Molecular Targets of Green Tea Catechin, EGCG, on Human Colorectal Carcinogenesis

This thesis explores the effect of major green tea catechin, EGCG, on colorectal carcinogenesis particularly on target genes, bFGF and NUDT6. Cancer is a second leading cause and colorectal cancer is a third leading cause of deaths in the Western World. Recently there is a lot of persuasive epidemiological and experimental evidence that phytochemical-enriched diet may be involved in the prevention of colorectal cancer. EGCG exhibits beneficial effects like anti-tumorigenic, anti-oxidant, anti-angiogenic, and pro-apoptotic activity. In studies presented here, we investigated the molecular mechanisms by which EGCG affects bFGF degradation (Chapter 3): EGCG increased the ubiquitination as well as trypsin like activity of 26S proteasome causing faster bFGF degradation. We also demonstrated that EGCG could decrease the tumor number and volume in mouse models of colorectal cancer. Our microarray data from catechin-treated colorectal cancer cells revealed down regulation of a gene called NUDT6, an anti-sense of bFGF, which we have shown to regulate the bFGF function. NUDT6 is a mitochondrial protein whose biological function is unknown. EGCG affected the mRNA stability of NUDT6 by decreasing the luciferase activity of its 3‘UTR (Chapter 4). NUDT6 stable cells showed increase in cell proliferation and soft agar colony formation. The electrical impedance bio-analytical assay showed increased attachment of NUDT6 cells to the surface media and to each other. NUDT6 cells showed increased invasion rate and decreased Caspase 3/7 activity. Injection of CONTROL and NUDT6 cells into nude mice complemented the in vitro data showing increase in tumor volume, mitotic index and decrease in caspase activity and apoptotic index. This was possibly due to binding of p53 to NUDT6 which led to degradation of p53. NUDT6 was highly expressed in human colon tumor tissue sample compared to normal and NUDT6 could be used as a marker in colon tumorigenesis. The findings presented in this thesis clearly show that EGCG is an effective chemopreventive dietary agent, decreasing the expression of pro-angiogenic and cell proliferation genes, bFGF and NUDT6 in colorectal cancer cells. Further characterization of NUDT6 as a potential biomarker in colorectal carcinogenesis may provide a better prediction of disease progression in the future

Assessment of potential anti-cancer stem cell activity of marine algal compounds using an in vitro mammosphere assay

Background: The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents. Methods: In this study, the tumoursphere assay was validated in MCF-7 cells and used to screen novel marine algal compounds for potential anti-cancer stem cell (CSC) activity in vitro. Results: MCF-7 breast cancer cells were observed to generate tumourspheres or mammospheres after 3-5 days growth in anchorage-independent conditions and an apparent enrichment in potential CSCs was observed by an increase in the proportion of CD44high/CD24low marker-bearing cells and Oct4 expression compared to those in the bulk population grown in regular adherent conditions. Using this assay, a set of algal metabolites was screened for the ability to inhibit mammosphere development as a measure of potential anti-CSC activity. We report that the polyhalogenated monoterpene stereoisomers RU017 and RU018 isolated from the red alga Plocamium cornutum, both of which displayed no cytotoxicity against either adherent MCF-7 breast cancer or MCF-12A non-transformed breast epithelial cells, were able to prevent MCF-7 mammosphere formation in vitro. On the other hand, neither the brown algal carotenoid fucoxanthin nor the chemotherapeutic paclitaxel, both of which were toxic to adherent MCF-7 and MCF-12A cells, were able to inhibit mammosphere formation. In fact, pre-treatment with paclitaxel appeared to enhance mammosphere formation and development, a finding which is consistent with the reported resistance of CSCs to traditional chemotherapeutic agents. Conclusion: Due to the proposed clinical significance of CSC in terms of tumour initiation and metastasis, the identification of agents able to inhibit this subpopulation has clinical significance

Assessment of potential anti-cancer stem cell activity of marine algal compounds using an in vitro mammosphere assay:

The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents

Epigallocatechin-3-gallate induces mesothelioma cell death via H2O2-dependent T-type Ca2+ channel opening

Malignant mesothelioma (MMe) is a highly aggressive, lethal tumour requiring the development of more effective therapies. The green tea polyphenol epigallocathechin-3-gallate (EGCG) inhibits the growth of many types of cancer cells. We found that EGCG is selectively cytotoxic to MMe cells with respect to normal mesothelial cells. MMe cell viability was inhibited by predominant induction of apoptosis at lower doses and necrosis at higher doses. EGCG elicited H2O2release in cell cultures, and exogenous catalase (CAT) abrogated EGCG-induced cytotoxicity, apoptosis and necrosis. Confocal imaging of fluo 3-loaded, EGCG-exposed MMe cells showed significant [Ca2+]irise, prevented by CAT, dithiothreitol or the T-type Ca2+channel blockers mibefradil and NiCl2. Cell loading with dihydrorhodamine 123 revealed EGCG-induced ROS production, prevented by CAT, mibefradil or the Ca2+chelator BAPTA-AM. Direct exposure of cells to H2O2produced similar effects on Ca2+and ROS, and these effects were prevented by the same inhibitors. Sensitivity of REN cells to EGCG was correlated with higher expression of Cav3.2 T-type Ca2+channels in these cells, compared to normal mesothelium. Also, Cav3.2 siRNA on MMe cells reduced in vitro EGCG cytotoxicity and abated apoptosis and necrosis. Intriguingly, Cav3.2 expression was observed in malignant pleural mesothelioma biopsies from patients, but not in normal pleura. In conclusion, data showed the expression of T-type Ca2+channels in MMe tissue and their role in EGCG selective cytotoxicity to MMe cells, suggesting the possible use of these channels as a novel MMe pharmacological target. \ua9 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd

A multi-targeted approach to suppress tumor-promoting inflammation

Cancers harbor significant genetic heterogeneity and patterns of relapse following many therapies are due to evolved resistance to treatment. While efforts have been made to combine targeted therapies, significant levels of toxicity have stymied efforts to effectively treat cancer with multi-drug combinations using currently approved therapeutics. We discuss the relationship between tumor-promoting inflammation and cancer as part of a larger effort to develop a broad-spectrum therapeutic approach aimed at a wide range of targets to address this heterogeneity. Specifically, macrophage migration inhibitory factor, cyclooxygenase-2, transcription factor nuclear factor-κB, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase B, and CXC chemokines are reviewed as important antiinflammatory targets while curcumin, resveratrol, epigallocatechin gallate, genistein, lycopene, and anthocyanins are reviewed as low-cost, low toxicity means by which these targets might all be reached simultaneously. Future translational work will need to assess the resulting synergies of rationally designed antiinflammatory mixtures (employing low-toxicity constituents), and then combine this with similar approaches targeting the most important pathways across the range of cancer hallmark phenotypes

Self-Calibrating Camera-Projection Systems: A Natural Interface for Presentation Control

This paper presents a system that enables the user to directly control the presentation in a more natural manner, either at a distance from the screen (using a laser pointer or shadow gestures), or at a distance from the computer (using a telescoping pointer). Figure 1 presents an overview of the camera-projector system. The camera may be placed anywhere in the room such that its field of view contains the presentation area. The system first calibrates itself by exploiting knowledge of the projected image. Subsequently, computer-vision algorithms determine where the user is pointing on the presentation surface, and this provides a correspondence between the user's actions (as seen by the camera), and active regions in the image being displayed on the screen as illustrated in Figure 1. The presentation software performs programmed actions in response to the location and characteristics of the user's pointer actions. This enables users to activate "virtual buttons" on the projection screen simply by pointing, and also to "draw" directly on the presentation surface