Molecular Mechanisms Study of Effective Constituents in Coptidis Rhizoma on Cancer-Specific Metabolic Reprogramming. TAN Wen

Transcription

1 Molecular Mechanisms Study of Effective Constituents in Coptidis Rhizoma on Cancer-Specific Metabolic Reprogramming by TAN Wen Doctor of Philosophy in Biomedical Sciences 2012 Institute of Chinese Medical Sciences University of Macau

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3 Molecular Mechanisms Study of Effective Constituents in Coptidis Rhizoma on Cancer-Specific Metabolic Reprogramming by TAN Wen SUPERVISOR: Prof. WANG Yitao Doctor of Philosophy in Biomedical Sciences 2012 Institute of Chinese Medical Sciences University of Macau

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5 Author s right 2012 by TAN, Wen

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7 Acknowledgements My deepest gratitude goes first and foremost to my supervisor Prof. WANG Yitao who has offered professional guidance and dedicated support throughout my work. There is no denying that the ultimate completion of study is closely attached to his full support and patient assistance. At the same time, I deeply appreciate Dr. LI Ning for her tireless and patient teaching, also for her real inspiration at all times. And I would also like to express my sincere gratitude to Prof. Simon Ming-Yuen LEE and Dr. YAN Ru for their kind help and recommendation letter in thesis advisory committee, also thank for our good cooperation and mutual understanding in the course of the experiment. Also, I greatly appreciate Dr. ZHANG Qinwen s isolated components and Prof. LI Shaoping s generous spirit of cooperation and valuable advices. The good suggestions and recommendations from them are very helpful and constructive. Besides, I want to thank Prof. ZHENG Ying, Dr. CHEN Xiuping, Dr. LU Jinjian, Dr. CHEN Meiwan, Dr. HOI Puiman, Dr. HU Hao, Dr. HU Yuanjia and Dr. CHENG Lijen, for their help and encouragement during my study in ICMS. And many thanks to the lab technicians, Ms Sandy, Ms Wing, and Mr. Leon, for maintaining a good lab environment and a good lab management, I m supported to carry out my thesis work safely and efficiently. i

8 Moreover, I would like to acknowledge and thank my ICMS colleagues, GAO Jianli, LI Yingbo, ZHANG Zaijun, ZHONG Zhangfeng, WANG Shengpeng and WU Guosheng for their assistances and supports during my thesis work; GUAN Jia, CHEN Xiajia, FENG Kun, QIAN Zhengming, DU Gang, XU Zengtao, CUI Guozhen and MENG Lanzhen for their help and encouragement during my study. Finally, I would like to thank my parents and my family for their full support and deep understanding. Ultimate completion of my study and these many supports were inseparable. The current study was supported by the Macao Science and Technology Development Fund (029/2007/A2, 045/2011/A) and the Research Fund of the University of Macau (UL016A/09-Y4/ICMS/WYT01/ICMS, MYRG208(Y2-L4)- ICMS11-WYT). ii

9 Abstract Berberine is an isoquinoline alkaloid with diversified pharmacological activities, mainly derived from Chinese material medica Coptidis Rhizoma (Huang Lian) and widely used as a gastrointestinal remedy in China. In modern pharmacology, the anti-tumor activity of berberine obtained a wealth of research results and strongly tied to the characteristics of dissipating heat and detoxifying from Chinese medicine principles. Meanwhile, the effect with communal characteristics on hypoglycemic, hypolipidemic and hypercholesterolemia, also on diabetes and obesity in clinic implied metabolic regulation potential of berberine. The combination of anti-tumor effect and metabolic regulation would give birth to cancer metabolism reprogramming. It was characterized by metabolic regulation dysfunction and existed as a state of homeostasis imbalance. Cancer metabolism reprogramming is a complex system with numerous biochemical pathways adequately responding to fast-changing metabolic state. And Chinese medicine principles also have an adaptation-eternal theme of biological evolution, emphasizing on holistic health and syndromes differentiation in a whole organism system. Therefore, energy homeostasis of cancer cells is similar to Chinese medicine principles, both are dynamic systems with regulatory networks. This relationship also supported hypothesis of anti-tumor effect of berberine through a metabolic control. Hence, we investigated anti-tumor effect from metabolic perspective and attempted to find the potential targets in berberine s effect on cancer cells metabolism from interdisciplinary view, in order to imply the communal pathways in cancer, diabetes and obesity and provide an in-depth perspective and various perspectives in mechanism research. Human breast cancer cell lines MCF-7 and MDA-MB-231 iii

10 were applied to investigate anti-tumor effect of Coptidis Rhizoma constitutes, and the effective constitute berberine was focused on the three aspects of cancer cells metabolism simultaneously. Mitochondrial function interference, glycolysis inhibition and macromolecular synthesis inhibition were illustrated in detail. Based on Chinese medicine principles, a better view of anti-tumor molecular mechanism of Chinese medicine from metabolic regulation perspective was obtained. Meanwhile a communal potential of solution for other anti-tumor drugs, anti-diabetes drugs and cholesterol reducing drugs was stimulated to explore further. The regulation of berberine on cancer cells reprogramming metabolism would also be benefit for therapeutic effect enhancement and side-effect alleviation, as well as a reference for individualized therapy in cancer treatment. iv

11 Declaration I declare that the thesis here submitted is original except for the source materials explicitly acknowledged and that this thesis as a whole, or any part of this thesis has not been previously submitted for the same degree or for a different degree. I also acknowledged that I have read and understood the Rules on Handling Student Academic Dishonesty and the Regulations of the Student Discipline of the University of Macau. v

12 Table of Contents Acknowledgements... i Abstract... iii Declaration... v List of Tables and Figures... ix List of Abbreviations... xii Chapter 1 Introduction General Background Homeostasis and Chinese medicine principles Cancer cells metabolism reprogramming Chinese medicine interfered with cancer cells metabolism reprogramming The effective constitutes of Coptidis Rhizoma Specific Background The implication of berberine in metabolic syndrome suggested the potential in cancer therapy Mitochondrial function alteration Glycolysis interference Macro-molecules synthesis inhibition Berberine s interference with cancer cells reprogramming Research Goals and Objectives Research Methodology and Design Potential Contributions Organization of the Thesis Statement of Originality Chapter 2 Mitochondrial function interference Introduction Materials and methods Reagents Cell culture Cell viability assay Cell cycle and apoptotic TUNEL assay Scratch assay Mitochondrial function assay Mitochondrial localization assay ATP assay Mitochondrial potential membrane assay ROS generation assay Western blot analysis Statistical analysis Results Effect of five constitutes in Coptidis Rhizoma on proliferation of human breast cancer cell lines Cell cycle and apoptosis alteration in human breast cancer cell lines after berberine and palmatine treatment vi

13 2.3.3 Migration and invasion alteration in human breast cancer cell lines after berberine and palmatine treatment Berberine treatment in a short term significantly restored the impaired mitochondrial induced by compound C Berberine increased ATP production with a long-term duration in MCF-7 cells Berberine induced mitochondrial membrane depolarization and ROS generation in MCF-7 cells and MDA-MB-231 cells Discussion Chapter 3 Glycolysis inhibition Introduction Materials and methods Reagents Cell culture Hoechst stain assay Lactate content assay Western blot assay Statistical analysis Results Berberine interfered with LDH expression accompanied with an increase of lactate content in MCF-7 cells PFKP and PKM was inhibited significantly by berberine treatment Discussion Chapter 4 Fatty acid synthesis inhibition Introduction Materials and methods Reagents Cell culture Oil-Red O stain assay DAPI stain assay Citrate content assay CoA content assay NADPH content assay Western blot assay Statistical analysis Results Berberine decreased lipid drops accumulation in MCF-7 cells The phosphor inhibition of ACC and a fluctuation of ACL induced by berberine were found in MCF-7 cells Berberine decreased citrate content in MCF-7 cells, a boost for acetyl coa accumulation Berberine significantly reduced NAPDH content alteration in MDA-MB- 231 cells Berberine attenuated TOFA-induced proliferation promotion in MCF-7 cells vii

14 4.4 Discussion Chapter 5 The involved mediated pathways in metabolism interference Introduction Materials and methods Reagents Cell culture Cell viability assay XTT assay Western blot assay Zebrafish assay Statistical analysis Results Proliferation inhibition effect of berberine on MCF-7 cells was reversed by Akt inhibitors for different durations The role of estrogen and tamoxifen in combination with berberine Combination effect of berberine with 5-Fluorouracil, cyclophosphamide monohydrate and methotrexate hydrate Effect of berberine and other constitutes on HUVEC cells viability and zebrafish toxicity Discussion Chapter 6 Conclusions Conclusions Limitations of Current Study Perspectives for Future Work References Curriculum Vitae viii

15 List of Tables and Figures Fig. 1.1 The conventional and novel approaches to cell homeostasis Fig. 2.1 Effect of berberine on cell viability of human breast cancer cell line MCF Fig. 2.2 Effect of palmatine on cell viability of human breast cancer cell line MCF Fig. 2.3 Effect of jatrorrhizine on cell viability of human breast cancer cell line MCF Fig. 2.4 Effect of coptisine on cell viability of human breast cancer cell line MCF Fig. 2.5 Effect of magnoflorine on cell viability of human breast cancer cell line MCF Fig. 2.6 Effect of berberine on cell proliferation of human breast cancer cell line MCF Fig. 2.7 Effect of palmatine on cell proliferation of human breast cancer cell line MCF Fig. 2.8 Effect of jatrorrhizine on cell proliferation of human breast cancer cell line MCF Fig. 2.9 Effect of coptisine on cell proliferation of human breast cancer cell line MCF Fig Proliferation inhibition curves of berberine in human breast cancer cell lines MCF-7 and MDA-MB Fig Proliferation inhibition curves of palmatine in human breast cancer cell lines MCF-7 and MDA-MB Fig Proliferation inhibition induced by berberine in ductal breast epithelial tumor cell line T47D, triple-negative breast cancer cell line MDA-MB-468 and non-tumorigenic epithelial cell line MCF-10A Fig Effect of berberine and palmatine on cell cycle of human breast cancer cell lines MCF-7 and MDA-MB Fig Effect of berberine and palmatine on cell apoptosis of human breast cancer cell lines MCF-7 and MDA-MB Fig Effect of berberine on cell migration of human breast cancer cell line MCF Fig Real time determination of berberine on cell migration in human breast cancer cell line MDA-MB Fig Effect of berberine and palmatine on cell invasion of human breast cancer cell line MCF Fig Effect of berberine and palmatine on cell migration and invasion of human breast cancer cell line MCF Fig Mitochondrial function alteration of human breast cancer cell lines after berberine treatment Fig Mitochondrial function alteration observation in human breast cancer cell line MCF-7 after berberine treatment Fig Mitochondrial function alteration of human breast cancer cell lines after compound C treatment Fig Mitochondrial function alteration of human breast cancer cell lines after compound C and berberine treatment Fig Mitochondrial function alteration after berberine treatment in human breast cancer cell line MCF ix

16 Fig Mitochondrial function alteration of MCF-7 cells after a short-term duration of berberine Fig Mitochondrial function alteration of MCF-7 cells after different durations of berberine Fig Mitochondrial function alteration of MCF-7 cells after a long-term duration of berberine Fig Mitochondrial function alteration of MCF-7 cells after combined treatment of berberine and AICAR Fig Mitochondrial function alteration of MCF-7 cells after combined treatment of berberine and LND Fig ATP content in MCF-7 cells after berberine treatment Fig Mitochondrial membrane potential alteration in human breast cancer cell lines after berberine treatment Fig Mitochondrial membrane potential alteration in human breast cancer cell lines after palmatine treatment Fig ROS generation in human breast cancer cell lines after berberine treatment Fig ROS generation in human breast cancer cell lines after palmatine treatment Fig. 3.1 Lactate content increased in MCF-7 cells after berberine treatment Fig. 3.2 Expression alteration of PFKP and phosphor PKM-2 after berberine treatment Fig. 3.3 Expression observation of PFKP and PKM-2 after berberine treatment Fig. 4.1 Berberine decreased lipid drops accumulation in MCF-7 cells Fig. 4.2 Berberine decreased lipid drops of MCF-7 cells Fig. 4.3 Expression alteration of phosphor ACC and phosphor ACL in MCF-7 cells after berberine treatment Fig. 4.4 Berberine phosphorylated ACC and inhibited phosphor ACL expression in MCF-7 cells Fig. 4.5 Alteration of citrate content and CoA content in MCF-7 cells after berberrine treatment Fig. 4.6 NADPH content alteration in MDA-MB-231 cells after berberine treatment Fig. 4.7 The TOFA-induced proliferation promotion was inhibited by berberine treatment in MCF-7 cells Fig. 4.8 Berberine attenuated TOFA-induced proliferation promotion in MCF-7 cells Fig. 4.9 Berberine decreased the TOFA-induced ROS generation in MCF-7 cells. 107 Fig Berberine regulated ACC and ACL to seek the balance Fig. 5.1 Proliferation inhibition effect of berberine on MCF-7 cells was reversed by Akt inhibitors for duration of 24 hours Fig. 5.2 Proliferation inhibition effect of berberine on MCF-7 cells was reversed by Akt inhibitors for duration of 48 hours Fig. 5.3 Proliferation inhibition effect of berberine on MCF-7 cells was reversed by Akt inhibitors for duration of 72 hours Fig. 5.4 Alteration of proliferation inhibition ratio induced by Akt inhibitors in MCF- 7 cells Fig. 5.5 Combination effect of berberine and tamoxifen on cell viability after 72-hour duration in stepwise manner of estradiol pre-treatment x

17 Fig. 5.6 Combination effect of berberine and tamoxifen on cell viability after 72-hour duration in continuous manner of estradiol pre-treatment Fig. 5.7 A continuous combination effect of berberine and tamoxifen on cell viability after 72-hour duration in stepwise or continuous manner of estradiol pretreatment Fig. 5.8 A stepwise combination effect of berberine and tamoxifen on cell viability after 72-hour duration in stepwise or continuous manner of estradiol pretreatment Fig. 5.9 Stepwise combination effect and continuous combination effect of berberine and tamoxifen on cell viability after 72-hour duration in stepwise manner of estradiol pre-treatment Fig Continuous combination effect and stepwise combination effect of berberine and tamoxifen on cell viability after 72-hour duration in continuous manner of estradiol pre-treatment Fig Combination effect of berberine with 5- Fluorouracil on human breast cancer cell line MCF Fig Combination effect of berberine with cyclophosphamide monohydrate on human breast cancer cell line MCF Fig Combination effect of berberine with methotrexate hydrate on human breast cancer cell line MCF Fig Combination effect of berberine with 5- Fluorouracil on human breast cancer cell line MDA-MB Fig Combination effect of berberine with cyclophosphamide monohydrate on human breast cancer cell line MDA-MB Fig Combination effect of berberine with methotrexate hydrate on human breast cancer cell line MDA-MB Fig Effect of berberine on HUVECs viability Fig Effect of palmatine on HUVECs viability Fig Effect of jatrorrhizine on HUVECs viability Fig Effect of coptisine on HUVECs viability Fig Effect of magnoflorine on HUVECs viability Fig Effect of berberine and palmatine on HUVECs viability Fig Effect of berberine, palmatine, jatrorrhizine and magnoflorine on zebrafish survival rate xi

18 List of Abbreviations 5-Flu 5- Fluorouracil ACC Acetyl-CoA carboxylase ACL ATP-citrate lyase AICAR 5-amino-1-β-D-ribofuranosyl-imidazole-4- carboxamide AMPK 5 adenosine monophosphate-activated protein Ber Berberine Cop Coptisine CYC Cyclophosphamide monohydrate DAPI 4-6-Diamidino-2-phenylindole DMSO Dimethyl sulfoxide E2 17-β-estradiol ELISA Enzyme-linked immunosorbent assay F12 medium Ham s F12 medium FACS analysis Fluorescence-activated cell sorting analysis FBS Fetal bovine serum FCS Fetal calf serum H 2 DCF-DA 2, 7 dichlorodihydrofluorescein diacetate HIF-I Hypoxia inducible factor -I HUEVCs Human umbilical vein endothelial cells Jat Jatrorrhizine LDH Latate dehydrogenase LND Lonidamine Mag Magnoflorine MMP Mitochondrial membrane potential MTT 3- (4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl tetrazolium bromide MTX Methotrexate hydrate NADPH Nicotinamide adenine dinucleotide phosphate OXPHO oxidative phosphorylation Pal Palmatine PBS Phosphate buffered saline PI Propidium iodide PI3K Phosphatidylinositol 3-kinase PMSF Phenylmethanesulfonyl fluoride PPP pathway pentose phosphate pathway ROS Reactive oxygen species RPMI1640 medium Roswell Park Memorial Institute 1640 medium TAM Tamoxifen TNF-α Tumor necrosis factor-α TOFA 5-(tetradecyloxy)-2-furancarboxylic acid VEGF Vascular endothelial growth factor xii