Toll样受体启动的信号通路在双歧杆菌脂磷壁酸诱导HCT-116细胞凋亡中作用
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摘要
双歧杆菌脂磷壁酸能在体内、外诱导肿瘤细胞凋亡，但其启动细胞凋亡的机制尚不清楚。Toll样受体是一组介导细胞天然免疫的受体，也是脂磷壁酸的天然受体。本实验通过体外研究Toll样受体在结肠癌细胞凋亡前后的表达，探讨Toll样受体在双歧杆菌脂磷壁酸体外诱导结肠癌细胞凋亡信号转导通路中的作用。实验通过RT-PCR检测脂磷壁酸适配的Toll样受体在结肠上皮细胞癌HCT-116细胞的基础表达及在脂磷壁酸作用后表达的变化，同时采用免疫细胞化学、Western blot检测该受体后续信号通路关键效应分子NF-κB在脂磷壁酸作用后表达的变化，欲确定双歧杆菌脂磷壁酸诱导结肠癌细胞凋亡信号途径的始末。结果发现Toll样受体2和Toll样受体4的mRNA在HCT-116细胞都有基础表达，且表达水平在脂磷壁酸作用后明显增加，而受体的后续信号分子NF-κB在脂磷壁酸作用后其蛋白表达水平却明显降低，并且都表现一定的剂量依赖性。由此揭示双歧杆菌脂磷壁酸在与Toll样受体结合后启动了NF-κB通路，从而激活Caspases，诱导了HCT-116细胞凋亡信号转导途径的发生，为脂磷壁酸抗肿瘤的机理研究提供了理论基础和新思路，也为双歧杆菌表面分子开发利用提供理论和技术支持。
Bifidobacterium lipoteichoic acid has the ability of inducing occurence of apoptosis in tumor cells in vitro and in vivo, but its mechanism is not clear. Toll-like receptors are a serial of receptors related to innate immunity and Toll-like receptor is the natural receptor of lipoteichoic acid. In the topic, we do experiments on Toll-like receptors in order to study the signal transduction pathway of bifidobacterium lipoteichoic acid in inducing apoptosis of colon cancer cells. After detecting the basal expression of TLRs, the matching receptor of LTA and alterating expression of it through RT-PCR, meanwhile detecting alteration of NF-κB protein expression, the continuous molecular of apoptosis pathway, through immunocytochemistry and western blot, we find that expression of TLR2 and TLR4 in mRNA level increases greatly. On the
contrary, expression of NF-κB in protein level decrease deeply, at the same time, switch on the Caspase pathway to apoptosis. Therefore we establish the cascade reaction of Toll-like receptor and NF-κB in apoptosis signal transduction pathway of lipoteichoic acid on colon cancer cells, which provide the theoretical base and idea for mechanism of lipoteichoic acid`s anti-tumor effect, utility and development of bifidobacterium surface molecular.
Bifidobacterium lipoteichoic acid has the ability of inducing occurence of apoptosis in tumor cells in vitro and in vivo, but its mechanism is not clear. Toll-like receptors are a serial of receptors related to innate immunity and Toll-like receptor is the natural receptor of lipoteichoic acid. In the topic, we do experiments on Toll-like receptors in order to study the signal transduction pathway of bifidobacterium lipoteichoic acid in inducing apoptosis of colon cancer cells. After detecting the basal expression of TLRs, the matching receptor of LTA and alterating expression of it through RT-PCR, meanwhile detecting alteration of NF-κB protein expression, the continuous molecular of apoptosis pathway, through immunocytochemistry and western blot, we find that expression of TLR2 and TLR4 in mRNA level increases greatly. On the
contrary, expression of NF-κB in protein level decrease deeply, at the same time, switch on the Caspase pathway to apoptosis. Therefore we establish the cascade reaction of Toll-like receptor and NF-κB in apoptosis signal transduction pathway of lipoteichoic acid on colon cancer cells, which provide the theoretical base and idea for mechanism of lipoteichoic acid`s anti-tumor effect, utility and development of bifidobacterium surface molecular.
引文
Sekine K, Ohta J, Hashimoto Y，etc, Analysis of antitumor properties of effector cells stimulated with a cell wall preparation (WPG) of Bifidobacterium infantis, Biol Pharm Bull, 1995,18(1): 148-153
Takada H, Kawabata Y, Arakaki R, etc. Molecular and structural requirements of a lipoteichoic acid from Enterococcus hirae ATCC 9790 for cytokine- inducing, antitumor, and antigenic activities. Infect Immun,1995，63(1):57-65
Okamoto M, Ohe G, Furuichi S, etc, Enhancement of anti-tumor immunity by lipoteichoic acid-related molecule isolated from OK-432, a streptococcal agent, in athymic nude mice bearing human salivary adenocarcinoma: role of natural killer cells. Anticancer Res. 2002 Nov-Dec; 22(6A): 3229-3239
Iain C. Sutcliffe, Stephen D. Hogg, Extraction of lipoteichoic acid from Streptococcus mutans with the non-ionic detergent Triton X-114, Journal of Microbiological Methods, 1993, 17(1993): 215-225
Ginsburg I, Role of lipoteichoic acid in infection and inflammation, Lancet Infect Dis, 2002, 2(3): 171-179
乐军, 胡宏，非离子型去垢剂Triton X—114提取两歧双歧杆菌的脂磷壁酸, 中国微生态学杂志, 1997,9(4): 58-59
Medzhitov R, Preston HP, Janeway CA Jr, A human homologue of the Drosophila Toll protein signals activation of adaptive immunity, Nature, 1997, 388(6640): 394-397
Janssens S, Burns K etc, Regulation of IL-1 and LPS induced NF-kappaB activation by alternative splicing of MyD88, Curr Biol, 2002, 12(6): 467-471
Jacinto R, Hartung T, McCall C, etc, Lipopolysaccharide- and lipoteichoic acid-induced tolerance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase, J Immunol, 2002,168(12): 6136-6141
Underhill DM, Ozinsky A, Smith KD, etc, Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages, Proc Natl Acad Sci U S A, 1999,96(25): 14459-14463
Hou L, Sasaki H, Stashenko P, Toll-like receptor 4-deficient mice have reduced bone destruction following mixed anaerobic infection, Infect Immun, 2000, 68(8): 4681-4687
Opitz B, Schroder NW, Ralf R Schumann etc, TLR2 mediates Treponemcx glycolipid and LTA-induced NF-kappaB translocation, J Biol Chem, 2001, 276(25): 22041-22047
Okamoto M, Oshikawa T etc, Severe impairment of anti cancer effect of LTA-related molecule isolated from a penicillin-killed streptococues pyogenes in TLR4-dificent mice, Int Immunpharmacal, 2001, 1(9-10): 1789-1795
Oshikawa T, Okamoto M, Ohe G, etc, Anti-tumor immune response induced by the fractions derived from OK-432, a streptococcal preparation, by using a monoclonal antibody TS-2 that neutralizes the interferon-gamma -inducing activity of OK-432: comparison between the TS-2-binding and TS-2-unbinding fraction, Int Immunopharmacol, 2003, 3(5): 643-655
Wang J, Kobayashi M, Han M, etc, MyD88 is involved in the signalling pathway for Taxol-induced apoptosis and TNF-alpha expression in human myelomonocytic cells, Br J Haematol, 2002, 118(2): 638-645
蒋虹，胡宏，双歧杆菌表面分子诱导大肠癌细胞分化的研究，中华微生物学和免疫学杂志，2001, 21(2) ：186-190
Otte JM, Cario E, Podolsky DK, Mechanisms of cross hyporesponsiveness to toll-like receptor bacterial ligands in intestinal epithelial cells, Gastroenterology, 2004, 126(4): 1054-1070
Yang RB, Mark MR, Gray A, etc, Toll-like receptor-2 mediates lipopolysaccharide- induced cellular signaling, Nature, 1998, 395(6699): 284-288
Alexander P, Xiaolong He etc, Defective LPS signaling in C3H/HeJ and
C57BL/10ScCr mice mutation in Tlr4 gene, Science, 1998, 282(5196): 2085-2088
Suzuki N, Suzuki S etc, Severe impairment of IL-1 and TLR signaling in mice lacking IRAK-4, Nature, 2002, 416(6882): 750-756
张秋玉, TOLL样受体家族与LPS信号转导, 国外医学：免疫学分册, 2001, 24(3): 142-146
Hasegawa T, Matsuguchi T etc, TLR2 is at least partly involved in the antitumor activity of glycoprotein from chlorella vulgris, Int Immunopharmacol, 2002, 2(4): 579-589
Sato S, Nomura F, Kawai T, etc, Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways, J Immunol, 2000, 165(12): 7096-7101
Debatin KM, Apoptosis pathways in cancer and cancer therapy, Cancer Immunol Immunother, 2004, 53(3): 153-159
Chung CS, Song GY, Moldawer LL, etc, Neither Fas ligand nor endotoxin is responsible for inducible peritoneal phagocyte apoptosis during sepsis/peritonitis, J Surg Res, 2000, 91(2): 147-153
Okada T, Lian ZX, Naiki M, etc, Murine thymic plasmacytoid dendritic cells, Eur J Immunol, 2003, 33(4): 1012-1019
Frantz S, Kelly RA, Bourcier T, Role of TLR-2 in the activation of nuclear factor kappaB by oxidative stress in cardiac myocytes, J Biol Chem, 2001, 276(7): 5197-5203
Bannerman DD, Erwert RD, Winn RK, etc, TIRAP mediates endotoxin- induced NF-kappaB activation and apoptosis in endothelial cells, Biochem Biophys Res Commun, 2002, 295(1): 157-162
Meyer T, Nindl I, Schmook T, etc, Induction of apoptosis by Toll-like receptor-7 agonist in tissue cultures, Br J Dermatol, 2003, 149 Suppl 66:9-14
Berkowitz B, Huang DB, Chen-Park FE etc, The x-ray crystal structure of the NF-kappa B p50.p65 heterodimer bound to the interferon beta -kappa B site, J Biol
Chem, 2002, 277(27): 24694-24700
Huxford T, Huang DB, Malek S, etc, The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation, Cell, 1998, 95(6): 759-770
Palozza P, Serini S, Torsello A, etc, Mechanism of activation of Caspase cascade during beta-carotene-induced apoptosis in human tumor cells, Nutr Cancer, 2003, 47(1): 76-87
Fenwick C，Na S Y，Voll R E，etc, A subclass of Ras protein that regulate the degradation of IkappaB, Science， 2000，287：869-873
Malek S, Huang DB, Huxford T, etc, X-ray crystal structure of an IkappaBbeta x NF-kappaB p65 homodimer complex, J Biol Chem, 2003, 278(25): 23094-23100
K1ement J F，Rice N R，Car B D，etc, IκBα deficiency results in a sustained NF-κB response and severe widespread dermatitis in mice, Mo1 Cell Biol，1996，16：2341-2349
Karin M，Ben-Neriah Y, Phosphorylation meets ubiquitination：the control of NF-κB activity, Annu Rev Immunol，2000，18：621-663
Chen G，Goeddel D V, TNF-R1 signaling：a beautiful pathway, Science, 2002，296：1634-1635
Lin CH, Kuan IH, Lee HM, etc, Induction of cyclooxygenase-2 protein by lipoteichoic acid from Staphylococcus aureus in human pulmonary epithelial cells: involvement of a nuclear factor-kappa B-dependent pathway, Br J Pharmacol, 2001, 134(3): 543-552
Into T, Kiura K, Shibata K etc, Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa B activation, Cell Microbiol, 2004, 6(2): 187-199
孙大业, 郭艳林，马力耕，细胞信号转导, 第二版，北京：科学出版社，1998，28-48
Huerta-Yepez S, Vega M, Jazirehi A, etc，Nitric oxide sensitizes prostate carcinoma cell lines to TRAIL-mediated apoptosis via inactivation of NF-kappaB and inhibition of Bcl-(xL) expression, Oncogene, Epub 2004, 29
Gewirtz AT, Intestinal epithelial toll-like receptors: to protect. And serve? Curr Pharm Des, 2003, 9(1): 1-5
Lotz S, Aga E, Wilde I, etc, Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2, J Leukoc Biol, 2004, 75(3): 467-477
Takada H, Kawabata Y, Arakaki R, etc. Molecular and structural requirements of a lipoteichoic acid from Enterococcus hirae ATCC 9790 for cytokine- inducing, antitumor, and antigenic activities. Infect Immun,1995，63(1):57-65
Okamoto M, Ohe G, Furuichi S, etc, Enhancement of anti-tumor immunity by lipoteichoic acid-related molecule isolated from OK-432, a streptococcal agent, in athymic nude mice bearing human salivary adenocarcinoma: role of natural killer cells. Anticancer Res. 2002 Nov-Dec; 22(6A): 3229-3239
Iain C. Sutcliffe, Stephen D. Hogg, Extraction of lipoteichoic acid from Streptococcus mutans with the non-ionic detergent Triton X-114, Journal of Microbiological Methods, 1993, 17(1993): 215-225
Ginsburg I, Role of lipoteichoic acid in infection and inflammation, Lancet Infect Dis, 2002, 2(3): 171-179
乐军, 胡宏，非离子型去垢剂Triton X—114提取两歧双歧杆菌的脂磷壁酸, 中国微生态学杂志, 1997,9(4): 58-59
Medzhitov R, Preston HP, Janeway CA Jr, A human homologue of the Drosophila Toll protein signals activation of adaptive immunity, Nature, 1997, 388(6640): 394-397
Janssens S, Burns K etc, Regulation of IL-1 and LPS induced NF-kappaB activation by alternative splicing of MyD88, Curr Biol, 2002, 12(6): 467-471
Jacinto R, Hartung T, McCall C, etc, Lipopolysaccharide- and lipoteichoic acid-induced tolerance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase, J Immunol, 2002,168(12): 6136-6141
Underhill DM, Ozinsky A, Smith KD, etc, Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages, Proc Natl Acad Sci U S A, 1999,96(25): 14459-14463
Hou L, Sasaki H, Stashenko P, Toll-like receptor 4-deficient mice have reduced bone destruction following mixed anaerobic infection, Infect Immun, 2000, 68(8): 4681-4687
Opitz B, Schroder NW, Ralf R Schumann etc, TLR2 mediates Treponemcx glycolipid and LTA-induced NF-kappaB translocation, J Biol Chem, 2001, 276(25): 22041-22047
Okamoto M, Oshikawa T etc, Severe impairment of anti cancer effect of LTA-related molecule isolated from a penicillin-killed streptococues pyogenes in TLR4-dificent mice, Int Immunpharmacal, 2001, 1(9-10): 1789-1795
Oshikawa T, Okamoto M, Ohe G, etc, Anti-tumor immune response induced by the fractions derived from OK-432, a streptococcal preparation, by using a monoclonal antibody TS-2 that neutralizes the interferon-gamma -inducing activity of OK-432: comparison between the TS-2-binding and TS-2-unbinding fraction, Int Immunopharmacol, 2003, 3(5): 643-655
Wang J, Kobayashi M, Han M, etc, MyD88 is involved in the signalling pathway for Taxol-induced apoptosis and TNF-alpha expression in human myelomonocytic cells, Br J Haematol, 2002, 118(2): 638-645
蒋虹，胡宏，双歧杆菌表面分子诱导大肠癌细胞分化的研究，中华微生物学和免疫学杂志，2001, 21(2) ：186-190
Otte JM, Cario E, Podolsky DK, Mechanisms of cross hyporesponsiveness to toll-like receptor bacterial ligands in intestinal epithelial cells, Gastroenterology, 2004, 126(4): 1054-1070
Yang RB, Mark MR, Gray A, etc, Toll-like receptor-2 mediates lipopolysaccharide- induced cellular signaling, Nature, 1998, 395(6699): 284-288
Alexander P, Xiaolong He etc, Defective LPS signaling in C3H/HeJ and
C57BL/10ScCr mice mutation in Tlr4 gene, Science, 1998, 282(5196): 2085-2088
Suzuki N, Suzuki S etc, Severe impairment of IL-1 and TLR signaling in mice lacking IRAK-4, Nature, 2002, 416(6882): 750-756
张秋玉, TOLL样受体家族与LPS信号转导, 国外医学：免疫学分册, 2001, 24(3): 142-146
Hasegawa T, Matsuguchi T etc, TLR2 is at least partly involved in the antitumor activity of glycoprotein from chlorella vulgris, Int Immunopharmacol, 2002, 2(4): 579-589
Sato S, Nomura F, Kawai T, etc, Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways, J Immunol, 2000, 165(12): 7096-7101
Debatin KM, Apoptosis pathways in cancer and cancer therapy, Cancer Immunol Immunother, 2004, 53(3): 153-159
Chung CS, Song GY, Moldawer LL, etc, Neither Fas ligand nor endotoxin is responsible for inducible peritoneal phagocyte apoptosis during sepsis/peritonitis, J Surg Res, 2000, 91(2): 147-153
Okada T, Lian ZX, Naiki M, etc, Murine thymic plasmacytoid dendritic cells, Eur J Immunol, 2003, 33(4): 1012-1019
Frantz S, Kelly RA, Bourcier T, Role of TLR-2 in the activation of nuclear factor kappaB by oxidative stress in cardiac myocytes, J Biol Chem, 2001, 276(7): 5197-5203
Bannerman DD, Erwert RD, Winn RK, etc, TIRAP mediates endotoxin- induced NF-kappaB activation and apoptosis in endothelial cells, Biochem Biophys Res Commun, 2002, 295(1): 157-162
Meyer T, Nindl I, Schmook T, etc, Induction of apoptosis by Toll-like receptor-7 agonist in tissue cultures, Br J Dermatol, 2003, 149 Suppl 66:9-14
Berkowitz B, Huang DB, Chen-Park FE etc, The x-ray crystal structure of the NF-kappa B p50.p65 heterodimer bound to the interferon beta -kappa B site, J Biol
Chem, 2002, 277(27): 24694-24700
Huxford T, Huang DB, Malek S, etc, The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation, Cell, 1998, 95(6): 759-770
Palozza P, Serini S, Torsello A, etc, Mechanism of activation of Caspase cascade during beta-carotene-induced apoptosis in human tumor cells, Nutr Cancer, 2003, 47(1): 76-87
Fenwick C，Na S Y，Voll R E，etc, A subclass of Ras protein that regulate the degradation of IkappaB, Science， 2000，287：869-873
Malek S, Huang DB, Huxford T, etc, X-ray crystal structure of an IkappaBbeta x NF-kappaB p65 homodimer complex, J Biol Chem, 2003, 278(25): 23094-23100
K1ement J F，Rice N R，Car B D，etc, IκBα deficiency results in a sustained NF-κB response and severe widespread dermatitis in mice, Mo1 Cell Biol，1996，16：2341-2349
Karin M，Ben-Neriah Y, Phosphorylation meets ubiquitination：the control of NF-κB activity, Annu Rev Immunol，2000，18：621-663
Chen G，Goeddel D V, TNF-R1 signaling：a beautiful pathway, Science, 2002，296：1634-1635
Lin CH, Kuan IH, Lee HM, etc, Induction of cyclooxygenase-2 protein by lipoteichoic acid from Staphylococcus aureus in human pulmonary epithelial cells: involvement of a nuclear factor-kappa B-dependent pathway, Br J Pharmacol, 2001, 134(3): 543-552
Into T, Kiura K, Shibata K etc, Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa B activation, Cell Microbiol, 2004, 6(2): 187-199
孙大业, 郭艳林，马力耕，细胞信号转导, 第二版，北京：科学出版社，1998，28-48
Huerta-Yepez S, Vega M, Jazirehi A, etc，Nitric oxide sensitizes prostate carcinoma cell lines to TRAIL-mediated apoptosis via inactivation of NF-kappaB and inhibition of Bcl-(xL) expression, Oncogene, Epub 2004, 29
Gewirtz AT, Intestinal epithelial toll-like receptors: to protect. And serve? Curr Pharm Des, 2003, 9(1): 1-5
Lotz S, Aga E, Wilde I, etc, Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2, J Leukoc Biol, 2004, 75(3): 467-477