Liver fibrosis is a wound healing process in liver with¡@chronic injury and is characterized by the excess production and accumulation of extracellular matrix (ECM) component. Liver injury of any etiology may lead to activation of hepatic stellate cells (HSCs), which are trans-differentiated from lipocyte-like cells to highly proliferative myofibroblast-like cells. Activation of HSCs is considered a crucial event that promotes increased ECM production and consequently hepatic fibrosis. Liver fibros is resulted from a net increased synthesis and decreased degradation of ECM proteins. Pluchea indica (Less) has been reported to have antipyretic, anti-ulcer, anti-inflammatory, anti-oxidant, diuretic and anti-amoebic activities. Our previous studies showed that the aqueous extract of roots from P. indica (PIRAE) showed that it can suppress the growth and migration of HeLa and GBM8401 cancer cell lines, and also significantly reduce serum glutamate pyruvate transaminase (GPT), alpha-smooth muscle actin (£\-SMA) and collagen type I expression in animal model of liver fibrosis induced by thioacetamide (TAA). In this study, we plan to investigate the effects of PIRAE on activation, proliferation and migration of rat culture activated HSCs. The results indicated that protein expression of £\-SMA and collagen type I of HSCs was decreased followed by treatment of either 0.5 or 1.0 mg/ml PIRAE for 48 hours. In addition, the effects of PIRAE on proliferation in culture activated HSCs were assessed by analyses of cell growth curve, MTT, WST-1 and BrdU, respectively. The results showed that PIRAE inhibited HSCs proliferation in a dose- and time-dependent manner. Moreover, wound healing assay and transwell assay showed that PIRAE prevented migration in activated HSCs. In conclusion, PIRAE may suppresse culture activated HSCs proliferation, migration, and activation of culture activated HSCs, as well as accumulation of collagen type I.

Neuroblastoma is a childhood solid tumor of a unique propensity to either regress spontaneously or grow relentlessly. Emerging evidence indicated that neuroblastoma contains heterogeneous populations of cells, and commitment of these cells to neuronal lineage may result in aggressive progression in patients, whereas to fibromuscular lineage may give a favorable outcome. However, mechanism(s) controlling the lineage commitment of neuroblastoma cells remains to be identified. Our preliminary data suggested that Kr?ppel-Like Factor 4 (KLF4) might promote neuroblastoma regression. KLF4 is a transcription factor regulating a variety of cellular functions, including proliferation and cell cycle progression. Recent studies have demonstrated that KLF4 may act as both tumor suppressor and oncogene in a cell-context dependent manner. Importantly, our preliminary data showed that low KLF4 expression is highly associated with poor clinical outcomes of the neuroblastoma patients. In addition, we found that overexpression of KLF4 suppresses neuroblastoma cell growth accompanied with loss of tumorigenicity. Morphologically, KLF4 overexpressing cells changed their morphologies to become epithelial-like, strongly substrate-adherent and expressing smooth muscle marker. Therefore, we hypothesized that KLF4 exerts its effects through two ways, it may (i) function to inhibit cell growth and reduce tumorigenicity; and (ii) promote differentiation of the neuroblastoma cells to the non-tumorigenic, fibromuscular-like cells. RT-PCR data revealed the differential expression of KLF4 in 11 neuroblastoma cell lines. In particular, a modest expression was found in Be(2)C, a cell line which was formerly demonstrated to differentiate and form tumor in mice xenograft assay. It was therefore chosen as the study model. To assess the effects of KLF4 knockdown on tumor growth, stable knockdown clones from Be(2)C cells were established by lentiviral transduction of KLF4-targeting shRNA. In parallel, clones that stably expressed non-target shRNA were used as controls. After the transduction, two stable knockdown clones showing significant KLF4 downregulation were isolated from single colony (monoclonal stable clones) and a pool of cells (polyclonal stable clones) respectively. The cell proliferation and growth rate of the stable clones were then measured by 5-bromo-2’-deoxyuridine (BrdU) proliferation assay and growth curve assay. The results have indicated that both monoclonal and polyclonal stable KLF4 knockdown clones grow faster than the control clones. In order to examine the tumorigenicity in vivo, the stable clones were xenotransplanted to severe combined immunodeficient mice. The stable KLF4 knockdown clones showed a significant higher growth rate and formed a larger tumor. The stable clones were also treated with BrdU for four weeks for differentiation towards fibromuscular lineage. As anticipated, the control clones showed fibromuscular features, like more flattened and epithelial-like morphology. In contrast, the stable KLF4 knockdown clones failed to present the fibromuscular features after treatment. In addition, immunocytochemistry staining of SMA and quantitative analysis of the immunocytochemistry further confirmed that only the control clones showed higher SMA expression after BrdU treatment, while there is no change in the SMA expression in the stable KLF4 knockdown clones. These results demonstrated that KLF4 functioned by inhibiting neuroblastoma cell proliferation and growth, reducing the tumorigenicity, and it was required for fibromuscular differentiation. / published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

Our study shows that the secretion of a major glycosylated, phosphoprotein with a molecular weight of 69kDa (pp69) is a specific marker for non-transformed NRK-49F cells. Antibody raised against pp69 recognizes, in addition to pp69, another major phosphoprotein with a molecular weight of 62kDa (pp62) secreted by RR1022 and spontaneously transformed NRK-49F cells (spt-NRK-49F). Immunoprecipitation of total cell lysates from both NRK-49F and RR1022 cells with anti-pp69 antibody detected only pp69. Treatments with: epidermal growth factor (EGF), transforming growth factor-$ beta$ (TGF-$ beta$) retinoic acid (RA), and TPA modulate the levels of pp69 present in the conditioned media. Furthermore, TPA and EGF induce the synthesis of 3 internal peptides with molecular weights of 58, 54, and 44 kDa which appear to be pre-processed forms of pp69. / Treatment of NRK-49F cells with insulin, EGF, TGF-$ beta$, PPA, levamisole and spermine clearly demonstrate alterations in the phosphorylation of pp69, concomitant with changes in extracellular phosphatase activity.

Arterial smooth muscle cells (SMCs) from the systemic and pulmonary circulations experience a broad range of oxygen concentrations under physiological conditions. The hypoxic response, however, has been inconsistent, with both enhanced proliferation and growth arrest being reported. This variability precludes a definitive conclusion regarding the role of oxygen tension in arterial disease. In the first part of this study, we determined if hypoxia elicits different proliferative and apoptotic responses in human aortic SMCs (HASMCs) incubated under conditions which do or do not result in cellular ATP depletion and whether these effects are relevant to vascular remodeling in vivo. Gene expression profiling was used to identify potential regulatory pathways. In HASMCs incubated at 3% O2, proliferation and progression through G1/S interphase are enhanced. Incubation at 1% O2 reduced proliferation, delayed G1/S transition, increased apoptosis and cellular ATP levels were reduced. In aorta and mesenteric artery from hypoxia exposed rats, both proliferation and apoptosis are increased after 48hrs. p53 and p21expression is differentially affected in HASMCs incubated at 1% and 3% O2. Hypoxia induces a state of enhanced cell turnover, conferring the ability to remodel the vasculature in response to changing tissue metabolic needs while avoiding the accumulation of mutations that may lead to malignant transformation or abnormal vascular structure formation. A unifying hypothesis in which events at the G1/S transition and apoptosis activation are coordinated by effects on p53, p21, their downstream effector genes and regulatory factors is proposed. Differences in the contractile responses of systemic and pulmonary arterial smooth muscle cells to hypoxia are well studied. Differences in proliferation and survival are anticipated because of differences in embryonal cell origin, oxygen concentrations within their respective microenvironments and in cellular energetics but these responses have not been directly compared. In the second part of the study, human pulmonary arterial SMCs (HPASMCs) proliferated at oxygen concentrations which inhibited cell growth in HASMCs. HPASMCs survived and maintained their intracellular ATP levels at levels of hypoxia sufficient to deplete ATP and induce apoptosis in HASMCs. In vivo studies in rats show proliferation and apoptosis in main or branch PASMCs only after 7 days of hypoxia. VSMCs are able to proliferate under hypoxic conditions as long as cellular ATP levels are maintained. HPASMCs have an enhanced capacity to maintain cellular energy status compared to HASMCs and hence their viability is preserved and the proliferative response predominates at lower oxygen concentrations.

Arterial smooth muscle cells (SMCs) from the systemic and pulmonary circulations experience a broad range of oxygen concentrations under physiological conditions. The hypoxic response, however, has been inconsistent, with both enhanced proliferation and growth arrest being reported. This variability precludes a definitive conclusion regarding the role of oxygen tension in arterial disease. In the first part of this study, we determined if hypoxia elicits different proliferative and apoptotic responses in human aortic SMCs (HASMCs) incubated under conditions which do or do not result in cellular ATP depletion and whether these effects are relevant to vascular remodeling in vivo. Gene expression profiling was used to identify potential regulatory pathways. In HASMCs incubated at 3% O2, proliferation and progression through G1/S interphase are enhanced. Incubation at 1% O2 reduced proliferation, delayed G1/S transition, increased apoptosis and cellular ATP levels were reduced. In aorta and mesenteric artery from hypoxia exposed rats, both proliferation and apoptosis are increased after 48hrs. p53 and p21expression is differentially affected in HASMCs incubated at 1% and 3% O2. Hypoxia induces a state of enhanced cell turnover, conferring the ability to remodel the vasculature in response to changing tissue metabolic needs while avoiding the accumulation of mutations that may lead to malignant transformation or abnormal vascular structure formation. A unifying hypothesis in which events at the G1/S transition and apoptosis activation are coordinated by effects on p53, p21, their downstream effector genes and regulatory factors is proposed. Differences in the contractile responses of systemic and pulmonary arterial smooth muscle cells to hypoxia are well studied. Differences in proliferation and survival are anticipated because of differences in embryonal cell origin, oxygen concentrations within their respective microenvironments and in cellular energetics but these responses have not been directly compared. In the second part of the study, human pulmonary arterial SMCs (HPASMCs) proliferated at oxygen concentrations which inhibited cell growth in HASMCs. HPASMCs survived and maintained their intracellular ATP levels at levels of hypoxia sufficient to deplete ATP and induce apoptosis in HASMCs. In vivo studies in rats show proliferation and apoptosis in main or branch PASMCs only after 7 days of hypoxia. VSMCs are able to proliferate under hypoxic conditions as long as cellular ATP levels are maintained. HPASMCs have an enhanced capacity to maintain cellular energy status compared to HASMCs and hence their viability is preserved and the proliferative response predominates at lower oxygen concentrations.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available in print.

Thesis (Ph.D.)--University of Western Australia, 2006.