published_or_final_version / abstract / Surgery / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 46-57)

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Wang, Ruizhi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 109-134). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Publications --- p.ii / Abstract --- p.iv / 中文摘要 --- p.viii / Abbreviations --- p.xi / List of tables --- p.xiv / List of figures --- p.xv / Contents --- p.xvii / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of gastric cancer --- p.2 / Chapter 1.2 --- Risk factors of gastric cancer --- p.3 / Chapter 1.3 --- Treatment of gastric cancer --- p.4 / Chapter 1.3.1 --- Surgical treatment --- p.4 / Chapter 1.3.2 --- Chemotherapy --- p.4 / Chapter 1.3.3 --- Targeted therapy --- p.5 / Chapter 1.4 --- "Phenotypes of cell death: apoptosis, oncosis and autophagy" --- p.9 / Chapter 1.4.1 --- Cell death --- p.9 / Chapter 1.4.2 --- Apoptosis --- p.10 / Chapter 1.4.2 --- Oncosis --- p.11 / Chapter 1.4.3 --- Autophagy --- p.12 / Chapter 1.4.4 --- p53 --- p.13 / Chapter 1.5 --- Heme oxygenase-1 --- p.14 / Chapter 1.5.1 --- General introduction of Heme oxygenase --- p.14 / Chapter 1.5.2 --- Anti-oxidant function of HO-1 --- p.15 / Chapter 1.5.3 --- Anti-inflammation function of HO-1 --- p.17 / Chapter 1.5.4 --- Pro-angiogenesis role of HO-1 --- p.18 / Chapter 1.5.5 --- HO-1 and cell proliferation --- p.19 / Chapter 1.5.6 --- HO-1 as a therapeutic target for tumors --- p.20 / Chapter 1.6 --- Objectives of study --- p.22 / Chapter Chapter Two: --- Methods and materials --- p.26 / Chapter 2.1 --- Gastric cancer cell lines --- p.27 / Chapter 2.2 --- Cell proliferation detection --- p.27 / Chapter 2.2.1 --- "MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay" --- p.27 / Chapter 2.2.1.1 --- Introduction of MTT assay --- p.27 / Chapter 2.2.1.2 --- Processes of MTT assay --- p.27 / Chapter 2.2.1.3 --- Cell proliferation and cytotoxicity of drugs --- p.28 / Chapter 2.2.2 --- Detection of apoptosis by TUNEL assay --- p.29 / Chapter 2.2.2.1 --- TUNEL (Terminal uridine deoxynucleotidyl transferase dUTP nick end labeling) --- p.29 / Chapter 2.2.2.2 --- Sample preparation --- p.29 / Chapter 2.3 --- Detection of cell cycle by flow cytometry --- p.32 / Chapter 2.3.1 --- Cell cycle --- p.32 / Chapter 2.3.2 --- Sample preparation --- p.33 / Chapter 2.3.3 --- Flow cytometry analysis --- p.34 / Chapter 2.4 --- Detection of mitochondrial membrane potential(ΔΨm) --- p.35 / Chapter 2.4.1 --- Sample preparation --- p.35 / Chapter 2.4.2 --- Mitochondrial membrane potential(ΔΨm) analysis by flow cytometry --- p.36 / Chapter 2.5 --- Detection of proteins investigated in the project --- p.37 / Chapter 2.5.1 --- Antibodies --- p.37 / Chapter 2.5.2 --- Sample Preparation --- p.39 / Chapter 2.5.2.1 --- Cell culture --- p.39 / Chapter 2.5.2.2 --- Protein extraction --- p.39 / Chapter 2.5.2.3 --- Protein assay --- p.41 / Chapter 2.5.2.4 --- Final loading protein --- p.42 / Chapter 2.5.3 --- Western blotting --- p.43 / Chapter 2.6 --- Statistical analysis --- p.45 / Chapter Chapter three: --- Roles of HO-1 in 5-FU treatment for gastric cancer cell lines --- p.47 / Chapter 3.1 --- Cell proliferations with drug treatments --- p.48 / Chapter 3.1.1 --- MTT assay --- p.48 / Chapter 3.1.1.1 --- Introduction --- p.48 / Chapter 3.1.1.2 --- Method and results --- p.49 / Chapter 3.1.2 --- TUNEL assay --- p.58 / Chapter 3.1.2.1 --- Introduction --- p.58 / Chapter 3.1.2.2 --- Method and results --- p.59 / Chapter 3.2 --- HO-1 expression with drug treatments --- p.63 / Chapter 3.2.1 --- Introduction --- p.63 / Chapter 3.2.2 --- Method and results --- p.64 / Chapter 3.3 --- Discussion --- p.72 / Chapter Chapter Four: --- Mechanism responsible for the additive effect of 5-FU and ZnPP --- p.77 / Chapter 4.1 --- Cell cycle arrest after drug treatments --- p.78 / Chapter 4.1.1 --- Introduction --- p.78 / Chapter 4.1.2 --- Method and results --- p.79 / Chapter 4.2 --- Mitochondrial dependent and independent pathway --- p.85 / Chapter 4.2.1 --- Introduction --- p.85 / Chapter 4.2.2 --- Method and results --- p.87 / Chapter 4.3 --- Alteration of apoptotic proteins in gastric cancer cell death after drug treatments --- p.91 / Chapter 4.3.1 --- Introduction --- p.91 / Chapter 4.3.2 --- Method and results --- p.94 / Chapter 4.4 --- Discussion --- p.101 / Chapter Chapter Five: --- Summary and future prospects --- p.107 / Chapter 5.1 --- Summary --- p.108 / Chapter 5.1.1. --- The inhibition of HO-1 enhances the sensitivity of gastric cancer cells to 5-FU --- p.108 / Chapter 5.1.2 --- Apoptosis induced by 5-FU plus HO-1 inhibitor ZnPP is through a mitochondrial-related pathway in MKN28 and MKN45 --- p.109 / Chapter 5.1.3 --- 5-FU plus ZnPP induces apoptosis in a caspase-dependent pathway in MKN45 while in both caspase-dependent and caspase-independent pathway in MKN28 --- p.110 / Chapter 5.2 --- Future prospects --- p.111 / References --- p.113

Essential hypertension (EH) is a risk factor for many cardiovascular disorders. Treatment of established EH, especially for prolonged control of this pathogenic process, represents a great challenge. Moreover, hypertension is considered an important risk factor for the development of many other diseases, e.g. erectile dysfunction. <p> Hemin and other heme derivatives, e.g. heme-L-lysinate (HLL) and heme-L-arginate, have been used extensively to upregulate expression of heme oxygenase (HO) and production of endogenous carbon monoxide (CO). Short-term hemin administration for 4-5 days has been shown to markedly decrease high blood pressure (BP) in spontaneously hypertensive rats (SHR), but not in normotensive Wistar-Kyoto (WKY) or Sprague Dawley (SD) rats. This short-term therapy was effective in treating young, but not adult SHR. In the present study, hemin (15 mg/kg/day) was administered to 12-week old adult SHR through subcutaneously implanted osmotic minipumps for 3 consecutive weeks (the hemin protocol). Into the second week of the hemin protocol, BP of SHR was normalized from 203.2 ± 2.5 to 123.4 ±1.9 mmHg (n=20, p<0.001). There was no further decrease of BP in the remaining days of the hemin protocol. Normalization of BP in these treated SHR was maintained for 9 months after the removal of hemin pumps. <p>To further investigate the metabolic characteristics of hemin during hemin protocol administration, we attempted to monitor circulatory heme levels. A valid standard calibration curve was established using hemin or HLL in <i>in vitro</i> experiments. It was established that the basal serum level of heme was negligible in all rat strains prior to hemin protocol initiation. During the hemin protocol, serum heme level of all rats was significantly elevated; however, it quickly dropped to basal levels thereafter. <p>At the end of the 3-week hemin protocol, HO-1 expression, HO activity, soluble guanylyl cyclase (sGC) expression, and cyclic guanosine monophosphate (cGMP) content were all increased, but phosphodiesterase-5 (PDE-5) expression was down-regulated in the mesenteric arteries. The hemin protocol also reversed SHRs decreased arterial lumen size, and increased expression levels of vascular endothelial growth factor. These changes lasted 9 months after the hemin protocol. The hemin protocol did not cause hepatic or renal toxicity. Our study thus unmasks a novel hemin protocol that will not only normalize BP in SHR with established hypertension but, more importantly, also provide long-lasting anti-hypertension protection. Sustained upregulation of the HO-regulated signaling pathways and reversal of vascular remodeling in small peripheral vessels in treated SHR are among potential underlying mechanisms for the anti-hypertensive effect of the hemin protocol.<p>We further studied if the beneficial effects of hemin protocol on BP of SHR could be extended to improvement of their penile erection. Intracavernosal pressure changes after electrical stimulation of the cavernous nerve (CN) were monitored in adult SHR and age-matched normotensive SD rats after administration of either hemin or hydralazine. Expression levels of HO-1, HO-2, sGC, and PDE-5 in penile tissues were also examined. Frequency-dependent intracavernosal pressure (ICP) changes were reduced in adult SHR. Three weeks after hemin treatment, ICP responses to CN stimulations were significantly increased to the level of normotensive rats, which was linked to normalization of BP of hemin-treated SHR. Hydralazine-treated SHR experienced normalization of BP but not ICP after 3 weeks of treatment. Expression of HO-1 and sGC was upregulated and that of PDE-5 downregulated in hemin-treated SHR. Decreased ICP response in adult SHR can be improved through chronic hemin treatment of SHR. Prolonged upregulation of HO-1 and sGC as well as lowered expression of PDE-5 may account for normalization of erectile dysfunction in SHR subsequent to hemin treatment. <p>This thesis reports for the first time that 21-day hemin administration led to a 9-month normalization of high BP of adult SHR. These effects were mediated through sustained stimulation of the HO/CO system and its downstream effectors targets. Increased activity of HO-1 led to normalization of the abnormally high expression level of VEGF in peripheral mesenteric arteries of adult SHR. Subsequently, this resulted in reversal of the eutrophic remodeling of these arteries, which seems to be the priniciple determinant of the long-term normalization of BP observed for 9 months after stoppage of hemin treatment. The invention of hemin protocol offers a new therapeutic approach for the clinical management of established hypertension for a long duration.<p>Our study, for the first time, correlated changes in serum heme levels with BP levels after injection of hemin or HLL in normotensive and hypertensive rats. Application of this heme monitoring technology will also pave the way for clinical application of hemin therapy in treatment of EH.<p> Another intriguing finding in this thesis is that upregulation of HO-1, through the hemin protocol, led to improvement of abnormally low ICP encountered in adult SHR. Upregulation of HO-1 may represent a novel therapeutic approach to treat hypertension-related erectile dysfunction.

Essential hypertension (EH) is a risk factor for many cardiovascular disorders. Treatment of established EH, especially for prolonged control of this pathogenic process, represents a great challenge. Moreover, hypertension is considered an important risk factor for the development of many other diseases, e.g. erectile dysfunction. <p> Hemin and other heme derivatives, e.g. heme-L-lysinate (HLL) and heme-L-arginate, have been used extensively to upregulate expression of heme oxygenase (HO) and production of endogenous carbon monoxide (CO). Short-term hemin administration for 4-5 days has been shown to markedly decrease high blood pressure (BP) in spontaneously hypertensive rats (SHR), but not in normotensive Wistar-Kyoto (WKY) or Sprague Dawley (SD) rats. This short-term therapy was effective in treating young, but not adult SHR. In the present study, hemin (15 mg/kg/day) was administered to 12-week old adult SHR through subcutaneously implanted osmotic minipumps for 3 consecutive weeks (the hemin protocol). Into the second week of the hemin protocol, BP of SHR was normalized from 203.2 ± 2.5 to 123.4 ±1.9 mmHg (n=20, p<0.001). There was no further decrease of BP in the remaining days of the hemin protocol. Normalization of BP in these treated SHR was maintained for 9 months after the removal of hemin pumps. <p>To further investigate the metabolic characteristics of hemin during hemin protocol administration, we attempted to monitor circulatory heme levels. A valid standard calibration curve was established using hemin or HLL in <i>in vitro</i> experiments. It was established that the basal serum level of heme was negligible in all rat strains prior to hemin protocol initiation. During the hemin protocol, serum heme level of all rats was significantly elevated; however, it quickly dropped to basal levels thereafter. <p>At the end of the 3-week hemin protocol, HO-1 expression, HO activity, soluble guanylyl cyclase (sGC) expression, and cyclic guanosine monophosphate (cGMP) content were all increased, but phosphodiesterase-5 (PDE-5) expression was down-regulated in the mesenteric arteries. The hemin protocol also reversed SHRs decreased arterial lumen size, and increased expression levels of vascular endothelial growth factor. These changes lasted 9 months after the hemin protocol. The hemin protocol did not cause hepatic or renal toxicity. Our study thus unmasks a novel hemin protocol that will not only normalize BP in SHR with established hypertension but, more importantly, also provide long-lasting anti-hypertension protection. Sustained upregulation of the HO-regulated signaling pathways and reversal of vascular remodeling in small peripheral vessels in treated SHR are among potential underlying mechanisms for the anti-hypertensive effect of the hemin protocol.<p>We further studied if the beneficial effects of hemin protocol on BP of SHR could be extended to improvement of their penile erection. Intracavernosal pressure changes after electrical stimulation of the cavernous nerve (CN) were monitored in adult SHR and age-matched normotensive SD rats after administration of either hemin or hydralazine. Expression levels of HO-1, HO-2, sGC, and PDE-5 in penile tissues were also examined. Frequency-dependent intracavernosal pressure (ICP) changes were reduced in adult SHR. Three weeks after hemin treatment, ICP responses to CN stimulations were significantly increased to the level of normotensive rats, which was linked to normalization of BP of hemin-treated SHR. Hydralazine-treated SHR experienced normalization of BP but not ICP after 3 weeks of treatment. Expression of HO-1 and sGC was upregulated and that of PDE-5 downregulated in hemin-treated SHR. Decreased ICP response in adult SHR can be improved through chronic hemin treatment of SHR. Prolonged upregulation of HO-1 and sGC as well as lowered expression of PDE-5 may account for normalization of erectile dysfunction in SHR subsequent to hemin treatment. <p>This thesis reports for the first time that 21-day hemin administration led to a 9-month normalization of high BP of adult SHR. These effects were mediated through sustained stimulation of the HO/CO system and its downstream effectors targets. Increased activity of HO-1 led to normalization of the abnormally high expression level of VEGF in peripheral mesenteric arteries of adult SHR. Subsequently, this resulted in reversal of the eutrophic remodeling of these arteries, which seems to be the priniciple determinant of the long-term normalization of BP observed for 9 months after stoppage of hemin treatment. The invention of hemin protocol offers a new therapeutic approach for the clinical management of established hypertension for a long duration.<p>Our study, for the first time, correlated changes in serum heme levels with BP levels after injection of hemin or HLL in normotensive and hypertensive rats. Application of this heme monitoring technology will also pave the way for clinical application of hemin therapy in treatment of EH.<p> Another intriguing finding in this thesis is that upregulation of HO-1, through the hemin protocol, led to improvement of abnormally low ICP encountered in adult SHR. Upregulation of HO-1 may represent a novel therapeutic approach to treat hypertension-related erectile dysfunction.

Melanoma is a cancer of the pigment producing cells, melanocytes, and is the most serious type of skin cancer. Cancer is a condition in which one type of cell grows without limit in a disorganized fashion, disrupting and replacing normal tissues and their functions. Normal melanocytes reside in the outer layer of the skin and produce a brown pigment called melanin, which is responsible for skin color. Melanoma occurs when melanocytes become cancerous, grow, and invade other tissues. Pro-opiomelanocortin (POMC) is a precursor polypeptide of 241 amino acids and the prohormone of various neuropeptide, including corticotropin (ACTH), £\-melanocyte-stimulating hormone (£\-MSH), and £]-endorphin (£]-EP). Recently, we demonstrated that systemic POMC overexpression potently suppresses the growth and metastasis of B16-F10 melanoma in vitro and in vivo. However, despite potent inhibition of tumor proliferation and angiogenesis, B16-F10 melanoma still managed to survive after POMC gene therapy. The underlying survival mechanism of B16-F10 melanoma remains unclear. Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix transcription factor that plays a key role not only in melanin synthesis, but also in melanocyte development and survival. Besides, MITF binds to the hypoxia-inducible factor-1£\ (HIF-1£\) promoter to stimulate its transcriptional activity. In this study, we investigate the influence of POMC gene delivery on the pro-survival MITF/HIF-1£\ pathway in B16-F10 melanoma cells. Quantitative RT-PCR and western blot analysis revealed that POMC gene delivery increased the MITF mRNA and protein level in B16-F10 melanoma cells. Besides, POMC gene delivery significantly enhanced the HIF-1£\-driven luciferase activities in melanoma cells. By transfection and puromycin selection, we generated and characterized a MITF-knockdown B16-F10 melanoma cells (MITF KD) stably expressing short hairpin RNA against MITF. The growth, invasion, and colonies formation of MITF-KD were similar to those of vector control. However, implantation of MITF-KD cells led to melanoma with significantly reduced tumor size compared with those in mice implanted with vector control cells. Histological analysis revealed a significant reduction of CD31-positive blood vessels in implantation of MITF-KD cells-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, POMC-mediated upregulation of MITF and HIF-1 £\ was significantly attenuated in MITF KD-B16-F10 cells. Acetylsalicylic acid (aspirin; ASA) is widely used as an analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. In our study, we found ASA enhanced cell proliferation. However, in invasion test, ASA had no effect on cell migration. POMC gene delivery elevated the mRNA and protein level of hemeoxygenase-1 (HO-1), a downstream effector of HIF-1£\ pathway and an enzyme catalyzing the converting reaction of heme to carbon monoxide, ion and biliverdine. Inhibition of HO-1 activities augmented the inhibitory effect of POMC gene delivery on proliferation, migration and anchorage-independent growth of B16-F10 melanoma cells. These studies indicated that activation of MITF/HIF-1£\/HO-1 indeed contributes to melanoma survival after POMC gene delivery.

Objective: Heme oxygenase-1 (HO-1) has been well established as a cytoprotective molecule, and has been shown to exert cardioprotective effects in both hypertension and cardiac hypertrophy. However, the precise mechanism of the cardioprotective effect of HO-1 has yet to be fully elucidated. The natriuretic peptide system (NPS) is also a key player in cardiovascular homeostasis and tissue dynamics, and has also been shown to be cardioprotective in a variety of pathologic conditions. This study examined the effect of high dietary salt treatment in genetic models of HO-1, and assessed the expression of the NPS in the left ventricle (LV), in order to gain insight into the relationship between varying levels of HO-1 expression with the development of cardiac hypertrophy and the expression of the NPS. Methods: Age-matched 12-week old male HO-1 knockout (HO-1-/-) and HO-1 cardiomyocyte-specific transgenic overexpressing (HO-1Tg) mice were treated with either normal salt (NS; 0.8%) or high salt (HS; 8.0%) chow for 5 weeks. LV mRNA expression was determined using quantitative real-time RT-PCR. Results: HO-1-/- mice fed HS diet had significantly higher left ventricle-to-body weight ratio (LV/BW) compared to HO-1+/+ mice fed NS diet. HO-1-/- mice had significantly reduced expression of the NPS compared to controls, and these mice did not exhibit a salt-induced increase in ANP expression. HS treatment had no effect on LV/BW in HO-1Tg mice compared to controls. HO-1Tg mice had significantly higher ANP and BNP expression compared to controls. Conclusions: The presence of HO-1 is required for normal salt-induced changes in the local cardiac NPS. HO-1 ablation resulted in significantly lower mRNA expression of the NPS, whereas HO-1 overexpression resulted in higher mRNA expression of the NPS. These data indicate that the detrimental effect of reduced HO-1 expression and the cardioprotective effect of increased HO-1 expression may be due, in part, to altered expression of the NPS. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2009-10-20 09:15:20.541

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Thesis advisor: Dr. William Durante. "December 2008" Includes bibliographical references.