ANTI-TUMOR PROPERTIES OF LACTOBACILLI ARE MEDIATED BY IMMUNO-MODULATION AND DIRECT CYTOTOXICITY CAI SHIRONG NATIONAL UNIVERSITY OF SINGAPORE 2010 ANTI-TUMOR PROPERTIES OF LACTOBACILLI ARE MEDIATED BY IMMUNO-MODULATION AND DIRECT CYTOTOXICITY CAI SHIRONG B.Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF SURGERY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements I would like to express my heartfelt gratitude to everyone who had helped me and made my pursuit for the PhD degree a pleasant and fulfilling experience. To my supervisor, Dr Ratha Mahendran, for her invaluable advice and guidance, without of which my PhD project would not have been so fruitful. To my co-supervisors, Prof Bay Boon Huat and A/Prof Lee Yuan Kun, for taking time off their busy schedules to sit through my updates and giving me advice and encouragement. To my fellow colleagues, Shih Wee, Mathu, Juwita and Rachel, thank you for all the support and patience you have shown me through the years. Your encouragement and help made my PhD journey a much sweeter one. Many thanks to Eng Shi, Kishore, Ms Chan Yee Gek and Mr Low Chin Seng, for all the technical help and advice given to me. To my CRCEC friends, Evelyn, Elaine, Delicia, Sally, Gaik Chin, Eric and Hafizah, thank you for your lovely company and for lending me a listening ear or a helping hand whenever I need it. I would like to thank my parents and brothers for their love, faith and support. Thank you for always believing in me and encouraging me to go a little further, dream a little bigger. Last but not least, my significant other, Terry, thank you for being my pillar of support and for being there with me always, through the high and lows of my PhD journey. i Table of Contents Page Acknowledgements……………………………………………………………………. i Table of Contents …………………………………………………………………… . ii Summary………………………………………………………………………………. x List of Tables.…………………………………………………………………………. xii List of Figures…………………………………………………………………………. xiv List of Abbreviations………………………………………………………………… xvi List of publications and conference papers…………………………………………… xix Chapter Introduction ……………………………………………………………… 1.1 Cancer…………………………………………………………………………… 1.1.1 Cancer and its prevalence……………………………………………… 1.1.2 Causes of cancer … ………………………………………………… 1.2 Cancer treatments.……………………………………………………………… 1.2.1 Surgery………………………………………………………………… 1.2.2 Chemotherapy…………………………………………………………. 1.2.3 Radiation………………………………………………………………. 1.2.4 Immunotherapy……………………………………………………… . 1.2.5 Future of cancer therapy………………………………………………. 1.3 Role of immune system in anti-tumor response……………………………… 1.4 Cell death pathways induced by chemotherapuetic drugs used in cancer therapy…………………………………………………………………… ii 1.5 Bacteria in cancer therapy………………………………………………………. 1.5.1 Bacteria as immunotherapeutic agents……………………………………… 1.5.2 Bacteria as a delivery vehicle……………………………………………… 1.5.3 Bacterial cytotoxic agents…………………………………………………… 1.5.4 Limitations………………………………………………………………… 10 1.6 Lactobacilli………………………………………………………………………. 10 1.6.1 Health benefits of Lactobacilli…………………………………………… . 11 1.6.2 Anti-tumor effects of lactobacilli………………………………………… . 14 1.6.2.1 Immunologically mediated anti-tumor effect……………………… 14 1.6.2.2 Non-immunologically mediated anti-tumor effect………………… 16 1.6.3 Lactobacilli immuno-modulatory potential.……………………………… 17 1.6.3.1 Lactobacilli modulate host immune response in vitro and in vivo…. 17 1.6.3.2 Receptor mediated interaction between lactobacilli and innate immune cells………………………………………………………. 20 1.6.3.2.1 Toll like receptors……………………………………… 20 1.6.3.2.2 Mannose receptors………………………………………. 21 1.6.3.2.3 NOD like receptors……………………………………… 21 1.6.3.3 Bridging innate and adaptive immunity………………………… . 22 1.6.3.3.1 Macrophages……………………………………………. 22 1.6.3.3.2 Dendritic cells………………………………………… . 22 1.6.3.3.3 Neutrophils…………………………………………… 23 1.6.3.3.4 Dendritic cell and neutrophil interaction……………… 23 1.7 Scope of study…………………………………………………………………. 25 iii Chaper Materials and Methods………………………………………………… 27 2.1 Bacteria Preparation………………………………………………………… 28 2.1.1 Live Lactobacilli………………………………………………………… 28 2.1.2 Lyophilized Lactobacilli………………………………………………… 29 2.1.3 Heat Killed LGG…………………………………………………………. 29 2.2 Ex vivo study of Lactobacilli interaction with immune cells……………… 30 2.2.1 Animals…………………………………………………………………… 30 2.2.2 Immune cells isolation……………………………………………………. 30 2.2.2.1 Isolation of bone marrow derived neutrophils and dendritic cells . 30 2.2.2.2 Isolation of T-cells……………………………………………… . 31 2.2.2.3 Splenocytes isolation…………………………………………… . 33 2.2.3 Co-culture of immune cells with Lactobacilli …………………………… 33 2.2.3.1 Co-culture of neutrophils or DCs with LGG …………………… 33 2.2.3.2 Study of neutrophil-neutrophil interaction……………………… 34 2.2.3.3 DC neutrophil co-culture…………………………………………. 35 2.2.3.4 DC or DC-neutrophil co-culture with T cells…………………… 35 2.2.3.5 Stimulation of splenocytes with live and lyophilized Lactobacilli 36 2.2.4 Interaction between immune cells and Lactobacilli……………………… 36 2.2.4.1 Uptake of Lactobacilli into immune cells………………………… 36 2.2.4.2 Blocking phagocytosis……………………………………………. 36 2.2.4.3 Cytokine and PGE2 ELISA………………………………………. 37 2.2.4.4 Blocking TLR2 and 9…………………………………………… 38 2.2.4.5 Flow cytometric analysis of surface markers and receptors on DCs and neutrophils……………………………………………… 39 iv 2.2.4.6 Blocking IL10 and COX2 in DC neutrophil co-culture…………. 40 2.2.4.7 Effect of LGG on neutrophil viability…………………………… 40 2.2.4.7.1 Annexin V-PI staining of neutrophils………………… 40 2.3 Cytotoxic effect of Lactobacilli on cancer cells……………………………… 41 2.3.1 Cancer and normal cell lines……………………………………………… 41 2.3.2 Direct co-culture of MGH with lactobacilli………………………………. 41 2.3.3 Production of cytotoxic molecule from LGG…………………………… 42 2.3.3.1 Cytotoxic molecule production in the culture supernatant……… 42 2.3.3.2 Extraction of LGG cytoplasmic fraction………………………… 42 2.3.3.3 Optimization of cytotoxic molecule production …………………. 42 2.3.3.4 Growth curve of LGG in media…………………………………… 43 2.3.3.5 Measurement of pH, lactate and glucose………………………… 43 2.3.4 Characterization of cytoxic molecule…………………………………… . 44 2.3.4.1 Stability of cytotoxic molecule……………………………………. 44 2.3.4.2 Molecular size of cytotoxic molecule…………………………… . 44 2.3.4.3 Nature of cytotoxic molecule……………………………………… 44 2.3.4.3.1 Proteinase K and trypsin digestion……………………… 44 2.3.4.3.2 Chloroform extraction…………………………………… 45 2.3.5 Effect of cytotoxic molecule on human cells……………………………… 45 2.3.5.1 Cell viability assays……………………………………………… 45 2.3.5.1.1 MTS and Multitox-fluor assay………………………… 45 2.3.5.1.2 Cell Count ………………………………………………. 46 2.3.5.2 Mechanism of cell death………………………………………… . 46 v 2.3.5.2.1 Caspase 3/7 activity……………………………………… 46 2.3.5.2.2 Lactate dehydrogenase (LDH) test………………………. 46 2.3.5.2.3 Cell cycle analysis……………………………………… 47 2.3.5.3 Effect of cytotoxic molecule on a panel of cancer and normal cells 47 2.3.5.4 Visualization of cell-lines…………………………………………. 48 2.3.5.4.1 Light and fluorescence microscopy…………………… . 48 2.3.5.4.2 Electron microscopy…………………………………… 48 2.3.5.5 Uptake of cytotoxic molecule…………………………………… . 49 2.3.5.6 Cellular Pathways activated by the cytotoxic molecule………… . 50 2.3.5.6.1 Total RNA extraction and cDNA conversion…………… 50 2.3.5.6.2 LDA…………………………………………………… . 51 2.3.5.6.3 Real-time PCR………………………………………… . 53 2.3.5.6.4 RT-PCR…………………………………………………. 53 2.3.5.6.5 Protein isolation from MGH cells………………………. 55 2.3.5.6.6 Western Blot of ACVR1C, pSMAD2 and SMAD.…… 55 2.3.6 Purification of cytotoxic molecule……………………………………… 56 2.3.6.1 High Performance Liquid Chromatography (HPLC)…………… 56 2.3.6.2 Gas chromatography TOF mass spectropmetry (GC-TOFMS)… 57 2.4 Statistical Analysis…………………………………………………………… 58 Chapter Results Part I: Immuno-stimulatory effect of lactobacilli………… 59 3.1 Interaction of Neutrophils and LGG………………………………………… 60 3.1.1 Internalization of LGG induces cytokine production in neutrophils…… . 60 vi 3.1.2 Role of toll-like receptor in LGG stimulation of neutrophils…………… 63 3.1.3 LGG induced cell death in neutrophils……………………………………. 64 3.1.4 Effect of LGG on surface marker expression in neutrophils……………… 66 3.1.5 Neutrophil-neutrophil interaction after exposure to LGG………………… 67 3.2 Effect of dose and exposure time of LGG on DC maturation and DC-neutrophils cross talk…………………………………………………… . 69 3.2.1 Maturation of dendritic cells is dependent on bacteria dose, exposure time and presentation by neutrophils…………………………… 70 3.2.2 Dose and duration of LGG exposure skews cytokine profile in DC and DC neutrophil co-culture…………………………………………………. 73 3.2.3 Effect of high LGG dose on IL12 production is dependent on IL10 levels but not Prostaglandin E2 (PGE2) levels…………………… 76 3.2.4 Downstream T-cell activation is dependent on the bacteria dose exposed to the DCs………………………………………………………. 78 3.3 Differential Immuno-stimulatory potential of live and lyophilized Lactobacillus species ……………………………………………………… 79 3.3.1 Different strains of lactobacilli induce different levels of TNF, IL10 and IL12p40 ………………………………………………………. 79 3.3.2 Lyophilized lactobacilli induced more TNF, IL10 and IL12p40 ……… 81 3.3.3 Contact is required for lactobacilli to stimulate spleen cells to produce cytokines……………………………………………………… . 83 3.3.4 Lactobacilli stimulate splenocytes through TLR2 but not TLR9……… . 84 3.3.5 Phagocytosis plays a role in cytokine induction by L.bulgaricus……… 85 Summary I……………………………………………………………………. 87 Chapter Results Part II: Direct cytotoxic effect of lactobacilli on cancer cells 89 3.4 Optimization of lactobacilli mediated direct cytotoxic effects on cancer cells…………………………………………………………………… . 90 vii 3.4.1 Effect of media pH on cytotoxic effect…………………………………… 90 3.4.2 Comparison of the cytotoxic effect of different lactobacillus strains ……. 91 3.4.3 LGG produced cytotoxic molecules are released into the culture supernatant 92 3.4.4 Other conditions that affect cytotoxicity of LGG………………………… 93 3.4.5 Glucose and amberlite enhances production of cytotoxic molecules by LGG 94 3.4.6 Culture media conditions at the end of 24 hours of incubation…………… 97 3.5 Characterizations of the cytotoxic molecule(s)………………………………… 98 3.5.1 Basic characterization of the cytotoxic molecule…………………………… 98 3.5.2 Purification of cytotoxic molecule from LGG supernatant…………………. 100 3.5.3 Possible identity of cytotoxic molecules, determined by GC-TOFMS ……. 101 3.6 Uptake of cytotoxic molecule into MGH cells…………………………………. 104 3.7 Cytotoxic and anti-proliferative effect of LGG supernatant and LCT……… 105 3.7.1 Cell cycle analysis with propidium iodide…………………………………. 106 3.7.2 LCT induced apoptosis in MGH but not LGG supernatant……………… . 106 3.7.3 Morphologies of MGH cells treated with LGG supernatant and LCT…… 109 3.8 LGG supernatant preferentially targets cancer cells and not normal cells… 112 3.9 Effect of LGG supernatant and LCT on gene expression in MGH cells…… 114 3.9.1 Confirmation of gene expression with real-time PCR ……………………. 114 3.9.2 Confirmation of gene expressions with RT-PCR…………………………. 116 3.10 Gene and protein expressions of ACVR1C…………………………………. 118 Summary II…………………………………………………………………… 120 Chapter Discussion………………………………………………………………. 122 4.1 Phagocytosis and TLR2 are important mediators of the interaction of lactobacilli with immune cells ………………………………………………. 123 viii 146. 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IIIB cervival cancer [75] Regular intake of lactobacilli can also reduce the risk of developing colorectal and bladder cancer [76, 77] 1.6.2.1 Immunologically mediated anti- tumor effect One explanation for the tumor suppressive effect of lactobacilli may be through immunomodulation of the host Intravesical instillations of lactobacilli recruited macrophages and neutrophils to the bladder mucosa [69,... reducing tumor growth in C3H mice [69] Aside from anti- tumor properties, lactobacilli also showed potential anti- metastatic properties as demonstrated by inhibition of lung and lymph node metastases by intrapleural and/ or intraveneous administration of L casei YIT9018 (LC 9018) [70, 71] in the tumor bearing mice Oral consumption of L casei strain Shirota (LcS) was found to suppress recurrence of bladder... cells like macrophages and neutrophils NK cells are able to provide surveillance against development of malignancies [13] and they can attack transformed tumor cells via NKG2D mediated cytotoxicity [14] CD8+ and NK cells are both cytotoxic lymphocytes and they induced apoptosis in cells similarly, by producing lytic molecules like perforin and granzymes and/ or trigger FasFas ligand (FasL) or TNF-related... LGG in media and the enhancement of cytotoxicity with increased glucose concentration and addition of amberlite……………… 96 3.15 Cytotoxicity of lactate on MGH cells………………………………………… 97 3.16 Purification of LGG supernatant using HPLC………………………………… 101 3.17 Comparison of GC-TOFMS chromatograms of LGG supernatant and control media…………………………………………………………………… 103 3.18 Effect of chemical inhibition of endocytotic... knowledge of tumor immunology and the immune system, other forms of cancer immunotherapy using monoclonal antibodies, cytokines (e.g interferon, interleukin-2), biological agents [e.g Mycobacterium bovis, Bacillus Calmette-Guérin (BCG)] and cancer vaccines have been developed Monoclonal antibodies used for cancer immunotherapy are raised against tumor antigens Once bound, the foreign cells are destroyed... with an anti- macrophage agent, carrageenan, and also in T-cell deficient athymic nude mice [66] These results suggest that the anti- tumor activity of lactobacilli may be macrophage and T cell dependent Recruitment and activation of immune cells is coupled with the production of cytokines that also contributes to the anti- tumor effect Matsuzaki et al showed that intrapleural injection of LcS into tumor. .. suppression in tumor bearing animals given BCG or Lactobacillus [29, 88] The former has potent anti- tumor and anti- metastatic effects against tumors by the stimulation of cytotoxic CD8+ T cells and natural killer cells while the latter activates macrophages to become cytotoxic to tumor cells [90] TNF is known to induce tumor cell apoptosis in vitro and enhance tumoricidal activity of macrophages [91,... host” They are part of the commensal microflora in animals and humans and they can be found in the oral, genital and gastrointestinal tracts They have also been conferred the GRAS (generally recognized as safe) status by the US FDA Many Lactobacillus species are associated with food production because of their nutritional benefits, ability to enhance flavor and preservation of food by production of lactic... acid Lactobacilli have also been associated with alleviating a host of diseases, ranging from a wide variety of gastrointestinal problems to allergies and the prevention of cancer More importantly, infection cases associated with use of lactobacilli are very rare and reported mostly in immuno- compromised individuals [43] As such, research on lactobacilli has much potential in both the food industry and . ANTI-TUMOR PROPERTIES OF LACTOBACILLI ARE MEDIATED BY IMMUNO-MODULATION AND DIRECT CYTOTOXICITY CAI SHIRONG B.Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. ANTI-TUMOR PROPERTIES OF LACTOBACILLI ARE MEDIATED BY IMMUNO-MODULATION AND DIRECT CYTOTOXICITY CAI SHIRONG NATIONAL UNIVERSITY OF SINGAPORE. duration and dose of lactobacilli exposure to DCs and neutrophils affect Th1 polarization of T cells. The different strains of lactobacilli showed differential immunostimulatory potential and their