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Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
Identification of novel mechanisms involved in pro-apoptotic and ...
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Identification of novel mechanisms involved in pro-apoptotic and ...

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  • 1. Identification of novel mechanisms involved in pro-apoptotic and anti-metastatic effects of Cryo-Shock Conditioned Media on prostate cancer cells Mini Varghese, MD PGY-3, Department of Urology Contributors: Seema Gupta, Mohammed M. Shareef Program Director: Daniel B. Rukstalis, MD Research Mentor: Mansoor M. Ahmed, PhD Resident Research Day-May 16, 2008
  • 2. Introduction <ul><li>Cryotherapy is a surgical technique in which freezing is used to destroy undesirable tissue </li></ul><ul><li>Cryotherapy has been recognized by the American Urological Association as a therapeutic option for prostate cancer </li></ul>
  • 3. Introduction <ul><li>Cryotherapy induces direct cellular injury and tissue death via 2 mechanisms </li></ul><ul><ul><li>Rapid cooling </li></ul></ul><ul><ul><li>Slow cooling </li></ul></ul><ul><li>Cryotherapy also proposed to invoke indirect pathways that eventually lead to necrosis and death </li></ul><ul><ul><li>Vascular injury </li></ul></ul><ul><ul><li>Apoptosis </li></ul></ul><ul><ul><li>Synergy with systemic therapy ie chemotherapy </li></ul></ul>
  • 4. Hypothesis <ul><li>Specific proteins are secreted by prostate cancer cells in response to freezing temperatures </li></ul><ul><li>These proteins have both direct and bystander effects in cancer control </li></ul>
  • 5. Experiment Model <ul><li>PC3 (androgen independent, p53 null prostate cancer cell line) cultures are exposed to cold temperatures of 4 º C to -20 º C </li></ul><ul><li>Cryo Shock Conditioned Media (CSCM) and cells are harvested for protein and RNA analysis </li></ul><ul><li>Media then used with untreated PC3 cells to assess bystander effect on cell growth </li></ul>
  • 6. PC3 Tissue Cultures Cold Shock x 20min, Photographed Culture media: Protein precipitation with 1% DOC and 100% TCA Cell Pellet: tx with cell lysis buffer, then centrifuged 13G x 5min, decanted to save supernatant Cell Flasks Centrifuged @3G x5min, decanted Bradford assay used to estimate protein conc, protein resuspended in SDS buffer to make 1ug/uL Bradford assay used to estimate protein conc, tx with TCA-DOC and then resuspended in SDS buffer to make 1ug/uL
  • 7. Control (0h) 20x Control (6h) 20x 4C (0h) 20x 4C (6h) 20x
  • 8. Control (0h) 20x Control (6h) 20x -20C (0h) 20x -20C (6h) 20x
  • 9. SDS-PAGE of culture media at 0 hrs Incubation after cold shock tx at target temp 20min SDS-PAGE of culture media at 6 hrs incubation after cold shock tx at target temp 20 min control MM -20C -10C -5C 0C 4C MM control -20C -10C -5C 0C 4C 12% Tris-HCl SDS-PAGE (BioRad Ready Gels)
  • 10. MM Ctrl-0h -10C-0h -20C-0h -20C-0h-2x Ctrl-6h -10C-6h -20C-6h -20C-6h-2x Ctrl-0h -10C-0h -20C-0h Ctrl-6h -10C-6h -20C-6h 2 nd experiment 1 st experiment 15% SDS-PAGE
  • 11. MM Ctrl-0h -10C-0h -20C-0h -20C-0h-2x Ctrl-6h -10C-6h -20C-6h -20C-6h-2x Ctrl-0h -10C-0h -20C-0h Ctrl-6h -10C-6h -20C-6h 2 nd experiment 1 st experiment Band A Band B 15% SDS-PAGE
  • 12. MALDI-TOF:peptide sequencing <ul><li>matrix assisted laser desorption/ionization time of flight mass spectrometry </li></ul><ul><li>an analytical method that allows the detection of the composition of various biological components, such as oligonucleotides </li></ul><ul><li>A molecule is embedded in a matrix on a metal surface and desorbed into the gas phase inside the machine by the force of a laser beam </li></ul><ul><li>An electric field accelerates the probe </li></ul><ul><li>The samples are classified by their weight, which is determined by the amount of time it takes to pass through the drift tube. </li></ul>http://usa.eurogentec.com/code/en/page_08_455_0.htm
  • 13. MALDI-TOF results: Band A, B <ul><li>Band A </li></ul><ul><ul><li>High rank: CypA (17.999kDa) (cyclophilin A, peptidylprolyl isomerase A) </li></ul></ul><ul><ul><li>Low rank: NM23B (17.286kDa) (nonmetastatic protein 23, nucleoside diphosphatase kinase B) </li></ul></ul><ul><li>Band B </li></ul><ul><ul><li>CypA (17.999kDa)(cyclophilin A, peptidylprolyl isomerase A) </li></ul></ul>
  • 14. Unique proteins secreted by PC3 cells treated at -20C <ul><li>CypA (17.999 kDa) (ie cyclophilin A) </li></ul><ul><ul><li>Chemoattractant to neutrophils, eosinophils </li></ul></ul><ul><ul><li>Overexpressed in NSC lung ca and pancreatic ca </li></ul></ul><ul><ul><li>Involvement in apoptosis by activating caspases </li></ul></ul><ul><li>NM23B (17.286 kDa) (ie nonmetastatic protein 23) </li></ul><ul><ul><li>Complexes with G proteins </li></ul></ul><ul><ul><li>Transcriptional regulator for c-myc gene </li></ul></ul><ul><ul><li>Potential suppressor protein for metastasis </li></ul></ul>
  • 15. Cytokine Expression Array <ul><li>CSCM analyzed by TransSignal Human Cytokine Antibody Array </li></ul><ul><li>Significant induction of IL-1  in PC3 cells treated at - 20 º C </li></ul><ul><li>Chemoluminescence used to show antigen-antibody interaction: shown is autoradiograph. </li></ul>
  • 16. Bystander effect of CSCM <ul><li>Cryo Shock Conditioned Media was then used in untreated exponentially growing PC3 cell cultures and growth assessed using Real-Time Cell Electronic Sensing </li></ul><ul><li>80% reduction in cell growth noted in PC3 cells exposed to CSCM from cells cryo shocked at -10 º C </li></ul>
  • 17. Bystander effect of CSCM
  • 18. RT-PCR Apoptotic Gene SuperArray <ul><li>Cell lysate from treated PC3 cell cultures were then treated for isolation of RNA </li></ul><ul><li>RT-PCR was used to conduct apoptotic gene analysis </li></ul><ul><li>Significant upregulation of NOD1, moderate upregulation of CASP6 and BIRC8 in PC3 cells treated at 20 º C </li></ul><ul><li>NOD-1 reported to be involved in caspase induced cell death </li></ul>
  • 19. Apoptotic gene array for PC3 cells treated at 20 º C  NOD1  CASP6  BIRC8 Fold Difference Apoptotic gene array
  • 20. Conclusions <ul><li>Cryo-treatment at -20  C resulted in secretion of at least two proteins, CypA and NM23B </li></ul><ul><li>Cryo-treatment at -20  C also showed significant release of cytokine IL-1  </li></ul><ul><li>Cryo-treatment at -20  C also showed significant upregulation of NOD1, CASP6 and BIRC8 in apoptotic gene analysis </li></ul><ul><li>Cryo-shock conditioned media exerted a bystander effect on untreated PC3 cells that resulted in &gt;80% reduction in cell growth </li></ul>
  • 21. References <ul><ul><li>Al-Fageeh et al. Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems Biochem J. 2006 Jul 15;397(2):247-59 </li></ul></ul><ul><ul><li>Luban et al. Human immunodeficiency virus type 1 gag protein binds to cyclophilins A and B. Cell 73: 1067-1078, 1993 </li></ul></ul><ul><ul><li>Campa et al. Protein expression profiling identifies macrophage migration inhibitory factor and cyclophilin a as potential molecular targets in non-small cell lung cancer. Cancer Res . 2003;63:1652–1656 </li></ul></ul><ul><ul><li>Yao et al. Roles of Cyclophilins in Cancers and Other Organ Systems World J. Surg . 29, 276–280 (2005) </li></ul></ul><ul><ul><li>Gilles et al. Nucleoside diphosphate kinase from human erythrocytes: structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme. J. Biol. Chem. 266: 8784-8789, 1991 </li></ul></ul><ul><ul><li>Montague JW, Hughes FM, Jr., Cidlowski JA. Native recombinant cyclophilins A, B, and C degrade DNA independently of peptidylprolyl cis-trans-isomerase activity. Potential roles of cyclophilins in apoptosis. J Biol Chem 1997;272:6677-84 </li></ul></ul><ul><ul><li>Lee HY, Lee H. Inhibitory activity of nm23-H1 on invasion and colonization of human prostate carcinoma cells is not mediated by its NDP kinase activity. Cancer Lett 1999;145:93-9 </li></ul></ul><ul><ul><li>Mullerad J, Cohen S, Benharroch D, Apte RN. Local delivery of IL-1 alpha polymeric microspheres for the immunotherapy of an experimental fibrosarcoma. Cancer Invest 2003;21:720-8 </li></ul></ul><ul><ul><li>Correia J, Miranda Y, Leonard N, Hsu J, and Ulevitch RJ. Regulation of Nod1-mediated signaling pathways. Cell Death and Differentiation 2007; 14: 830-839 </li></ul></ul><ul><ul><li>Correia J, Miranda Y, Austin-Brown N, Hsu J, Mathison J, Xiang R, Zhou H, Li Q, Han J, and Ulevitch R. Nod1-dependent Control of Tumor Growth. PNAS 2006; 103: 1840-1845 </li></ul></ul><ul><ul><li>Boulos S, Meloni B, Arthur P, Majda B, Bojarski C, and Knuckey N. Evidence that intracellular cyclophilin A and cyclophilin A/CD147 receptor-mediated ERK1/2 signaling can protect neurons against in vitro oxidative and ischemic injury. Neurobiology of Disease 2007; 25: 54-64 </li></ul></ul><ul><ul><li>Choi KJ, Piao YJ, Lim MJ, Kim JH, Ha J, Choe W, Kim SS. Overexpressed cyclophilin A in cancer cells renders resistance to hypoxia- and cisplatin-induced cell death. Cancer Res 2007; 67: 3654-62 </li></ul></ul><ul><ul><li>Ricote M, Garcia-Tunon I, Bethencourt F, Fraile B, Paniagua R, and Royuela M. Interleukin-1 (IL-1a and IL-1b) and Its Receptors in Prostate Carcinoma. Cancer 2004; 100 (7): 1388-96 </li></ul></ul>

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