Jsi 124 pathway


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Jsi 124 pathway

  1. 1. Presented by: Khuram Aziz <ul><li>Discovery of JSI-124, a selective JAK/STAT3 inhibitor with antitumor activity against human and murine cancer cells in MICE </li></ul>
  2. 4. introduction <ul><li>Discovery of disrupter in abberant activation of STAT </li></ul><ul><li>Supression of levels of phosphotyrosine STAT3 will inhibit proliferation and induce apoptosis </li></ul><ul><li>Use of JSI-124 </li></ul><ul><li>Which are inhibited by it; </li></ul><ul><li>A549 tumors, v-src-transformed NIH 3T3 tumors, MDA-MB-468 </li></ul>
  3. 6. Which are not affected <ul><li>Ras transformed tumors, akt pathways, c-jun nh2 terminal kinase, extracellular signal regulated kinases ½, calu-1 </li></ul>
  4. 8. <ul><li>STATS are of diferent types. </li></ul><ul><li>They have dual role </li></ul><ul><li>They are involved in many physiological functions </li></ul>
  5. 10. <ul><li>For the biological functioning, these must be tyrosine phosphorylated </li></ul><ul><li>Tyrosine kinases that are involved to phosphorylate are non-RTKs, JAKs and peptide growth factors </li></ul><ul><li>Regulated by SHP-1 and SHP-2 </li></ul>
  6. 11. STAT3 invovlment in oncogenesis is most thoroughly.. <ul><li>First, STAT3 is constitutively tyrosine phosphorylated </li></ul><ul><li>Second, for expression of activated mutant STAT3, stable dimerization was forced </li></ul><ul><li>Its validation as an anticancer drug target </li></ul>
  7. 13. Materials and Methods <ul><li>Cell Lines. All human and murine tumor cell lines used were obtained </li></ul><ul><li>from the American Type Culture Collection (Manassas, VA). Stably transfected </li></ul><ul><li>v-Src, oncogenic H-Ras, and vector NIH 3T3 cell lines have been described previously . </li></ul>
  8. 14. NCI Diversity Set. <ul><li>The NCI Structural Diversity Set is a library of 1,992 </li></ul><ul><li>compounds selected from the approximately 140,000-compound NCI drug </li></ul><ul><li>depository. These compounds were selected based on various criteria including </li></ul><ul><li>availability, drug-like structure, uniqueness of pharmacophore, and anticancer </li></ul><ul><li>activity as determined by cell growth inhibition assays against a panel of </li></ul><ul><li>human tumor cell lines. In-depth data on the selection, structures, and activities </li></ul><ul><li>of these diversity set compounds can be found on the NCI Developmental </li></ul>
  9. 15. Cytoblot Screening for Phospho-STAT3 Inhibition. <ul><li>NIH 3T3 cells stably transfected with v-Src or NIH 3T3 vector control cells (11) were plated into sterile, opaque, 96-well tissue culture plates at 25,000 cells/well. </li></ul><ul><li>After overnight growth at 37°C, the cells were treated for 4 h in the presence of either vehicle control or 10 _M of NCI Diversity Set compounds. </li></ul><ul><li>After treatment,cells were washed in 100 _l of cold TBS and </li></ul><ul><li>then fixed for 1 h at 4°C with cold 3.7% formaldehyde in TBS (190 _l/well) as described previously for a similar cytoblot for phospho-nucleolin (20). </li></ul>
  10. 16. . <ul><li>Membranes were permeabilized during a 5-min incubation in ice-cold methanol at 4°C. </li></ul><ul><li>Cells were washed with 3% milk in TBS (180 _l/well) and </li></ul><ul><li>then rocked overnight at 4°C with 3% milk in TBS (50 _l/well) containing a 1:1,000 dilution of anti-phospho-STAT3 (P-Tyr 705; Cell Signaling Technology, Beverly, MA) and a 1:2,000 dilution of horseradish peroxidase-conjugated goat antirabbit IgG (Jackson Immuno Research, West Grove, PA). </li></ul><ul><li>Antibodies were aspirated, and then plates were washed twice with TBS (180 _l/well). </li></ul>
  11. 17. . <ul><li>Results were visualized by adding Western blot chemiluminescence reagent directly to the wells of the plates, incubating at room temperature for 5 min, and </li></ul><ul><li>then placing X-ray film directly on top of the plate in a dark room for 1–5 min. Quantification of results was done using a GS-700 scanning densitometer. </li></ul>
  12. 18. Western Blotting <ul><li>Treated cell samples were lysed in 30 mM HEPES (pH 7.5), 10 mM NaCl, 5 mM MgCl2, 25 mM NaF, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 2 mM sodium orthovanadate, 10 _g/ml aprotinin, 10 _g/ml soybean trypsin inhibitor, 25 _g/ml leupeptin, 2 mM PMSF, and 6.4 mg/ml p -nitrophenyl phosphate. Phospho-STAT3, phospho-Akt, phospho-MEK, and </li></ul><ul><li>phospho-p42/p44 mitogen-activated protein kinase antibodies were obtained from Cell Signaling Technologies. </li></ul><ul><li>Antibodies to STAT3, JAK2, and phospho-JNK were purchased. </li></ul>
  13. 19. <ul><li>Phospho-JAK2 antibody came from Upstate Biotechnology. Membranes were blocked in either 5% milk in PBS (pH 7.4) containing 0.1% Tween 20 or 1% BSA in TBS (pH 7.5) containing 0.1% Tween 20. </li></ul><ul><li>Phospho-specific antibodies (except phospho-mitogen-activated </li></ul><ul><li>protein kinase and phospho-JNK) were incubated in 1% BSA in TBS (pH 7.5) containing 0.1% Tween 20, </li></ul><ul><li>whereas all other antibodies were diluted in 5% milk in PBS (pH 7.4) containing 0.1% Tween 20 for either 2 h at room temperature or overnight at 4°C. </li></ul>
  14. 20. <ul><li>Horseradish peroxidase-conjugated secondary </li></ul><ul><li>antibodies (Jackson ImmunoResearch) were diluted in 5% milk in either PBS (pH 7.4) containing 0.1% Tween 20 or TBS (pH 7.5) containing 0.1% Tween 20 at a 1:1000 dilution for 1 h at room temperature. </li></ul><ul><li>Western blots were visualized using enhanced chemiluminescence </li></ul>
  15. 21. Immunoprecipitation of STAT3 <ul><li>A549 cells were treated for 4 h with vehicle or JSI-124 and then lysed in 150 mM HEPES (pH 7.5), 150 mM NaCl, </li></ul><ul><li>1 mM EDTA, 0.5% NP40, 10% glycerol, 5 mM NaF, 1 mM DTT, 1 mM PMSF, 2 mM sodium orthovanadate, and 5 _g/ml leupeptin. </li></ul>
  16. 22. <ul><li>Sample lysates were collected and cleared, and then 500 _g of lysate were immunoprecipitated with50 ng of STAT3 antibody overnight at 4°C and rocked with 25 _l of protein A/G PLUS-agarose (Santa Cruz Biotechnology) for 1 h at 4°C. </li></ul><ul><li>Samples were washed four times with lysis buffer and then boiled in 2_ SDS-PAGE sample buffer and run on 10% SDS-PAGE gel. Protein was transferred to nitrocellulose </li></ul><ul><li>blotted as described above for both phospho-specific STAT3 and STAT3. </li></ul>
  17. 23. <ul><li>DNA Binding and Transcription </li></ul><ul><li>Transfection and Generation of Stable Clones </li></ul><ul><li>Preparation of Cytosolic Extracts </li></ul><ul><li>Nuclear Extract Preparation and Gel Shift Assay </li></ul>
  18. 24. JAK Kinase Assays <ul><li>A549, MDA-MB-468, and v-Src-transformed NIH3T3 cells were harvested, </li></ul><ul><li>washed three times in PBS [10 mM sodium phosphate (pH 7.4), 137 mM NaCl, and 1 mM sodium orthovanadate] </li></ul><ul><li>lysed for 30 min on ice in JAK kinase lysis buffer [25 mM HEPES (pH 7.4), 0.1% Triton X-100, 0.5 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, 10 _g/ml aprotinin, and 10 _g/ml leupeptin]. </li></ul>
  19. 25. <ul><li>Samples were spun at high speed to clear, and 800-1000 _g of protein were immunoprecipitated per treatment condition with 50 ng of either JAK1 or JAK2 antibody </li></ul><ul><li>with rocking overnight at 4°C. Twenty-five _l of protein A/G PLUSagarose were then added, and rocking continued for 1 h at 4°C. </li></ul><ul><li>Samples were spun to collect agarose pellet, and pellet was washed twice in wash buffer [50 mM HEPES (pH 7.4), 0.1% Triton X-100, 0.5 mM DTT, and 150 mM NaCl] </li></ul><ul><li>and once in phosphorylation buffer [50 mM HEPES (pH 7.4), 0.1% Triton X-100, 0.5 mM DTT, 6.25 mM manganese chloride, and 100 mM NaCl]. Kinase reactions were performed at 30°C for 15 min in a final volume of 100 _l of phosphorylation buffer. </li></ul>
  20. 26. <ul><li>Samples were pretreated with DMSO control, JSI-124, and control compounds [AG490 (100 _M) and PD180970 (2 _M)] before addition of 20 _Ci/sample [_-32P]ATP. </li></ul><ul><li>The reaction was halted using stop buffer (wash buffer _ 10 mM EDTA), samples were spun to collect pellet, </li></ul><ul><li>then pellet was washed once with stop buffer and twice with wash buffer. </li></ul><ul><li>Samples were then placed in 2_ SDS-PAGE sample buffer, boiled at 100°C </li></ul>
  21. 27. <ul><li>run on 8% SDS-PAGE gels to separate proteins. Autophosphorylation </li></ul><ul><li>results were visualized by autoradiography. </li></ul>
  22. 28. <ul><li>Src Kinase Assay </li></ul><ul><li>In Vitro Cellular Proliferation and TUNEL Assays </li></ul>
  23. 29. Antitumor Activity in the Nude Mouse Tumor Xenograft Model. <ul><li>Nude mice and C57 BL-6 mice (NCI, Bethesda, MD) were maintained in accordance with the Institutional Animal Care and Use Committee procedures and guidelines. </li></ul><ul><li>v-Src-transformed and oncogenic H-Ras-transformed NIH 3T3, A549, MDA-MB-468, and Calu-1 cells were harvested, resuspended in PBS, and injected s.c. into the right and left flank (10 _ 106 cells/flank) of 8-week-old female nude mice as reported previously (25). </li></ul>
  24. 30. <ul><li>Similarly, murine B16-F10 melanoma cells were injected s.c. into the right and left flank (106 cells/flank) of C57 black mice. </li></ul><ul><li>When tumors reached about 150 mm3, animals were randomized (5 animals/group; 2 tumors/animal) and dosed i.p. with 0.2 ml vehicle of drug once daily. Control animals received DMSO (20%) vehicle, </li></ul><ul><li>whereas treated animals were injected with JSI-124 (1 mg/kg/day) in 20% DMSO in water. The tumor volumes were determined by measuring the length ( l ) and the width ( w ) and calculating the volume ( V _ lw 2/2) </li></ul>
  25. 31. RESULTS <ul><li>Development of Phosphotyrosine STAT3-specific Cytoblot </li></ul><ul><li>High Throughput Assay and Identification of JSI-124. </li></ul><ul><li>Experiments in animal models using gene therapy with a dominant negative form of STAT3 and a constitutively active mutant of STAT3, as well as the prevalence of constitutively activated STAT3 in many human cancers, strongly suggest STAT3 as having a causal role in oncogenesis </li></ul>
  26. 32. <ul><li>constitutive activation of STAT3 induces genes such as cyclin D1, c-myc, and bcl-xl that are intimately involved in oncogenesis and tumor survival, coupled with the fact that constitutively activated STAT3 is required for survival of some human cancer cells, further validates the STAT3 signaling pathway as a selective cancer drug discovery target </li></ul>
  27. 33. <ul><li>we have identified JSI-124, a selective JAK/STAT3 signaling pathway inhibitor with potent antitumor activity against human tumors in nude mice, from the NCI Diversity Set of 1,992 compounds. </li></ul>
  28. 34. <ul><li>cucurbitacin I (JSI-124) reduced the levels of phosphotyrosine of constitutively activated STAT3 in many human cancer cell lines including pancreatic, lung, and breast carcinomas. This suppression in the levels of constitutively activated STAT3 resulted in blockade of STAT3 DNA-binding activity and STAT3-mediated gene transcription. JSI-124 was highly selective for disrupting STAT3 signaling over other pivotal oncogenic and tumor survival pathways. </li></ul>
  29. 35. <ul><li>JSI-124 may be more selective toward inhibiting the growth of tumors with constitutively activated STAT3. </li></ul><ul><li>Reduction in phosphotyrosine levels could be a result of either inhibition of protein tyrosine kinases or activation of protein phosphotyrosine phosphatases. STAT3 is known to be phosphotyrosine dephosphorylated by two protein phosphotyrosine phosphatases, </li></ul><ul><li>SHP-1 and SHP-2 </li></ul>