Objetivo: Avaliar as vitaminas e as enzimas envolvidas na defesa antioxidante e os marcadores de peroxidação lipídica durante o tratamento oncológico de pacientes com neoplasia de estômago ou de esôfago Casuística: O estudo prospectivo longitudinal foi conduzido com 14 pacientes com neoplasia de estômago ou de esôfago [62,1 anos (IC95% 55,6-68,6)], sob tratamento oncológico em unidade especializada. O estudo incluiu também 15 voluntários saudáveis [61,3 anos (IC95% 57,3-65,3)]. Métodos: Foram aplicados os questionários de ingestão alimentar (Recordatórios de 24h), de qualidade de vida (FACT G), de fadiga (FACIT Fatigue) e inquéritos relacionados com efeitos adversos e de toxicidade (CTCAE) que potencialmente interferem na ingestão alimentar e no estado nutricional. Foram feitas as medidas antropométricas, a impedância bioelétrica e coleta de sangue para os exames laboratoriais. Foram dosadas as vitaminas antioxidantes C e E, as enzimas superóxido dismutase (SOD) e glutationa peroxidase (GPx), além dos marcadores de peroxidação lipídica, malondialdeído (MDA) e 8-isoprostano. No Grupo Câncer, os procedimentos foram feitos antes do início, na metade e ao término do tratamento oncológico; o Grupo Controle foi submetido às mesmas avaliações em apenas uma ocasião. A análise estatística foi feita por meio do software Statistica 8.0, usando testes estatísticos não paramétricos. Resultados: Não foram observadas diferenças nos escores de qualidade e de fadiga. As reações adversas relacionadas ao tratamento oncológico foram redução da ingestão de alimentos, saliva espessa com alteração no paladar e náuseas. Antes do início do tratamento, os pacientes com câncer já haviam perdido 17% do peso em relação ao usual; o peso corporal e o IMC reduziram entre a primeira e a terceira avaliação, mas não houve alteração na composição de massa corporal magra e gorda, na ingestão energética e da maioria dos macronutrientes no decorrer do estudo. Quando comparados ao Grupo Controle, o Grupo Câncer apresentou menores valores de vitamina C [10,8 (IC95%4,5-17,0) vs. 25,5 (IC95%19,9-30,7) mol/L], maiores valores da SOD [2056 (IC95%1933-2178) vs. 1595 (IC95%1430-1761) USOD/gHb] e MDA [0,73 (IC95%0,50-0,88) vs. 0,29 (IC95%0,25-0,33) mol/]. No decorrer do estudo, houve diminuição nos níveis de SOD entre a primeira e a segunda [2056 (IC95%1933-2178) vs. 1973 (IC95%1826-2120) USOD/gHb] e entre a primeira e a terceira avaliação [2056 (IC95%1933-2178) vs. 1827 (IC95%1687-1967) USOD/gHb]. A concentração da vitamina C permaneceu baixa e os valores da GPx, vitamina E, MDA e 8-isoprostano permaneceram inalterados durante o estudo. Conclusões: O tratamento oncológico reduziu o peso, mas não alterou os escores de qualidade de vida e de fadiga. No início do estudo, a baixa concentração da vitamina C e os valores aumentados da SOD e do MDA sugerem que o processo tumoral induz o estresse oxidativo e a peroxidação lipídica. A redução da atividade da SOD e os valores inalterados da GPx, vitamina C, vitamina E, MDA e isoprostano ao longo do estudo, sugerem que o tratamento oncológico não intensifica o estresse oxidativo. / Objective: To evaluate the vitamins and the enzymes involved in antioxidant defense and lipid peroxidation markers for cancer treatment of patients with gastric or esophageal cancer. Subjects: The prospective longitudinal study was conducted with 14 patients with gastric or esophageal cancer [62,1 years (IC95% 55,6-68,6)], under cancer treatment in a specialized unit. The study also included 15 healthy volunteers [61,3 years (IC95% 57,3-65,3)]. Methods: The questionnaires about dietary intake (24-hour Dietary Recall), quality of life (FACT-G), fatigue (FACIT-Fatigue) and inquiries related adverse effects and toxicity (CTCAE) that potentially interfere with food intake and nutritional status were applied. The anthropometric measurements, the bioelectrical impedance and blood collection for laboratory tests were made. The antioxidant vitamins C and E, the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) were dosed, beyond the markers of lipid peroxidation, malondialdehyde (MDA) and 8-isoprostane. In the Cancer Group, the procedures were done before the start, in the middle and at the end of cancer treatment; the Control Group underwent the same evaluations on only one occasion. The statistical analysis was performed using Statistica 8.0 software, using non-parametric statistical tests. Results: There were no differences in the quality and fatigue scores. The adverse reactions related to cancer treatment were the reduction of dietary intake, thick saliva with altered taste and nausea. Before the treatment, the patients with cancer had already lost 17% of weight with respect to the usual; the body weight and IMC reduced between the first and the third evaluation, but there was no change in the composition of lean and fat mass, energy intake and most micronutrient during the study. When compared to the Control Group, the Cancer Group had lower vitamin C [10,8 (IC95% 4,5-17,0) vs. 25.5 (IC95% 19,9-30,7) mol/L], higher values of SOD [2056 (IC95% 1933-2178) vs. 1595 (IC95% 1430-1761) USOD/gHb] and MDA [0,73 (IC95% 0,50-0,88) vs. 0,29 (IC95% 0,25-0,33) mol/L]. During the study, there was a decrease in the levels of SOD between the first and the second [2056 (IC95% 1933-2178) vs. 1973 (IC95% 1826-2120) USOD/gHb] and between the first and the third evaluation [2056 (IC95% 1933-2178) vs. 1827 (IC95% 1687-1967) USOD/gHb]. The concentration of vitamin C remained low and values of GPx, vitamin E, MDA and 8-isoprostane were unchanged during the study. Conclusions: The cancer treatment reduced the weight but did not change the quality of life scores and fatigue. At the beginning of the study, the low concentration of vitamin C and increased levels of SOD and MDA suggest that the tumor process induced oxidative stress and lipid peroxidation. The reduction of the SOD activity and the unchanged values GPx, vitamin C, vitamin E, MDA and isoprostane throughout the study suggest that the cancer treatment does not enhance oxidative stress.

Glioblastoma is the most common adult malignant primary brain tumor with one of the worst prognosis. With a survival of 10 to 12 months, glioblastoma remains one of the most challenging disease to treat. The standard treatment method involves maximal possible resection of the tumor followed by radiation and chemotherapy. However, the short half-life of most chemotherapeutic drugs, high systemic toxicity and inability to cross the blood brain barrier inhibits effective delivery of the chemotherapeutics to the tumor. An ideal drug delivery system can reach the tumor site with high efficiency and continuously release the drug at the tumor site for an extended period. Adult stem cells including neural stem cells (NSC) and mesenchymal stem cells (MSC) have inherent tumor trophic properties allowing for site-specific delivery of chemotherapeutics. They can also be genetically engineered to secrete the chemotherapeutic drug continuously making them ideal candidates for cell-based delivery system for treating glioblastoma. MSC have been isolated from a wide range of sources including bone marrow, umbilical cord, adipose tissue, liver, multiple dental tissues and induced pluripotent stem cells. MSC are also easily amenable to viral modification allowing for easy manipulation to produce chemotherapeutic drugs. Additionally, more than 350 clinical trials using MSC have successfully established the safety of using MSC for cell-based therapies. Collectively these factors have led to the widespread use of MSC in cancer therapy. MSC have been successfully transduced to produce chemotherapeutic drugs to treat glioma, melanoma, lung cancer, ovarian cancer and breast cancer. Despite the multitudes of advantages that cell therapy provides they are limited in three main domains (1) Low cell retention and survival at the site of the tumor (2) In ability to co-deliver multiple therapeutics and (3) In ability to deliver drugs other than peptide based drugs. This thesis details the work to engineer mesenchymal stem cells to tackle these three issues and develop a system that can increase the efficacy of glioblastoma treatment. To increase the cellular retention and survival we engineered MSC to form multicellular spheroids and cell sheets. To co-delivery multiple therapeutics we engineered MSC to form MSC/DNA-templated nanoparticle hybrid cluster to co-deliver drugs for cancer therapy. The system showed superior performance due to the increased retention of the cells and nanoparticle at the tumor site. Finally, to deliver drugs other peptide based we engineered graphene oxide cellular patches for mesenchymal stem cells. Graphene oxide can carry diverse therapeutics and can kill the cancer cells without affecting the cellular viability of MSC.

Breast cancer is the most common cancer and the second leading cause of cancer death among women. About 1 in 8 U.S. women (about 12%) will develop invasive breast cancer over the course of their lifetime. Existing methods of early detection of breast cancer include mammography and palpation, either by patient self-examination or clinical breast exam. Palpation is the manual detection of differences in tissue stiffness between breast tumors and normal breast tissue. The success of palpation relies on the fact that the stiffness of breast tumors is often an order of magnitude greater than that of normal breast tissue, i.e., breast lesions feel ''hard'' or ''lumpy'' as compared to normal breast tissue. A mammogram is an x-ray that allows a qualified specialist to examine the breast tissue for any suspicious areas. Mammography is less likely to reveal breast tumors in women younger than 50 years with denser breast than in older women. When a suspicious site is detected in the breast through a breast self-exam or on a screening mammogram, the doctor may request an ultrasound of the breast tissue. A breast ultrasound can provide evidence about whether the lump is a solid mass, a cyst filled with fluid, or a combination of the two. An invasive needle biopsy is the only diagnostic procedure that can definitely determine if the suspicious area is cancerous. In the clinic, 80% of women who have a breast biopsy do not have breast cancer. Most women with breast cancer diagnosed will have some type of surgery to remove the tumor. Depending on the type of breast cancer and how advanced it is, the patient might need other types of treatment as well, such as chemotherapy and radiation therapy. Image-guided minimally-invasive treatment of localized breast tumor as an alternative to traditional breast surgery, such as high intensity focused ultrasound (HIFU) treatment, has become a subject of intensive research. HIFU applies extreme high temperatures to induce irreversible cell injury, tumor apoptosis and coagulative necrosis. Compared with conventional surgical procedures the main advantages of HIFU ablation lie in the fact that it is non-invasive, less scarring and less painful, allowing for shorter recovery time. HIFU can be guided by MRI (MRgFUS) or by conventional diagnostic ultrasound (USgFUS). Worldwide, thousands of patients with uterine fibroids, liver cancer, breast cancer, pancreatic cancer, bone tumors, and renal cancer have been treated by USgFUS. In this dissertation, the objective is to develop an integrated Harmonic Motion Imaging guided Focused Ultrasound (HMIgFUS) system as a clinical monitoring technique for breast HIFU with the added capability of detecting tumors for treatment planning, evaluation of tissue stiffness changes during HIFU ablation for treatment monitoring in real time, and assessment of thermal lesion sizes after treatment evaluation. A new HIFU treatment planning method was described that used oscillatory radiation force induced displacement amplitude variations to detect the HIFU focal spot before lesioning. Using this method, we were able to visualize the HMIgFUS focal region at variable depths. By comparing the estimated displacement profiles with lesion locations in pathology, we demonstrated the feasibility of using this HMI-based technique to localize the HIFU focal spot and predict lesion location during the planning phase. For HIFU monitoring, a HIFU lesion detection and ablation monitoring method was first developed using oscillatory radiation force induced displacement amplitude variations in real time. Using this method, the HMIgFUS focal region and lesion formation were visualized in real time at a feedback rate of 2.4 Hz. By comparing the estimated lesion size against gross pathology, the feasibility of using HMIgFUS to monitor treatment and lesion formation without interruption is demonstrated. In order to reduce the imaging time, it is shown in this dissertation that using the steered FUS beam, HMI can be used to image a 2.3 times larger ROI without requiring physical movement of the transducer. Using steering for HMI can be used to shorten the total imaging duration without requiring physical movement of the transducer. For the application of breast tumor, HMI and HMIgFUS were optimized and applied to ex vivo breast tissue. The results showed that HMI is experimentally capable of mapping and differentiating stiffness in normal and abnormal breast tissues. HMIgFUS can also successfully generate thermal lesions on normal and pathological breast tissues. HMI has also been applied to post-surgical breast mastectomy specimens to mimic the in vivo environment. In the end, the first HMI clinical system has been built with added capability of GUP-based parallel beamforming. A clinical trial has been approved at Columbia University to image breast tumor on patient. The HMI clinical system has shown to be able to map fibroadenoma mass on two patients with valid HMI displacement. The study in this dissertation may yield an early-detection technique for breast cancer without any age discrimination and thus, increase the survival rate.

In 2018, it is estimated that 55,440 Americans will be diagnosed with pancreatic cancer and this figure is expected to continue to rise with increased life expectancy. Despite some measurable progress over the past few decades, pancreatic cancer remains one of the most lethal malignancies with five-year survival rate of 8.7%. Novel therapies, and their timely translation to the clinic, are urgently needed. As part of an effort to identify and characterize novel therapeutic strategies for pancreatic ductal adenocarcinoma, we began a study of the role of Bmi1 in tumor maintenance and progression. While Bednar and colleagues showed that Bmi1 is critical for the development of pancreatic cancer, and that its pancreas-specific deletion impairs PanIN formation, we were interested in assessing its function in established tumors. During the course of this work, we acquired a novel compound, PTC596, developed by PTC Therapeutics as a post-translational inhibitor of BMI1. Treatment with PTC596 leads to hyperphosphorylated BMI1, and this modification is associated a loss of protein activity. We planned to study this compound, in vitro and in vivo, as a complement to genetic perturbations of Bmi1. Initial characterizations of the effects of PTC596 on human and murine-derived pancreatic cancer cell lines revealed a potent anti-proliferative effect, accompanied by BMI1 hyperphosphorylation, and followed by polyploidy and cell death after prolonged treatment. Further analysis showed a clear G2/M arrest and elevated levels of phospho-histone H3. Bmi1 is known to play a role the cell cycle, but its inhibition in pancreatic cancer cell lines has been shown to induce G1 arrest. We decided to further explore the mechanism of PTC596’s antiproliferative effects by carrying out RNA sequencing on Aspc1 cells treated with PTC596. We found that 8 of the ten most down-regulated genes were members of the tubulin family and began to study this compound’s effect on microtubules. Compelling results from a cell-free tubulin polymerization assay support inhibition of tubulin polymerization as the mechanism of action for PTC596. These data are further supported by evidence that PTC596 increases the fraction of free-tubulin in treated cells, as well as dramatically alters the cell’s microtubule network. Given our laboratory’s interest in identifying novel therapies for pancreatic cancer, and the fact that PTC596 has already begun clinical trials, we continued to characterize this compound in vivo. We found PTC596 to have properties favorable for in vivo administration. PTC596 is orally available, has a plasma half-life of approximately 22 hours following oral administration, and accumulates in tumor tissue where it has an expected pharmacodynamic effect. Furthermore, it is well tolerated in vivo in combination with gemcitabine. We carried out a four-arm intervention study in tumor-bearing KPC mice, examining PTC596 alone and in combination with gemcitabine. We found that PTC596 synergizes with gemcitabine to significantly reduce tumor growth rates and provide a 3-fold extension of survival as compared to vehicle. These findings are, to our knowledge, the first evidence of in vivo synergy between a microtubule-destabilizing agent and gemcitabine for the treatment of pancreatic cancer. Importantly, this study identifies an alternative mechanism for PTC596 and implicates its efficacy in a novel treatment regimen for pancreatic ductal adenocarcinoma.

Liu Bonnie Pui-ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 134-163). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abbreviations --- p.ii / Abstract / English --- p.1 / Chinese --- p.3 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Colorectal Cancer Statistics --- p.5 / Chapter 1.2 --- Colon Cancer --- p.5 / Chapter 1.3 --- Treatment --- p.6 / Chapter 1.4 --- Effects of Cytotoxic Drug Treatment --- p.7 / Chapter 1.5 --- Cell Cycle --- p.8 / Chapter 1.6 --- Oxidases --- p.9 / Chapter 1.7 --- Chemistry of Novel β-carboline: Tangutorine --- p.11 / Chapter 1.8 --- Aim of Study --- p.14 / Chapter Chapter 2 --- Cytotoxicity / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and Methods --- p.18 / Chapter 2.3 --- Results --- p.23 / Chapter 2.4 --- Discussion --- p.44 / Chapter Chapter 3 --- Oxidase Activity and Protein Oxidation / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Materials and Methods --- p.54 / Chapter 3.3 --- Results --- p.60 / Chapter 3.4 --- Discussion --- p.80 / Chapter Chapter 4 --- Cell Cycle / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Materials and Methods --- p.93 / Chapter 4.3 --- Results --- p.96 / Chapter 4.4 --- Discussion --- p.118 / Chapter Chapter 5 --- General Discussion --- p.126 / References --- p.134

研究背景： / 肝癌是全球常見的惡性腫瘤之一，世界上每年大約有50萬死亡病例，並且呈逐年上升之勢， 是全球第3位的腫瘤死亡原因。慢性乙型和丙型肝炎病毒感染是肝癌的主要成因。肝癌惡性程度高、預後差，並且目前的治療手段非常有限，術後易復發和轉移，迄今尚無正式獲准有效治療藥物。現階段，治療肝癌的主要方法是手術切除，但是隨之引起的併發症以及較高的復發機率嚴重影響了治療的療效，大大降低肝癌病人的存活期。 / 研究目的： / 分析TDP對肝癌細胞和肝腫瘤旁細胞生長的影響；分析TDP抑癌的分子靶標蛋白及其分子機理；驗證TDP對肝癌動物模型的抑制效果。開發一種新型有效的肝癌治療藥物。 / 研究方法： / 首先用MTT法從102種來源於嶺南山竹子的純複合物中分離出了TDP,它是一種甾醇類化合物。採用MTT法檢測TDP對腫瘤細胞生長的影響；流式細胞實驗驗證TDP能否引起腫瘤細胞的凋亡；採用蛋白組學和質譜分析找出TDP抑癌的分子靶標；進一步的蛋白功能增加和缺失實驗證明Hsp27的功能和作用；生物資訊學驗證HSP27和TDP的作用結果；最後利用動物模型驗證TDP對肝腫瘤的治療效果。 / 結果： / TDP不但能效率極高的抑制肝癌細胞的生長而且可以大量誘發肝癌細胞的凋亡，而對正常的肝癌旁細胞沒有影響。二維電泳以及質譜分析TDP處理的肝癌細胞對比DMSO處理的肝癌細胞發現了具有不同表達水準的18種蛋白，Hsp27是其中一個在TDP誘導下調變化倍數較大並且與細胞凋亡有密切關係的蛋白，Hsp27的過表達以及Knock-down都充分驗證了TDP通過調節Hsp27的表達參與了依賴於caspase的線粒體凋亡途徑，在Western Blotting以及RT-PCR中得到了充分的驗證。生物資訊學預測TDP可以與Hsp27結合，實驗結果表明TDP可以誘導Hsp27的聚集並導致功能喪失。動物實驗腫瘤生長結果以及免疫組化結果證明，TDP可以在很大程度上對肝癌有抑製作用。 / 結論： / 本研究首次表明，TDP如果不是完全的,最起碼也是部分通過誘導依賴於caspase的線粒體凋亡的途徑來抑制肝癌細胞的增值和分化, 具有明顯的抗腫瘤的功效，特別是對Hsp27高表達的腫瘤細胞有比較明顯的作用，是一種值得繼續深入研究的有較高潛在價值的藥物。 / Background： / Hepatocellular carcinoma (HCC), the most common primary hepatic malignancy, is a global public health problem that accounts for approximately 500,000 deaths annually. Chronic hepatitis B and hepatitis C infections are the major risk factors for the development of HCC. Due to the high rate of these infections, the incidence of HCC remains alarmingly high globally. Although great advances have been made in HCC treatment, poor prognosis and high risk of recurrence have been major challenges to patients. Currently, surgical resection is the main treatment option for HCC patients; however, complications arising from surgery can threaten its therapeutic effect and patients’ survival. / Objectives： / To characterize the functions of 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b]Xanthone (TDP) in cell proliferation of HepG2 cells; to discover the molecular target genes and elucidate the underlying molecular mechanism of TDP; to examine the in vivo function of TDP in a nude mouse tumor model of HCC. Finally, to investigate TDP’s potential as an anti-HCC drug candidate. / Methods and Results： / In this study, we discovered that TDP, isolated from the Chinese medicinal herb, Garcinia oblongifolia, strongly inhibited cell growth and induced caspase-dependent mitochondrial apoptosis in HCC, as evidenced from MTT assay and flow cytometry analysis. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to find the molecular targets of TDP in HCC cells, and eighteen proteins were identified with altered expression, with Hsp27 protein being one of the proteins most significantly down-regulated by TDP. Further Hsp27-siRNA knockdown and Lenti-Hsp27 overexpression studies found that Hsp27 was involved in TDP induced mitochondrial apoptosis, with bioinformatics predictions and biological results revealing that TDP might cause Hsp27 protein form dimer and consequent degradation via the ubiquitin-proteasome system. Finally, subcutaneously injecting cancer cells with Hsp27 expression vector into the dorsal flank of nude mice tumor model also demonstrated the suppressive effect of TDP on HCC. / Conclusions： / In summary, our study discovered that TDP, a natural xanthone, was a potent inhibitor of Hsp27 in HCC. TDP inhibited cell growth and induced apoptosis by inducing Hsp27 degradation, which stimulated mitochondrial cytochrome C release which resultantly activated caspase-3 and caspase-9. These data combined with the results of the animal model strongly supported TDP’s potential as a novel anti-cancer drug candidate, especially for cancers with an abnormally high expression of Hsp27. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fu, Weiming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 111-151). / Abstract also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iiv / Acknowledgment --- p.vi / Publications --- p.viii / List of Contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.1.1 --- Overview of HCC --- p.1 / Chapter 1.1.2 --- Epidemiology of HCC in China and Hong Kong --- p.3 / Chapter 1.2 --- Etiology of HCC --- p.7 / Chapter 1.2.1 --- Cirrhosis --- p.8 / Chapter 1.2.2 --- HBV infection --- p.9 / Chapter 1.2.3 --- HCV infection --- p.10 / Chapter 1.2.4 --- Viral Co-Infection --- p.11 / Chapter 1.2.5 --- Fatty Liver Disease and Cryptogenic Cirrhosis --- p.12 / Chapter 1.2.6 --- Alcohol --- p.13 / Chapter 1.2.7 --- Iron --- p.13 / Chapter 1.2.8 --- Aflatoxin --- p.14 / Chapter 1.2.9 --- Others --- p.14 / Chapter 1.3 --- Diagnosis of HCC --- p.14 / Chapter 1.4 --- Prognosis of HCC --- p.17 / Chapter 1.5 --- Treatment of HCC --- p.19 / Chapter 1.5.1. --- Early stage --- p.19 / Chapter 1.5.2. --- Intermediate and advanced stage --- p.24 / Chapter 1.5.3. --- Terminal stage --- p.28 / Chapter 1.6 --- Signaling pathways in HCC --- p.28 / Chapter 1.6.1 --- Proliferation signaling pathways --- p.29 / Chapter 1.6.2 --- Signaling pathways frequently dysregulated in HCC --- p.30 / Chapter 1.6.3 --- Pathways involved in liver development and cell differentiation --- p.34 / Chapter 1.6.4 --- Inflammation pathways involved in hepatocarcinogenesis --- p.35 / Chapter 1.6.5 --- Pathways involved in neoangiogenesis --- p.37 / Chapter 1.6.6 --- The P53 tumor suppressor --- p.38 / Chapter 1.6.7 --- Heat shock proteins in HCC --- p.39 / Chapter 1.7 --- The roles of microRNAs in liver cancer progression --- p.42 / Chapter 1.8 --- TCM in the treatment of HCC --- p.45 / Chapter 1.8.1 --- Introduction --- p.45 / Chapter 1.8.2 --- Garcinia --- p.49 / Chapter 1.9 --- Objectives of the study --- p.51 / Chapter Chapter 2 --- Materials and Methods --- p.52 / Chapter 2.1 --- Preparation of the pure compounds --- p.52 / Chapter 2.2 --- Liver cell lines and tissue culture --- p.52 / Chapter 2.3 --- Human tissue samples --- p.52 / Chapter 2.4 --- Cell viability assessment with MTT assay --- p.53 / Chapter 2.5 --- Apoptosis analysis --- p.53 / Chapter 2.6 --- Two-dimensional electrophoresis (2-DE), protein visualization and image analysis --- p.54 / Chapter 2.6.1 --- Materials --- p.54 / Chapter 2.6.2 --- Protein extraction --- p.54 / Chapter 2.6.3. --- 2-DE protein profiling --- p.55 / Chapter 2.6.4. --- Gel staining and image analysis --- p.55 / Chapter 2.6.5. --- In-gel protein digestion with trypsin --- p.56 / Chapter 2.6.6. --- MALDI-TOF mass spectrometric analysis --- p.56 / Chapter 2.6.7. --- Database search --- p.57 / Chapter 2.7.1 --- Sample preparation --- p.58 / Chapter 2.7.2 --- SDS-PAGE --- p.58 / Chapter 2.7.3 --- Protein transfer --- p.58 / Chapter 2.7.4 --- Blocking --- p.59 / Chapter 2.7.5 --- Incubation with primary and secondary antibodies --- p.59 / Chapter 2.7.6 --- Proteins Visualization --- p.59 / Chapter 2.8 --- Real-time PCR --- p.60 / Chapter 2.9 --- Vector construction and lentivirus production --- p.61 / Chapter 2.9.1 --- Lenti-vector construction for Hsp27 expression --- p.61 / Chapter 2.9.2 --- Lentivirus production --- p.62 / Chapter 2.9.3 --- Lentivirus infection --- p.63 / Chapter 2.10 --- SiRNAs transfection. --- p.63 / Chapter 2.11 --- Identification of potential protein targets for TDP --- p.64 / Chapter 2.12 --- In Vivo Tumorigenesis --- p.64 / Chapter 2.13 --- Assay of chaperone activity of Hsp27 using lysozyme as substrate --- p.65 / Chapter 2.14 --- Mitochondria and cytosolic proteins preparation --- p.66 / Chapter 2.15 --- Immunohistochemistry (IHC) --- p.67 / Chapter 2.15.1 --- Preparation of paraffin tissue sections --- p.67 / Chapter 2.15.2 --- Immunostaining --- p.67 / Chapter 2.16 --- Methodology of this study --- p.68 / Chapter 2.17 --- Statistical analysis --- p.68 / Chapter CHAPTER 3 --- Results --- p.69 / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- TDP significantly suppressed cell growth and induced apoptosis in HCC cells. --- p.69 / Chapter 3.2.1 --- TDP was identified from 102 pure compounds by using MTT assay --- p.69 / Chapter 3.2.2 --- TDP significantly suppressed HCC cell growth --- p.73 / Chapter 3.2.3 --- TDP induced the apoptosis of HCC cells --- p.74 / Chapter 3.3 --- Study of the molecular mechanism of TDP on HCC --- p.76 / Chapter 3.3.1 --- The comparative proteomic profiling --- p.76 / Chapter 3.3.2 --- Hsp27 was one of the molecular targets of TDP in HepG2 cells. --- p.80 / Chapter 3.3.3 --- TDP induced apoptosis through the caspase-dependent mitochondrial pathway. --- p.82 / Chapter 3.3.4 --- Hsp27 involved in the mitochondrial apoptosis induced by TDP --- p.84 / Chapter 3.3.5 --- Enforced Hsp27 overexpression rescued the mitochondrial apoptosis induced by TDP in HepG2 cells --- p.87 / Chapter 3.3.6 --- The possible regulatory signaling by TDP --- p.91 / Chapter 3.4 --- TDP directly targeted Hsp27 and destroyed its chaperone action --- p.92 / Chapter 3.5 --- Degradation of Hsp27 aggregation stimulated by TDP was mediated by ubiquitin-proteasome system (UPS) pathway --- p.96 / Chapter 3.6 --- Nude mice model demonstrated the suppressive effect of TDP on HCC --- p.97 / Chapter Chapter 4 --- Discussion and Conclusions --- p.100 / Chapter 4.1 --- Discussion --- p.100 / Chapter 4.2 --- Conclusion --- p.110 / Reference --- p.111

Tang Wan-yee. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 192-212). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Celecoxib is a COX-2 selective non-steroidal anti-inflammatory drug which has been shown to inhibit growth and induce apoptosis in various cancer cell lines. Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and an apoptosis detection kit, we demonstrated that celecoxib was able to induce growth inhibition and apoptosis in a dose-dependent manner in 3 NPC cell lines: HK-1, Hone-1, and C666-1. Afterwards, a proteomic approach was used to study the underlying mechanisms involved in celecoxib-mediated effects on two COX-2 positive NPC cell lines (HK-1 and C666-1). Results showed that a total of 18 protein spots were differentially expressed in the HK-1 and C666-1 cells. On the other hand, we also compared the proteomic expression profile between an NPC cell line (C666-1) and a normal nasopharynx cell line (NP69) in order to study whether those differentially expressed proteins after celecoxib treatment were also involved in NPC carcinogenesis. Proteomics results with confirmation using Western blotting discovered that HSP27 phosphorylated of serine 82 (HSP27-pSer82) protein was up-regulated in C666-1 cells when compared with that in NP69 cells. After treatment with celecoxib, expression of HSP27-pSer82 protein was down-regulated in both HK-1 and C666-1 cells. These findings suggest that down-regulation of HSP27-pSer82 protein expression may have mediated the growth-inhibitory effects of celecoxib in HK-1 and C666-1 cells. Finally, other differential expressed proteins identified from proteomics with confirmation by immunocytochemical staining in the 2 NPC cell lines and 40 NPC patient specimens showed that down-regulation of annexin 2 and beta2-tubulin may be important in NPC formation. / COX-2 over-expression has been found in various cancers such as colorectal cancer, liver cancer and lung cancer. In vivo studies have shown that mice overexpressing COX-2 developed breast cancer whereas COX-2 knockout mice had reduced rates of cancer formation in the intestines and skin. In the present study, COX-2 expression in NPC patient biopsies was examined and correlated with the clinicopathological data of the patients. Immunocytochemical staining showed that COX-2 protein was over-expressed in 84.6% (66/78) of non-metastatic NPC patients and was associated with an advanced nodal stage (P<0.05). All these data support an important role for COX-2 in NPC pathogenesis. / In summary, this study is the first to identify HSP27-pSer82 protein as a potential target of celecoxib in NPC cells. Detailed investigations of the functional role of molecular targets identified in this study would improve our understanding of the chemotherapeutic effects of celecoxib and, in the long run, may lead to a more effective chemotherapeutic treatment to this common cancer. / Nasopharyngeal carcinoma (NPC) is prevalent in southern China. Although early stage patients have a high rate of cure with radiotherapy alone, the prognosis for those with stage III or IV disease remains poor due to subsequent development of distant metastases. Therefore there is an urgent need to develop novel biologic agents to improve treatment outcomes. / Chan, Ming Lok. / Adviser: Anthony T.C. Chan. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3418. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 141-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Five traditional Chinese medicines were screened for their antiangiogenic activities through zebrafish angiogenic assay. Two of them, Tripterygium wilfordii and Rheum palmatum showed potential in the primary screening. T. wilfordii was selected in further study. / In the further investigation of antiangiogenic activity of triptolide on mammal systems, triptolide showed potent activity in human umbilical vein endothelial cells (HUVECs) assays including proliferation, migration and tube formation assay. It inhibited HUVEC proliferation with IC50 as low as 34 nM. It also showed more potency in HUVEC migration and tube formation assay at as low concentration as nanomolar level than SU5416, a putative VEGFR-2 inhibitor currently in clinic trials. RT-PCR and western blotting analysis showed that the underlying mechanism of triptolide correlated with down-regulation of VEGFR-2 and Tie2 expression and production. Tie2 inhibition appeared to be a later event as compared with VEGFR-2. Tie2 overexpression in HUVEC could attenuate the inhibitory effect of triptolide on HUVEC proliferation. Tie2 knockdown mimicked the inhibition activity of triptolide in tube formation assay. These phenomemon revealed that Tie2 signaling pathway plays a crucial role in triptolide-mediated angiogenesis inhibition. In in vivo Matrigel Plug assay, triptolide showed inhibition effect at as low as 100 nM. / T. wilfordii is an immune-suppressive, anti-inflammatory herb used in China for centuries. Through bioassay-guided purification, three antiangiogenic terpenoids were isolated from the ethyl acetate fraction, namely, celastrol, cangoronine and triptolide. Among them, triptolide manifested the most potent antiangiogenic activities against vessel formation. As low as 0.31microM, triptolide inhibited 20% of vessel formation, and the inhibition reached a plateau of 50% at 1.2 microM. Celatrol reduced vessel formation by more than 30% at 0.62microM, but killed 50% of the embryos at higher concentrations. Cangoronine was much weaker, inhibiting vessel formation by 20% at 2.5microM. These three components all showed stronger antiangiogenic activities than 2-methoxyestradiol, a putative compound currently under clinical trials as an antiangiogenic agent for cancer treatment, as the latter inhibited angiogenesis in zebrafish embryos by 34% at 10microM. The loss of vessel formation in the embryos treated with triptolide was further confirmed using endogenous alkaline phosphatase staining. Semi-quantitative RT-PCR analysis revealed that triptolide dose- and time-dependently reduced the mRNA expression of angiopoietin (angpt2) and tie2 in zebrafish, indicating the involvement of angpt2/tie2 signaling pathway in the antiangiogenic action of triptolide. / This research revealed that zebrafish model is a promising antiangiogenic model for both the screening of antiangiogenic agents from Chinese herbal medicine and the subsequent discovery for the drug targets. Triptolide, an anti-inflammatory component from T. wilfordii, is a potent angiogenic inhibitor through targeting VEGFR-2 and Tie2 pathways in mammal models whereas targeting ang2-tie2 pathway in zebrafish model. The anti-tumor action of triptolide was demonstrated to be partly through inhibition of tumor angiogenesis. Moreover, the potent antiangiogenic action exerted by triptolide at nanomolar dosage level gives proof that it is a promising lead compound for the development of antiangiogenic drug for cancer treatment. (Abstract shortened by UMI.) / He, Mingfang. / Adviser: Paul Pui-Hey Bot. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0247. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 84-106). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

In conclusion, our study has demonstrated that TGZ, a synthetic PPARgamma ligand, inhibits lung cancer cells growth through cell-cycle arrest, increased cell differentiation and induction of apoptosis. In this pathway, the activation of ERK by TGZ plays a central role in promoting apoptosis, which appears to be mediated via a mitochondria-related mechanism and functions in a PPARgamma-dependent manner. The interaction between PPARgamma and ERK may create an auto-regulatory and positive feedback loop to enhance the effect of ERK whereas the activation of Akt may generate a negative regulation to control the degree of apoptosis occurred in lung cancer cells. TGZ may counteract NNK function to inhibit lung cancer cell growth in the PPARgamma-dependent manner. / Lung cancer is the world's leading cause of cancer death. Currently there is not an acceptable adjuvant or palliative treatment modalities that have been conclusively shown to prolong survival in lung cancer. Therefore, translational research to improve outcomes with this disease is critical. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription. PPARgamma ligands have been demonstrated to inhibit growth of cancer cells. The role of the PPARgamma in cell differentiation, cell cycle arrest and apoptosis has attracted increasing attention. Our study focused on the role of PPARgamma and its ligand troglitazone (TGZ) in the cell death of human lung cancer and the interaction between PPARgamma system and 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major tobacco-specific carcinogen. / The epidemic of lung cancer is directly attributable to cigarette. However, it is still not completely known the molecular pathway of cigarette smoking in the pathogenesis of lung cancer. Among the carcinogenoic chemicals of cigarette smoking, 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent, which induces lung cancer in all animal species tested. Unlike PPARgamma ligands, NNK can promote cell proliferationa and growth. It is interesting to know whether PPARgamma ligands can inhibit the growth-promoting function of NNK. To address this question, we used NCI-H23 lung cancer cells as the model to study how TGZ influenced the function of NNK. Results showed that NNK stimulated cell proliferation, induced the DNA binding activity of nuclear factor-kappaB (NF-kappaB), down-regulated Bad expression, and up-regulated PPARgamma protein expressions. Inhibition of NF-kappaB nuclear translocation led to the suppression of NNK-mediated Bad expression, indicating that NNK may regulate Bad expression through the activation of NF-kappaB. TGZ significantly inhibited cell proliferation induced by NNK. Though TGZ did not affect nuclear factor-kappaB (NF-kappaB) activity, it up-regulated Bad expression. Taken together, TGZ can efficiently inhibit the proliferation of lung cancer cells induced by NNK via Bad- and PPARgamma- related pathways, which may not be directly relevant to the activity of NF-kappaB. / To elucidate the mechanism responsible for the effect of PPARgamma and TGZ on lung cancer cells, we further studied the PPARgamma molecular pathway in NCIH23 treated by TGZ. The result demonstrated that TGZ induced PPARgamma and ERK1/2 accumulation in the nucleus, where the co-localization of both proteins was found. It showed that the activation of ERK1/2 resulted in apoptosis via the mitochondrial pathway, reflecting by reduction of mitochondria membrane potential, change in Bcl-2 family members, release of cytochrome c into cytosol, and activation of caspase 9. Both PPARgamma siRNA and U0126, a specific inhibitor of ERK1/2, were able to block these effects of TGZ, suggesting that apoptosis induced by TGZ was PPARgamma- and ERK1/2-dependent. Inhibition of ERK1/2 by U0126 also led to a significant decrease in the level of PPARgamma, indicating that there was probably a positive cross-talk between PPARgamma and ERK 1/2 or an auto-regulatory feedback mechanism to amplify the effect of ERK1/2 on cell growth arrest and apoptosis. In addition to ERK1/2, TGZ also activated Akt. Interestingly, inhibition of ERK1/2 prevented the activation of Akt whereas suppression of Akt had no effect on ERK1/2, suggesting that Akt was not necessary for TGZ-PPARgamma-ERK pathway. However, the inhibition of Akt promoted the release of cytochrome c. Thus, the activation of Akt may have a negative effect on apoptosis induced by TGZ. Wortmannin, a PI3K inhibitor, inhibited TGZ-induced ERK1/2 and Akt activation, indicating that PI3K may function at the up-stream of ERK and Akt. In conclusion, our study has demonstrated that TGZ induced apoptosis in NCI-H23 lung cancer cells via a mitochondrial pathway and this pathway was PPARgamma-and ERK1/2-dependent. / We first investigated the effect of PPARgamma ligand TGZ on two human lung cancer cells (NCI-H23 and CRL-2066) and one human lung normal cell (CCL-202). The results showed that in consistence with the loss of cell viability, TGZ induced apoptosis in CRL-2066 and NCI-H23 cells but not in CCL-202 cells. TGZ up-regulated PPARgamma expression in all these three lung cell lines, especially in the cancer cells. In association of the time-dependent inhibition of the cell proliferation, TGZ down-regulated the expression of Bcl-w and Bcl-2 but activated ERK1/2 and p38, suggesting that the growth-inhibitory effect of TGZ is associated with the reduction of Bcl-w and Bcl-2 and the increase of ERK1/2 and p38 activation. SAPK/JNK activation assay showed a decreased activity in all these three cell lines treated by TGZ. It was also demonstrated that TGZ was able to activate PPARgamma transcriptionally. We conclude that TGZ inhibits the growth of human lung cancer cells via the induction of apoptosis, at least in part, in a PPARgamma-relevant manner. / Li Mingyue. / "June 2006." / Advisers: George Gong Chen; Anthony Ping Chuen Yim. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6202. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 174-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.