Carvacrol is a phenol monoterpene found in aromatic plants specially in Lamiaceae family, which has been evaluated in an experimental model of breast cancer. However, any proposed mechanism based on its antitumor effect has not been reported. In our previous study, carvacrol showed a protective effect on 7,12-dimethylbenz[α]anthracene- (DMBA-) induced breast cancer in female rats. The main objective in this research was to evaluate by using in silico study the carvacrol on HER2, PI3Kα, mTOR, hERα, PR, and EGFR receptors involved in breast cancer progression by docking analysis, molecular dynamic, and drug-likeness evaluation. A multilevel computational study to evaluate the antitumor potential of carvacrol focusing on the main targets involved in the breast cancer was carried out. The in silico study starts with protein-ligand docking of carvacrol followed by ligand pathway calculations, molecular dynamic simulations, and molecular mechanics energies combined with the Poisson–Boltzmann (MM/PBSA) calculation of the free energy of binding for carvacrol. As result, the in silico study led to the identification of carvacrol with strong binding affinity on mTOR receptor. Additionally, in silico drug-likeness index for carvacrol showed a good predicted therapeutic profile of druggability. Our findings suggest that mTOR signaling pathway could be responsible for its preventive effect in the breast cancer. / Revisión por pares

The human pancreatic cancer cell line T3M4, is known to produce transforming growth factor-alpha (TGF-alpha); as well as overexpress the receptor for this ligand, epidermal growth factor (EGF) receptor. TGF-alpha messenger RNA (mRNA) levels were assayed using northern blot, after addition of epidermal growth factor or TGF-alpha. The level of TGF-alpha mRNA was found to increase 2-fold at 2 hours and then return to near basal levels at 10 hours, after treatment with either ligand. Both ligands were also equipotent in a 2 hour dose response assay, with half maximal stimulation seen at 1 nM and maximal stimulation reached at 4 nM. Furthermore, there appeared to be a 2-fold increase in TGF-alpha transcription as determined by nuclear runoff experiments. Induction of TGF-alpha mRNA coupled with the overexpression of the EGF receptor, may result in a potent autocrine cycle; which may be found in other cancers.

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Oocyte maturation (OM) in teleosts is under precise hormonal control by estrogens and progestins. We show here that estrogens activate an epidermal growth factor receptor (EGFR) signaling pathway through the G protein-coupled estrogen receptor (GPER) to maintain meiotic arrest of full-grown zebrafish (Danio rerio) oocytes in an in vitro germinal vesicle breakdown (GVBD) bioassay. A GPER- specific agonist decreased OM and a GPER-specific antagonist increased spontaneous OM, whereas specific nuclear estrogen receptor (ERα and ERβ) agonists did not affect OM, which suggests the inhibitory action of estrogens on OM are solely mediated through GPER. Furthermore, a peptide-bound estrogen, which cannot enter the oocyte, decreased GVBD, showing that these estrogen actions are mediated through a membrane receptor. Treatment of oocytes with actinomycin D, a transcription inhibitor, did not block the inhibitory effects of estrogens on OM, indicating that estrogens act via a nongenomic mechanism to maintain oocyte meiotic arrest. EGFR mRNA was detected in denuded zebrafish oocytes by reverse transcription polymerase chain reaction (RT-PCR). Therefore, the potential role of transactivation of EGFR in estrogen inhibition of OM was investigated. The matrix metalloproteinase inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor (HB-EGF), increased spontaneous OM. Moreover, specific EGFR1 (ErbB1) inhibitors and inhibitors of extracellular-related kinase 1 and 2 (ERK1/2) increased spontaneous OM. Previously, estrogens have been shown to increase 3’-5’-cyclic adenosine mono phosphate (cAMP) levels through GPER in zebrafish oocytes during meiotic arrest. Taken together these present results suggest that estrogens also act through GPER to maintain meiotic arrest through a second signaling pathway involving transactivation of EGFR and activation of ERK 1 and 2. / text

The introduction of tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) inhibitors to the clinic has resulted in an improvement in the treatment of non small cell lung cancer (NSCLC). However, many patients treated with EGFR TKIs do not respond to therapy. The burden of failed treatment is largely placed on the healthcare field, limiting the effectiveness of EGFR TKIs. Furthermore, responses are hindered by the emergence of resistance. Thus, two questions must be addressed to achieve maximum benefit of EGFR inhibitors: How can patients who will benefit from EGFR TKIs be selected a priori? How can patients who respond achieve maximal benefit? To answer these questions, two hypotheses were formed. First, the EGFR-dependent phenotype, which is displayed by the tumors cells of those patients who respond clinically to EGFR TKIs, can be captured by genomic profiling of NSCLC cell lines stratified by sensitivity to EGFR TKIs. This gene signature may be used to predict the outcome of EGFR TKI therapy in unknown samples. Secondly, the predictive signature of response to EGFR TKI could provide insights into the underlying biology of the phenotype of EGFR-dependency. This information could be exploited to identify inhibitors which could be combined with EGFR inhibitors to elicit a greater effect, thereby minimizing resistance. The work herein describes the testing of these hypotheses. Pharmacogenomics was utilized to define a signature of EGFR-dependency which effectively predicted response to EGFR TKI in vitro and in vivo. Furthermore, the signature was analyzed by bioinformatic approaches to identify the RAS/MAPK pathway as a candidate target in EGFR-dependent NSCLC. The RAS/MAPK pathway regulates expression and activation of EGF-like ligands. Furthermore, the RAS/MAPK pathway modulates EGFR stability in the EGFR-dependent phenotype. Further biochemical analyses demonstrated that the RAS/MAPK pathway mediates proliferation and survival of EGFR-dependent NSCLC cells. Finally, combinatorial treatment of EGFR-dependent NSCLC cell lines with small molecules targeting EGFR and the RAS/MAPK pathway yielded cytotoxic synergy. Thus, we have used pharmacogenomics methods to potentially improve NSCLC treatment by developing a method of predicting response and identifying an additional target to combine with EGFR TKIs to maximize responses.

Feline oral squamous cell carcinomas (FOSCCs) are locally aggressive tumours and a common cause of mortality and morbidity. Current treatment options are rarely successful and animals are frequently euthanised upon diagnosis due to their grave prognosis. Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase receptor which is frequently dysregulated in SCC of the head and neck (HNSCC) in man. Recent advances in human medicine have identified EGFR as a therapeutic target in HNSCC. In this study the role of EGFR in FOSCC was investigated. Sixty seven biopsy samples were immunohistochemically labelled for EGFR and Ki67, a proliferation marker. The tyrosine kinase region of feline EGFR was cloned and sequenced, and six small interfering RNAs (siRNAs) targeting the tyrosine kinase region were developed. The most effective siRNA as well as an EGFR specific tyrosine kinase inhibitor, gefitinib, was then used on a feline SCC cell line (SCCF1), and the effect of EGFR targeting alone, or in combination with irradiation, on the cell line was determined. The majority of the biopsy samples were labelled positively for EGFR and Ki67, and high proliferation corresponded with poor prognosis. The siRNA caused reduction in EGFR mRNA by Real-Time Polymerase Chain Reaction and protein levels as assessed by western blot analysis. Reduced cell proliferation and migration were also observed by proliferation assays and scratch assays respectively. Combining EGFR knockdown with irradiation caused an additive effect on the ability of the cell line to form colonies. These results support the role of EGFR as a potential therapeutic target in FOSCCs.

Cell polarity is an important regulator of cellular processes and is vital in helping to prevent metaplasia and tumorigenesis. There are three many polarity complexes that regulate and maintain epithelial cellular polarity. The Par and Crumbs complexes locate to the apical membrane of the cell, while the Scribble complex is located basolaterally. Of the Scribble complex components, the polarity protein Hugl1, also known as Mgl1 in mice, is especially important in helping to maintain apical basolateral and planar polarity, and is lost in multiple types of cancer. When Hugl1 expression is lost in epithelial cells, it results in a mesenchymal phenotype. We now show that the loss of Hugl1 fundamentally shifts the cellular phenotype and specifically alters EGFR trafficking and signaling. Loss of Hugl1 results in the nuclear translocation of Taz and Slug, increased migration, and the mislocalization of EGFR (Epidermal Growth Factor Receptor), driving cellular growth. Hugl1 regulates the expression of multiple cell identity markers and its loss results in stem cell characteristics, including the increased expression of CD44, and a decrease of CD49f and CD24 expression. The loss of Hugl1 also results in increased growth in soft-agar and prolonged survival when transplanted into NOD-SCID mice; its loss also results in EGF-dependent migration which aids in increasing mammosphere survival. Furthermore, isolated EGFR mislocalization via a point mutation (P667A) also drives these same phenotypes, including activation of Akt and Taz nuclear translocation, indicating the importance of Hugl1 in the regulation of EGFR localization and its signaling. In mice, the loss of total Mgl1 is lethal within days of birth due to hydrocephaly and results in the formation of rosette like structures in the brain that are reminiscent of neuroectodermal tumors. We designed a targeted Mgl1 knockout in the mammary epithelial cells using the Cre/Lox system to evaluate the effects of Mgl1 loss in murine mammary gland development and tumorigenesis. The loss of Mgl1 expression in mice inhibits ductal outgrowth, increases side branching and epithelial layers, and results in the mislocalization of EGFR. While overt mammary tumors did not develop, some individuals did develop hyperplastic nodules that could progress into cancer. The knockdown of Hugl1 in vitro and Mgl1 in vivo reveal how the loss of polarity and presence of Hugl1 results in cancer stem cell characteristics, increased migration, and abnormal signaling due to the mislocalization of EGFR. While these changes result in metaplasia and a potential pre-cancerous state, the loss of Hugl1 alone is not enough to drive the cancer progression, indicating that other mutations or factors are necessary for the development of breast cancer. Because of the key role polarity plays in the prevention of breast cancer development we investigated if the addition of Hugl1 back into breast cancer cells could revert the cancerous cells to a normal epithelial phenotype. Most of the breast cancer cells transfected with Hugl1 expression did not survive, indicating that the re-expression of polarity regulators forces cancer cells to die. The small percentage of cells that did survive re-expression of Hugl1 had retarded growth in soft agar and a decrease in EGFR expression. Together, these data indicate that Hugl1 expression and EGFR activity are closely related and that Hugl1 is required for the proper localization and signaling of EGFR. When Hugl1 is lost, EGFR is mislocalized and fails to be degraded properly, promoting pre-neoplastic changes.

Pang Yee Man Flora. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 111-128). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.v / Table of Contents --- p.vii / List of Figures --- p.xii / Symbols and Abbreviations --- p.xv / Scientific Names --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Vitellogenesis --- p.2 / Chapter 1.2 --- Vitellogenin --- p.3 / Chapter 1.2.1 --- Structure --- p.3 / Chapter 1.2.2 --- Vitellogenin synthesis in the liver --- p.4 / Chapter 1.3 --- Regulation of vitellogenin synthesis --- p.5 / Chapter 1.3.1 --- Estradiol --- p.5 / Chapter 1.3.1.1 --- Mechanism of action --- p.6 / Chapter 1.3.1.2 --- Estradiol-stimulated vitellogenin expression --- p.7 / Chapter 1.3.1.3 --- Memory effects --- p.9 / Chapter 1.3.2 --- Testosterone --- p.10 / Chapter 1.3.3 --- Cortisol --- p.13 / Chapter 1.3.4 --- Progesterone --- p.14 / Chapter 1.3.5 --- Growth Hormone --- p.14 / Chapter 1.3.6 --- Prolactin --- p.15 / Chapter 1.3.7 --- Thyroid hormone --- p.15 / Chapter 1.4 --- Growth factors --- p.16 / Chapter 1.4.1 --- Activin --- p.16 / Chapter 1.4.1.1 --- Structure --- p.16 / Chapter 1.4.1.2 --- Functions --- p.17 / Chapter 1.4.2 --- Epidermal growth factors (EGF) --- p.18 / Chapter 1.4.2.1 --- Structure --- p.18 / Chapter 1.4.2.2 --- Functions --- p.19 / Chapter 1.5 --- Objectives of the present study --- p.20 / Chapter Chapter 2 --- Expression of Goldfish Vitellogenin in vivo and in vitro --- p.25 / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Materials --- p.26 / Chapter 2.2.2 --- Sequencing --- p.27 / Chapter 2.2.3 --- Cell culture --- p.28 / Chapter 2.2.4 --- RNA extraction --- p.29 / Chapter 2.2.5 --- Northern hybridization --- p.31 / Chapter 2.2.6 --- Slot blot hybridization --- p.32 / Chapter 2.2.7 --- Data analysis --- p.33 / Chapter 2.2.8 --- SDS-PAGE analysis --- p.33 / Chapter 2.2.9 --- in situ hybridization --- p.34 / Chapter 2.3 --- Results --- p.37 / Chapter 2.3.1 --- Validation of vitellogenin mRNA detection --- p.37 / Chapter 2.3.2 --- Basal and estradiol-stimulated vitellogenin expression and production invivo --- p.38 / Chapter 2.3.3 --- Localization of vitellogenin expression in the liver --- p.39 / Chapter 2.3.4 --- Expression of vitellogenin in vitro --- p.40 / Chapter 2.4 --- Discussion --- p.54 / Chapter Chapter 3 --- Effects of Steroids on the Expression of Goldfish Vitellogenin in vitro --- p.60 / Chapter 3.1 --- Introduction --- p.60 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Materials --- p.62 / Chapter 3.2.2 --- Animal --- p.62 / Chapter 3.2.3 --- Primary culture of dispersed hepatic cells --- p.62 / Chapter 3.2.4 --- Drug treatment --- p.64 / Chapter 3.2.5 --- Total RNA isolation --- p.64 / Chapter 3.2.6 --- Messenger RNA isolation --- p.65 / Chapter 3.2.7 --- Slot blot analysis --- p.66 / Chapter 3.2.8 --- Data analysis --- p.68 / Chapter 3.2.9 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.68 / Chapter 3.2.10 --- Cloning of aromatase cDNA --- p.69 / Chapter 3.2.11 --- Sequencing --- p.70 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- Effect of 17-β estradiol on vitellogenin mRNA expression --- p.71 / Chapter 3.3.2 --- Effect of testosterone on vitellogenin mRNA expression --- p.71 / Chapter 3.3.3 --- Detection of aromatase mRNA expression in the liver by RT-PCR --- p.72 / Chapter 3.3.4 --- Effect of aromatase inhibitors on testosterone-stimulated vitellogenin expression --- p.73 / Chapter 3.4 --- Discussion --- p.81 / Chapter Chapter 4 --- Effects of Epidermal Growth Factor (EGF) and Activin on the Expression of Vitellogenin in the Goldfish Hepatic Cells in vitro --- p.86 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and Methods --- p.88 / Chapter 4.2.1 --- Materials --- p.88 / Chapter 4.2.2 --- Primary culture of dispersed hepatic cells --- p.89 / Chapter 4.2.3 --- Slot blot analysis --- p.91 / Chapter 4.2.4 --- Data analysis --- p.91 / Chapter 4.3 --- Results --- p.92 / Chapter 4.3.1 --- Effect of activin on vitellogenin mRNA expression --- p.92 / Chapter 4.3.2 --- Effect of EGF and TGF-α on vitellogenin mRNA expression --- p.93 / Chapter 4.4 --- Discussion --- p.99 / Chapter Chapter 5 --- General Discussion --- p.104 / Chapter 5.1 --- Overview --- p.104 / Chapter 5.2 --- Contribution of the present study --- p.106 / Chapter 5.2.1 --- Expression of goldfish vitellogenin in vivo and in vitro --- p.106 / Chapter 5.2.2 --- Effects of steroids on the expression of goldfish vitellogenin in vitro --- p.106 / Chapter 5.2.3 --- Effects of EGF and activin on the expression of vitellogenin in the goldfish hepatic cells in vitro --- p.107 / Chapter 5.3 --- Future prospects --- p.108

by Chung Lung Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 149-157). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.ii / Abstracts --- p.v / List of figures --- p.ix / List of tables --- p.xii / Contents --- p.xiii / Contents / General Introduction --- p.1 / Chapter CHAPTER ONE --- CISPLATIN RESISTANCE MECHANISMS / Chapter 1.1 --- INTRODUCTION --- p.3 / Chapter 1.1.1 --- History of Cisplatin as An Anticancer Drug --- p.3 / Chapter 1.1.2 --- Active Mechanisms of Cisplatin --- p.8 / Chapter 1.1.3 --- Formation of DNA Adducts --- p.8 / Chapter 1.1.4 --- Cisplatin Resistance Mechanisms --- p.9 / Chapter 1.1.4.1 --- Intracellular Accumulation of Cisplatin --- p.11 / Chapter 1.1.4.2 --- Glutathione-S-transferase and Glutathion --- p.12 / Chapter 1.1.4.3 --- Metallothionein --- p.16 / Chapter 1.1.4.4 --- Cell Cycle Perturbation --- p.16 / Chapter 1.1.4.5 --- P-glycoprotein --- p.17 / Chapter 1.1.4.6 --- Multidrug Resistant Protein --- p.19 / Chapter 1.1.4.7 --- Topoisomerase II --- p.20 / Chapter 1.1.4.8 --- DNA Repair --- p.22 / Chapter 1.1.4.9 --- Induction of Programme Cell Death --- p.23 / Chapter 1.2 --- OBJECTIVES --- p.27 / Chapter 1.3 --- MATERIALS AND METHODS / Chapter 1.3.1 --- Materials --- p.28 / Chapter 1.3.2 --- Methods --- p.31 / Chapter 1.3.2.1 --- Cell Lines --- p.31 / Chapter 1.3.2.2 --- Drug Sensitivity Assay --- p.31 / Chapter 1.3.2.3 --- Platinum Uptake --- p.32 / Chapter 1.3.2.4 --- Cell Cycle Analysis --- p.32 / Chapter 1.3.2.5 --- Western Blot Analysis --- p.33 / Chapter 1.3.2.6 --- Glutathione Content Determination --- p.36 / Chapter 1.3.2.7 --- DNA Fragmentation --- p.36 / Chapter 1.3.2.8 --- JC-1 Staining --- p.37 / Chapter 1.3.2.9 --- HE and DCF Staining --- p.38 / Chapter 1.3.2.10 --- Quantitative RT-PCR --- p.38 / Chapter 1.4 --- RESULTS / Chapter 1.4.1 --- Cisplatin Sensitivity of A431 Cells by MTT Assay --- p.40 / Chapter 1.4.2 --- Cross-resistance to Anti-cancer Drugs --- p.40 / Chapter 1.4.3 --- Quantitation of Cisplatin Accumulation in A431 Cells by AAS --- p.44 / Chapter 1.4.4 --- Drug Detoxification Agent --- p.45 / Chapter 1.4.5 --- Detection of Cell Cycle Arrest by Flow Cytometer --- p.47 / Chapter 1.4.6 --- Expression of Drug Resistance Related Genes --- p.48 / Chapter 1.4.7 --- Detection of Apoptosis by DNA Fragmentation --- p.50 / Chapter 1.4.8 --- Role of Mitochondria and Reactive Oxygen Species by Flow Cytometer --- p.52 / Chapter 1.4.9 --- Detection of Apoptotic mRNA Level by Quantitative RT-PCR --- p.57 / Chapter 1.4.10 --- Detection of Apoptotic Protein Level by Western Blot Analysis --- p.57 / Chapter 1.5 --- DISCUSSIONS --- p.59 / Chapter CHAPTER TWO: --- THE INTERACTION BETWEEN DRUG RESISTANCE MECHANISMS AND GROWTH FACTOR SIGNALLING PATHWAY / Chapter 2.1 --- INTRODUCTION --- p.63 / Chapter 2.1.1 --- Structure of EGF and EGFR --- p.64 / Chapter 2.1.2 --- Growth Factor Signal Transduction Pathway --- p.69 / Chapter 2.1.3 --- Biological Effect of EGF --- p.69 / Chapter 2.1.3.1 --- Modification of Drug Sensitivity by EGF --- p.71 / Chapter 2.2 --- OBJECTIVES --- p.74 / Chapter 2.3 --- MATERIALS AND METHODS / Chapter 2.3.1 --- Materials --- p.75 / Chapter 2.3.2 --- Methods / Chapter 2.3.2.1 --- Cell Lines --- p.76 / Chapter 2.3.2.2 --- Drug Sensitivity Assay --- p.77 / Chapter 2.3.2.3 --- Northern Blot Analysis --- p.77 / Chapter 2.3.2.4 --- Southern Blot Analysis --- p.78 / Chapter 2.3.2.5 --- Others --- p.78 / Chapter 2.4 --- RESULTS / Chapter 2.4.1 --- Sensitivity to EGF --- p.79 / Chapter 2.4.2 --- EGFR Expression Levels --- p.80 / Chapter 2.4.3 --- EGF Induced Protein Phosphorylation Pattern --- p.84 / Chapter 2.4.4 --- Effect of EGF on A431 Cells --- p.86 / Chapter 2.4.5 --- Response of Cells to Agents Targeting on EGF Signalling Pathway --- p.91 / Chapter 2.4.6 --- Response of Cells to Other Growth Factors --- p.97 / Chapter 2.4.7 --- Sensitivity of Cells to Different Anti-cancer Drugs --- p.99 / Chapter 2.4.8 --- Drug Resistance Mechanisms --- p.103 / Chapter 2.4.9 --- 5-Fluorouracil Sensitivity in A431 Cells --- p.108 / Chapter 2.4.10 --- Cisplatin Sensitivity in A431 Cells --- p.113 / Chapter 2.5 --- DISCUSSIONS --- p.117 / Chapter CHAPTER THREE: --- IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENE IN A431 CELLS BY DIFFERENTIAL DISPLAY / Chapter 3.1 --- INTRODUCTION --- p.122 / Chapter 3.2 --- MATERIALS AND METHODS / Chapter 3.2.1 --- Materials --- p.128 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Identification of Differentially Expressed Genes by RT-PCR / Chapter 3.2.2.2 --- Cloning of a Differentially Expressed cDNAs --- p.129 / Chapter 3.2.2.3 --- Screening and Sequencing of cDNA Inserts --- p.130 / Chapter 3.2.2.4 --- Rapid Amplification of cDNA Ends (RACE) --- p.131 / Chapter 3.2.2.5 --- Amplifcation Reaction --- p.131 / Chapter 3.2.2.6 --- Cloning and Sequencing of the RACE Fragment --- p.132 / Chapter 3.3 --- RESULTS / Chapter 3.3.1 --- Identification of novel cDNA by mRNA differential display --- p.133 / Chapter 3.4 --- DISCUSSIONS --- p.145 / General Conclusion --- p.147 / References --- p.149