人参皂苷Rh2提高肿瘤细胞对桦木酸敏感性的研究
摘要
恶性肿瘤现已经成为威胁人类健康和生命,导致死亡的主要原因之一。目前,化学疗法,放射性治疗,免疫疗法是肿瘤术后治疗的方法,其中化学治疗是辅助术后治疗的主要手段。治疗肿瘤的方法都是利用药物诱导肿瘤细胞发生凋亡起作用的。化疗药物的疗效在一定范围内与药物剂量成正比。随着用药剂量的增加,机体产生毒副作用的风险也相应增加。发现有协同作用的抗肿瘤药物可以使药物在治疗安全窗内产生更好的疗效。本研究中,我们使用天然成分人参皂苷Rh2(G-Rh2)和桦木酸(Bet A)同时作用于人肝癌细胞HepG2和人肺癌细胞A549,并且广泛的测定与分析人参皂苷Rh2(G-Rh2)和桦木酸(Bet A)对肿瘤细胞的杀伤效果。我们采用细胞形态学测定、流式细胞仪检测分析、caspase-3酶活性测定分析、免疫印记法分析等实验技术,证明了人参皂苷Rh2(G-Rh2)和桦木酸(Bet A)两者可诱导癌症细胞发生凋亡并且二者同时作用于癌症细胞时对癌症细胞的促凋亡作用更加明显。随着药物联合作用时间的延长,细胞内发生明显的caspase-9前体活化,PARP断裂、caspase-3激活及凋亡。研究结果表明人参皂苷Rh2(G-Rh2)能有效提高肿瘤细胞对桦木酸(Bet A)的敏感性。
At present, cancer is a serious threat to human and animal health, which is a serious threat to the health of people. Fragnent, the main treatment for cancer is surgery, supplemented by radiotherapy, chemotherapy and other methods. The efficacy of chemotherapy is proportional to the dose within a certain range. As the increases the risk of side effects occurred a corresponding increase in the tumor cells to chemotherapeutic drugs, tolerance, often lead to treatment failure. Found a synergistic effect of anticancer drugs can make drugs safe in therapeutic doses to generate better results. Most of the anti-tumor treatment, mainly including chemotherapy through induced apoptosis, and to prevent the body from drug tolerance, two and multi-drug combination therapy in the treatment of malignant tumors is better than a single anti-cancer drug. Because of synergies between the various drugs help to enlarge the original weak death signal. Therefore, the drug combination treatment may be a strong therapeutic strategy.
Ginsenoside Rh2 (ginsenoside Rh2, G-Rh2) is extracted from ginseng with natural active, the study confirmed that the composition has a strong anti-tumor activity. According to reports, G-Rh2 can induce human breast cancer cells, human melanoma cells, human prostate cancer cells, human leukemia cells, human liver cancer cells and other tumor cells. Ginsenoside Rh2 also induces tumor cell differentiation, enhance immunity, and improve the ability of anti-tumor effect.
Betulinic acid [3β-Hydroxy-lup-20(29)-en-28-oic acid] is a lup-type pentacyclic triterpenoids. Betulin acid is the carboxylic acid derivatives of C-28 of betulinol. However, betulinic acid with more people expected pharmacological activity which diference with betulin, such as anti-inflammatory, anti-cancer, anti-HIV and so on. Betulinic acid has anti-tumor, anti-HIV, anti-inflammatory effects, anti-malarial in vitro and other diseases.
In this study, we found ginsenoside Rh2 significantly increased sensitivity to betulinic acid in A549 cells and HepG2 cells. Through analysis of cell morphology of two cell lines joining the two drugs, the morphological change significantly, there bubble membrane, nuclear condensation phenomena such as apoptosis. DAPI nuclear staining can be more easily observed phenomenon. Based on the above phenomenon, we analyzed by flow cytometry showed that, cells in the Sub-G1 significantly increased the percentage of the number when G-Rh2 and Bet A join to A549 cells and HepG2 cells simultaneously,. Caspase-3 activity analysis showed that, cells joined G-Rh2 and Bet A at the same time increased the activity of caspase-3, compared with G-Rh2 and Bet A were added alone, and the activity of caspase-3 increased significantly as the time. Using Western-Blot analysis PARP that is substrate of caspase-3 , the results show that the fracture of PARP when G-Rh2 and Bet A added at the same time increased significantly. Using Western-Blot analysis of caspase-9 activation, the results showed that activation of caspase-9 that G-Rh2 and Bet A all in tumor cells is higher than G-Rh2 and Bet A alone in A549 cells and HepG2 cells, and with time, activation is more evident.
In summary, we found that ginsenoside Rh2 can improve the A549 cells and HepG2 cells to betulinic acid sensitivity, and confirmed that the result is caused by tumor cells through programmed cell death occurred.
At present, cancer is a serious threat to human and animal health, which is a serious threat to the health of people. Fragnent, the main treatment for cancer is surgery, supplemented by radiotherapy, chemotherapy and other methods. The efficacy of chemotherapy is proportional to the dose within a certain range. As the increases the risk of side effects occurred a corresponding increase in the tumor cells to chemotherapeutic drugs, tolerance, often lead to treatment failure. Found a synergistic effect of anticancer drugs can make drugs safe in therapeutic doses to generate better results. Most of the anti-tumor treatment, mainly including chemotherapy through induced apoptosis, and to prevent the body from drug tolerance, two and multi-drug combination therapy in the treatment of malignant tumors is better than a single anti-cancer drug. Because of synergies between the various drugs help to enlarge the original weak death signal. Therefore, the drug combination treatment may be a strong therapeutic strategy.
Ginsenoside Rh2 (ginsenoside Rh2, G-Rh2) is extracted from ginseng with natural active, the study confirmed that the composition has a strong anti-tumor activity. According to reports, G-Rh2 can induce human breast cancer cells, human melanoma cells, human prostate cancer cells, human leukemia cells, human liver cancer cells and other tumor cells. Ginsenoside Rh2 also induces tumor cell differentiation, enhance immunity, and improve the ability of anti-tumor effect.
Betulinic acid [3β-Hydroxy-lup-20(29)-en-28-oic acid] is a lup-type pentacyclic triterpenoids. Betulin acid is the carboxylic acid derivatives of C-28 of betulinol. However, betulinic acid with more people expected pharmacological activity which diference with betulin, such as anti-inflammatory, anti-cancer, anti-HIV and so on. Betulinic acid has anti-tumor, anti-HIV, anti-inflammatory effects, anti-malarial in vitro and other diseases.
In this study, we found ginsenoside Rh2 significantly increased sensitivity to betulinic acid in A549 cells and HepG2 cells. Through analysis of cell morphology of two cell lines joining the two drugs, the morphological change significantly, there bubble membrane, nuclear condensation phenomena such as apoptosis. DAPI nuclear staining can be more easily observed phenomenon. Based on the above phenomenon, we analyzed by flow cytometry showed that, cells in the Sub-G1 significantly increased the percentage of the number when G-Rh2 and Bet A join to A549 cells and HepG2 cells simultaneously,. Caspase-3 activity analysis showed that, cells joined G-Rh2 and Bet A at the same time increased the activity of caspase-3, compared with G-Rh2 and Bet A were added alone, and the activity of caspase-3 increased significantly as the time. Using Western-Blot analysis PARP that is substrate of caspase-3 , the results show that the fracture of PARP when G-Rh2 and Bet A added at the same time increased significantly. Using Western-Blot analysis of caspase-9 activation, the results showed that activation of caspase-9 that G-Rh2 and Bet A all in tumor cells is higher than G-Rh2 and Bet A alone in A549 cells and HepG2 cells, and with time, activation is more evident.
In summary, we found that ginsenoside Rh2 can improve the A549 cells and HepG2 cells to betulinic acid sensitivity, and confirmed that the result is caused by tumor cells through programmed cell death occurred.
引文
[1] Chou T-C. The median-effect principle and the combination index for quantitation of synergism and antagonism. In: Chou T-C, Rideout D, editors. Synergism and antagonism in chemotherapy. San Diego: Academic Press 1991; 4:61-102.
[2] Chou T-C, Rideout D, Chou JH, Bertino JR.Chemotherapeutic synergism, potentiation and antagonism.In: Dulbecco R, editor. Encyclopedia of human biology (vol2). 2nd ed. San Diego: Academic Press 1997:675-83.
[3] Chou T-C, Motzer RJ, Tong Y, Bosl GJ. Computerized quantitation of synergism and antagonism of taxol, topotecan and cisplatin against teratocarcinoma cell growth: a rational approach to clinical protocol design. J Natl Cancer Inst 1994; 86:1517-24.
[4] Bertino JR, Chon T-C. Chemotherapy: synergism and antagonism.In: Bertino JR, editor. Encyclopedia of cancer (vol1). San Diego: Academic Press 1997; 41:368-79.
[5] Guo J, Reidenberg MM. Inhibition of 11-[3 hydroxysteroid dehydrogenase by bioflavonoids and their interaction with furosemide and gossypol. J Lab Clin Med 1998; 131:32-38.
[6] Permuterm Subject Index, Science Citation Index.Philadelphia: Institute for Scientific Information 1997; 17:12-15.
[7] Chou T-C, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Rdgul 1984; 22:27-55.
[8] Chou T-C. Derivation and properties of Michaelis-Menten type and Hill type equations for reference ligands. J Theor Biol 1996; 22:253-76.
[9] Chou J, Chou T-C. Dose-effect analysis with microcomputers: quantitation of EDs0, LDs0, synergism, antagonism, lowdoserisk, receptor-ligand binding andenzyme kinetics.Manual and software. Cambridge, England: Biosoft 1987; 39:34-54.
[10] Chou T-C, Hayball, M. CalcuSyn for Windows, multiple-drug dose-effect analyzer and manual. Cambridge, England: Biosoft 1996; 115:23-30.
[11] Colussi P. A. et al. Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery. J. Cell Biol 2000; 39: 703–714.
[12] Zhang H, et al. Drosophila Pro-apoptotic Bcl-2/Bax homologue reveals evolutionary conservation of cell death mechanisms. J. Biol. Chem 2000: 27303–27306.
[13] Kelekar A & Thompson C, B. Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol 1998:324–330.
[14] Vaux, D. L, Weissman, I. L & Kim, S. K. Prevention of programmed cell-death in Caenorhabditis elegans by human bcl-2. Science 258:1992:1955–1957.
[15] White K. et al. Genetic control of programmed cell death in Drosophila. Science 1994; 264:677–683.
[16] Ollmann M. et al. Drosophila p53 is a structural and functional homolog of the tumor suppressor p53. Cell 101 2000; 148:91–101.
[17] Jin S. et al. Identification and characterization of a p53 homologue in Drosophila melanogaster. Proc.Natl Acad. Sci. USA 97 2000:7301–7306.
[18] Brodsky M. H. et al. Drosophila p53 binds a damage response element at the reaper locus. Cell 2000; 101:103–113.
[19] Bergmann A, Agapite J, McCall, K. & Steller, H. The Drosophila gene hid is a direct molecular target of Ras-dependent survival signaling. Cell 1998; 95:331–341.
[20] Kurada P & White K Ras promotes cell survival in Drosophila by down-regulating hid expression.Cell 1998; 95:319–329.
[21] Foley, K. & Cooley, L. Apoptosis in late stage Drosophila nurse cells does not require genes within the H99 deficiency. Development 1998; 125:1075–1082.
[22] Du C, Fang M Li, Y Li L & Wang X. Smac, a mitochondrial protein that promotes cytochrome cdependent caspase activation by eliminating IAP inhibition. Cell 2000; 102:33–42.
[23] Verhagen A. M. et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000; 102: 43–53.
[24] Chai J, Du C, Wu J-W Wang, X & Shi Y. Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 2000; 406:855–862.
[25] Goyal L, Mc Call K, Agapite J, Hartwieg E. & Steller H. Induction of apoptosis by Drosophila reaper, hid and grimthrough inhibition of IAP function. EMBO J. 2000; 19:589–597.
[26] Mitchell J A, Paul-Clark M, J. Clarke G.W. et al. Critical role of toll-like receptors and nucleotide oligomerisation domain in the regulation of health and disease. J Endocrinol 2007:323-30.
[27] Meier P, Silke J, Leevers S J & Evan G I. The Drosophila caspase DRONC is regulated by DIAP1.EMBO J19 2000:598–611.
[28] Wang S L, Hawkins, C J, Yoo S J, Muller H A & Hay B A. The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 1999; 98:453–463.
[29] Chen P & Abrams J. M. Drosophila apoptosis and Bcl-2 genes: outliers fly in. J. Cell Biol. 2000; 148:625–627.
[30] Bergmann A, Agapite J & Steller H. Mechanisms and control of programmed cell death in invertebrates. Oncogene 1998; 17:3215–3223.
[31] McCall K & Steller H. Requirement for DCP-1 caspase during Drosophila oogenesis. Science 1998; 279:230–234.
[32] Jiang C, Baehrecke E H & Thummel C S. Steroid regulated programmed cell death during Drosophila metamorphosis. Development 1997; 124: 4673–4683.
[33] Kiess W & Gallaher B. Hormonal control of programmed cell death/apoptosis. Eur. J. Endocrinol 1998; 138:482–491.
[34] Mikami F, Gu H, Jono H et al. Epidermal growth factor receptor acts as a negative regulator for bacterium nontypeable Haemophilus influenzae-induced Toll-like receptor 2 expression via an Src-dependent p38 mitogen-activated protein kinase signaling pathway. J Biol Chem 2005; 36:185-94.
[35] Merino R, Ganan Y, Macias D, Rodriguez-Leon J & Hurle J. M. Bone morphogenetic proteins regulate interdigital cell death in the avian embryo. Ann. NY Acad. Sci 887 1999:120–132.
[36] Hu S & Yang X. dFADD, a novel death domain-containing adapter protein for the Drosophila caspase DREDD. J. Biol. Chem. (in the press).
[37] Kondo T, Yokokura T & Nagata S. Activation of distinct caspase-like proteases by Fas and reaper in Drosophila cells. Proc. Natl Acad. Sci. USA 94 1997; 20:11951–11956.
[38] Zou H, Li Y, Liu X & Wang X. An APAF-1.Cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem 1999; 274:11549–11556.
[39] Zhang Q, Bagrade L, Bernatoniene J. et al. Low CD4 T cell immunity to pneumolysin is associated with nasopharyngeal carriage of pneumococci in children. J Infect Dis 2007; 14:1194-202.
[40] Varkey J, Chen P, Jemmerson R & Abrams J M. Altered cytochrome c display precedes apoptotic cell death in Drosophila J Cell Biol 1999; 144:701–710.
[41] Jain V, Halle A, Halmen K A et al. Phagocytosis and intracellular killing of MD-2 opsonized Gram-negative bacteria depend on TLR4 signaling. Blood 2008; 122:4637-45.
[42] Davey M, Liu X, Ukai T et al. Bacterial fimbriae stimulate proinflammatory activation in the endothelium through distinct TLRs. J Immunol 2008; 28:2187-95.
[43] Koziczak-Holbro M, Joyce C, Gluck A et al. IRAK-4 kinase activity is required for interleukin-1 (IL-1) receptor- and tolllike receptor 7-mediated signaling and gene expression. J Biol Chem 2007; 356:13552-60.
[44] Yarden Y, Sliwkowski M X. Untangling the ErbB signaling network. Nat Rev Mol Cell Biol 2001:127-37.
[45] Barres B A & Raff M C. Axonal control of oligodendrocyte development. J Cell Biol 1999; 147:1123–1128.
[46] Raff M C. Social controls on cell survival and cell death. Nature 1992; 356:397–400.
[47] Raff M C et al. Programmed cell death and the control of cell survival: lessons from the nervous system. Science 1993; 262:695–700.
[48] Weil M, Jacobson M D & Raff M C. Is programmed cell death required for neural tube closure? Curr Biol 1997; 7:281–284.
[49] Kuida K et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.Nature 1996; 384:368–372.
[50] Kuida K et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 1998; 94:325–337.
[51] Hakem R et al. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 1998; 94:339–352.
[52] Yoshida H et al. Apaf-1 is required for mitochondrial pathways of apoptosis and brain development.Cell 1998; 94:739–750.
[53] Cecconi F, Alvarez-Bolado G, Meyer B I, Roth K A & Gruss P. Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 1998; 94:727–737.
[54] Middleton G, Cox S W, Korsmeyer S & Davies A M. Differences in Bcl-2- and Bax-independent function in regulating apoptosis in sensory neuron populations. Eur J Neurosci 2000; 12:819–827.
[55] Motoyama N et al.Massive cell death of immature hematopoietic cells and neurons in Bcl-xdeficient mice. Science 1995; 267:1506–1510.
[56] Braun J S, Hoffmann O, Schickhaus M et al. Pneumolysin causes neuronal cell death through mitochondrial damage. Infect Immun 2007; 42: 45-54.
[57] Mitchell J A, Paul-Clark M J, Clarke G W et al. Critical role of toll-like receptors and nucleotide oligomerisation domain in the regulation of health and disease. J Endocrinol 2007;3: 23-30.
[58] Zhang Q, Bagrade L, Bernatoniene J et al. Low CD4 T cell immunity to pneumolysin is associated with nasopharyngeal carriage of pneumococci in children. J Infect Dis 2007; 11: 94-202.
[59] Jono H, Lim J H, Chen L F et al. NF-kappaB is essential for induction of CYLD, the negative regulator of NF-kappaB: evidence for a novel inducible autoregulatory feedback pathway.J Biol Chem 2004; 267:36171-4.
[60] Shuto T, Imasato A, Jono H et al. Glucocorticoids synergistically enhance nontypeable Haemophilus influenzae-induced Tolllike receptor 2 expression via a negative cross-talk with p38 MAP kinase. J Biol Chem 2002; 172: 63-70.
[2] Chou T-C, Rideout D, Chou JH, Bertino JR.Chemotherapeutic synergism, potentiation and antagonism.In: Dulbecco R, editor. Encyclopedia of human biology (vol2). 2nd ed. San Diego: Academic Press 1997:675-83.
[3] Chou T-C, Motzer RJ, Tong Y, Bosl GJ. Computerized quantitation of synergism and antagonism of taxol, topotecan and cisplatin against teratocarcinoma cell growth: a rational approach to clinical protocol design. J Natl Cancer Inst 1994; 86:1517-24.
[4] Bertino JR, Chon T-C. Chemotherapy: synergism and antagonism.In: Bertino JR, editor. Encyclopedia of cancer (vol1). San Diego: Academic Press 1997; 41:368-79.
[5] Guo J, Reidenberg MM. Inhibition of 11-[3 hydroxysteroid dehydrogenase by bioflavonoids and their interaction with furosemide and gossypol. J Lab Clin Med 1998; 131:32-38.
[6] Permuterm Subject Index, Science Citation Index.Philadelphia: Institute for Scientific Information 1997; 17:12-15.
[7] Chou T-C, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Rdgul 1984; 22:27-55.
[8] Chou T-C. Derivation and properties of Michaelis-Menten type and Hill type equations for reference ligands. J Theor Biol 1996; 22:253-76.
[9] Chou J, Chou T-C. Dose-effect analysis with microcomputers: quantitation of EDs0, LDs0, synergism, antagonism, lowdoserisk, receptor-ligand binding andenzyme kinetics.Manual and software. Cambridge, England: Biosoft 1987; 39:34-54.
[10] Chou T-C, Hayball, M. CalcuSyn for Windows, multiple-drug dose-effect analyzer and manual. Cambridge, England: Biosoft 1996; 115:23-30.
[11] Colussi P. A. et al. Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery. J. Cell Biol 2000; 39: 703–714.
[12] Zhang H, et al. Drosophila Pro-apoptotic Bcl-2/Bax homologue reveals evolutionary conservation of cell death mechanisms. J. Biol. Chem 2000: 27303–27306.
[13] Kelekar A & Thompson C, B. Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol 1998:324–330.
[14] Vaux, D. L, Weissman, I. L & Kim, S. K. Prevention of programmed cell-death in Caenorhabditis elegans by human bcl-2. Science 258:1992:1955–1957.
[15] White K. et al. Genetic control of programmed cell death in Drosophila. Science 1994; 264:677–683.
[16] Ollmann M. et al. Drosophila p53 is a structural and functional homolog of the tumor suppressor p53. Cell 101 2000; 148:91–101.
[17] Jin S. et al. Identification and characterization of a p53 homologue in Drosophila melanogaster. Proc.Natl Acad. Sci. USA 97 2000:7301–7306.
[18] Brodsky M. H. et al. Drosophila p53 binds a damage response element at the reaper locus. Cell 2000; 101:103–113.
[19] Bergmann A, Agapite J, McCall, K. & Steller, H. The Drosophila gene hid is a direct molecular target of Ras-dependent survival signaling. Cell 1998; 95:331–341.
[20] Kurada P & White K Ras promotes cell survival in Drosophila by down-regulating hid expression.Cell 1998; 95:319–329.
[21] Foley, K. & Cooley, L. Apoptosis in late stage Drosophila nurse cells does not require genes within the H99 deficiency. Development 1998; 125:1075–1082.
[22] Du C, Fang M Li, Y Li L & Wang X. Smac, a mitochondrial protein that promotes cytochrome cdependent caspase activation by eliminating IAP inhibition. Cell 2000; 102:33–42.
[23] Verhagen A. M. et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000; 102: 43–53.
[24] Chai J, Du C, Wu J-W Wang, X & Shi Y. Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 2000; 406:855–862.
[25] Goyal L, Mc Call K, Agapite J, Hartwieg E. & Steller H. Induction of apoptosis by Drosophila reaper, hid and grimthrough inhibition of IAP function. EMBO J. 2000; 19:589–597.
[26] Mitchell J A, Paul-Clark M, J. Clarke G.W. et al. Critical role of toll-like receptors and nucleotide oligomerisation domain in the regulation of health and disease. J Endocrinol 2007:323-30.
[27] Meier P, Silke J, Leevers S J & Evan G I. The Drosophila caspase DRONC is regulated by DIAP1.EMBO J19 2000:598–611.
[28] Wang S L, Hawkins, C J, Yoo S J, Muller H A & Hay B A. The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 1999; 98:453–463.
[29] Chen P & Abrams J. M. Drosophila apoptosis and Bcl-2 genes: outliers fly in. J. Cell Biol. 2000; 148:625–627.
[30] Bergmann A, Agapite J & Steller H. Mechanisms and control of programmed cell death in invertebrates. Oncogene 1998; 17:3215–3223.
[31] McCall K & Steller H. Requirement for DCP-1 caspase during Drosophila oogenesis. Science 1998; 279:230–234.
[32] Jiang C, Baehrecke E H & Thummel C S. Steroid regulated programmed cell death during Drosophila metamorphosis. Development 1997; 124: 4673–4683.
[33] Kiess W & Gallaher B. Hormonal control of programmed cell death/apoptosis. Eur. J. Endocrinol 1998; 138:482–491.
[34] Mikami F, Gu H, Jono H et al. Epidermal growth factor receptor acts as a negative regulator for bacterium nontypeable Haemophilus influenzae-induced Toll-like receptor 2 expression via an Src-dependent p38 mitogen-activated protein kinase signaling pathway. J Biol Chem 2005; 36:185-94.
[35] Merino R, Ganan Y, Macias D, Rodriguez-Leon J & Hurle J. M. Bone morphogenetic proteins regulate interdigital cell death in the avian embryo. Ann. NY Acad. Sci 887 1999:120–132.
[36] Hu S & Yang X. dFADD, a novel death domain-containing adapter protein for the Drosophila caspase DREDD. J. Biol. Chem. (in the press).
[37] Kondo T, Yokokura T & Nagata S. Activation of distinct caspase-like proteases by Fas and reaper in Drosophila cells. Proc. Natl Acad. Sci. USA 94 1997; 20:11951–11956.
[38] Zou H, Li Y, Liu X & Wang X. An APAF-1.Cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem 1999; 274:11549–11556.
[39] Zhang Q, Bagrade L, Bernatoniene J. et al. Low CD4 T cell immunity to pneumolysin is associated with nasopharyngeal carriage of pneumococci in children. J Infect Dis 2007; 14:1194-202.
[40] Varkey J, Chen P, Jemmerson R & Abrams J M. Altered cytochrome c display precedes apoptotic cell death in Drosophila J Cell Biol 1999; 144:701–710.
[41] Jain V, Halle A, Halmen K A et al. Phagocytosis and intracellular killing of MD-2 opsonized Gram-negative bacteria depend on TLR4 signaling. Blood 2008; 122:4637-45.
[42] Davey M, Liu X, Ukai T et al. Bacterial fimbriae stimulate proinflammatory activation in the endothelium through distinct TLRs. J Immunol 2008; 28:2187-95.
[43] Koziczak-Holbro M, Joyce C, Gluck A et al. IRAK-4 kinase activity is required for interleukin-1 (IL-1) receptor- and tolllike receptor 7-mediated signaling and gene expression. J Biol Chem 2007; 356:13552-60.
[44] Yarden Y, Sliwkowski M X. Untangling the ErbB signaling network. Nat Rev Mol Cell Biol 2001:127-37.
[45] Barres B A & Raff M C. Axonal control of oligodendrocyte development. J Cell Biol 1999; 147:1123–1128.
[46] Raff M C. Social controls on cell survival and cell death. Nature 1992; 356:397–400.
[47] Raff M C et al. Programmed cell death and the control of cell survival: lessons from the nervous system. Science 1993; 262:695–700.
[48] Weil M, Jacobson M D & Raff M C. Is programmed cell death required for neural tube closure? Curr Biol 1997; 7:281–284.
[49] Kuida K et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.Nature 1996; 384:368–372.
[50] Kuida K et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 1998; 94:325–337.
[51] Hakem R et al. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 1998; 94:339–352.
[52] Yoshida H et al. Apaf-1 is required for mitochondrial pathways of apoptosis and brain development.Cell 1998; 94:739–750.
[53] Cecconi F, Alvarez-Bolado G, Meyer B I, Roth K A & Gruss P. Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 1998; 94:727–737.
[54] Middleton G, Cox S W, Korsmeyer S & Davies A M. Differences in Bcl-2- and Bax-independent function in regulating apoptosis in sensory neuron populations. Eur J Neurosci 2000; 12:819–827.
[55] Motoyama N et al.Massive cell death of immature hematopoietic cells and neurons in Bcl-xdeficient mice. Science 1995; 267:1506–1510.
[56] Braun J S, Hoffmann O, Schickhaus M et al. Pneumolysin causes neuronal cell death through mitochondrial damage. Infect Immun 2007; 42: 45-54.
[57] Mitchell J A, Paul-Clark M J, Clarke G W et al. Critical role of toll-like receptors and nucleotide oligomerisation domain in the regulation of health and disease. J Endocrinol 2007;3: 23-30.
[58] Zhang Q, Bagrade L, Bernatoniene J et al. Low CD4 T cell immunity to pneumolysin is associated with nasopharyngeal carriage of pneumococci in children. J Infect Dis 2007; 11: 94-202.
[59] Jono H, Lim J H, Chen L F et al. NF-kappaB is essential for induction of CYLD, the negative regulator of NF-kappaB: evidence for a novel inducible autoregulatory feedback pathway.J Biol Chem 2004; 267:36171-4.
[60] Shuto T, Imasato A, Jono H et al. Glucocorticoids synergistically enhance nontypeable Haemophilus influenzae-induced Tolllike receptor 2 expression via a negative cross-talk with p38 MAP kinase. J Biol Chem 2002; 172: 63-70.