Dokument

Zusammenfassung:
An den beiden Ovarialkarzinomzelllinien SKOV-3 und BG1 wurden neben dem bereits in der Therapie etablierten Carboplatin die Proteasomen-Inhibitoren Bortezomib und MG-115 und der CDK-Inhibitor Flavopiridol mittels Durchflusszytometrie auf ihre antiproliferative Wirkung untersucht. Dabei konnte im Rahmen der Zellzyklusanalyse und des Annexin-V-Assays für alle vier Medikamente eine zeit- und konzentrationsabhängige antiproliferative Wirkung nachgewiesen werden. Da die Signaltransduktionswege der Apoptose mögliche Angriffpunkte in der modernen Tumortherapie darstellen, wurden aus den beiden Hauptsignalwegen exemplarisch Proteine ausgewählt, deren Detektion mittels Western Blot über eine mögliche Aktivierung durch die Medikamente Auskunft geben soll. Während bei beiden Zelllinien die zeit- und konzentrationsabhängige Apoptoseinduktion über den extrinsischen Weg durch Bortezomib nachgewiesen werden kann, gelingt dies nach Inkubation mit Flavopiridol lediglich bei den BG1-Zellen. Neben der Wirkung der Reinsubstanzen auf die Ovarialkarzinomzelllinien wurde der Frage nach einem möglichen Synergismus von Flavopiridol und den Proteasomeninhibitoren in Kombination mit Carboplatin nachgegangen. Dabei zeigt nur die Kombination von Flavopiridol mit Carboplatin konzentrationsabhängig eine signifikant synergistische antiproliferative Wirkung. Nach simultaner Behandlung mit Bortezomib und Carboplatin lässt sich bei der SKOV-3-Zelllinie, sowohl durchflusszytometrisch als auch im Western Blot, sogar ein deutlich antagonistischer Effekt dokumentieren. Als Schaltstelle vieler Signaltransduktionswege in der Zelle bietet die Interaktion mit dem NFκB-Protein einen viel versprechenden Ansatz in der Entwicklung neuer Strategien der Tumortherapie. Mittels ELISA wurde deshalb dessen Aktivität im Zytosol beider Karzinomzelllinien nach Inkubation mit den vier Substanzen untersucht. Während sich bei den BG1-Zellen eine Zunahme der NfĸB-Aktivität sowohl durch Carboplatin als Reinsubstanz, wie auch in Kombination mit Flavopiridol und den beiden Proteasomen-Inhibitoren abzeichnet, kommt es bei den SKOV-3-Zellen lediglich durch Flavopiridol zu einer signifikanten Modulation des Aktivitätslevels. Auch nach Kombination mit Carboplatin zeigt nur jener mit Flavopiridol inkubierte SKOV-3-Ansatz einen signifikanten Synergismus durch den Zusatz des Platinderivats.

Bibliographie / References

1. Gerschenson L, Rotello R. Apoptosis: a different type of cell death. FASEB J. 1992; 6: 2450-5.
2. Perren T, Swart A, Pfisterer J, Ledermann J, Pujade-Lauraine E, Kristensen G et al. A Phase 3 Trial of Bevacizumab in Ovarian Cancer. N Engl J Med. 2011; 365: 2484- 96.
3. Goff B, Mandel L, Melancon C, Muntz H. Frequency of Symptoms of Ovarian Cancer in Women Presenting to Primary Care Clinics. JAMA. 2004; 291: 2705-12.
4. Huwe A, Mazitschek R, Giannis A. "Small Molecules as Inhibitors of Cyclin- Dependent Kinases," Angewandte Chemie, International Edition, Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim. Angew Chem Int Ed Engl. 2003;42: 2122-38.
5. Li Y, Tanaka K, Li X, Okada T, Nakamura T, Takasaki M et al. Cyclin-dependent kinase inhibitor, flavopiridol, induces apoptosis and inhibits tumor growth in drug- resistant osteosarcoma and Ewing's family tumor cells. Int J Cancer. 2007; 121: 1212- 8.
6. Ahn H, Kim Y, Kim J, Han S, Shin J, Yang H. Mechanism of taxol-induced apoptosis in human SKOV3 ovarian carcinoma cells. J Cell Biochem. 2004; 91: 1043-52.
7. Bruening A, Mylonas I. New emerging drugs targeting the genomic integrity and replication machinery in ovarian cancer. Arch Gynecol Obstet. 2011; 283: 1087-96.
8. Mayer F, Mueller S, Malenke E, Kuczyk M, Hartmann J, Bokemeyer C. Induction of apoptosis by flavopiridol unrelated to cell cycle arrest in germ cell tumour derived cell lines. Invest New Drugs. 2005; 23: 205-11.
9. Singh S, Bhat M. Carboplatin induces apoptotic cell death through downregulation of constitutively active nuclear factor-kappaB in human HPV-18 E6-positive HEp-2 cells. Biochem Biophys Res Commun. 2004; 318: 346-53.
10. Synergistic interaction between the novel histone deacetylase inhibitor ST2782 and the proteasome inhibitor bortezomib in platinum-sensitive and resistant ovarian carcinoma cells. J Inorg Biochem. 2012; 113: 94-101.
11. Morice P, Joulie F, Camatte S, Atallah D, Rouzier R, Pautier P et al. Lymph node involvement in epithelial ovarian cancer: analysis of 276 pelvic and paraaortic lymphadenectomies and surgical implications. J Am Coll Surg. 2003; 197: 198-205.
12. Sharma R, Graham J, Mitchell H, Brooks A, Blagden S, Gabra H. Extended weekly dose-dense paclitaxel/carboplatin is feasible and active in heavily pre-treated platinum- resistant recurrent ovarian cancer. Br J Cancer. 2009; 100: 707-12.
13. Aghajanian C. Clinical update: Novel targets in gynecologic malignancies. Semin Oncol. 2004; 31: 22-6
14. Proteasome inhibitors induce death but activate NF-kappaB on endometrial carcinoma cell lines and primary culture explants. J Biol Chem. 2006; 281: 22118-30.
15. Kim H, Park N, Chung H, Kim J, Song Y, Kang S. Significance of preoperative serum CA-125 levels in the prediction of lymph node metastasis in epithelial ovarian cancer. Acta Obstet Gynecol Scand. 2008; 87: 1136-42.
16. Weinberg R. The biology of cancer, New York, NY [u.a.]: GS Garland Science, 2007. 1: 255-356
17. Bazzaro M, Lee MZA, Stirling W, Santillan A, Shih I, Roden R. Ubiquitin- proteasome system stress sensitizes ovarian cancer to proteasome inhibitor-induced apoptosis. Cancer Res. 2006; 66: 3754-63.
18. Dose Finding Study for Combination Treatment with Topotecan and Gemcitabine of Patients with Recurrent Ovarian Cancer after Failure of First-Line Chemotherapy with Paclitaxel and Platinum. Onkologie. 2004; 27: 58-64.
19. Schmerwitz U, Sass G, Khandoga A, Joore J, Mayer B, Berberich N et al. Flavopiridol protects against inflammation by attenuating leukocyte-endothelial interaction via inhibition of cyclin-dependent kinase 9. Arterioscler Thromb Vasc Biol. 2011; 31: 280-8.
20. Havrilesky L, Darcy M, Hamdan H, Priore R, Leon J, Bell J et al. Prognostic significance of p53 mutation and p53 overexpression in advanced epithelial ovarian cancer: a Gynecologic Oncology Group Study. J Clin Oncol. 2003; 21: 3814-25.
21. Randomized Study of the Arbeitsgemeinschaft Gynaekologische Onkologie Ovarian Cancer Study Group Comparing Quality of Life in Patients With Ovarian Cancer Treated With Cisplatin/Paclitaxel Versus Carboplatin/Paclitaxel. J Clin Oncol. 2006 ; 24: 579-86.
22. Wang L, Ren D. Flavopiridol, the first cyclin-dependent kinase inhibitor: recent advances in combination chemotherapy. Mini Rev Med Chem. 2010; 10: 1058-70.
23. Sethi G, Sung B, Aggarwal B. Nuclear Factor-kappa B Activation: From Bench to Bedside. Exp Biol Med (Maywood). 2008; 233: 21-31.
24. Arlt A, Schaefer H. NFkappaB-dependent chemoresistance in solid tumors. Int J Clin Pharmacol Ther. 2002; 40 :336-47.
25. Phillips A, Vousden K. E2F-1 induced apoptosis. Apoptosis. 2001; 6: 173-82.
26. Evan G, Vousden K. Proliferation, cell cycle and apoptosis in cancer. Nature. 2001; 411: 342-8.
27. Reed E, Kohn E, Sarosy G, Dabholkar M, Davis P, Jacob J, Maher M. Paclitaxel, cisplatin, and cyclophosphamide in human ovarian cancer: molecular rationale and early clinical results. Semin Oncol. 1995; 22: 90-6.
28. Burger R, Brady M, Bookman M, Fleming G, Monk B, Huang H, et al. Incorporation of bevacizumab in the primary treatment of ovarian cancer. N Engl J Med. 2011; 365: 2473-83.
29. Schutte B. Detailed Analysis of Cell Cycle Kinetics Upon Proteasome Inhibition.
30. Johnson N, Shapiro G. Cyclin-dependent kinases (cdks) and the DNA damage response: rationale for cdk inhibitor-chemotherapy combinations as an anticancer strategy for solid tumors. Expert Opin Ther Targets. 2010; 14: 1199-212.
31. Aghajanian C, Dizon D, Sabbatini P, Raizer J, Dupont J, Spriggs D. Phase I Trial of Bortezomib and Carboplatin in Recurrent Ovarian or Primary Peritoneal Cancer. J Clin Oncol. 2005; 23: 5943-9.
32. Ramaswamy B, Phelps M, Baiocchi R, Bekaii-Saab T, Ni W, Lai J et al. A dose- finding, pharmacokinetic and pharmacodynamic study of a novel schedule of flavopiridol in patients with advanced solid tumors. Invest New Drugs. 2012; 30: 629- 38.
33. Abb. 28 Auswertung Annexin-V-Assay der BG1-Zellen nach Inkubation mit 1µmol Flavopiridol und Zugabe von 25µmol Carboplatin 63
34. Abb. 29 Änderung derZellvitalität der BG1-Zelllinie 65
35. Abb. 30 Western Blot-Analyse Caspasen 8
36. Abb. 31 Western Blot-Analyse Caspasen 8, Kombinationsversuche 67
37. Abb. 32 Western Blot-Analyse Caspasen 3
38. Abb. 33 Western Blot-Analyse Caspasen 3, Kombinationsversuche 69
39. Abb. 34 Western Blot-Analyse Cytochrom C bei SKOV-3-Zellen 69
40. Abb. 35 Western Blot-Analyse Cytochrom C bei BG1-Zellen 70
41. Abb. 36 relative Änderung der NfĸB-Aktivität der SKOV-3-Zelllinie 71
42. Abb. 37 relative Änderung der NfĸB-Aktivität der BG1-Zelllinie 72
43. Dai Y, Rahmani M, Pei X, Dent P, Grant S. Bortezomib and flavopiridol interact synergistically to induce apoptosis in chronic myeloid leukemia cells resistant to imatinib mesylate through both Bcr/Abl-dependent and -independent mechanisms.
44. Bortezomib treatment of ovarian cancer cells mediates endoplasmic reticulum stress, cell cycle arrest, and apoptosis Bortezomib treatment of ovarian cancer cells mediates endoplasmic reticulum stress, cell cycle arrest, and apoptosis. Invest New Drugs. 2009; 27: 543-51.
45. Wagstaff A, Ward A, Benfield P, Heel R. Carboplatin: A Preliminary Review of its Pharmacodynamic and Pharmacokinetic Properties and Therapeutic Efficacy in the Treatment of Cancer. Drugs. 1989; 37: 162-90.
46. [18] Bruesselbach S, Nettelbeck D, Sedlacek H, Mueller R. Cell cycle-independent induction of apoptosis by the anti-tumor drug. Int J Cancer. 1998;77: 146-52.
47. Bible K, Kaufmann S. Cytotoxic synergy between flavopiridol and various antineoplastic agents: importance of sequence of administration. Cancer Res. 1997; 57: 3375-80.
48. Tallarida R. Drug Synergism: Its Detection and Applications. J Pharmacol Exp Ther. 2001; 298: 865-72.
49. Cory A, Chen J, Cory J. Effects of PRIMA-1 on Wild-type L1210 Cells Expressing Mutant p53 and Drug-resistant L1210 Cells Lacking Expression of p53: Necrosis vs.
50. Permuth-Wey J, Sellers T. Epidemiology of ovarian cancer. Methods Mol Biol. 2009; 472: 413-37.
51. Takada Y, Aggarwal B. Flavopiridol Inhibits NF-kappaB -Activation Induced by Various Carcinogens and Inflammatory Agents through Inhibition of I?Ba-Kinase and p65 Phosphorylation. J Biol Chem. 2004; 279: 4750-9.
52. Christian B, Grever M, Byrd J, Lin T. Flavopiridol in the treatment of chronic lymphocytic leukemia. Curr Opin Oncol. 2007; 19: 573-8.
53. Boyd J, Rubin S. Hereditary Ovarian Cancer: Molecular Genetics and Clinical 7 Literaturangaben 94
54. Histopathological typing of ovarian tumours. World Health Organisation, Geneva. 1973.
55. Foti C, Demarchi F, Brancolini C. Inhibitors of the ubiquitin-proteasome system are not all alike. Autophagy. 2009; 5: 543-5.
56. Serov S, Scully R. Internationaö Histological Classification of Tumours, No. 9
57. Ligation, Hysterectomy, and Risk of Ovarian Cancer. JAMA. 1993; 270: 2813-8.
58. Al-Eisawi Z, Beale P, Chan C, Yu J, Huq F. Modulation of cisplatin cytotoxicity due to its combination with bortezomib and the nature of its administration. Anticancer Res. 2011; 31: 2757-62.
59. Fischer U, Schulze-Osthoff K. New Approaches and Therapeutics Targeting Apoptosis in Disease. Pharmacol Rev. 2005; 57: 187-215.
60. Pfizer Corporation Austria. Carboplatin " Pfizer " CS 150mg-Konzentrat zur Infusionsbereitung. 2013
61. Shapiro G. Preclinical and Clinical Development of the Cyclin-Dependent Kinase Inhibitor Flavopiridol. Clin Cancer Res. 2004; 10: 4270s-4275s.
62. Dou Q, McGuire T, Peng Y, An B. Proteasome Inhibition Leads to Significant Reduction of Bcr-Abl Expression and Subsequent Induction of Apoptosis in K562 Human Chronic Myelogenous Leukemia Cells. J Pharmacol Exp Ther. 1999; 289: 781- 90.
63. Post P, Casparie M, Blaauwgeers J, De Blok S. Reduced risk of ovarian cancer after hysterectomy. Acta Obstet Gynecol Scand. 2005; 84: 1024
64. Bast R. Status of Tumor Markers in Ovarian Cancer Screening. J Clin Oncol. 2003; 21: 200s-205s.
65. Wolf B, Green D. Suicidal Tendencies: Apoptotic Cell Death by Caspase Family Proteinases. J Biol Chem. 1999; 274: 20049-52.
66. Liu Z, MA N, Jiang Z, Yang G, Wang J, Zhao D. The experimental study of bortezomib induce ovarian cancer SKOV-3, SKOV-3/DDP cell proliferation and drug resistance reversion. J Clin Oncol (Meeting Abstracts) 2011; 29
67. Kelland L. Flavopiridol, the first cyclin-dependent kinase inhibitor to enter the clinic: current status. Expert Opin Investig Drugs. 2000; 9: 2903-11
68. Sterz J, von Metzler I, Hahne J, Lamottke B, Rademacher J, Heider U et al. The potential of proteasome inhibitors in cancer therapy. Expert Opin Investig Drugs. 2008; 17:879-95.
69. Song Y, Shen K, Tang P. Therapeutic effect of flavopiridol, a small molecular cyclin-dependent kinase inhibitor, in human ovarian carcinoma. Zhonghua Fu Chan Ke Za Zhi. 2007; 42: 761-4.
70. Banerjee S, Gore M. The Future of Targeted Therapies in Ovarian Cancer.
71. Armstrong M, Schumacher K, Mody R, Yanik G, Opipari AJ, Castle V. Bortezomib as a therapeutic candidate for neuroblastoma. J Exp Ther Oncol. 2008; 7: 135-45.
72. Budihardjo I, Holt O, Lutter M, Luo X, Wang X. Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol. 1999; 15: 269-90.
73. Krebs in Deutschland 2003 – 2004 Häufigkeiten und Trends. 2008. 6 ed. Robert Koch- Institut, Berlin [11] Baumann K, Kim H, Rinke J, Plaum T, Wagner U, Reinartz S. Effects of alvocidib and carboplatin on ovarian cancer cells in vitro. Exp Oncol. 2013; 35: 168-73.
74. Gazos Lopes U, Erhardt P, Yao R, Cooper G. p53-dependent Induction of Apoptosis by Proteasome Inhibitors. J Biol Chem. 1997; 272: 12893-6.
75. Comparative analysis of cell injury after exposure to antitumor platinum derivatives in kidney tubular epithelial cells. Chemotherapy. 2008; 54: 217-23.
76. Cohen G. Caspases: the executioners of apoptosis. Biochem J. 1997; 326: 1-16.
77. Fung-Kee-Fung M, Oliver T, Elit L, Oza A, Hirte H, Bryson P. Optimal chemotherapy treatment for women with recurrent ovarian cancer. Curr Oncol. 2007; 14: 195-208.
78. Kerr J, Wyllie A, Currie A. Apoptosis: a basic biological phenomenon with wide- ranging implications in tissue kinetics. Br J Cancer. 1972; 26: 239-57.
79. Lagunas V, Meléndez-Zajgla J. Nuclear Factor-kappa B as a Resistance Factor to Platinum-Based Antineoplasic Drugs. Met Based Drugs. 2008; 2008: 576104.
80. Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri E, Baehrecke E et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ. 2009; 16: 3-11
81. Vaseva A, Moll U. The mitochondrial p53 pathway. Biochim Biophys Acta. 2009; 1787: 414-20.
82. Chen D, Frezza M, Schmitt S, Kanwar J, Dou Q. Bortezomib as the First Proteasome Inhibitor Anticancer Drug: Current Status and Future Perspectives. Curr Cancer Drug Targets. 2011;11: 239-53.
83. Kao C, Chao A, Tsai C, Lin C, Chuang W, Chen H et al. Phosphorylation of signal transducer and activator of transcription 1 reduces bortezomib-mediated apoptosis in cancer cells. Cell Death Dis. 2013; 4: 512.
84. Singh S, Kumar Upadhyay A, Kumar Ajay A, Kumar Bhat M. p53 regulates ERK activation in carboplatin induced apoptosis in cervical carcinoma: A novel target of p53 in apoptosis. FEBS Lett. 2007; 581: 289-95.
85. Biliary excretion of flavopiridol and its glucuronides in the isolated perfused rat liver: role of multidrug resistance protein 2 (Mrp2). Life Sci. 2003; 73: 2841-54.
86. Meijer L, Leclerc S, Leost M. Properties and potential-applications of chemical inhibitors of cyclin-dependent kinases. Pharmacol Ther. 1999;82: 279-84.
87. Aggressive surgical strategies in advanced ovarian cancer: a monocentric study of 203 stage IIIC and IV patients. Eur J Surg Oncol. 2009;35: 135-43.
88. Boulikas T, Vougiouka M. Cisplatin and platinum drugs at the molecular level.
89. Xiong X, Sui MFW, Kraft A. Cell Cycle Dependent Antagonistic Interactions between Paclitaxel and Carboplatin in Combination Therapy. Cancer Biol Ther. 2007; 6:1067-73.

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