Dissertations / Theses on the topic 'Colon cell lines'

Colorectal and pancreatic cancer are leading causes of cancer related mortality, suggesting the need for further development of treatment approaches. Gnaphalin, a flavone derived from Gnaphalium gracile H. B. K. which is found in the Andean regions of South America, has shown anti-proliferative properties in solid tumors. Further investigation has shown this compound interferes with signaling conducive to proliferation and cell adhesion, inducing the cell to undergo apoptosis. The primary objective of the study was to look at key regulatory proteins in the cell survival and proliferative pathways to determine Gnaphalin’s mechanism of action. Cytotoxic activity was measured using MTT analysis on the colon cancer cell lines Caco2 and HCT-116, and on the pancreatic cancer cell lines MIA PaCa and Panc28. Apoptosis was determined by the presence of fragmented DNA via TUNEL and cleaved effector caspase 3. Finally, immunoblots were used to determine the mechanism of action using key proteins involved in both the intrinsic and extrinsic apoptotic pathways. Gnaphalin showed the highest activities in colon cancer HCT-116 and pancreatic cancer Panc 28 cells with a half maximal effective concentrations of 25.82±1.0887 and 30.07 ± 1.553 µM respectively. Gnaphalin impediment of cell viability involves the inhibition of phospho-ERK proliferation of the MAPK pathway along with phospho-FAK and c-Met, which are adhesion molecules. Gnaphalin has shown cytotoxic activity towards several colon cancer and pancreatic cancer cell lines by targeting cell proliferation and adhesion, and ultimately causing apoptosis.

Background: Isolation of respiratory viruses routinely requires a panel of cell lines. Management of these cell lines is usually considered complex, cumbersome, long turnaround time and high cost. In order to improve the detection rate and cost, there is a need to evaluate any other cell line that could be as sensitive as the routine cell line for the detection of respiratory viruses. The human colon adeno-carcinoma (Caco-2) cell line has been shown to support the growth of enteroviruses, enteric viruses, and influenza A virus, and ability to grow coronavirus NL63 and SARS from culture isolates. In the present study, the isolation rate of respiratory viruses in Caco-2 from clinical specimens was studied and compared with the conventional panel of cell lines for the diagnosis of respiratory virus disease. Material and methods: The study was performed in two stages. In the first stage, one hundred and eighty-nine nasopharyngeal aspirates confirmed positive by direct immunofluorescence were used to inoculate onto Caco-2, and HEp-2, A549, MDCK, LLC-MK2 cell lines at the same time. In the second stage, fifty-six nasopharyngeal aspirates were randomly selected and cultured on Caco-2, HEp-2, A549, MDCK and LLC-MK2. Infected cells were examined daily for cytopathic effect for up to 10 days. Virus was further identified by performing direct immunofluorescence test for detection of eight common respiratory viruses (respiratory syncytial virus, influenza A and B viruses, parainfluenza type 1, 2, 3, 4 viruses and adenovirus). Results: Caco-2 (84%) was found to be the most efficient cell line to propagate the respiratory viruses from nasopharyngeal aspirate when compared with any positive by these conventional panel cells (78%) or individual cell line MDCK (38%), HEp-2 (21%), LLC-MK2 (27%) and A549 (37%). The latter differences were statistically significant (p = <0.000001). Furthermore, Caco-2 recovered 86% (36/42) viruses of conventional panel cells negative samples, while conventional panel cells recovered 80% (24/30) viruses of Caco-2 cells negative samples. Conclusion: Caco-2 is sensitive to a wide range of virus and can greatly simplify routine cell culture service for isolation of respiratory viruses.
published_or_final_version
Microbiology
Master
Master of Medical Sciences

Colorectal cancer is one of the most prevailing cancers worldwide with particularly high incidence and mortality rates in the Western countries. Compelling evidence indicates that diet is considered as an important etiological factor of colorectal cancer risk. Consumption of vegetables has been shown in numerous epidemiological, case-control and prospective studies to be inversely associated with the pathogenesis of several cancers including cancers of colon and rectum. Leguminous and cruciferous vegetables abundant in every day diet are a rich source of many bioactive compounds. Some of those phytochemicals such as glucosinolates and their derivatives, flavonoids - especially isoflavones and saponins have been demonstrated to be potent chemoprotective agents with an ability to inhibit carcinogenesis. Recently, sprouting seed of many plants has been popularized as a healthy and nutritious food plentiful in proteins, minerals, vitamins and a range of phytochemicals. The germination process results in significant increases in concentrations of particular compounds as compared to the mature plants and may contribute to enhanced anticancer activity of sprouts. The focus of this study was primarily to evaluate the anti-cancer effects of the extracts prepared from leguminous (alfalfa, red clover) and cruciferous (Raab broccoli, Daikon radish) sprouts on key stages of colon carcinogenesis, namely initiation, promotion and invasion using in vitro model systems. The shoots were investigated as a commercial product - BroccoShoots sprouts mixture which were processed following extraction methods to obtain crude juice, methanol and in vitro digested extracts to assess how changes in the chemical characteristics could affect the biological activity of the samples. BroccoShoots extract at the highest concentrations (100, 150, 200 ug/ml) inhibited growth of CaCo-2 cells. At non-toxic range of concentrations (0 - 50 ug/ml) BroccoShoots extracts reduced H202 induced DNA damage in CaCo-2 cells and significantly inhibited migration and invasion of the HT115 cell-line. These effects appeared to be stronger for the crude juice extracts than for the other two types of extracts. There were no evident changes in the epithelial integrity of CaCo-2 cells (measured as trans-epithelial electrical resistance) and in cell cycle progression (24 hours) after exposure to the extracts. Then individual plants were prepared as crude juice extracts to examine the extent to which particular species contributed to the overall activity of the mixture. It was found that anti-genotoxic, anti-proliferative, anti-migrative and anti-invasive effects observed for Daikon radish extracts were the highest among all individual sprouts. Further, Daikon radish extracts at non-toxic concentrations (35, 50 ug/ml) were shown to induce G2/M phase cell cycle arrest in CaCo-2 cells and significantly reduce cell proliferation (over 144 hours) due to cytostatic effects. Thus, it was postulated that anti- cancer effects exerted by BroccoShoots mixture were mainly mediated by activity of Daikon radish extracts. These results demonstrate that sprouts and notably Daikon radish sprouts were effective in inhibiting several stages of colon carcinogenesis in vitro. Also, hypothesis was proposed that lymphocytes could be used as a surrogate biomarker of colonic tissue in assessment of DNA hypermethylation status of genes involved in colon carcinogenesis and that supplementation with folate rich watercress could affect promoter methylation of these genes. However, no detectable levels in promoter methylation ofp16INK4a, MGMT, CDX-2 and C-Myc genes were found in lymphocytes from healthy volunteers. Moreover, there was no evidence that watercress consumption could change methylation status of investigated genes. This indicates that lymphocytes may not be a suitable marker for measurement of DNA hypermethylation in the selected genes.

Colorectal cancer (CRC) is the third largest cause of cancer deaths worldwide. Short-chain fatty acids (SCFA) are reported to be chemoprotective against CRC and are beneficial to colon epithelia by virtue of being their preferred energy source. Despite being essential to human metabolism and health, SCFAs are only accessible to humans as nutritional by-products of the anaerobic fermentation of dietary fibre by gut bacteria. Identifying novel chemotherapeutic roles for SCFAs is attractive due to their high tolerance by colonocytes, however the underlying metabolic actions are not fully understood. This project took a Systems Biology approach by employing high-throughput, quantitative proteomic and cellomic experimentation to investigate whether the three principle SCFAs in colon epithelia, butyrate, propionate and valerate, display unique roles with potentially chemoprotective actions. A hypothesised anti-mitotic pathway was formulated in which odd-chain SCFAs at above physiological concentrations induce downstream epigenetic acetylation of transcriptional regulators to differentially regulate β-tubulin isotypes. This creates an aberrant tubulin code leading to the disruption of microtubule (MT) dynamics, failure of critical MT cellular functions and eventual cell death. The pathway was simulated by computational dynamical modelling to predict the behaviour of SCFAs in relation to MT dynamics under both treatment and physiological conditions. This verified the plausibility of the hypothesis and provided valuable insights into the underlying mechanisms. Bioinformatic searches, combined with proteomic evidence, indicated that propionate and valerate, the odd-chain SCFAs, differentially regulated pro-tumourigenic β-tubulin isotypes. The alteration of the β-tubulin expression pattern countered potential metabolic adaptions in colon cancer cells, suggesting a chemopreventive action. Anti-microtubule drugs (AMD) are among the most successful chemotherapies to date, however their toxicity and drug resistance increase with successive rounds of treatment. This project has demonstrated that odd-chain SCFAs may act as novel chemotherapeutics by reducing the negative effects of AMDs while enhancing their efficacy.

Darmkrebszellen und T-Zellen regulieren ihren zentralen Kohlenstoffmetabolismus um ihren anabolen Bedarf zu erfüllen. Tumorzellen mit einer KRAS- oder BRAF-Mutation zeigen ein schnelles Wachstum, welches eine Umprogrammierung des Metabolismus vor aussetzt. Der mitochondriale T-Zellen-Aktivierungsinhibitor (TCAIM) ist bekannt dafür die mitochondriale Zellstruktur zu beeinflussen. Der Einfluss auf den Metabolismus nicht klar. In dieser Arbeit präsentiere ich erstmalig ein mathematische Model des zentralen Kohlen stoffmetabolismus in Darmkrebszellen und T-Zellen. Mithilfe dieses Modells analysiere ich, wie sich die Regulation in ähnlichen Zelllinien unterscheidet. In Bezug auf die Darm krebszellen vergleiche ich BRAF-(CaCO2-BRAFV600E), KRAS-(CaCO2-KRASG12V) mu tierte Zelllinien mit einer Basiszelllinie (CaCO2-control) und zeige, dass der Kohlenstoff metabolismus in BRAF-mutierten Zellen im Vergleich zu den beiden übrigen Zelllinien herabreguliert ist. Das Modell bestätigt außerdem, dass der Monocarboxylattransporter (MCT) in den Darmkrebszellen eine wichtige Rolle, insbesondere in den KRAS mu tierten Zellen, spielt. In T-Zellen zeigt der Vergleich von Wildtypzellen (CD8 T-Zellen) mit TCAIM homozygoten Zellen (TCAIM homozygote CD8 T-Zellen), dass der Kohlen stoffmetabolismus in zweiteren überwiegend herabreguliert und weniger aktiv ist. Diesen Effekt konnte ich durch die Analyse von RNASeq-Daten der jeweiligen Zelltypen bestä- tigen. Des Weiteren stelle ich fest, dass sich der Tricarbonsäurezyklus umkehrt, wenn durch die Glykolyse nicht ausreichend Laktat exportiert und die Biomasseproduktion unterstützt werden kann. Meine Arbeit stellt damit insgesamt einen neuartigen Ansatz zur Integration von Meta bolomik und RNAseq Daten dar, um die Regulation des zentralen Kohlenstoffmetabo lismus zu verstehen.
Colon cancer cells and T cells regulate central carbon metabolism to meet their anabolic needs. In KRAS and BRAF tumors, metabolic reprogramming is a premise to support rapid proliferation. In T cells, the mitochondrial T cell activation inhibitor (TCAIM) is known to affect mitochondrial morphology but its effect on cellular metabolism is not well understood. Via mathematical modelling, I investigate the differential regulation of closely related cell lines. I present the first mathematical model for colon cancer and T cell metabolism, unraveling differential regulation between related cell lines. The model shows that CaCO2-BRAFV600Ecells are mostly downregulated compared to CaCO2-KRASG12Vand CaCO2-control. Additionally, it demonstrates the critical role of monocarboxylate transporter (MCT), especially for CaCO2-KRASG12V. Concerning T cells, I compare wild-type T cells to homozygous TCAIM T cells. This unveils that TCAIM homozygous cells have a mostly downregulated TCA cycle, validated by RNASeq data, and are less metabolically active than wild-type T cells. Furthermore, if the glycolytic flux is not sufficient to support lactate export and biomass production, the model reveals that the TCA cycle is reversed as it requires less regulation. Taken together, this work presents a novel approach to integrate data referring to metabolic and genetic regulation of metabolism. On this basis, we can now better discriminate the metabolic capacity of CaCO2-control, CaCO2-BRAFV600E, CaCO2-KRASG12V, wildtype CD8 T cells, and homozygous TCAIM CD8 T cells.

Chromobacterium violaceum (CV) produces a violet color pigment known as Violacein. It has been reported that violacein has anticancer activity. This compound is produced by CV a gram-negative facultatively anaerobic bacterium found in soil and water environmental samples. The purpose of this study was to determine the effect of purified violacein on select cancer cell lines. Violacein used in this study was purified from CV strain (14N23), a strain isolated from environmental samples collected in the Tennessee Copper Basin. The previous reports used a crude extract preparation of violacein; thus, it was of interest to determine the effect of the pure compound on cancer cell growth was similar to that of the crude extracts. The compound purified following the method of Mehta, et al. was exposed to cancer cells and cell death assessed using the Alamar Blue procedure. It was found that violacein had no effect on A549, BT549, and PC3 cancer cell growth; however, there was a significant effect on Colo-320 cancer cells. It was concluded that further studies are required to assess the effect of violacein on enzymes and proteins involved in the cancer cell apoptotic pathways. Such studies will explain why cancer cell death was observed in certain cancer cells and not others.

The methods, experimentations, philosophies and influential artists mentioned in this thesis all form part of my artistic exploration and art from the level of cells to the large oceanic water world. As an artist of Fine Arts at the University of Texas, Rio Grande Valley, my main emphasis is to associate these concepts with my personal experiences, cultural traditions and artistic perspectives with my likes for lines, colors and spaces in what I called, Nature, Essence and Spirit. Furthermore, my artistic approach was mostly influenced by the views of artists such as, Vasily Kandinsky, Frank Stella, Piet Mondrian, Paul Klee, Dale Chihuly and Tauba Auerbach.

L'extraction par CO2 supercritique démontre les avantages d'un procédé de chimie verte en comparant ce procédé à la méthode d'extraction par solvant organiques et en tenant compte du degré de toxicité et de pollution du solvant. L'extraction par solvants organiques met en évidence l'influence du solvant d'extraction alors que l'extraction par CO2-SC met en évidence l'influence de différents paramètres dont la pression, la température, le temps de contact entre la matrice végétale et le CO2-SC, le diamètre moyen des particules et l'ajout d'un co-solvant. L'analyse chromatographique a permis d'identifier et de quantifier les flavonolignanes (silychristine, silydianine, silybine, taxifoline) dans les extraits de graines obtenus par solvants organiques et par CO2-SC avec co-solvant. A 220 bar, les concentrations en silydianine (38,87 mg/g) et en silybine (45,91mg/g) sont les plus élevés et à 40°C les concentrations en silychristine (31,97mg/g), en silydianine (38,87 mg/g) et en silybine (45,91mg/g) sont les plus importantes. Les extraits huileux obtenus à 220 bar et à 40°C des graines de Silybum marianum sont riches en acides gras : acide linoléique (65,22%), acide oléique (27,01%), acide palmitique (12,12%). L'activité antioxydante a été évaluée par deux tests : test DPPH et test ABTS. Ces deux tests sont complémentaires et ont permis de conclure que l'extrait ayant un effet antioxydant le plus important est l'extrait obtenu par CO2-SC à 220 bar et à 40°C. L'activité biologique de cet extrait est mise en évidence par rapport à une lignée cellulaire cancéreuse du colon Caco-2. La silychristine, la silydianine et la silybine ainsi que l'extrait obtenu par CO2-SC avec co-solvant (éthanol) à 220 bar et à 40°C ont été testés vis à vis de cette lignée cancéreuse. Ces expérimentations in vitro reflètent une activité cytotoxique quantifiable et une mortalité cellulaire des Caco-2 des flavonolignanes allant jusqu'à 71%
The supercritical CO2 extraction demonstrates the benefits of green chemistry process comparing with the method of organic solvents extraction and depending to toxicity and pollution solvent degree. Organic solvents extraction shows the solvent extraction influence, so that the SC-CO2 extraction highlights different parameters including pressure, temperature, contact time between the plant matrix and CO2 SC, the average particle diameter and the addition of a cosolvent. Chromatographic analysis identified and quantified four flavonolignans (silychristin, silydianin, silybin, taxifolin) in seed extracts obtained by organic solvents and SC-CO2 with cosolvent. At 220 bar, silydianin (38.87 mg / g) and silybin (45.91 mg / g) have highest concentrations and at 40°C silychristin (31.97 mg / g), silydianin (38.87 mg / g) and silybin (45.91 mg / g) have the most important concentrations. The oily extracts obtained at 220 bar and 40°C of Silybum marianum seeds are rich in fatty acids: linoleic acid (65.22%), oleic acid (27.01%), palmitic acid (12.12%). The antioxidant activity measured by two tests: DPPH and ABTS test. These two tests are complementary and confirm that the extract with the higher antioxidant effect is the extract obtained by SC-CO2 at 220 bar and 40°C. The biological activity of this extract is demonstrated with respect to a colon cancer cell line Caco-2. Silychristin, silydianin and silybin and the extract obtained by CO2-SC with co-solvent (ethanol) at 220 bar and 40°C were tested with respect to this line cancer. These experiments in vitro cytotoxic activity reflect estimable and cell death of Caco-2 flavonolignans of up to 71%

碩士
國立中央大學
化學工程與材料工程學系
106
Tumors contain a small subpopulation of cells, i.e., cancer-initiating cells or cancer stem cells (CSCs), which exhibit stem cell properties, possess a self-renewing capacity and are responsible for tumor generation and metastasis. Cancer stem cells persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Furthermore, it is expected to establish primary colon cancer cell lines in vitro, since patient specific colon cancer cell lines is in great advantages of developing patient specific therapy in clinical application. We are developing a method of establishing cancer cell lines from primary patient colon cancer tissue. Colon cancer tissue is digested by collagenase to generate colon cancer tissue solution. Subsequently, primary colon cancer cell lines were established by (a) culture method on specific culture materials, and (b) the membrane migration method through Nylon mesh filter.1 We investigate which factor is more important to establish the cancer cell lines from minimum amount of colon cancer tissue and determine the optimal conditions to establish patient specific colon cancer cell lines from primary colon cancer tissues. The patient specific colon cancer cell line will be useful for the patient-specific therapy in the future. In this study, we designed cell sorting dishes from two combined concepts, which are physical cues and biological cues. Different extracellular matrix (ECM) and cell binding domain oligopeptides (biological cues) having different elasticity (physical cues) are immobilized on the cell sorting dishes as biological cues and physical cues for capturing CSCs. Optimal ECM/ECM-derived oligopeptide and elasticity of the dishes for (a) establishment of the patient-specific cancer cell lines and (b) isolation or depletion of CSCs have been shown in this study. Moreover, CSCs identification is quantified by colony forming assay and/or tumor generation in serial xenotransplantation model.

Recent experiments are bringing to the fore more and more information about the effects of different treatments on the gene expression of different genes. The results obtained from these experiments show that some definite trends are observed in different genes in the Human Embryonic Kidney and Human Colon Adenocarcinoma Grade II cell lines. The difference in the gene expressions of the two cell lines motivates the problem in this thesis. The thesis provided intervention methods to make the colon cancer cell line genes behave more like their Human Embryonic Kidney cell line counterparts. Two methods of intervention were introduced. The first method was the simpler on-off control intervention while the second method used a more advanced proportional integral control to meet the goal. A comparison of these two intervention methods showed the clear implementational advantages of proportional integral control over on-off control.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Masters of Science in Medicine (Pharmacology) Johannesburg, 2013
Colon cancer is the third most common cancer worldwide and the second most common in the western world. More than 40 % of colon cancer sufferers develop metastases and chemotherapy is often used alone or in combination with radiotherapy as adjunctive therapy for the advanced disease. A major effort has been made in the past decade to develop anticancer agents through both empiric screening and rational design of new compounds. These attempts are made to improve the survival rate, reduce the severe adverse effects associated with existing cancer chemotherapeutic agents as well as to reduce the development of drug resistance. In the present study, two colon cancer cell lines were exposed to novel imidazo[1,2-a]pyridines and novel nucleoside analogues, aiming to investigate the cytotoxic efficacy on the cells, the mode of cell death, and to explore the pathways by which cell death was induced.

碩士
國立成功大學
藥理學研究所
107
In addition to epidermal growth factor receptor (EGFR), overexpression of its downstream substrate EGFR pathway substrate number 8 (Eps8) occurs in human colorectal cancer (CRC). Eps8 overexpression promotes the kinase activity of Src and FAK, leading to the enhancement of cell proliferation and motility in cancer cells. Insulin Receptor tyrosine kinase Substrate Protein of 53 kDa (IRSp53) is an adaptor protein that links Rho-family small GTPases such as Rac and Cdc42 to the actin cytoskeleton reorganization. Previously, our study indicated that Eps8-IRSp53S complex participated in v-Src-mediated tumorigenesis. To substantiate the importance of Eps8-IRSp53 interaction in CRC, we generated cells expressing both Eps8 and IRSp53S in SW480 cells that exhibits much lower level of these proteins as compared to their metastasized counterpart SW620 cells. Unfortunately, we were unable to obtain cell lines constitutively expressing both Eps8 and IRSp53 in SW480 cells suggestting that Eps8-IRSp53 interaction might cause growth arrest and/or apoptosis in SW480 cells. To confirm this, we generate Tet-Off control and Tet-Off regulated IRSp53S-expressing SW480 cells first. Then, IRSp53S-inducible cells were transfected with EGFP-expressing or EGFP-Eps8 epressing plasmid DNA. In this way, we observed enhanced chromatin condensation only in IRSp53S/EGFP-Eps8-overexpressing cells, but not in IRSp53S/EGFP-expressing cells, nor in doxycycline-treated IRSp53S/EGFP-Eps8 overexpressing cells. In addition, MTT assay revealed that double-overexpressing Eps8 and IRSp53S reduced cell viability/proliferation in SW480 cells. Flow cytometry studies also showed that double-expressing Eps8 and IRSp53S retarded cell cycle progression as indicated by increased G1 phase and reduced S phase population. However, our prelimery study in animal xenografted tumor model revealed that due to low percentage (less than 10%) of cells really expressing both Eps8 and IRSp53S at the same time, tumors derived from this double overexpressing cell line were not statistically different from those derived from IRSp53S-expressing cells nor from vector control cells. Nevertheless, our data highlighted IRSp53S-Eps8 interaction might play a sophisticated role in regulating colon cancer progression, which deserves further study.

碩士
中國醫藥大學
營養學系碩士班
98
Colorectal cancer is one of the most common causes of cancer death in many countries including Taiwan. Several studies have indicated that leptin may enhance the growth of tumor cancers via multiple signaling pathways. The upregulation of circulating leptin level was shown to correlate with malignant progression of colon cancer cells. In the other hand, MMP-7 (matrix metalloproteinase-7) has been demonstrated as an important malignant biomarker in human colonic cancer patients. Overexpression of MMP-7 has been shown to increase the metastasis ability in a variety of cancer cell lines. However, the relationship of leptin mediated cellular metastasis on human colon cancer has not been demonstrated well yet. Our results demonstrated that leptin (0, 0.0125, 0.0625, 0.25 μM) induced cellular invasion in a dose-dependent manner, which was accompanied with enhanced MMP-7 expression in human colon cancer HT-29 cells. The molecular mechanism was leptin regulated cellular invasion through GSK3β/β-catenin signaling pathways in human colon cancer HT-29 cells. Besides, leptin induce active β-catenin accumulated in nucleus, enhanced transcription factor β-catenin bind to MMP-7 promoter, and result in increased cellular invasion in colon cancer HT-29 cells. Next, we would like to examine the mechanism of MMP-7 induced cellular invasion. By using si-RNA (Small interfering RNA), we found that protein expression of MMP-7 was reduced and decreased leptin-mediated cellular invasion in HT-29 colon cancer cells. These findings suggest that leptin increased MMP-7 expression through β-catenin singaling pathway, result in induced colon cancer HT-29 cells invasion. Our results demonstrated that MMP-7 act as biological marker in leptin-mediated malignant progression of colon cancer cells.

碩士
國立成功大學
微生物及免疫學研究所
90
Natural antitumor agent Justicidin-A (JA), isolated from J. procumbens, induced apoptotic responses in HT-29 and HCT 116 human colorectal cancer cells in a dose- and time-dependent manner. JA inhibited the proliferation of these cancer cells and IC50 ( 50％ of effective dose ) on day 6 of exposure to JA was 0.11μM for HT-29, 0.4μM for HCT 116 and 23μM for human peripheral blood mononuclear cells (PBMC). The translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane, increased sub-G1 DNA contents, and fragmentation of DNA were observed in JA-treated cells. Both cells treated with JA resulted in mitochondrial perturbation including losses of mitochondrial membrane potential, releases of Smac/DIABLO and cytochrome c from mitochrondria into the cytosol, decrease the XIAP protein contents and promotes the activation of caspase-9 and caspase-3. Besides, the anti-apoptotic members of Bcl2 family, Bcl-2 and Bcl-XL, were decreased and pro-apoptotic Bax was up-regulation in JA treated cells. These may be associated with mitochondrial membrane potential which contributes to cytochrome c released from mitochondria. Furthermore, the activated caspase-3 cleavaged poly (ADP-ribose) polymerase (PARP) and DNA fragmentation factor-45(DFF45)/ICAD. All of these finially led to fragmentation of DNA. Our findings suggest that JA induces the loses of mitochondrial membrane potential, releases of Smac/DIABLO and cytochrome c from mitochondria to the cytosol, inhibits XIAP content, increases the activities of caspase-9 and –3, cleavages PARP protein and DFF45, and fragmentation of DNA. This research indicates JA may become a novel potent drug for the treatments of colorectal cancer.

Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) is a cytokine of TNF family, which participates in the non-exclusive regulation of survival and proliferation of mainly hematopoietic cells. Shortly after its discovery it also brought significant attention as specific and potent inducer of apoptosis of cancer cells of various origins, and since then it has been investigated as a potential novel anti-tumor therapeutics. Recently, cancer stem cells (CSCs) were suggested to be a distinct subset of tumor cells that could be responsible at least in some tumors for their sustainment, recurrence and drug resistance. These cells in the "hierarchic" model of tumorigenesis thus represent an important and attractive target for efficient tumor therapy. In this study we use several colorectal adenocarcinoma cell lines as an experimental model for the analysis of CSC-prone cultivation conditions on TRAIL-induced apoptosis of these cells. For enrichment of eventual cancer stem cells we cultivated cell lines in a serum-free medium, originally developed for cultivation of neural stem cells, and assessed the expression of putative CSC markers CD133 and ABCG2 by flow cytometry (FACS). Simultaneously, we tested the expression of TRAIL receptors and susceptibility to TRAIL-induced apoptosis in these cells. We...

碩士
國立中興大學
生物醫學研究所
101
Colon cancer is the third leading cause of cancer mortality in Taiwan. Metfomin is a widely used drug to treat type II diabetes. A number of epidemiological studies have demonstrated that patients taking metfomin shows substantially lower cancer incidence and mortality than those not receiving this agent, but studies about the effect of metformin on the incidence and treatment efficacy of colon cancer are limited. Using human colon adenocarcinoma cell lines HT-29, HCT116 and SW620 as the cellular model, in this study we found that metfomin treatment led to inhibition of cell colony formation and cell viability, induction of cell cycle arrest at G1, and down-regulation of cyclin D1, CDK4, CDK6, SKP2, p27KIP1 and p21CIP1 in all cell lines examined. It is known that two signaling pathways leading to the G1 arrest, namely, the SKP2-p27KIP1 pathway and the, cyclin D-CDK4/6 pathway. We found that overexpression of SKP2 and knockdown of p27KIP1 protein in HT29 or HCT116 cells showed limited effect to rescue cells from metformin-induced repression of colony formation, suggesting that the SKP2-p27KIP1 pathway does not appear to be important for metformin-induced anti-colon cancer effect and thus implicating a potential role of the cyclin D-CDK4/6 pathway in this process. Additionally, we found that metformin induced apoptosis in SW620 cells, suggesting an additional mechanism underlying the anti-colon cancer effect of metformin.

碩士
國立中央大學
化學工程與材料工程學系
107
Cancer stem cells (CSCs, cancer-initiating cells) typically comprise 1%–5% of the total tumor cell population. This cell population is considered to be primarily responsible for tumor initiation, growth, and metastasis. However, it is hard to distinguish cancer cells from other cells (i.e. fibroblasts, blood cells, etc.) in primary tissue after enzyme digestion process. Therefore, We develop membrane filtration and migration method to target and purify rare primary CSCs based on their high migration mobility characteristics compared with other tissue cells. It is expected to establish the primary colon cancer cell line from primary tumor tissue by the membrane filtration and migration method for the development of patient specific therapy in clinical application. We designed a membrane filtration method to purify and further establish primary colon cancer cell line from human colon cancer tissue. Nylon mesh filters with pore size of 11 μm and 20 μm and PLGA (poly (lactic-co-glycolic acid))-silk screen membranes with different PLGA concentration 3%, 5%, and 10% as the membranes are used in this study where PLGA has biodegradation and biocompatibility properties. With increasing PLGA concentration, the thickness of the PLGA-silk screen membranes also increased. Nylon has unique physical and chemical properties. These properties can also apply to cell culture process. In the primary cancer cell extraction procedures, the colon cancer tissue was digested by collagenase type IV at first. After digestion of the tissue, the primary colon cancer cell solution was permeated through the membranes with different pore size and different degree of biocompatibility. These membranes were used to capture primary colon cancer cells, and the cells on the membranes were expected to migrate into the culture dishes during the cultivation of the membranes in the culture medium (i.e., membrane migration method) after the filtration of colon cancer tissue solution through the membranes. Afterwards, the expression of cancer stem cell markers, CD133 and CD44 was evaluated by flow cytometry. Furthermore, the malignancy index of primary cells and the cells purified by the membrane filtration and migration method was evaluated by soft agarose colony forming assay. It is expected that the cells migrate from the membranes will have higher expression of the colon cancer stem cell surface markers. The similar results may be obtained in soft agarose colony forming assay where the cells show higher colony forming efficiency and bigger colony size. The goal of this project is to establish a patient-specific colon cancer cell line by the membrane filtration and/or migration method for precision medicine in the future.

碩士
中國醫藥大學
中國藥學研究所
99
The molecular mechanism of extracts of Solanum lyratum THUNBERG induced cytotoxicity and apoptosis in human colon adenocarcinoma cell lines (Colo 205) Jih-Hung Lu Institute of Chinese Pharmaceutical Science, China Medical University, Taichung, Taiwan Abstract Solanum lyratum THUNBERG is a tranditional herbal medicine for diminishing inflam- mation, anti-oncosis and -cancer. Several studies have indicated that Solanum lyratum THUNB. exhibits anti-tumor properties, but underlying mechanism is not well known. Therefore, we try to investigate the mechanism of anticancer activity and evaluate the chemopreventive role of the extracts from Solanum lyratum THUNB. Our data shown that the 50% ethanol extract (SLE) and n-butanol layer extract exerted cytotoxic effects, and inhibited significantly proliferation of colo 205 cells. They induced S arrest in earlier 24 hours after treatment and G2/M arrest in later 48 hours and apoptosis in Colo 205 cells. The DNA gel electrophoresis array shown the occurrence of typical DNA ladder parttern. Investigation on the gene expression of cyclins, CDKs and CDKIs by RT-PCR (Polymer- ase chain reaction) showed that p21, cyclin B1, p53, cdc25b, cdc25c, Wee1 were unaffected, whereas Cdk1, p27 is up-regulation, and Chk1, Chk2 down-regulation. The gene expression levels changed with increasing the doses of SLE in Colo 205 cells. Investigation on the levels of cyclins, Cdks and CdkIs by immunoblot analysis showed that cyclin E and p53 levels were unaffected, whereas Cdk1 and cylin B1 decreased, p27 levels increased with the time of treatment in Colo 205 cells. The increase of the levels p27, Cdk1 up- regulation, and Chk1, Chk2 down-regulation could be the major factor for SLE to cause G2/M arrest in this examined cells. Flow cytometric assays and DNA fragmentation gel electrophoresis also confirmed that SLE induced apoptosis in Colo 205 cells. Investigation on the levels of members of Bcl-2 family, caspases and NF-κB levels by RT-PCR shown that Bad, caspase-3, caspase-6, cas- pase-8, and caspase-9 levels were unaffected, whereas Bcl-2 and NF-κB levels decreased with increasing the doses of SLE in Colo 205 cells. By immunoblot analysis shown that caspase-8 were unaffected, whereas caspase-3 and caspase-9 levels increased and NF-κB (p65), NF-κB(p50) levels decreased with increasing SLE in Colo 205 cells. Investigation on the gene expression of topoisomerase by RT-PCR showed that topoiso- mer aseⅡβ were unaffected, whereas topoisoneraseⅠis up-regulation, and topoisoneraseⅡα down-regulation. The gene expression levels changed with increasing the doses of SLE in Colo 205 cells. The down-regulation of topoisoneraseⅡα could be the major factor for SLE to cause cytotoxicity in this examined cells. In conclusion, SLE appears to exert its anticarcinogenic properties by cytotoxicity and inducing cell cycle arrest and apoptosis. These results suggest that Solanum lyratum THUNB. could be used as a potent agent for colon cancer therapy and chemoprevention.

Thesis (Ph. D.), Faculty of Health Sciences, University of the Witwatersrand, 2009
With the high prevalence and high mortality rate of cancer in the global community, it is increasingly essential to accelerate our understanding of the disease, to identify new genetic targets for therapy, and to pursue avenues for improving on the therapies in development and in current use. The aim of this study is to identify cell cycle-related genes whose expression is influenced by the chemotherapeutic drugs curcumin, SAHA, lycopene and thalidomide in breast and colon cancer and normal cell lines. These drugs are currently not in clinical use for cancer in South Africa, and while there have been investigative studies of these chemotherapeutic agents, this study aims to identify the specific genes that are influenced by the drugs. The result of this is that several genes that were not previously documented as targets of these drugs are highlighted. The cell cycle pathway is the area of focus as loss of regulation in the cell cycle is one of the important factors involved in promoting cancer initiation and progression. In the first instance, flow cytometry was used to identify optimal drug concentrations relative to the cell cycle stages. Following this, alterations in gene expression were assessed using a PCR-based differential display after each drug treatment. Subsequently, a more focussed approach was taken in a PCR-array analysis of panels of cell cyclerelated genes. A subset of genes is identified that is implicated in oncogenic transformation in breast cancer. This has the potential to inhibit the genetic pathways involved in breast malignancy by providing targets that perhaps may not be manipulated in current therapies. The gene expression studies here suggest that lycopene and thalidomide function in inhibiting this transformation, and play significant roles in suppressing the oncogenic state of breast cancer. Curcumin and SAHA also exhibit important functions in inhibiting tumourigenesis in colon cancer. While the results propose that the drugs have clear roles in inhibiting breast and colon cancer, they are also implicated in promoting cancer. This research has defined the genes that must be carefully monitored during drug administering as they may promote these and other cancers. The availability of these results to researchers will aid in selecting the criteria for assessing the success rate of these drugs.

A dissertation submitted to the Faculty of Heath Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science Johannesburg, 2019
Colorectal cancer (CRC) is the fourth most common and the sixth most lethal cancer in South Africa. Although, there have been advances in anticancer drug development, there is nevertheless a need for the development of novel cost-effective drugs for treatment. Several reports have suggested that the indole ring contained in various natural and synthetic compounds, is an important chemical moiety with certain biological activities that confers anticancer properties against several cancers. Thus, the aim of this study was to evaluate the potential apoptotic effects of a synthesized Indoline molecule (N-(2-hydroxy-5-nitrophenyl (4methylphenyl) methyl) (HNPMPI) on HT-29 (mid stage) and DLD-1 (late stage) colon cancer cell lines. In particular, the possible effects of HNPMPI on cell morphology and apoptosis (Annexin V) were assessed. After establishing the apoptotic profile of the drug treated cells, changes in the expression of key apoptotic proteins were evaluated using a focused human apoptosis protein array. Based on the identification of differentially regulated proteins identified from the array study, gene expressions of selected genes were analyzed using RTPCR. Further, the subcellular localization of key proteins was determined using Confocal Microscopy. After treatment with HNPMPI, the Annexin V profiling demonstrated that HNPMPI more effectively induced apoptosis in HT-29 cells as compared to the DLD1 cell line. The protein profiler assay revealed differential expression of the apoptotic proteins and it showed that the extrinsic pathway of apoptosis is triggered in HT 29 cells; and Caspase mediated apoptosis was activated in DLD-1 cells, after the treatment. With regards to gene expression levels, the transcription levels of the pro-apoptotic genes BAD, BAX and p53 decreased in DLD-1 cells with HNPMPI treatment. In comparison in the HT-29 cells, while mRNA levels of BAX and p53 decreased, BAD levels were increased. The transcription of the two anti-apoptotic genes, Bcl-2 and CASP-3, had diverse responses to the drug treatment in the two cell lines CASP-3 transcription levels increased in both cell lines; while Bcl-2 levels were constant in the HT-29 cells, but decreased in the DLD-1 cells. In support of this, immunofluorescence confirmed that the intracellular protein expression of Bcl-2 and P53 reflected the results obtained from the protein array, after drug treatment. The indole derivative HNPMPI showed effective apoptotic activity against both cell lines, representing mid and late stage colon cancer. A balance between an over-expression of proapoptotic genes and under expression of anti-apoptotic genes is responsible for carcinogenesis. Although gene expression of selected genes were altered, the protein expression results obtained here confirm that HNPMPI has promising pro-apoptotic properties acting to induce both the intrinsic and extrinsic pathways of cell death in a stage specific manner in the two colon cancer cell lines
MT 2019

碩士
國立中興大學
生物醫學研究所
104
Colorectal cancer is the most commonly diagnosed cancer around the world and the third cause of cancer mortality in Taiwan. Up-regulation of prosurvival BCL-2 family members has been shown to be correlated with early colorectal cancer formation. Obatoclax (a.k.a GX15-070) is a pan-BCL-2 inhibitor. Previous studies have pointed out the antiproliferative effect of obatoclax, but the mechanisms remain unclear. In this study, we used colorectal cancer cell lines HCT116, HT-29 and LoVo as the cellular model to study the antiproliferative action of obatoclax. We found that obatoclax causes cell cycle arrest at the G1 phase and also marked decrease in the clonogenic capacity in dose-dependently. Immunoblotting analyses indicated that a decline of cyclin D1 levels following obatoclax treatment. Importantly, overexpression of cyclin D1 conferred cells resistance to obatoclax-induced G1 arrest and suppression of clonogenicity, indicating that cyclin D1 decrease is a fundamental mechanism for obatoclax to induce antiproliferation. To further understand how obatoclax down-regulates cyclin D1, we found that the levels of the cyclin D1 mRNA and promoter activity were not significantly affected. In contrast, pre-treatment with the proteasome inhibitor MG132 markedly rescued cyclin D1 protein levels in obatoclax-treated cells. Additionally, cycloheximide chase analyses revealed that the half-life of cyclin D1 protein was evidently decreased following obatoclax stimulation, indicating that obatoclax reduces cyclin D1 expression mainly by destabilizing cyclin D1 protein. It is known that a major mechanism of cyclin D1 destabilization is through glycogen synthase kinase-3β (GSK3β)-mediated phosphorylation of Thr286 of cyclin D1 (p-Cyclin D1 (Thr286). However, we found that obatoclax induced ser473 phosphorylation and thus activation of AKT, which then causes inactivation of GSK3β as evidenced by the increase in the levels of p-GSK3β (Ser9), suggesting that GSK3β activity is not required for obatoclax-induced cyclin D1 destabilization. In summary, we herein provide the first evidence that obatoclax induced antiproliferation in human colorectal cancer cells by down-regulating cyclin D through cyclin D1 protein destabilization.

碩士
國立中興大學
生物醫學研究所
102
Abstract Colorectal cancer is the third most commonly diagnosed cancer in the world and the third leading cause of cancer mortality in Taiwan. Inducing apoptosis represents an important mode of action of anti-cancer drugs. Pro-survival Bcl-2 is known to play a key role in neoplastic progression in the large intestine. Recently, a number of BH3 mimetic small molecules, which mimic the action of proapoptotic BH3-only proteins, have been developed to counteract pro-survival Bcl-2 proteins such as BCL-2. Obatoclax is one of the BH3 mimetics. It is an intravenously-administered drug under investigation in Phase I and II clinical trials as a novel anticancer therapeutic for hematological malignancies and solid tumors. Survivin is a member of the inhibitor-of-apoptosis proteins (IAPs) family. Overexpression of survivin generally correlates with tumor progression and induces anticancer drug resistance. It has been revealed in past studies that patients with survivin overexpression had a more aggressive and metastatic colorectal carcinoma. We herein found that Obatoclax can downregulate survivin in human colon cancer cell lines, but studies about the effect of Obatoclax on the incidence and treatment efficacy of colon cancer are limited. Using human colon cancer cell lines HCT116, DLD-1, LoVo and WiDr as the cellular model, we found that Obatoclax dose-dependently induced down-regulation of survivin. This down-regulation is mainly regulated at the level of transcription, as Obatoclax reduced the levels of both survivin mRNA and survivin promoter activity but failed to rescue survivin expression when proteasome-mediated degradation is abolished. Furthermore, overexpression of survivin showed significant effect to rescue cells from Obatoclax-induced repression of colony formation. Additionally, Obatoclax can inhibit the endogenously activated Wnt/ β-catenin signaling in HCT116, DLD-1, LoVo and WiDr by β-catenin/TCF transcription reporter assay. Furthermore, overexpression of β-catenin showed significant effect to rescue cell viability. Taken together, we conclude that survivin is one of the molecular target of Obatoclax through which to induce apoptosis in human colon cancer cell lines.

碩士
國立中央大學
系統生物與生物資訊研究所
98
In order to understand the complexity of regulation, expression and feedback mechanism of genes, the methodology of system biology through the high-throughput analysis and information analysis must be utilized to elucidate the gene regulation network. The advance in microarray experiment and its high throughput analysis has set up an important milestone in the system biology. In this study, we used the exon array and high throughput data analysis software developed by Partek Corporation to observe the change of EGFR downstream targets after EGF and Cetuximab blockade in Sw480, Caco2 and Ht29 cells. By using SAM biochip significance analysis method, 3052 significantly different genes were selected. These genes were grouped by different functions and relativity between different cell lines by correlating these genes with KEGG mapping .We analyze the heat map of each cell line before and after stimulation by using R package. We also compare the difference of control and experiment group and similarity between other two groups by using Euclidean distance and Pearson correlation. Using KegArray in conjunction with KEGG biological information pathway data，we found the different downstream pathway transformation.of the Sw480、 Ht29 、Caco2 cell line after treatment with Cetuximab to block EGFR and EGFR-Ras pathway. In the furture, we hope to detect different gene mutation after treated with Cetuximab using colon cancer cell lines and observe gene alteration in various EGFR-Ras pathway. We want to integrate complex gene interaction by using system biology. It is valuable to study and investigate the regulation mechanism of upstream and downstream interaction in a pathway.

碩士
國立成功大學
藥理學研究所
85
Colon cancer is one of the primary resistant neoplasia that basically does not respond on its first exposure to the currently available standard agents. Nevertheless, 5-fluorouracil (5-FU), alone or together with leucovorin, has been taken as the last resort for treating colon cancer. However, the response rate of 5-FU is approximately 30% and the remission is usually temporary. The present investigation aims at knowing why the colon tumor does not respond on the first exposure to 5-fluoro-uracil。The cells used were from ten established colon cell lines that were obtained originally from ATCC (American type culture collection), and a primary cell line that we established ourselves. The primary cell line was derived from one of the 20 colon tumor specimens and was subjected to further characterization. On the soft agar test, the primary line was found to have the property of anchorage-independent growth; the eight strains cloned from the primary line all have normal chromosome number of 46. The cell lines, that from ATCC and ours, can be categorized into 3 groups according to their resistance to 5-FdUR. As for the p53 gene status, SW 480 has one mutations (codon 273) in one allele and the other mutation (codon 309)in the other allele; the line SW 480 is more resistant to 5-FdUR than the others. In addition, WiDr, being hemizygous at codon 273 with mutation; is resistant to 5-FdUR. We therefore conclude that there exist some correlation between p53 gene mutation and the resistance to 5-FdUR. In other cytotoxicity experiments, we found that lines SW 480, WiDr and Colo 201 showed cross resistance between 5-FU and 5-FdUR. When verapami, which is an antagonist of p-glycoprotein, was added to the growth medium, the cytotoxicity of 5-FdUR to cell line did not increase, suggesting that multi-drug resistance (MDR) probably is not involved in the cytotoxicity of 5-FdUR. We plowed further into the sensitivities of the cell lines to (-ray irradiation. In the low therapeutic dose of 1 Gy, the cell lines all still possessed 70% to 80% of survivabilities, indicating that all the cell lines have the propensity of radiation resistance. However, at high dose of radiation (20Gy), the survival rate of all of the cell lines declined to 5 ~10%, suggesting cell death due to either cell damage or necrosis.In addition, we did not find correlation between the chromosome number of cell lines and 5-FU drug resistance. As for the correlation between 5-FU resistance and the thymidylate synthase (TS) gene, we used polymerase chain reaction (PCR) to amplify a fragment of 250 bp DNA of the exon 5 of TS gene that encodes the catalytic site of the TS enzyme; then subjected the PCR product to the single-strand conformation poly-morphism (SSCP) testing. We did not find any SSCP band shifting between normal control and the tumor lines, suggesting that there is no mutation in the region of thymidylate synthase gene encoding catalytic site. The result of Southern blotting and dot blotting did not show quantitative difference of thymidylate synthase gene between the control and the specimen lines. They suggest that gene amplification might have no contribution to the primary resistance of tested cancer lines to 5-FdUR.

碩士
中華醫事科技大學
生物醫學研究所
100
Lactobacillus species is one of the important probiotic used for functional food. To evaluate the effect of Lactobacillus on intestinal inflammation, the different UV-killed Lactobacillus strains (L1 ~ L9 or S10) co-culture with an intestinal cell line-HT-29 cells was as a experimental model, and its cell cytokine secretion and regulation mechanism were measured in this study. According to the results, when HT-29 cells was treated with different Lactobacillus strains and co-culture with or without TNF-α for 24 hours, the cell viability was not significantly change. The cell number and shape of HT-29 cells were not affected. The secretion levels of cytokine IL-10 was not significantly change in all of Lactobacillus strains groups. But, the IL-8 secretion in the L1, L3 or L9 groups were significantly decreased as compared with TNF-α control group (P<0.05) when various Lactobacillus strains co-culture with HT-29 cells induced by TNF-α for 6 hours. In addition, the cytoplasmic IκB phosphorylation and nucleic NF-κB-DNA binding activity were significantly decreased induced by TNF-α. We identified the specific Lactobacillus strains can reduce cytokine IL-8 secretion levels. The regulate mechanism maybe through the inhibition of cytoplasmic IκB phosphorylation and reduce NF-κB-DNA binding activity in HT-29 cells induced by TNF-α（P<0.05）.

碩士
國立中興大學
生物醫學研究所
103
ABT-199 is a BH3 mimetic derived from navitoclax (ABT-263). ABT-199 inhibits the anti-apoptotic activity of Bcl-2, but not BCL-xL and MCL-1. MCL-1 is an anti-apoptotic Bcl-2 family protein. Overexpression of MCL-1 causes cancer cell evasion of apoptosis and increases drug resistance. Previous reports have shown that navitoclax upregulates MCL-1 in various cancer cells, but the effect of ABT-199 on MCL-1 expression is still unknown. In this study, we use human colon adenocarcinoma cell lines HCT116, DLD-1, HT-29, LoVo and SW620 as the cell model. Here, we found that ABT-199 up-regulates MCL-1 levels. This up-regulation is ascribed to increased levels of MCL-1 mRNA and an increase in MCL-1 protein stability, which is likely through ERK-induced phosphorylation of MCL-1Thr163 and AKT-mediated inactivation of GSK-3.Additionally, MCL-1 inhibition appears to increase ABT-199-induced cytotoxicity. Taken together, we conclude that ABT-199 could cause colon cancer cell resistant to its cytotoxicity by increasing MCL-1 protein stability and mRNA expression.

碩士
靜宜大學
食品營養研究所
94
Cancer is the major death cause in Taiwan. Recently, there is an increasing interest in the study how to prevention or delay the colon cancer development. Zanthoxylum ailanthoides Sieb & Zucc. is a traditional Chinese herbal medicine that is belonging to the Rutaceae family. In this study, the effects of 50% ethanolic extracts from the stem of Z. ailanthoides (SE) on the growth and apoptosis indution of human colon adenocarcinoma cell lines (colo 205) will be studied. The results demonstrated that SE can inhibit the cell growth, induce cell cycle arrest on G2/M phase. The morphology, DAPI staining, comet assay and DNA fragmentation assay were conducted and they indicated that SE induced colo 205 cells apoptosis. Western blotting analysis, SE decreased the protein level of cyclin A, B, cdc2 and cdc25c and increased the levels of CHK2, Wee1 and p21 of cells. Flow cytometry assay demonstrated that SE induced the change of mitochondrial membrane potential, reactive oxygen species (ROS), and calcium releasing. SE inhibited the expression of Bcl-2 and Bcl-XL and promoted the levels of Bax, and Bid. Furthermore, SE can increase the expression of caspase-3, -8, -9 and cytochrom c. In MAPK family, SE can decrease the expression levels of ERK and increase the levels of JNK, p38 and p-p38, however, SE increace the protein level of p-c-jun and Fas/FasL. SE decreased the expression levels of COX-2, iNOS and NF-κB and induced apoptosis. SE also can inhibit the invasiveness of human colon cancer cells by suppressing MMP-2, -7 and -9 expressions. SE decreased the glutathione peroxidase activity and glutathione levels, however, SE increased the malondialdehyde levels in colo 205 cells. The results of this study we suggested that SE might inhibite cell growth and induced G2/M phase arrest. SE induced apotptosis throught the activation of JNK/Jun/Fas/FasL and caspase-8, which lead to changes on mitochondriall membrane permeability and cytochrome c releasing. Subsequently, caspase-9 and -3 are activated and decrease iNOS, COX-2, NF-κB, MMP-2, MMP-7 and MMP-9 expression. Furthermore, our results demonstrate that SE can change GSH antioxidant system. These results support that Zanthoxylum ailanthoides Sieb & Zucc. stem can inhibit the cell growth, indue apoptosis and inhibite invasiveness.

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2012
O cancro é uma doença complexa que envolve numerosas alterações na fisiologia da célula que conduzem, em última instância, a tumores malignos. Os processos biológicos através dos quais as células normais são transformadas em células cancerígenas malignas têm sido alvo de vasta investigação durante várias décadas (Seyfried & Shelton, 2010). Existem seis alterações essenciais na fisiologia celular que estão na base do crescimento celular maligno e são a auto-suficiência em termos de sinais de crescimento, a insensibilidade a sinais inibidores de crescimento, a evasão à morte celular programada (apoptose), um potencial replicativo ilimitado, a angiogénese sustentada e a invasão tecidular e metastização (Hanahan & Weinberg, 2000). Contudo, recentemente, têm sido propostos outros atributos distintos às células cancerígenas funcionalmente importantes para o desenvolvimento de cancro, que incluem a reprogramação do metabolismo energético celular, por forma a suportar o crescimento celular e a proliferação contínua, e a evasão ao controlo imunitário (Hanahan & Weinberg, 2011). Ao longo da última década, os tumores têm sido identificados como órgãos cuja complexidade pode exceder a dos tecidos saudáveis normais, contrastando com a anterior visão reducionista dos tumores como um conjunto relativamente homogéneo de células cancerígenas. Desta forma, a biologia de um tumor só pode ser entendida analisando os tipos celulares especializados que o constituem bem como a microambiente tumoral criado durante o processo de tumorigénese (Seyfried & Shelton, 2010) . Em termos de incidência global, estima-se que 12.7 milhões de novos casos de cancro e 7.6 milhões de mortes causadas por cancro ocorram por ano (Ferlay et al., 2010). O cancro do colo do útero é o terceiro cancro mais comum entre as mulheres, e o sétimo globalmente (Ferlay et al., 2010; Yugawa & Kiyono, 2009), sendo a quarta causa de morte por cancro entre as mulheres, mundialmente (Jemal et al., 2011; Ferlay et al., 2010). Apesar de a incidência deste tipo de tumor ter diminuído nos países desenvolvidos, a incidência mundial estimada ronda os 500 000 novos casos por ano (Jemal et al., 2011; Yugawa & Kiyono, 2009). Em Portugal, o cancro do colo do útero surge como o quarto mais frequente entre as mulheres (Globocan, 2008). Em termos histológicos, existem duas variantes de cancro do colo do útero com significado clínico, os adenocarcinomas (AC) (que compreendem 10 a 25 % dos casos) e os carcinomas pavimento celulares (SCC) (que compreendem cerca de 85 a 90% dos casos) (Chan et al., 2003; Smith et al., 2000). Sabe-se que em algumas doentes, o tumor apresenta um crescimento endofítico, invadindo o endométrio, enquanto noutros casos, o tumor cresce exofíticamente para o canal da vagina (Nguyen & Averette, 1999). Além disso, neste tipo de tumor, a composição da flora vaginal, concretamente, a presença de Doderlein bacilli, bacilo que possui a capacidade de converter o glicogénio presente na mucosa vaginal em ácido láctico, contribui para a criação e manutenção de um microambiente rico em ácido láctico que pode desempenhar um papel na selecção das células cancerígenas e na progressão tumoral (Gupta, 2011; Redondo-Lopez et al., 1990). O virus do papiloma humano (HPV) é o agente etiológico do cancro do colo do útero, contudo, a infecção por HPV não é o único factor envolvido na carcinogénese do carcinoma do colo do útero (Yeasmin et al., 2011). Alterações genómicas em oncogenes e genes supressores de tumor nas células do colo uterino são também essenciais ao desenvolvimento de carcinoma do colo do útero (Yeasmin et al., 2011). A amplificação genética em certas regiões cromossómicas é um dos mecanismos de activação de vários oncogenes e outros genes relacionados com o desenvolvimento e progressão tumoral (Schwab, 1999). Estudos em carcinoma do colo do útero, revelaram que os receptores do factor de crescimento epidérmico (EGF) e o proto-oncogene c-Myc estão frequentemente activados por amplificação e estão claramente associados ao fenótipo maligno (Hale et al., 1993; Pfeiffer et al., 1989). As células tumorais reprogramam o seu metabolismo energético de forma a sustentar as elevadas taxas proliferativas (Tennant et al., 2009; DeBerardinis et al., 2008). Além disso, esta reprogramação permite-lhes resistir a alguns sinais de morte celular, particularmente aqueles mediados por danos oxidativos (King & Gottlieb, 2009). Para se dividir uma célula necessita de aumentar em tamanho mas também de replicar o seu DNA – processos que são altamente exigentes do ponto de vista metabólico e que requerem grandes quantidade de proteínas, lípidos e nucleótidos, tal como energia no forma de ATP, o que implica um aumento no consumo de glucose e aminoácidos (Tennant et al., 2010). O fenótipo metabólico melhor caracterizado que se observa nas células tumorais é o efeito de Warburg, que consiste no cancelamento do ciclo de Krebs e cadeia transportadora de electrões e aumento da taxa de glicólise na presença de concentrações normal de oxigénio (Vander Heiden et al., 2009; Warburg, 1956). Consequentemente, ao contrário da maioria das células normais, muitas células tumorais produzem quantidades substanciais da sua energia através da glicólise aeróbia, convertendo a maior parte da glucose a lactato ao invés de a metabolizarem mediante fosforilação oxidativa (Semenza, 2008; Warburg, 1956). Em suma, as células tumorais apesentam maior consumo de nutrientes e também maior produção de subprodutos do que os tecidos normais, resultando numa acumulação de metabolitos no interior da célula e na criação de um ambiente mais hostil no exterior da mesma. As alterações metabólicas e as adaptações desenvolvidas pelas células tumorais, extensivamente estudadas durante o último século, criam um fenótipo que é essencial ao crescimento tumoral e à sobrevivência da célula tumoral, alterando o fluxo ao longo de vias metabólicas chave, tais como a glicólise e a glutaminólise (Tennant et al., 2009). Estas alterações incluem a alteração da expressão (Semenza et al., 1996), mutação e inactivação pós-traducional de uma enzima (Kim et al., 2006) ou a substituição de uma enzima por uma isoforma diferente (Atsumi et al., 2002). Por outro lado, embora os factores chave envolvidos na criação do fenótipo metabólico das células tumorais estejam por elucidar, a literatura actual indica que o c-Myc, p53 o HIF-1 são cruciais neste processo e que existe uma acção combinada desta tríade de factores de transcrição na regulação do metabolismo tumoral (Yeung et al., 2008). Assim, as observações referidas anteriormente colocam a glicólise como contribuinte chave para o fenótipo maligno e suportam a procura de novos tratamentos para o cancro que têm como alvo esta via metabólica essencial. Na verdade, a maioria das adaptações metabólicas desenvolvidas pelas células tumorais são únicas e exclusivas relativamente aos tecidos saudáveis(Porporato et al., 2011). De forma a estabelecer novos alvos terapêuticos os transportadores de compostos carboxilatos, principalmente de glucose, lactato e piruvato devem ser estudados bem como as enzimas que catalisam passos chave em vias metabólicas centrais, como é o caso da lactato e da piruvato desidrogenase. A reacção reversível de redução do piruvato a lactato é catalisada pela família de enzimas lactato desidrogenases (LDH), formadas pelo rearranjo de até quatro cópias de duas subunidades diferentes: a subunidade LDH-H codificada no gene LDHB e a subunidade LDH-M codificada no gene LDHA para formar tetrâmeros activos que conduzem à formação de cinco enzimas, LDH1 a LDH5. A LDH5/LDH-4M (LDHA) catalisa preferencialmente a redução de piruvato a lactato e a LDH1/LDH-4H (LDHB) a conversão de lactato em piruvato (Porporato et al., 2011). A família de genes LDH também o gene LDHC, expresso nos testículos e espermatozóides, que codifica uma terceira isoenzima, a LDHC (Holmes & Goldberg, 2009). A reacção catalisada pela LDH5/LDH-4M produz concentrações equimolares de lactato e protões, então, de forma a evitar a acidificação intracelular e a morte, as células glicolíticas exportam protões através de sistemas adaptados ao transporte dos mesmos, em que se incluem os transportadores de monocarboxilatos (MCT), entre os quais o MCT1, MCT2, MCT3 e MCT4 efectuam o simporte passivo de lactato e protões (Halestrap & Meredith, 2004). Tem sido reportada a sobre-expressão e activação do gene LDHA no tecido tumoral comparativamente com o tecido normal (Walenta & Mueller-Klieser, 2004; Lewis et al., 2000; Shim et al., 1997). Estudos anteriores revelaram transactivação do promotor deste gene pelo factor de transcrição c-Myc (Shim et al., 1997), aumentando directamente a sua expressão. Em relação ao LDHB não foi ainda estabelecida uma relação definitiva entre a expressão deste gene e o cancro, com alguns autores a reportarem o aumento (Chen et al., 2006) da mesma no tumor e outros, o seu silenciamento transcricional no tumor (Leiblich et al., 2006; Maekawa et al., 2003). Recentemente, tem sido também identificada expressão do gene LDHC num largo espectro de tumores (Koslowski et al., 2002). Sabe-se que o MCT4 transporta preferencialmente o lactato para o exterior da célula e o MCT1 regula preferencialmente a entrada do mesmo em células tumorais (mas também o efluxo) e células endoteliais tumorais (Draoui & Feron, 2011). Existem estudos que demonstram a sobre-expressão de MCT1 em carcinoma da mama (Pinheiro et al., 2010) e outros em que se verificou a sobre-expressão de MCT1 e MCT4 em neuroblastoma, cancro do colo do útero e colorectal (Pinheiro et al., 2009; Pinheiro et al., 2008; Sonveaux et al., 2008; Fang et al., 2006). Com base no trabalho científico que vem sendo realizado na área do metabolismo tumoral, foi definido como objectivo desta tese a caracterização do perfil metabólico de duas linhas celulares de cancro de colo do útero de dois tipos histológicos diferentes, adenocarcinoma (HeLa) e carcinoma pavimento celular (SiHa), determinando a relevância das enzimas LDHA, LDHB e LDHC e dos transportadores MCT1 e MCT4. Especificamente, compreender o papel do factor de crescimento EGF e do lactato de sódio (NaLac) na regulação da expressão de LDHA, LDHB, LDHC, MCT1 e MCT4 nas linhas celulares HeLa e SiHa. Os resultados revelaram que em células HeLa, a presença de NaLac promove a sobre-expressão de LDHA através da interacção do factor de transcrição c-Myc com o promotor do gene. Nesta linha celular foi também identificada amplificação genética de c-Myc. Em células SiHa a presença de NaLac promove igualmente um aumento na expressão de LDHA, a nível do mRNA, contudo não por intermédio da acção de c-Myc. Nesta linha celular, observou-se também um aumento no número de cópias de c-Myc, porém em resultado de aneuploidia, uma vez que estas células apresentaram mais de dois cromossomas 8, onde se localiza o c-Myc. Relativamente ao gene LDHB, verificou-se que, em ambas as linhas celulares, a presença de NaLac promove a sobre-expressão do mesmo. Em relação às células SiHa observou-se ainda que a estimulação com EGF promove a expressão de LDHB sendo este efeito activador potenciado pela presença de NaLac. Em termos de regulação da expressão de LDHB propõe-se que esta seja mediada pelo factor de transcrição STAT3. No que concerne à expressão dos transportadores de monocarboxilatos, verificou-se que a expressão de MCT1 em células HeLa na presença de NaLac é regulada pelo factor de transcrição c-Myc que actuará, neste cenário, como repressor. Já no que diz respeito às células SiHa os dados apontam para a existência de repressão da expressão do gene pelo c-Myc, em condições controlo, e para a sua activação na presença de NaLac. Em termos de perspectivas futuras, a elucidação do papel do c-Myc como activador ou repressor da expressão de MCT1 dependerá do estudo dos seus parceiros nos diferentes contextos. Em relação ao MCT4, este encontra-se sobre-expresso em ambas as linhas celulares quando expostas a NaLac. Os resultados obtidos na análise de casos de AC e SCC do colo do útero evidenciaram o papel crucial do microambiente tumoral na selecção de células tumorais. No contexto oxidativo em que os tumores pavimentosos se desenvolvem, a sobre-expressão de MCT1 pode conferir vantagens às células tumorais, dada a sua importância na importação de lactato. Além disso, esta sobre-expressão pode estar na base do switch do consumo de glucose para o de lactato neste tipo de tumor, tal como foi descrito em células SiHa. Nos casos de AC, as baixas concentrações de lactato que caracterizam o microambiente tumoral dos AC, parecem promover a diminuição da expressão de MCT1. Por outro lado, se considerarmos o papel do microambiente tumoral como agente de selecção, os menores níveis de lactato podem selecionar as células tumorais que expressam menos MCT1 dada a sua menor necessidade de importar lactato, comparativamente com as células tumorais de carcinomas pavimentosos. O estudo da migração e proliferação in vitro revelou que a estimulação com EGF e a presença de NaLac promove a migração e proliferação das células SiHa. Propomos que o factor de transcrição c-Myc possa estar implicado neste fenótipo via regulação da expressão de MCT1, cuja função permite fornecer à célula tumoral fontes de energia alternativas de modo a sustentar a sua avidez energética. Assim sendo, o factor de transcrição c-Myc pode estar implicado na regulação de vários genes alvo regulando processos não só relacionados com o metabolismo mas também com o controlo do ciclo celular. O estabelecimento de modelos in vivo, nomeadamente para estudo das consequências da inibição de MCT1 a nível do crescimento e progressão tumoral surge como futura abordagem.
Emerging evidence indicates that impaired cellular energy metabolism is the defining characteristic of nearly all cancers regardless their cellular or tissue origin. Tumor cells have a remarkably different metabolism from that of the tissues from which they are derived. Although lactate is generally considered a waste product, several studies show that it is a prominent substrate that fuels the oxidative metabolism of oxygenated tumor cells. The study of the enzymes involved in lactate production (LDHA) and consumption (LDHB), as well as its transporters (MCTs), will help to define new therapeutic targets. The main objective of this thesis is to characterize the metabolic profile of two uterine cervix cancer cell lines from two different histological types, adenocarcinoma (HeLa) and squamous cell carcinoma (SiHa), namely LDHs and MCTs expression regulation by sodium lactate (NaLac) and Epidermal Growth Factor (EGF). Our findings suggested c-Myc activation of LDHA expression in HeLa cells grown in the presence of NaLac. Regarding SiHa cells, it was observed LDHA upregulation, at mRNA levels, by NaLac, however not c-Myc mediated. NaLac promotes LDHB expression in both cell lines. In SiHa cells, EGF promotes LDHB expression too, being this activator effect increased by NaLac. We proposed NaLac-mediated c-Myc repression of MCT1 expression in HeLa cells. In SiHa cells, NaLac activates MCT1 expression, decreasing c-Myc interaction with MCT1 promoter or promoting an alternative role of c-Myc as an activator. NaLac also promotes MCT4 expression. In the oxidative context where SCC cancer cells develop, the upregulation of MCT1 confer crucial advantages to these cells, given its importance in lactate uptake and could be in the basis of a switch from glucose to lactate consumption in SCC tumors, as described in SiHa cells. We suggested that c-Myc could also be involved in cell migration and proliferation in vitro, via MCT1.

博士
國立陽明大學
生物藥學研究所
102
Cancer stem cells (CSCs) are known to play critical roles in tumor initiation and progression. Accumulating evidence also shows that CSCs are responsible for tumor relapse after chemotherapy. The purpose of this study was to isolate CSCs from established human colorectal carcinoma (CRC) cell lines, characterize them thoroughly and dissect the mechanism of their stemness. Additionally, identifying novel agents capable of specifically targeting colorectal cancer stem cells (CRSCs) that may improve CRC treatment is the other goal of this work. To reach the first aim, freshly isolated CD44+ and CD44- cells from the HCT-15 human CRC line were subjected to various analyses. Interestingly, CD44+ cells exhibited higher soft agar colony-forming ability and in vivo tumorigenicity than the CD44- ones. In addition, a significant upregulation of the protein level of Snail and a marked downregulation of miR-203, a stemness inhibitor, were found in CD44+ HCT-15 cells which suggested that this EMT activator and microRNA might be crucial for the generation and/or maintenance of CRSCs. Moreover, the levels of several other EMT inducers and miR-203 were found to be positively and negatively correlated with those of CD44, respectively, in both HCT-15 and HCT-116 cells. Interestingly, further analyses revealed that miR-203 levels were repressed by Snail, which was shown to bind directly to specific E-box(es) present in the miR-203 promoter. In agreement, silencing miR-203 expression in wild-type HCT-116 cells also resulted in an increase of their stemness. Finally, I discovered that c-Src kinase activity was required for the downregulation of miR-203 in HCT-15 cells, which was stimulated by the interaction between hyaluronan (HA) and CD44. With respect to the identification of agent(s) capable of selectively targeting CRSCs, I found that Compound X, was the most effective one among the compounds provided kindly by Professor Chi-Yin Huang of our Institute after a quick primary screening. In fact, Compound X could dose-dependently diminish the CRSC populations present in both HCT-15 and HT-29 cells as well as induce apoptosis of the CRSCs-enriched oxaliplatin-resistant HT-29 cells. Additionally, this Compound not only inhibited spheroid- and colony-forming abilities of HCT-15 and HT-29 cells drastically but also suppressed the expression of several well-defined pluripotency factors such as Oct4, Nanog, and Bmi1 as well as EMT inducers Snail and Twist. Moreover, Compound X could act synergistically with oxaliplatin to kill HCT-15 and HT-29 cells, especially their CD44+ populations. Finally, I showed that not only was the percentage of SC population decreased significantly but also the sensitivity to Compound X was notably reduced in both HT-29 and HCT-116 lines by RNAi-mediated silencing of transcriptional factor Y, an oncogenic transcription factor, a SC self-renewal promoter, and a well-defined target of this antibiotic. Taken together, this study shows that CD44 is critical for modulating stemness in CSCs, at least those present in HCT-15 and HCT-116 human CRC lines. More importantly, my results demonstrate that the downregulation of miR-203 mediated by HA/CD44 signaling is the main reason for stemness maintenance in the aforementioned CRSCs. In the meantime, I identify and validate the selective inhibitory effects of Compound X on various CRSCs although this antibiotic has already been shown to be active against a wide variety of types of tumors. Judging by its good synergism with oxaliplatin, Compound X may be used in combination with the clinically available chemotherapeutic drugs to improve CRC treatments.

PURPOSE: Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, whereas its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706, could be activated by CYP2W1 into potent antitumor agents. EXPERIMENTAL DESIGN: The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on severe combined immunodeficient mice bearing CYP2W1-positive colon cancer xenografts. RESULTS: Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism shown in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue. CONCLUSION: Our findings suggest a novel approach for treatment of colon cancer that uses a locoregional activation of systemically inactive prodrug by the tumor-specific activator enzyme CYP2W1.