A successful therapy for colorectal cancer (CRC), one of the most common malignancies worldwide, requires the greatest possible research effort. Of critical importance is an understanding of the relevant intracellular networks of signaling cascades, their activation, and the resulting cellular changes that are a prerequisite for a more successful CRC therapy. Vascular endothelial growth factor (VEGF) and the appropriate VEGF receptors represent molecular targets that have already been successfully implemented in the clinic (i.e. using monoclonal antibodies, tyrosine kinase inhibitors). However, for platelet derived growth factor (PDGF) and the relevant PDGF receptors, there are currently no clinically approved molecular therapeutics available. However, there are preliminary data to show that PDGF and its associated signaling pathways play an important role in CRC progression. In particular, the PI3K/Akt/mTOR pathway is emerging as an important intracellular partner of PDGF with which to control proliferation, migration, and angiogenesis in tumor cells. Therefore it was the objective of this work to investigate the multifactorial influence of PDGF on proliferation and metabolism, depending on CRC mutation status. The intention was to identify new therapeutic targets for future cancer therapy through analyses of PDGF-induced intracellular changes. For this purpose two human colorectal cancer cell lines were analyzed at gene and/or protein level for components of the PI3K/Akt/mTOR and MAPK signaling pathway, c-Myc, p53, and HIF1α (hypoxia-inducible-factor 1α). Changes in proliferation and metabolism, either during stimulation with PDGF and/or PI3K/Akt/mTOR inhibition, were also investigated. Experiments conducted at protein level during PDGF stimulation and/or PI3K/Akt/mTOR inhibition revealed changes in signaling pathways and crosstalk. The influence of the tumor suppressors (retinoblastoma, Rb), oncogenes (c-Myc, p53mut), and HIF1α during stimulation with PDGF, and their interactions in the tumor cell with respect to proliferation and glycolysis warrant further examination in terms of clinical treatment options. Investigations at the gene level of ex vivo samples (UICC I-IV) complete the study with regards to the clinical relevance of PDGF. PDGF stimulation increases tumor cell proliferation in HT29 cells via the PI3K/Akt/mTOR pathway rather than the MAPK pathway. However, if the PI3K/Akt/mTOR pathway is pharmacologically blocked, PDGF stimulation is mediated by inhibitory crosstalk through the MAPK pathway. Further analyses revealed that specific Akt inhibition impedes tumor cell growth, while PI3K inhibition had little effect on proliferation. Inhibitory crosstalk was found to be responsible for these different effects. Careful intervention strategies are therefore required if future therapies intend to make use of these specific signaling pathways. One aim of future research should be to gain a better understanding of the crosstalk between these signaling pathways. In this fashion, “over-inhibition” of the signal pathways, which would result in additional clinical side effects for patients, could be prevented. In late stage UICC, more mutation events occur, with tumorigenicity promoted by an increased mutation rate. Given that PDGF is increasingly expressed in the late UICC stages, our data would indicate that PDGF's effects are amplified with increasing malignancy. The activating effect of PDGF on the PI3K/Akt/mTOR pathway and subsequent changes in the activity of p53mut, Rb, c-Myc, and HIF1α, lead to an unfavorable prognosis for colon cancer patients. PDGF acts on colon cancer cells in an Akt-activating, glycolysis-dependent manner. PDGF increases glycolysis and the ability of CRC cells to adjust their energy metabolism. These activities should be taken as possible starting points with which to design therapeutic interventions for CRC therapy. PDGF, as another representative of the growth factor family, seems to play a similar role to VEGF in CRC. The data from this study underline the importance of the PDGF - PI3K/Akt/mTOR pathway-axis and its potential as a possible target in colorectal cancer. Thus PDGF represents an attractive therapeutic target, besides the VEGF/EGFR-based therapies already used in CRC. / Die erfolgreiche Therapie von Darmkrebs, eine der häufigsten malignen Erkrankungen weltweit, erfordert den größtmöglichen Forschungsaufwand. Von entscheidender Bedeutung ist das Verständnis der maßgeblichen intrazellulären Vernetzungen der Signalkaskaden, deren Aktivierung und die daraus resultierenden zellulären Veränderungen als Vorrausetzung einer erfolgreicheren Darmkrebstherapie. Der Vascular Endothelial Growth Factor (VEGF) und die entsprechenden VEGF Rezeptoren stellen molekulare Ziele dar, die im Kolorektalkarzinom bereits erfolgreich in die Klinik implementiert wurden (wie zum Beispiel monoklonale Antikörper oder Tyrosinkinaseinhibitoren). Für den Wachstumsfaktor Platelet Derived Growth Factor (PDGF) und den entsprechenden PDGF Rezeptoren stehen jedoch noch keine klinisch zugelassenen molekularen Therapeutika zu Verfügung. Allerdings zeigen erste Daten, dass PDGF und seine zugehörigen Signalwege auch eine wichtige Rolle in der Tumorprogression spielen. Insbesondere der PI3K/Akt/mTOR Signalweg kristallisiert sich als wichtiger intrazellulärer Partner von PDGF heraus, um die Proliferation, Migration und Angiogenese in Tumorzellen zu steuern. Deshalb war es Ziel dieser Arbeit, den multifaktoriellen Einfluss von PDGF auf die Proliferation und den Metabolismus, abhängig vom Mutationsstatus, im CRC näher zu untersuchen. Neue therapeutische Angriffsziele für eine zukünftige Tumortherapie sollen durch Analyse der PDGF-bedingten intrazellulären Veränderungen gefunden werden. Hierfür wurden zwei humane Kolorektalkarzinom Zelllinien auf Gen- und/oder Proteinebene auf Komponenten des PI3K/Akt/mTOR- und des MAPK-Signalwegs, auf c-Myc, p53 und HIF1α (Hypoxia-inducible-factor 1α) analysiert. Ferner wurden Änderungen der Proliferation und des Metabolismus jeweils unter Stimulation mit PDGF bzw. PI3K/Akt/mTOR Inhibition untersucht. Durchgeführte Proteinuntersuchungen unter PDGF Stimulation bzw. PI3K/Akt/mTOR Inhibition offenbarten Veränderungen in den Signalwegen und des Crosstalks. Auch die Beeinflussung der Tumor Suppressoren (Retinoblastoma, Rb), Onkogenen (c-Myc, p53mut) und HIF1α unter PDGF Stimulation und deren Zusammenspiel in der Tumorzelle hinsichtlich Proliferation und Glykolyse rechtfertigen im Hinblick auf klinische Therapiemöglichkeiten weitere Untersuchungen. Die Genuntersuchung von ex vivo Gewebeproben (UICC I-IV) komplettieren die Untersuchung unter Beachtung der klinischen Relevanz von PDGF. Die PDGF Stimulation steigert die Tumorzellproliferation in den HT29 Kolonkarzinom Zellen über den PI3K/Akt/mTOR Signalweg anstatt über den MAPK Signalweg. Allerdings wird die Stimulation mit PDGF durch den inhibitorischen Crosstalk über den MAPK Signalweg vermittelt, sollte der PI3K/Akt/mTOR Signalweg durch pharmakologische Inhibition blockiert sein. Die weitergehende Analyse hat gezeigt, dass eine spezifische Akt Inhibition das Tumorzellwachstum hemmt, während eine PI3K Inhibition kaum Einfluss auf die Proliferation besitzt. Der inhibitorische Crosstalk ist für diese unterschiedlichen Effekte verantwortlich. Sorgfältige Interventionsstrategien sind daher erforderlich, wenn man diese spezifischen Signalwege zukünftig therapeutisch ausnutzen möchte. Daher sollte ein besseres Verständnis der Crosstalks zwischen diesen Signalwegen Ziel zukünftiger Forschung sein. Auf diese Weise könnte auch eine „Über-Inhibition“ der Signalwege verhindert werden, die zusätzliche klinische Nebenwirkungen für die Patienten zur Folge hätte. In den späten UICC Stadien treten vermehrt Mutationen auf, die die Kanzerogenität mit steigender Mutationsrate fördert. Angesichts der Tatsache, dass PDGF in den späten UICC Stadien verstärkt exprimiert wird, deuten unsere Daten darauf hin, dass die Effekte von PDGF die erhöhte Malignität verstärken. Der aktivierende Effekt von PDGF auf den PI3K/Akt/mTOR Signalweg und nachfolgende Aktivitätsänderungen von p53mut, Rb, c-Myc und HIF1α führen zu einem ungünstigen Verlauf bei Patienten mit Kolorektalkarzinom. PDGF wirkt auf Darmkrebszellen in einer Akt- aktivierenden, Glykolyse-abhängigen Art und Weise. PDGF steigert die Glykolyse und die Fähigkeit der kolorektalen Karzinomzellen, ihren Energiemetabolismus unter PDGF Stimulation anzupassen. Diese Aktivitäten sollten als mögliche therapeutisch nutzbare Ausgangspunkte verwendet werden, um Therapieansätze für das kolorektale Karzinom zu entwerfen. PDGF, als weiterer Vertreter aus der Familie der Wachstumsfaktoren, scheint eine vergleichbare Rolle wie VEGF im kolorektalen Karzinom zu spielen. Die Daten dieser Studie unterstreichen die Wichtigkeit der PDGF - PI3K/Akt/mTOR Signalwegsachse und deren Potential für ein mögliches Angriffsziel im kolorektalen Karzinom. PDGF stellt somit ein interessantes therapeutisches Ziel neben der bereits genutzten VEGF/EGFR – basierten Therapie im kolorektalen Karzinom dar.

This thesis described the chemical investigations of natural products from Australian marine sponge-derived fungi. Sponge samples were collected from the Great Barrier Reef, Queensland, Australia, by Queensland Museum. The thesis is divided into eight chapters and can be devided into two major parts. The first three chapters comprised the first part of the thesis: Chapter 1 outlined the research background, literature review of marine fungal secondary metabolites; Chapter 2 introduced fungal culture and storage background knowledge, and the list of isolated marine fungal strains. Chapter 3 introduced the background of the thrombin inhibition assay and assay results. The second part (Chapter 4 to 7) of this thesis is focused on chemical isolation and structure elucidation of secondary metabolites from isolated fungal strains, mostly active strains against thrombin. An unidentified fungal strain, FS-G315858 (T)-Y, isolated from the frozen sponge sample Dysidea sp.1400 produced five peptide compounds (chapter 4, 16-20). Compound 16 is a polypeptide which features the same relative configuration with a known compound unguisine A, and compounds 17-20 are diketopiperazines. Active fungal strains FS-G315695 (T)-Y and FDPS-61732-YB were isolated from different sponge samples. However, they were identified to be the identical fungal strain Eurotium rubrum; the chemical isolation of FS-G315695 (T)-Y from its mycelia EtOAc extract resulted in three compounds (chapter 5, 17-19). Compounds 18 and 19 were identified to be flavoglaucin and iso-dihydroauroglaucin. Compound 17 was identified to have the same relative configuration with a known compound neo-echinulin A. The chemical isolation of FDPS-61732-YB from its broth EtOAc extract resulted in several diketopiperazines (chapter 5, 27-29). Another active fungal strain FS-G315695 (T)-WY was identified as Aspergillus ochraceous, the chemical isolation of its mycelia EtOAc extract resulted in one benzodiazepine compound (chapter 6, 18), together with two fatty acids (chapter 6, 16-17). The structure of compound 18 was elucidated and identified to have same relative configuration with the known compound circumdatin E. Media comparison for active fungal strain FS-G315695 (T)-Y was conducted and this work resulted in producing several neo-echinulin analogues (chapter 7, 1-3). The isolation and structure elucidation of these compounds were reported in chapter 7.

The transmembrane isoform of mucin 1 (MUC1/TM) is a well recognized tumor antigen, contributing to tumorigenesis and immune evasion. While MUC1/TM has been correlated with malignancy, it appears that a secreted splice variant of MUC1 (MUC1/sec) has antitumor properties and prevents tumor development. It was discovered that MUC1/sec expressing tumor cells (DA-3/sec) have a significant reduction in expression of urokinase plasminogen activator (uPA) relative to the parental tumor line, and tumor cells expressing MUC1/TM (DA-3/TM). The serine protease uPA, has been found to be involved in growth promoting signaling, angiogenesis, and induction of matrix remodeling leading to metastasis. Furthermore, the tumor suppressive and interferon responsive Stat1 transcription factor is dramatically upregulated in DA-3/sec cells. In addition, treatment of various murine and human cell lines with conditioned media containing MUC1/sec results in up-regulation of Stat1. DA-3/sec tumor cells are also sensitized to the anti-proliferative effects of IFN-g. Furthermore, transfection of the Stat1 gene into DA-3 tumor cells leads to a downregulation of uPA, and delays tumor progression. Since myeloid-derived suppressor cells (MDSC) play a critical role in tumor-induced immunosuppression, we investigated their recruitment by DA-3/sec and DA-3/TM cells. DA-3/sec tumor cells recruit dramatically lower levels of MDSC, relative to DA-3/TM cells. Since MUC1/sec down-regulates tumor expression of uPA, its potential role in MDSC recruitment was investigated. Tumor-derived uPA is capable of recruiting MDSC, and correlates with tumor development. In addition to diminishing recruitment of MDSC, the effect of MUC1/sec on MDSC suppressive mechanisms was investigated. MUC1/sec, or its unique immunoenhancing peptide (IEP), is capable of blocking expression of arginase 1 and production of reactive oxygen species (ROS) in MDSC, implicated in the suppression of T cells. These findings demonstrate a new mechanism of MDSC recruitment, and provide evidence that MUC1/sec has antitumor properties affecting both tumor cells and MDSC. Furthermore, it was discovered that MDSC home to the liver in addition to the tumor, bone marrow, blood, and spleen of tumor bearers, as previously described. The liver is thus an organ where MDSC accumulate and can contribute to immunosuppression directly and indirectly, via interactions with a variety of immune cells.

Background: Human salivary esterases have been shown to degrade dental resin composite restorations in vivo. Objective: To optimize in vivo protocols to recover biodegradation products and to compare the biostability of urethane-modified-bisGMA- (ubis) and bisGMA-based (bis) commercial resin composites. Methods: Class V and III composite restorations were placed in patients using adhesive and composite resin. Gingival crevicular fluid (GCF), plaque and a 2-minute oral rinse with 20% ethanol in saline (n=10) were collected immediately and 7-days after restoration placement. Samples were analyzed for biodegradation products using high performance liquid chromatography. The oral rinse protocol was then used to compare the bis and ubis composite resins (Z250, 3M; TPH, Dentsply) (n=58). Results and conclusions: The bisGMA composite matrix derived product, bishydroxypropoxyphenylpropane (BisHPPP) was only detected from oral rinse collected immediately after restoration placement. There was no statistical difference in the amount of bisHPPP collected from bis and ubis composite resins. This research was supported by CIHR (MOP 68947).

Schwann cells are the glial cells of the peripheral nervous system. When transplanted into the injured central or peripheral nervous systems they promote repair. Traditionally Schwann cells have been isolated from the sciatic nerve, creating nerve-SC. An alternative Schwann cell source is from the differentiation of skin-derived precursors (SKPs), stem cells found in the skin, to Schwann cells (SKP-SC). SKP-SC have shown enhanced regenerative ability compared to nerve-SC. This study compares nerve-SC with SKP-SC at the functional and gene expression level to determine their degree of similarity and find their sources of variance. The functional ability of both Schwann cell types appeared similar. Their gene expression, as assessed by microarray, was similar but not identical. Genes that differed between nerve-SC and SKP-SC may represent differences important to regeneration. The similarity of SKP-SC to nerve-SC supports the use of SKP-SC for repair, and reasons for enhanced regeneration by SKP-SC are suggested.

Background: Human salivary esterases have been shown to degrade dental resin composite restorations in vivo. Objective: To optimize in vivo protocols to recover biodegradation products and to compare the biostability of urethane-modified-bisGMA- (ubis) and bisGMA-based (bis) commercial resin composites. Methods: Class V and III composite restorations were placed in patients using adhesive and composite resin. Gingival crevicular fluid (GCF), plaque and a 2-minute oral rinse with 20% ethanol in saline (n=10) were collected immediately and 7-days after restoration placement. Samples were analyzed for biodegradation products using high performance liquid chromatography. The oral rinse protocol was then used to compare the bis and ubis composite resins (Z250, 3M; TPH, Dentsply) (n=58). Results and conclusions: The bisGMA composite matrix derived product, bishydroxypropoxyphenylpropane (BisHPPP) was only detected from oral rinse collected immediately after restoration placement. There was no statistical difference in the amount of bisHPPP collected from bis and ubis composite resins. This research was supported by CIHR (MOP 68947).

Schwann cells are the glial cells of the peripheral nervous system. When transplanted into the injured central or peripheral nervous systems they promote repair. Traditionally Schwann cells have been isolated from the sciatic nerve, creating nerve-SC. An alternative Schwann cell source is from the differentiation of skin-derived precursors (SKPs), stem cells found in the skin, to Schwann cells (SKP-SC). SKP-SC have shown enhanced regenerative ability compared to nerve-SC. This study compares nerve-SC with SKP-SC at the functional and gene expression level to determine their degree of similarity and find their sources of variance. The functional ability of both Schwann cell types appeared similar. Their gene expression, as assessed by microarray, was similar but not identical. Genes that differed between nerve-SC and SKP-SC may represent differences important to regeneration. The similarity of SKP-SC to nerve-SC supports the use of SKP-SC for repair, and reasons for enhanced regeneration by SKP-SC are suggested.

The goniothalamin (GTN) and related styryl-lactones were found to be cytotoxic and apoptotic to a variety of tumor cell lines including breast cancer MCF-7, cervical cancer HeLa and leukemia HL60. In HL60 cells, GTN triggers mitochondria dysfunction, caspase activation and eventually, apoptosis. In this study, we demonstrated that GTN was able to induce apoptosis in hepatocellular carcinoma derived cells, SK-Hep1 and Hep-3B cells via upregulation of the phorbol-12-myristate-13-induced 1 (PMAIP1) protein , alternation of mitochondrial membrane potentials (P < 0.05) via TP53-dependent and -independent transactivations. Treatment with GTN induced DNA damage, formation of reactive oxygen species (ROS, P < 0.05) and activated cleaved CASP8, CASP9, CASP3 and poly (ADP-ribose) polymerase 1 proteins. However, cleaved BH3 interacting domain (BID) death agonist protein was not identified, suggesting that an intrinsic cellular stress was produced after GTN treatments in both cell lines. A pan caspase inhibitor, z-VAD-FMK, suppressed GTN-induced apoptotic cell percentages (P < 0.05), demonstrating that GTN-induced apoptosis was mediated by the activation of the caspase cascade protein. The GTN induced ROS formation prior to DNA damage in SK-Hep1, yet in reverse order in Hep-3B cells. Moreover, GTN induced DNA damages through activation of the gamma H2A histone family member X (£^H2AFX, Ser139)/phospho-CHK1 checkpoint homolog (pCHEK1, Ser345)/phospho-CHK2 checkpoint homolog (pCHEK2, Thr68)/phosphos-tumor protein p53 (pTP53, Ser15), and £^H2AFX/pCHEK2 (Thr68), in SK-Hep1 and Hep-3B cells,respectively. Among five integral outer mitochondrial membrane proteins that blocks or induces apoptotic death, PMAIP1 protein and PMAIP1 mRNA levels were upregulated after GTN treatments for 4 to 6 h in both cell lines (P < 0.05). Transfection of shRNA interference plasmids targeting PMAIP1 gene downregulated PMAIP1 mRNA (P < 0.05) and PMAIP1 protein (P < 0.05) levels, as well as GTN-induced apoptotic cell percentages (P < 0.05) in both cell lines. In parallel, transfection of the shRNA interference plasmid targeting TP53 gene in SK-Hep1 cells, downregulated TP53 and PMAIP1 mRNA (P < 0.05) and TP53, pTP53, PMAIP1, cleaved PARP1 protein levels and apoptotic cell percentages (P < 0.05). Treatment with the TP53 inhibitor, PFT-£\ or transfection of a dominant negative TP53 plasmid, pTP53mt135, repressed TP53, pTP53 and PMAIP1 protein and/or TP53, PMAIP1 mRNA levels (P < 0.05), however, significantly augmented GTN-induced apoptotic cell percentages (P < 0.05). Cytosol/mitochondria fractionation identified that TP53, along with PMAIP1 proteins, were translocated to mitochondria after GTN treatment in time-dependent manners. Taken together, our studies demonstrated that GTN induced apoptosis via PMAIP1 via both TP53-dependent and -independent transactivation pathways. In TP53-positive SK-Hep1 cells, PMAIP1 and TP53 proteins conducted synergic effects.

Neuronal cell death may be responsible for the pathogenesis of neuropathic pain. Glial cell line-derived neurotrophic factor (GDNF) protects sensory neurons after injury and offers a promising alternative for the management of intractable pain. However, continuous administration of trophic factors into the central nervous system is costly and difficult to maintain. Therefore, we evaluated the potential of intrathecal GDNF gene delivery for the treatment of neuropathic pain. Recombinant adenovirus encoding GDNF (Ad-GDNF) was characterized and shown to enhance viability of neuronal cultures. After intrathecal injection of Ad-GDNF, an elevated GDNF level was observed in spinal cord for four weeks. In rats with sciatic nerve axotomy,intrathecal injection of Ad-GDNF significantly ameliorated the duration of neuropathic pain. However, animals treated with Ad-GDNF developed hyperalgesia in the early stage of treatment. Immunofluorescence analysis indicated that intrathecal GDNF gene delivery prominently attenuated the neuronal loss due to nerve injury. Unexpectedly, varying degrees of hair loss was found in some rats receiving Ad-GDNF. Histological analysis revealed that hair loss resulted from severe degeneration of hair follicles in skin from Ad-GDNF-treated animals. In summary, the present study demonstrate the feasibility and limitations of GDNF gene delivery for the management of neuropathic pain.

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression during melanoma carcinogenesis remains unclear. In this study, adding exogenous HDGF stimulated the invasion and colonies formation of B16-F10 melanoma cells. Adenovirus vectors encoding HDGF and HDGF-RNAi were generated and characterized to up- and down-regulated HDGF expression in B16-F10 melanoma cells. It was found that HDGF overexpression stimulated the proliferation, invasiveness, anchorage-independent growth of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects. In lung-metastasis model, intravenous injection of HDGF-overexpressing melanoma cells resulted in increased metastasis while HDGF-downregulated melanoma cells caused decreased metastasis. Similarly, in primary melanoma model, subcutaneous injection of HDGF-overexpressing melanoma cells enhanced while HDGF-downregulated melanoma cells reduced the tumor burden in mice. Histological analysis revealed increased tumor proliferation and neovascularization with concomitant reduction of apoptosis in HDGF-overexpressing melanoma. Moreover, HDGF-overexpressing melanoma also exhibited enhanced propensity to metastasize from the primary tumors to lymph node and lung. Finally, it was found that HDGF overexpression increased nuclear factor kappa B (NF£eB) activities and Akt phosphorylation up and down stream alternation like PI3K, PTEN, I£eB and it¡¦s subunit IKK£\, IKK£], IKK£^ in melanoma cells. It also found that HDGF overexpression influenced MITF and HIF1£\ in melanoma after gene delivery. HDGF also altered EMT changes like E,N-cadherin, vimentin, and £],£^-catenin. The present study provides conclusive evidence that HDGF upregulation promotes the growth and metastasis of melanoma by promoting the survival and vascularization. Besides, HDGF knockdown may constitute a novel strategy for melanoma control.