Studies on the anti-tumor effects of conjugated fatty acids on murine macrophage-like leukemia cells.

• Other Authors: Liu, Wai Nam.
• Format: Others
• Language: English; Chinese
• Published: 2012
• Subjects: Leukemia > Treatment; Fatty acids; Leukemia > therapy; Fatty Acids
• Online Access: http://library.cuhk.edu.hk/record=b5549197; http://repository.lib.cuhk.edu.hk/en/item/cuhk-328560

白血病由血液或骨髓中的癌細胞所形成，基於其造血幹細胞（HSC）的增殖和分化出現偶聯或不平衡的情況而產生的結果。白血病是香港最常見的兒童癌症，報告指出，於2005年至2009年期間，平均每年約有270名患者死於該病。傳統治療白血病的方法包括化療，放射性治療，骨髓或外周血幹細胞移植，至於採用哪種療法，則要視乎白血病的類型和階段。然而，這些療法會為患者帶來各種副作用，因此，在過去十年間，研發新型治療白血病的藥物引起了越來越多人的關注。 === 共軛脂肪酸（CFA），是指一群位置及幾何異構體的多元不飽和脂肪酸（PUFA），於它們的化學結構中，最少有一組共軛雙鍵。天然的共軛脂肪酸包括，存在於反芻動物的肌肉及乳製品中的共軛雙烯酸（CLA），存在於植物種子油中的共軛三烯酸（CLN），以及存在於海藻中的共軛四烯酸，共軛五烯酸（CEPA）和共軛六烯酸（CDHA）。過往的研究證實了共軛雙烯酸擁有各種生理及醫藥功效，包括抗脂肪分化，抗動脈硬化，抗糖尿病，免疫調節和抗腫瘤作用。根據體外實驗報告指出，共軛三烯酸和共軛四烯酸對多種腫瘤細胞株皆具有生長抑制作用，然而，它們對小鼠巨噬細胞樣的白血病細胞之調節作用和機制仍有待研究。因此，在這篇論文中，共軛三烯酸和共軛四烯酸對小鼠巨噬細胞樣的白血病細胞的抗增殖作用，以及它們引起的相關機制將會被探討。 === 本實驗計劃研究了九個不同的多元不飽和脂肪酸異構體對小鼠巨噬細胞樣的白血病細胞PU5-1.8細胞的抗增殖能力，當中包括三烯酸、四烯酸、共軛雙烯酸、共軛三烯酸和共軛四烯酸的異構體。結果清楚地表明，共軛三烯酸和共軛四烯酸異構體皆能對白血病細胞表現出劑量依賴性的生長抑制作用。於十個異構體當中，順式-8，反式-10，順式-12共軛三烯酸(蘭花酸)和順式-9，反式-11，反式-13，順式-15共軛四烯酸(杷茬酸)較其他共軛脂肪酸異構體更有效抑制白血病細胞的生長，因此，他們被選定為主要的研究對象，以便對它們所引起的相關機制作進一步了解。此外，蘭花酸和杷茬酸對其他小鼠巨噬細胞樣的白血病細胞，包括J774 A.1細胞和P388D1細胞，也具備抗增殖作用，表現其生長抑制作用並不是純粹針對單一種腫瘤細胞株的。有趣的是，蘭花酸和杷茬酸對PU5-1.8細胞的生長抑制作用是可以局部逆轉的，但只限以低濃度的共軛脂肪酸培養白血病細胞以及培養的時間不多於24小時，否則，隨著培養的時間增加或共軛脂肪酸的濃度增加時，該生長抑制作用幾乎是不可逆轉的。另一方面，結果也表明蘭花酸和杷茬酸在其抑制白血病細胞增殖率為五十百分比之濃度下，它們對腫瘤細胞以及小鼠正常細胞的毒性作用是很少的。除了在體外研究，預先以蘭花酸處理的PU5-1.8細胞於BALB/c小鼠內導致白血病細胞生長的能力也以劑量依賴方式被抑制。 === 幾種不同的機制也能解釋蘭花酸和杷茬酸對PU5-1.8細胞的生長抑制能力，當中包括阻礙腫瘤細胞週期的前進，增加腫瘤細胞內活性氧（ROS）的生產或誘導腫瘤細胞的凋亡。研究結果指出，蘭花酸和杷茬酸可以抑制PU5-1.8細胞週期的進程並將其停留在G₀/G₁時相，換來的是減少處於S時相的細胞的比例。此外，透過西方蛋白質印跡分析，細胞週期蛋白E的表達有所下調，同時，幾個細胞週期調控蛋白的表達，包括p21，p27及p53蛋白則被上調，跟上述的實驗結果吻合。此外，經蘭花酸和杷茬酸處理後，PU5-1.8細胞內的ROS濃度和線粒體質量也有所增加，而它們對白血病細胞的生長抑制作用則會被抗氧化劑所減弱，這一點說明了蘭花酸和杷茬酸對PU5-1.8細胞的抗增殖作用可能與細胞內的脂質過氧化物濃度和線粒體質量有關。最後，通過各種測試細胞凋亡的實驗，包括利用細胞死亡檢測的ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40}試劑盒，Annexin-V和JC-1染色等方法，清楚地表明了蘭花酸和杷茬酸能誘導PU5-1.8細胞的凋亡。加上西方蛋白質印跡分析，PU5-1.8細胞內Bcl-2和Bcl-XL的蛋白的表達水平有所下降，而相反Bax蛋白的表達水平則有所提升，足以證明蘭花酸和杷茬酸能引發PU5-1.8細胞的凋亡。 === 總括來說，蘭花酸和杷茬酸對PU5-1.8細胞的抗增殖作用呈現時間和劑量依賴性，該作用可能基於阻礙腫瘤細胞週期的前進，增加腫瘤細胞內ROS的生產或誘導腫瘤細胞的凋亡。由於蘭花酸和杷茬酸分別在植物種子油和海藻的含量相當高，再加上它們對正常細胞無直接毒性，若果能夠對它們的抗腫瘤作用以及其分子機制有更透徹的理解，它們有望發展成為未來治療白血病的藥物。 === Leukemia is a cancer of the blood or bone marrow which is the result of uncoupling or imbalance of the proliferation and differentiation of hematopoietic stem cells (HSC). It is the most common childhood cancer in Hong Kong and it claims the lives of around 270 patients per year from 2005 to 2009 in average. Conventional approaches to leukemia therapy include chemotherapy, radiotherapy and bone marrow or peripheral blood stem cell transplantation, depending on the types and stages of leukemia. Nevertheless, these therapies are accompanied by a number of undesirable effects to the patients, hence, the development and research in novel treatments of leukemia are attracting increasing attention in the past decades. === Conjugated fatty acids (CFA) refer to the positional and geometric isomers of polyunsaturated fatty acids (PUFA) with conjugated double bonds. Naturally-occurring CFA include conjugated linoleic acids (CLA) from meat and dairy products of ruminant animals, conjugated linolenic acids (CLN) from plant seed oils, conjugated tetraenoic acids, conjugated eicosapentaenoic acids (CEPA) and conjugated docosahexaenoic acids (CDHA) from seaweeds. CLA have been shown to possess various biological and pharmacological activities, including anti-adipogenic, anti-atherogenic, anti-diabetogenic, immunomodulatory and anti-tumor effects. Furthermore, previous researches have demonstrated the growth-inhibitory effects of CLN and conjugated tetraenoic acids on a wide variety of cancer cell lines in vitro, however, their modulatory effects and action mechanisms on murine macrophage-like leukemia cells remain poorly understood. In this thesis project, the anti-proliferative effects of CLN and conjugated tetraenoic acids on the murine macrophage-like leukemia cells, as well as their action mechanisms will be elucidated. === Nine different PUFA isomers, including linolenic acid, tetraenoic acid, CLA, CLN and conjugated tetraenoic acids were examined for their anti-proliferative effects on the murine macrophage-like leukemia PU5-1.8 cells. The results clearly showed that all CLN isomers and cis-parinaric acid could exhibit growth-inhibitory effects on the leukemia cells in a dose-dependent manner. It was found that jacaric acid and cis-parinaric acid were relatively more potent than the other isomers used in the present study, hence, they were chosen to be the specific targets for more in-depth mechanistic studies. In addition, the anti-proliferative effects of jacaric acid and cis-parinaric acid were observed in other murine macrophage-like leukemia cell lines, including J774 A.1 cells and P388D1 cells, suggesting that the effects were not cell-line specific. Interestingly, the growth-inhibitory effects were partially reversible at lower concentrations of CFA used within 24 hours of incubation, but the effects were almost irreversible when either the incubation time or the concentration of CFA used was increased. Furthermore, the results showed that both jacaric acid and cis-parinaric acid at their IC₅₀ growth-inhibitory concentrations on PU5-1.8 cells exerted minimal, if any, direct cytotoxic effects on the tumor cells as well as the murine normal cells. Apart from the in vitro studies, it was also demonstrated that pre-treatment of PU5-1.8 cells with jacaric acid could significantly decrease the leukemic cell growth in syngeneic BALB/c mice in a dose-dependent manner. === Several mechanisms were proposed for the anti-proliferative effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells, including the triggering of cell cycle arrest, increasing the production of intracellular reactive oxygen species (ROS) or induction of apoptosis in the tumor cells. The results showed that jacaric acid and cis-parinaric acid could inhibit the cell cycle progression since an accumulation of PU5-1.8 cells at the G₀/G₁ phase was observed, together with a decrease in the cell population at the S phase. This finding was supported by the down-regulation of cyclin E protein and up-regulation of several cell cycle regulatory proteins, including the p21, p27 and p53 proteins. Apart from that, the intracellular ROS concentration and the mitochondrial mass were found to be increased in jacaric acid- or cis-parinaric acid-treated PU5-1.8 cells, and their growth-inhibitory effects were alleviated after the addition of antioxidants. Therefore, the anti-proliferative effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells might be correlated with the intracellular concentration of lipid peroxides and the mitochondrial mass. Furthermore, the results clearly demonstrated that both jacaric acid and cis-parinaric acid exhibited dose-dependent apoptosis-inducing effects on PU5-1.8 cells, as revealed by the Cell Death Detection ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40} kit, annexin V assay and JC-1 dye staining method. In addition, it was found that the expression levels of Bcl-2 and Bcl-xL proteins were decreased, whereas the expression level of Bax protein was increased in PU5-1.8 cells, further confirming that apoptosis occurred in PU5-1.8 cells after treatment with jacaric acid and cis-parinaric acid. === Collectively, the results showed that jacaric acid and cis-parinaric acid could exhibit their anti-proliferative effects on PU5-1.8 cells in a time- and dose-dependent manner, through the triggering of cell cycle arrest, increasing the production of intracellular ROS or induction of apoptosis in the tumor cells. Owing to their high abundance in plant seed oils and seaweeds, and being relatively non-cytotoxic, they might be potential candidates for the treatment of leukemia. Further investigations are required in order to develop a better understanding on the molecular action mechanisms underlying the anti-tumor effects of jacaric acid and cis-parinaric acid on leukemia cells before they could be developed as the therapeutic drugs for leukemia. === 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. === Liu, Wai Nam. === Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. === Includes bibliographical references (leaves 169-182). === Abstracts also in Chinese. === Abstract --- p.i === 摘要 --- p.v === Acknowledgements --- p.viii === List of Abbreviations --- p.ix === List of Figures and Tables --- p.xiii === Publications --- p.xvii === Chapter Chapter 1 --- General introduction === Chapter 1.1 --- Introduction to hematopoiesis and leukemia --- p.1 === Chapter 1.1.1 --- Introduction to hematopoiesis --- p.1 === Chapter 1.1.2 --- Introduction to leukemia --- p.7 === Chapter 1.1.2.1 --- Classification of leukemia --- p.7 === Chapter 1.1.2.2 --- Epidemiology of leukemia --- p.10 === Chapter 1.1.2.3 --- Conventional approaches to leukemia therapy --- p.12 === Chapter 1.1.2.4 --- Alternative approaches to leukemia therapy --- p.17 === Chapter 1.2 --- Introduction to conjugated fatty acids --- p.19 === Chapter 1.2.1 --- An overview of polyunsaturated fatty acids and conjugated fatty acids --- p.19 === Chapter 1.2.2 --- Chemical structures and physical properties of CLN and conjugated tetraenoic acids --- p.21 === Chapter 1.2.3 --- Natural occurrence of CLN and conjugated tetraenoic acids --- p.26 === Chapter 1.2.4 --- Synthesis of CLN and conjugated tetraenoic acids --- p.28 === Chapter 1.2.5 --- Metabolism of CLN --- p.30 === Chapter 1.2.6 --- Major biological and pharmacological activities of CLN and conjugated tetraenoic acids --- p.30 === Chapter 1.2.6.1 --- Anti-obese and hypolipidemic property --- p.31 === Chapter 1.2.6.2 --- Anti-carcinogenic property --- p.32 === Chapter 1.2.6.2.1 --- Anti-proliferative effect --- p.32 === Chapter 1.2.6.2.2 --- Apoptosis-inducing effect --- p.33 === Chapter 1.3 --- Aims and scopes of this thesis --- p.36 === Chapter Chapter 2 --- Materials and methods === Chapter 2.1 --- Materials --- p.39 === Chapter 2.1.1 --- Animals --- p.39 === Chapter 2.1.2 --- Cell lines --- p.39 === Chapter 2.1.3 --- Cell culture media and reagents --- p.40 === Chapter 2.1.4 --- Fatty acids --- p.44 === Chapter 2.1.5 --- Reagents and buffers for flow cytometry --- p.48 === Chapter 2.1.6 --- Reagents and buffers for Western blotting --- p.51 === Chapter 2.1.7 --- Cell Death Detection ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40} kit --- p.60 === Chapter 2.2. --- Methods --- p.62 === Chapter 2.2.1 --- Culture of tumor cell lines --- p.62 === Chapter 2.2.2 --- Isolation and culture of murine normal cells --- p.63 === Chapter 2.2.3 --- Determination of cell proliferation by CyQUANT® NF cell proliferation assay --- p.65 === Chapter 2.2.4 --- Determination of cell viability --- p.66 === Chapter 2.2.5 --- Cytotoxicity test of CFA on normal cells --- p.67 === Chapter 2.2.6 --- In vivo tumorigenicity assay --- p.68 === Chapter 2.2.7 --- Analysis of cell cycle profile --- p.69 === Chapter 2.2.8 --- Measurement of DNA fragmentation by Cell Death Detection ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40} kit --- p.70 === Chapter 2.2.9 --- Analysis of Annexin V-GFP/PI dual staining profile --- p.71 === Chapter 2.2.10 --- Determination of mitochondrial membrane potential by JC-1 staining --- p.72 === Chapter 2.2.11 --- Determination of intracellular reactive oxygen species generation --- p.72 === Chapter 2.2.12 --- Determination of mitochondrial mass --- p.73 === Chapter 2.2.13 --- Protein expression study --- p.74 === Chapter 2.2.14 --- Statistical analysis --- p.78 === Chapter Chapter 3 --- Studies on the anti-proliferative effects of jacaric acid and cis-parinaric acid on murine macrophage-like leukemia cells === Chapter 3.1 --- Introduction --- p.79 === Chapter 3.2 --- Results --- p.82 === Chapter 3.2.1 --- Anti-proliferative effects of CFA isomers on murine macrophage-like leukemia PU5-1.8 cells in vitro --- p.82 === Chapter 3.2.2 --- Kinetic and reversibility studies of the anti-proliferative effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells --- p.92 === Chapter 3.2.3 --- Cytotoxic effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells --- p.97 === Chapter 3.2.4 --- Cytotoxic effects of jacaric acid and cis-parinaric acid on murine normal cells in vitro --- p.99 === Chapter 3.2.5 --- Effect of jacaric acid on the in vivo tumorigenicity of PU5-1.8 cells --- p.102 === Chapter 3.3 --- Discussion --- p.104 === Chapter Chapter 4 --- Mechanistic studies on the anti-tumor effects of jacaric acid and cis-parinaric acid on murine macrophage-like leukemia cells === Chapter 4.1 --- Introduction --- p.111 === Chapter 4.2 --- Results --- p.117 === Chapter 4.2.1 --- Effects of jacaric acid and cis-parinaric acid on the cell cycle profile of murine macrophage-like leukemia PU5-1.8 cells --- p.117 === Chapter 4.2.2 --- Effects of jacaric acid and cis-parinaric acid on the expression of cell cycle regulatory proteins in murine macrophage-like leukemia PU5-1.8 cells --- p.121 === Chapter 4.2.3 --- Effects of jacaric acid and cis-parinaric acid on the generation of reactive oxygen species in murine macrophage-like leukemia PU5-1.8 cells --- p.124 === Chapter 4.2.4 --- Effects of antioxidants on the anti-proliferative effects of jacaric acid and cis-parinaric acid on the murine macrophage-like leukemia PU5-1.8 cells --- p.128 === Chapter 4.2.5 --- Effects of jacaric acid and cis-parinaric acid on the mitochondrial mass in murine macrophage-like leukemia PU5-1.8 cells --- p.131 === Chapter 4.2.6 --- Effects of jacaric acid and cis-parinaric acid on the induction of apoptosis in murine macrophage-like leukemia PU5-1.8 cells --- p.135 === Chapter 4.2.7 --- Effects of jacaric acid and cis-parinaric acid on the induction of phosphatidylserine externalization in murine macrophage-like leukemia PU5-1.8 cells --- p.139 === Chapter 4.2.8 --- Effects of jacaric acid and cis-parinaric acid on the mitochondrial membrane potential in murine macrophage-like leukemia PU5-1.8 cells --- p.144 === Chapter 4.2.9 --- Effects of jacaric acid and cis-parinaric acid on the expression of apoptosis-regulatory proteins in murine macrophage-like leukemia PU5-1.8 cells --- p.149 === Chapter 4.3 --- Discussion --- p.152 === Chapter Chapter 5 --- Conclusions and future perspectives === Chapter 5.1 --- Conclusions --- p.161 === Chapter 5.2 --- Future perspectives --- p.164 === References --- p.169