that metformin prevents ADT-induced metabolic deterioration in men with newly diagnosed metastatic disease, including improved fasting insulin and HOMA, which supports its use as adjuvant therapy with androgen deprivation.

The identification and segmentation of target objects from medical images is often confused by other more salient objects in the image. This is a specific problem for X-ray projection images where the shadows of semi-transparent objects are overlaid. A bone shadow may confuse the automated detection of other crossing bones and of important soft tissue findings in the lung like lung nodules. We present a method to identify and remove such bone shadows from a chest radiograph for the purpose of suppressing all bone shadows overlapping with the lung field in a standard posterior-anterior view. In this context an elegant novel approach to the problem of identifying and segmenting overlaid objects is followed: Disturbing objects are identified first and literally removed from the image, therefore no longer confusing the detection of other more subtle objects. This method allowed the identification, segmentation, and suppression of the clavicles, the posterior and the anterior parts of the ribs - one after another. In a clinical study the detection of lung nodules by experienced radiologists was improved after bone suppression. © 2016 IEEE.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Natural Sciences, Science, Environment, Engineering and Technology, The lack of a cure for advanced prostate cancer (PCa) highlights the need for better therapies to fight this disease, which is the second most common cause of cancer-related death in men. Nature is a rich source of potent anticancer agents, as 75% of the current drugs are derived from natural products, some being microtubule-targeting drugs like vinblastine and docetaxel. While proven to increase overall survival time of PCa patients, docetaxel treatment ultimately fails in part due to the development of drug resistance, presenting a major clinical challenge, given the limited number of alternative treatments currently available. As part of a research program aimed at identifying new anticancer compounds from nature for drug discovery and development, a pre-fractionated plant library was screened for cytotoxic activity in the PCa cell line LNCaP. This cell line is positive for the androgen receptor (AR) and is androgen-dependent; LNCaP cells are representative of advanced PCa sensitive to AR-targeted therapies. Only plants native to Australia were considered for the pre-fractionated library. A taxonomically diverse set of plants were used with 12 botanical families and 16 genera represented. The library consisted of 38 fractions obtained from C18 HPLC fractionation of 38 MeOH plant extracts, with one fraction collected for each extract. A growth inhibition assay based on metabolic activity (alamarBlue) was used to screen the 38 fractions against the PCa cell line LNCaP. Of the 38 fractions screened, six displayed inhibition of more than 50% at 10 µg/mL, which was the cut-off selected for pursuing hit fractions. Bioassay-guided fractionation and/or mass-directed isolation were performed on two plant samples, Anopterus macleayanus and Hernandia albiflora. Several cytotoxic natural products were purified and their chemical structures determined using a combination of 1D/2D NMR, MS, and UV data.