Jsi 124 pathway

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Text of Jsi 124 pathway

  • 1. Presented by: Khuram Aziz
    • Discovery of JSI-124, a selective JAK/STAT3 inhibitor with antitumor activity against human and murine cancer cells in MICE

2. 3. 4. introduction

  • Discovery of disrupter in abberant activation of STAT
  • Supression of levels of phosphotyrosine STAT3 will inhibit proliferation and induce apoptosis
  • Use of JSI-124
  • Which are inhibited by it;
  • A549 tumors, v-src-transformed NIH 3T3 tumors, MDA-MB-468

5. 6. Which are not affected

  • Ras transformed tumors, akt pathways, c-jun nh2 terminal kinase, extracellular signal regulated kinases , calu-1

7. 8.

  • STATS are of diferent types.
  • They have dual role
  • They are involved in many physiological functions

9. 10.

  • For the biological functioning, these must be tyrosine phosphorylated
  • Tyrosine kinases that are involved to phosphorylate are non-RTKs, JAKs and peptide growth factors
  • Regulated by SHP-1 and SHP-2

11. STAT3 invovlment in oncogenesis is most thoroughly..

  • First, STAT3 is constitutively tyrosine phosphorylated
  • Second, for expression of activated mutant STAT3, stable dimerization was forced
  • Its validation as an anticancer drug target

12. 13. Materials and Methods

  • Cell Lines.All human and murine tumor cell lines used were obtained
  • from the American Type Culture Collection (Manassas, VA). Stably transfected
  • v-Src, oncogenic H-Ras, and vector NIH 3T3 cell lines have been described previously .

14. NCI Diversity Set.

  • The NCI Structural Diversity Set is a library of 1,992
  • compounds selected from the approximately 140,000-compound NCI drug
  • depository. These compounds were selected based on various criteria including
  • availability, drug-like structure, uniqueness of pharmacophore, and anticancer
  • activity as determined by cell growth inhibition assays against a panel of
  • human tumor cell lines. In-depth data on the selection, structures, and activities
  • of these diversity set compounds can be found on the NCI Developmental

15. Cytoblot Screening for Phospho-STAT3 Inhibition.

  • 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.
  • After overnight growth at 37C, the cells were treated for 4 h in the presence of either vehicle control or 10 _M of NCI Diversity Set compounds.
  • After treatment,cells were washed in 100 _l of cold TBS and
  • then fixed for 1 h at 4C with cold 3.7% formaldehyde in TBS (190 _l/well) as describedpreviously for a similar cytoblot for phospho-nucleolin (20).

16. .

  • Membranes were permeabilized during a 5-min incubation in ice-cold methanol at 4C.
  • Cells were washed with 3% milk in TBS (180 _l/well) and
  • then rocked overnight at 4C 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).
  • Antibodies were aspirated, and then plates were washed twice with TBS (180 _l/well).

17. .

  • Results were visualized by adding Western blot chemiluminescence reagent directly to the wells of the plates, incubating at room temperature for 5 min, and
  • then placing X-ray film directly on top of the plate in a dark room for 15 min. Quantification of results was done using a GS-700 scanning densitometer.

18. Western Blotting

  • 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/mlp -nitrophenyl phosphate. Phospho-STAT3, phospho-Akt, phospho-MEK, and
  • phospho-p42/p44 mitogen-activated protein kinase antibodies were obtained from Cell Signaling Technologies.
  • Antibodies to STAT3, JAK2, and phospho-JNK were purchased.

19.

  • 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.
  • Phospho-specific antibodies (except phospho-mitogen-activated
  • protein kinase and phospho-JNK) were incubated in 1% BSA in TBS (pH 7.5) containing 0.1% Tween 20,
  • 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 4C.

20.

  • Horseradish peroxidase-conjugated secondary
  • 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.
  • Western blots were visualized using enhanced chemiluminescence

21. Immunoprecipitation of STAT3

  • 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,
  • 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.

22.

  • Sample lysates were collected and cleared, and then 500 _g of lysate were immunoprecipitated with50 ng of STAT3 antibody overnight at 4C and rocked with 25 _l of protein A/G PLUS-agarose (Santa Cruz Biotechnology) for 1 h at 4C.
  • 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
  • blotted as described above for both phospho-specific STAT3 and STAT3.

23.

  • DNA Binding and Transcription
  • Transfection and Generation of Stable Clones
  • Preparation of Cytosolic Extracts
  • Nuclear Extract Preparation and Gel Shift Assay

24. JAK Kinase Assays

  • A549, MDA-MB-468, and v-Src-transformed NIH3T3 cells were harvested,
  • washed three times in PBS [10 mM sodium phosphate (pH 7.4), 137 mM NaCl, and 1 mM sodium orthovanadate]
  • 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].

25.

  • 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
  • with rocking overnight at 4C. Twenty-five _l of protein A/G PLUSagarose were then added, and rocking continued for 1 h at 4C.
  • 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]
  • 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 30C for 15 min in a final volume of 100 _l of phosphorylation buffer.

26.

  • 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.
  • The reaction was halted using stop buffer (wash buffer _ 10 mM EDTA), samples were spun to collect pellet,
  • then pellet was washed once with stop buffer and twice with wash buffer.
  • Samples were then placed in 2_ SDS-PAGE sample buffer, boiled at 100C

27.

  • run on 8% SDS-PAGE gels to separate proteins. Autophosphorylation
  • results were visualized by autoradiography.

28.

  • Src Kinase Assay
  • In VitroCellular Proliferation and TUNEL Assays

29. Antitumor Activity in the Nude Mouse Tumor Xenograft Model.

  • 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.
  • v-Src-transformed and oncogenic H-Ras-transformed NIH 3T3, A549, MDA-MB-468, and Calu-1 cells were harvested, resuspended in PBS, andinjected s.c. into the right and left flank (10 _ 106 cells/flank) of 8-week-old female nude mice as reported previously (25).

30.

  • Similarly, murine B16-F10 melanoma cells were injected s.c. into the right and left flank (106 cells/flank) of C57 black mice.
  • 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,
  • 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)