Investigating the chemopreventive effect of hesperetin, luteolin and cyclooxygenase inhibitors in a mouse model of breast cancer.

乳腺癌是女性最常見的腫瘤之一，多發生在女性絶經後，並具有雌激素依賴性。芳香化酶(CYP19)是雌激素生物合成過程中的關鍵酶，而芳香化酶抑製劑(AI)則被用於替代治療雌激素依賴性的乳腺癌。然而，AI在降低雌激素水平的同時能夠引起骨質酥鬆。此項研究的目的是找尋AI替代物。黃酮類化合物是一種多酚化合物，廣泛分佈于植物中。我們先前的研究發現二氢黄酮陈皮素能夠抑制芳香化酶的生物活性，并且抑制芳香化酶高表達的乳腺癌生長。在本研究中，我們發現陳皮素在抑制腫瘤生長的同時能夠降低来曲唑引起的骨質流失。木犀草素是另外一種黄酮类化合物，它同樣能夠抑制芳香化酶的活性并減少骨流失。而與陳皮素不同的是，它能夠抑制芳香化酶的表達。在芳香化酶高表達的乳腺癌細胞(MCF-7 aro)中，木犀草素抑制芳香化酶活性的IC50是3 μM。在MCF-7 細胞中，5 μM的木犀草素能夠抑制CYP19 mRNA 的表達，螢光素酶報告實驗顯示木犀草素是通過作用于啟動子I.3和II來抑制CYP19的表達。蛋白印跡實驗表明木犀草素抑制CYP19表達的分子機制可能通過調節JNK信號通路進而減少AP-1的活性來實現。動物實驗結果顯示木犀草素能夠抑制MCF-7aro腫瘤的生長并改善來曲唑引起的骨流失。環氧化酶(COX)是花生四烯酸轉化為前列腺素途徑中的一種關鍵酶。研究發現COX-2在乳腺癌組織中廣泛表達。本實驗研究了COX抑製劑在裸鼠動物模型中對乳腺癌腫瘤的作用機制。研究結果表明塞來昔布和阿司匹林在不影響血液中雌激素水平的情況下抑制乳腺癌腫瘤的生長。蛋白印迹實驗顯示這兩種藥物能夠降低腫瘤中COX-2，Cyclin A和Bcl-xL的表達。miR-98, miR-222和miR-145也能夠被塞來昔布和阿司匹林影響。本研究表明陳皮素，木犀草素及COX抑制劑有潛力成為替代AI的化學治療藥物或共同治療藥物。Breast cancer is one of the most prevalent cancers affecting women. The majority of breast tumor growth occurred in the post-menopausal period are estrogen dependent. Aromatase (CYP19) catalyzes the rate-limiting step in the synthetic reaction of estrogen and aromatase inhibitors (AIs) are contemporary treatment for estrogen-positive breast cancer. However, estrogen-lowering drugs may promote osteoporosis. Our objective of this study further identified some alternatives for AIs.Flavonoids are polyphenolic compounds that are ubiquitously distributed in plants. We have previously found that the flavanone hesperetin can inhibit the activity of aromatase and suppress aromatase-expressing breast tumor growth. In this project, we investigated the potential interaction between hesperetin and the AI letrozole in a mouse model. Our results showed that hesperetin could inhibit the tumor growth and reduce bone loss induced by letrozole. Similarly, another flavonoid luteolin also inhibited aromatase and prevented bone deterioration as observed in this project. In cells stably transfected with CYP19 (MCF-7aro), luteolin inhibited the aromatase activity with an IC50 value of 3μM. In addition, 5μM luteolin significantly reduced CYP19 mRNA expression in MCF-7 cells. Luciferase reporter assay revealed that luteolin could suppress CYP19 transcription at promoter regions I.3 and II. Western analysis illustrated that JNK signaling pathway was involved and deactivation of AP-1 could be the underlying molecular mechanism. Subsequently, we examined the effect in vivo. Our results showed that luteolin could inhibit the MCF-7aro tumor growth and improved bone loss induced by letrozole.Cyclooxygenase (COX) is an enzyme responsible for the conversion of arachidonic acid into prostaglandins. It is over-expressed in breast cancer tissue and an increased expression of COX-2 was also observed in the xenograft model employed in this project. In the last study we evaluated the importance of COX-2 in breast tumor growth in this model. Our data showed that celecoxib and aspirin could significantly suppress the tumor growth without changing the plasma estrogen level. Western analysis illustrated that COX-2, Cyclin A, Bcl-xL and ER were reduced in celecoxib- and aspirin- treated tumor samples and miR-98, miR-222 and miR-145 were altered by celecoxib or aspirin.After all, this project demonstrated that hesperetin, luteolin and COX-inhibitors could be potential chemopreventive or co-therapeutic agents.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Li, Fengjuan.Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 131-148).Abstract also in Chinese.ACKNOWLEDGEMENTS --- p.IABSTRACT --- p.II摘要 --- p.IVLIST OF ABBREVIATIONS --- p.VTABLE OF CONTENTS --- p.VIICHAPTER 1 --- p.1GENERAL INTRODUCTION --- p.1Chapter 1.1 --- Types of Breast Cancer --- p.3Chapter 1.2 --- Nuclear Receptor Signaling Pathways in Breast Cancer --- p.5Chapter 1.3 --- Estrogen and Breast Cancer --- p.7Chapter 1.4 --- Estrogen and Bone Health --- p.8Chapter 1.5 --- Estrogen Biosynthesis and Aromatase --- p.10Chapter 1.6 --- Tissue Specific Promoter for Aromatase Expression --- p.13Chapter 1.7 --- Nuclear Receptors and Aromatase Promoter Regulation --- p.15Chapter 1.8 --- Signaling Pathway and Aromatase Expression --- p.17Chapter 1.9 --- Cell Cycle in Breast Cancer --- p.20Chapter 1.10 --- Cell Apoptosis --- p.23Chapter 1.11 --- Treatment of breast cancer --- p.25Chapter 1.12 --- Phytoestrogens --- p.29Chapter 1.13 --- Aim of My Study --- p.32CHAPTER 2 --- p.33MATERIALS AND METHODS --- p.33Chapter 2.1 --- Chemicals and Materials --- p.33Chapter 2.1.1 --- Chemicals --- p.33Chapter 2.1.2 --- Plasmids --- p.33Chapter 2.2 --- Cell Culture --- p.33Chapter 2.3 --- Aromatase Activity Assay --- p.34Chapter 2.4 --- Quantitative Real Time PCR --- p.36Chapter 2.4.1 --- RNA Isolation and cDNA Synthesis --- p.36Chapter 2.4.2 --- Quantitative Real Time PCR Assay --- p.37Chapter 2.4.3 --- MiRNA Quantitative Real Time PCR Assay --- p.38Chapter 2.5 --- Western Blot --- p.39Chapter 2.6 --- Measurement of Promoter Activity --- p.41Chapter 2.6.1 --- Plasmid Preparation --- p.41Chapter 2.6.2 --- Transient Transfection and Dual-Luciferase Assay --- p.42Chapter 2.7 --- Electrophoretic Mobility Shift Assay (EMSA) --- p.43Chapter 2.7.1 --- Nuclear protein extraction --- p.43Chapter 2.7.2 --- Electrophorectic Mobility Shift Assay --- p.44Chapter 2.8 --- Animal Experiment Design --- p.45Chapter 2.8.1 --- Animal Model for Hesperetin Study --- p.45Chapter 2.8.2 --- Animal Model for Luteolin Study --- p.46Chapter 2.8.3 --- Animal Model for Cycooxygenase Inhibitors Study --- p.48Chapter 2.8.4 --- Serum Estradiol Determination --- p.49Chapter 2.8.5 --- Analysis of serum lipoproteins --- p.49Chapter 2.8.6 --- Bone Image Acquisition and Region of Interest Selection --- p.50Chapter 2.9 --- Statistical Analysis --- p.50CHAPTER 3 --- p.51The citrus flavonone hesperetin prevents letrozole- induced bone loss in a mouse model of breast cancer --- p.51Chapter 3.1 --- Introduction --- p.51Chapter 3.2 --- Results --- p.54Chapter 3.2.1 --- Murine Body Weight and Liver Weight --- p.54Chapter 3.2.2 --- Effect of Hesperetin and Letrozole on Xenograft Growth in Ovariectomized Mice --- p.55Chapter 3.2.3 --- Hesperetin Reduced Plasma Estradiol Concentration --- p.58Chapter 3.2.4 --- PS2 mRNA Expression in Tumor --- p.59Chapter 3.2.5 --- Uterine Wet Weight --- p.60Chapter 3.2.6 --- Hesperetin Prevent Bone Deterioration Induced by Letrozole --- p.61Chapter 3.3 --- DISCUSSION --- p.63CHAPTER 4 --- p.66dIETARY FLAVONOID LUTEOLIN ON cyp19 transcription in the breast cancer cells mcf-7 --- p.66Chapter 4.1 --- Introduction --- p.66Chapter 4.2 --- Results --- p.68Chapter 4.2.1 --- Inhibitory Effect of Luteolin on Aromatase Activity --- p.68Chapter 4.2.2 --- Luteolin Reduced Aromatase mRNA Expression in MCF-7 Cells --- p.70Chapter 4.2.3 --- Effect of Luteolin on Promoter I.3/II Activity of CYP19 in MCF-7 Cells --- p.71Chapter 4.2.4 --- The Effect of Luteolin on Truncation CYP19 Gene Reporter Assay --- p.72Chapter 4.2.5 --- Luteolin Reduced AP-1 Binding in Promoter I.3/II DNA Fragment --- p.74Chapter 4.2.6 --- Inhibitory Effect of Luteolin on Protein Kinase Signaling --- p.76Chapter 4.3 --- Discussion --- p.78CHAPTER 5 --- p.83interaction OF LUTEOLIN and letrozole in a postmenopausal breast cancer model --- p.83Chapter 5.1 --- Introduction --- p.83Chapter 5.2 --- Results --- p.86Chapter 5.2.1 --- Luteolin and letrozole treatment had no effect on mouse body weight and liver weight --- p.86Chapter 5.2.2 --- Effect of luteolin and Letrozole on Xenograft Growth in Ovariectomized Mice --- p.88Chapter 5.2.3 --- Luteolin reduced plasma estradiol concentration --- p.91Chapter 5.2.4 --- Luteolin Counteracted Uterine Weight Reduction under Letrozole Treatment --- p.92Chapter 5.2.5 --- Luteolin Prevented Bone Deterioration Induced by Letrozole --- p.93Chapter 5.2.6 --- The Effect of Luteolin on Plasma TC and TG --- p.95Chapter 5.2.7 --- Luteolin Increased HDL Level and Reduced the Ratio of LDL/HDL --- p.97Chapter 5.2.8 --- Effect of Luteolin on Cell Cycle and Apoptotic Protein Expression --- p.99Chapter 5.3 --- DISCUSSION --- p.104CHAPTER 6 --- p.107cyclooxygenase inhibitors suppresse breast tumor growth in NUDE MICE --- p.107Chapter 6.1 --- Introduction --- p.107Chapter 6.2 --- Results --- p.109Chapter 6.2.1 --- Celecoxib and aspirin treatment had no effect on mouse body weight and liver weight --- p.109Chapter 6.2.2 --- Effect of celecoxib and aspirin on Xenograft Growth in Ovariectomized Mice --- p.111Chapter 6.2.3 --- Celecoxib and aspirin had no effect on plasma estradiol concentration --- p.113Chapter 6.2.4 --- Celecoxib and Aspirin Had no Effect on Uterine Weight --- p.114Chapter 6.2.5 --- Protein expression of COX-2, Cell cycle-related and cell Apoptotic Genes --- p.115Chapter 6.2.6 --- Detection of Related miRNA Expression Level in Tumors --- p.118Chapter 6.2.7 --- c-Myc mRNA Expression Level were Regulated in Tumors --- p.121Chapter 6.3 --- DISCUSSION --- p.124CHAPTER 7 --- p.127SUMMARY --- p.127REFERENCE --- p.13