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Signal Transduction

Signal Transduction. Terry Moody, Ph.D. Bldg. 31, Rm. 4A48 301-451-9451. SIGNAL TRANSDUCTION Low doses of RNS and ROS may stimulate proliferation of cancer cells. High doses of RNS and ROS may cause apoptosis of cancer cells.

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Signal Transduction

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  1. Signal Transduction Terry Moody, Ph.D. Bldg. 31, Rm. 4A48 301-451-9451

  2. SIGNAL TRANSDUCTIONLow doses of RNS and ROS may stimulate proliferation of cancer cells.High doses of RNS and ROS may cause apoptosis of cancer cells.

  3. Elevated cytosolic Ca2+ activates nitric oxide synthase (NOS) leading to cGMP. • NO synthase uses arginine as a substrate to make the products NO and citrulline. • Soluble guanylyl cyclase uses GTP as a substrate to make the product cGMP.

  4. CYCLIC GMP PRODUCTIONCalcium channel Atrial Natriuretic Peptide Receptor-A,B PLASMA MEMBRANE ATP dependent kinase Ca2+ Membrane bound Guanylate Cyclase NO synthase NO Soluble Guanylate Cyclase-α,β cGMP cGMP is degraded by phosphodiesterase 5 CYTOSOL

  5. Atrial Natriuretic Peptide Receptor A B # amino acids 1061 1047 Molecular weight 118,918 117,021 Signal sequence 1-32 1-22 Extracellular 33-473 23-458 Transmembrane 474-494 459-478 Kinase 528-805 513-786 Guanylate cyclase 876-1006 861-991

  6. Soluble guanylate cyclase α2 β1 # amino acids 732 619 Molecular weight 81,749 70,514 Guanylate cyclase 521-648 421-554

  7. In soluble guanylate cyclase, the Fe is nitrosylated by NO. This increases enzymatic catalysis 400-fold NO Fe + NO Fe sGC has 3 domains HemeDimer-Catalytic bindingization Domain domaindomain

  8. Elevated cGMP has 4 protein targets • cGMP dependent protein kinase (PKGI) a 76 kDa serine/threonine kinase which ultimately leads to vasodilation • PKGII which phosphorylates the cystic fibrosis transmembrane conductance regulator • Cyclic nucleotide gated channel which translate visual signals to nerve impulses • Phosphodiesterases (PDE). Viagra selectively inhibits PDE 5

  9. The NO delivery agent SPER/NO increases cGMP and ERK activation. • SPER/NO increases cGMP 30 min. after addition to cells. • Increased P44/P42 MAPK (ERK) tyrosine phosphorylation is observed after 30 min. • Thomas et al., PNAS 101:8894 (2004).

  10. SUMMARY Low NO cGMP ERK tyrosine phosphorylation Proliferation

  11. High NO causes apoptosis of cancer cells. • NO can induce stress proteins, disrupt mitochondria, release cytochrome c and activate caspases.

  12. SPER/NO causes phosphorylation of p53. • The phosphorylated p53 results in less G1 to S transitions in the cell cycle, leading to increased apoptosis.

  13. NO and apoptosis. • The NO donars S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) cause apoptosis of lung cancer cells.

  14. Table I. NO inhibits lung cancer cellular proliferation. Addition % Proliferation Nitrite, uM None 100 3 SNAP, 0.4 mM 70 35 SNAP, 0.8 mM 60 55 SNP, 1 mM 80 35 SNP, 2 mM 55 45 SNAP and SNP were added to NCI-H1299 cells for 24 hr.Chao et al., JBC, 20267 (2004).

  15. NO delivery agents inhibit lung cancer cellular proliferation using the MTT assay. Addition Absorbance at 540 nm None .332 + .057 DEA/NO .201 + .021* PAPA/NO .193 + .025* The mean absorbance + S.D. of 8 determinations is indicated using NCI-H1299 cells; p < 0.05, *.

  16. DAF reactive chemicals form in cells within minutes after the addition of PAPA/NO.

  17. PAPA/NO inhibits lung cancer colony formation.

  18. Macrophages inhibit colony formation. Addition Colony number None 929 + 72 Macrophages, 0.5 M 756 + 98 Macrophages, 1 M 586 + 117 Macrophages, 2 M 474 + 58* Macrophages, 5 M 456 + 37* The mean number + S.D. of 3 determinations is indicated; p < 0.05, *.

  19. SNP causes phosphorylation of p38 MAPK. • P38 MAPK is a mediator of NO induced caspase-3 associated apoptosis. • The p38 MAPK inhibitor SB202190 protects cells from NO-mediated cell death.

  20. SNP and SNAP decrease survivin and Bcl-2 levels. • Survivin is critical for cell cycle progression. • Bcl-2 is critical for cellular survival.

  21. SUMMARY High NO Bcl-2 (-) p38-MAPK (+) Cytochrome c + ATP + Apaf1 Caspase-9 IAP (-) Caspase-3 Cell Death (Apoptosis)

  22. Two signaling pathways can be activated on exposure to oxidants. MAPK/AP-1 NF-κB (Activator Protein-1) (Nuclear Factor) Proliferation Inflammation Apoptosis Survival

  23. MAPK cascade Growth Factors Cellular Stress (ROS/RNS) (ROS) Raf MEKK MEK1/2 MEK3 MEK4 ERK1/2 p38 JNK1/2 Growth Stress Responses

  24. NO autoxidation results in protein nitrosylation.NO reacts with O2 and targets nucleophile

  25. Hydrophobic catalysis of NO autoxidation. NO reacts with O2 generating NO2 which reacts with NO to form N2O3. Membrane protein Cys amino acids undergo nitrosation. membrane protein

  26. Numerous cellular proteins are nitrosylated. • Ras, the p21 monomeric GTPase is nitrosylated at Cys118 resulting in activation of MAPK and PI3-K. • Denitrosylation of caspase-3 is essential for apoptosis.

  27. Low concentrations of H2O2 transiently stimulate increases in cytosolic free [Ca2+](B) and NOS activity (A) Barrett, D. M. et al. J. Biol. Chem. 280:14453-14461, 2005

  28. How do cells sense and transduce a cytoplasmic oxidative event?NO synthase activation leads to NO which forms metal nitrosyl complexes in cytochrome C oxidase and guanylate cyclase; Cys nitrosylation in PTP, caspase, Zn proteins and ATM; and RNS causing tyrosine nitration in NF-kB, Bcl, pte and Keap1/Nrf2.

  29. Protein Tyrosine Phosphatases can be oxidized.

  30. Effects of PGE2 on cancer cells EP2R NO PGE2 VEGF COX-2 EP2R EGFR

  31. Lung Cancer cells produce LTs and PGs. Phospholipids PLA2 Arachidonic Acid LOX COX Leukotrienes Prostaglandins

  32. Arachidonic acid is metabolized slowly by the rate limiting enzyme COX. Arachidonic acid Cyclooxygenase PGG2 PG endoperoxide Syn. PGH2 TXA2 PGI2 PGE2 Motility Sprouting Multiple effects

  33. Two subtypes of COX are present, COX-1 and COX-2 ●COX-1 is a constitutive house keeping enzyme expressed in the normal kidney, platelets and GI tract. COX-1 is inhibited by non-steroidal antiinflammatory drugs (NSAIDs). ●COX-2 is induced in inflammation and neoplasia by EGF, TGFβ, TNFα, hypoxia and uv B light. COX-2 is inhibited by NSAIDs and celecoxib.

  34. Cyclooxygenase (COX) COX-2 COX-1 # amino acids 604 599 Molecular weight 68,996 68,656 Distal His 193 206 Fe binding site 374 387 Aspirin acetylated Ser 516 529

  35. COX-2 immunostaining inthe A/J mouse lung.

  36. The A/J mouse represents an animal model for lung carcinogenesis. ●COX-2 is present in all lung compartments including the alveoli, bronchi and bronchioles.

  37. Lung adenomas develop 4 months after injections of carcinogen.

  38. Indomethacin, a NSAID, reduces lung adenoma number in A/J mice.

  39. Celecoxib (CELEBREX) is approved by the FDA for arthritis and treatment of colorectal polyps in FAP patients. ●Oral celecoxib inhibited corneal angiogenesis and PGE2 levels by 79% ●Oral celecoxib reduced endothelial cell proliferation by 2.5-fold and increased apoptosis 2.7 fold ●In lung cancer patients treated with celecoxib paclitaxel and carboplatin, serum VEGF declined.

  40. S-NSAIDs and a COX-2 inhibitor reduce PGE2 in NSCLC cells. Table I. S-NSAIDs and PGE2. __________________________________________ Addition Relative PGE2, % _________________ A549 H1299___________ None 100 + 13 100 + 8 NO-Asa, 1 ug/ml 18 + 3** 23 + 4** S-Valproate, 1 ug/ml 26 + 8** 33 + 8** S-Dicofenac, 1 ug/ml 18 + 6** 25 + 4** S-Sulindac, 1 ug/ml 64 + 9* 75 + 10 DuP-697, 1 ug/ml 30 + 7** 21 +4** __________________________________________ The mean value + S.D. of 4 determinations is indicated; (p < 0.05, *; p < 0.01, ** using student’s t-test).

  41. EP2 receptor Amino acids 358 Molecular weight 39,760 Transmembrane 24-47, 66-91, 112-132, 152- 176, 199-223, 263-286, 300- 323 Extracellular 1-23 Intracellular 324-358 N-glycosylation 3, 6, 96, 287

  42. PGE2 binds to EP2-Rs which are present in lung cancer cell lines

  43. PGE1, PGE2, PGF2α and AH6809 bind with high affinity. Compound IC50, ųM Arachidonic acid >10 AH6809 5 + 0.7 PGD2 >10 PDE1 0.2 + .03 PGE2 0.04 + .01 PGF2α 2 + 0.2 PGG2 >10 PGI2 >10 Casibang, M. et al., Lung Cancer 2001; 31: 203

  44. The EP2 receptor is coupled to adenylylcyclase. ●PGE2 is an agonist which increases the cAMP in lung cancer ●AH6809 is an antagonist which reversibly blocks the receptor

  45. EP2 receptor antagonists block the increase in cAMP caused by PGE2.

  46. NO causes increased VEGF mRNA.

  47. VEGF mRNA is increased by PGE2 in a PKA-dependent manner Addition Relative VEGF mRNA None 100 + 5 PGE2, 1 uM 200 + 17* EGF, 0.1 ug/ml 185 + 16* H89, 50 uM 104 + 3 PGE2 + H89 110 + 6 The mean value + S.D. of 4 determinations is indicated; p < 0.05, *

  48. COX-2 and VEGF expression are intimately linked. ●In Apc/COX-2 double knockout mice, VEGF protein is reduced by 94%. ●In NSCLC patients, COX-2 mRNA expression correlates with VEGF mRNA, increased microvessel density, decreased patient survival and early relapse.

  49. EGF causes increased COX-2 expression in NSCLC cells.

  50. Tyrosine kinase receptorsMolecular Biology of the Cell, Alberts etl al., 2001.

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