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Apoptosis sensitizers enhance cytotoxicity in hepatoblastoma cells

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Abstract

Purpose

Drug resistance remains a major challenge for the treatment of high-risk hepatoblastoma (HB). To enhance effectiveness of chemotherapy we modulate apoptosis in HB cells in vitro.

Methods

Viability was monitored in HB cells (HuH6, HepT1) and fibroblasts in monolayer and spheroid cultures treated with ABT-737, obatoclax, HA14-1, and TW-37 and each in combination with CDDP, etoposide, irinotecan, paclitaxel, and DOXO in a MTT assay. Western blot analyses were performed to determine expressions of pro- and anti-apoptotic proteins.

Results

Obatoclax and ABT-737 led to a dose-dependent decrease of viability in HB cells at concentrations above 0.3 μM. TW-37 and HA14-1 were less effective. ABT-737 and obatoclax had additive effects when combined with CDDP, etoposide, irinotecan, paclitaxel, or DOXO. This was also observed for fibroblast, however, for higher drug concentrations. In spheroid cultures, relative expression of Bcl-XL was increased, Bax was decreased, Mcl-1 was low, and Bcl-2 was not detected compared to 2D cultures, denoting an anti-apoptotic state in spheroids. Obatoclax and ABT-737 have overcome the resistance to CDDP. HuH6 cells have shown higher susceptability for apoptosis sensitizers than HepT1.

Conclusion

The data provide evidence that ABT-737 and obatoclax might improve treatment results in children with HB.

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References

  1. Marin JJ, Romero MR, Martinez-Becerra P, Herraez E, Briz O (2009) Overview of the molecular bases of resistance to chemotherapy in liver and gastrointestinal tumours. Curr Mol Med 9(9):1108–1129 (CMM#09)

    Article  PubMed  CAS  Google Scholar 

  2. Fuchs J, Rydzynski J, von Schweinitz D, Bode U, Hecker H, Weinel P, Burger D, Harms D, Erttmann R, Oldhafer K, Mildenberger H (2002) Pretreatment prognostic factors and treatment results in children with hepatoblastoma: a report from the German Cooperative Pediatric Liver Tumor Study HB 94. Cancer 95(1):172–182. doi:10.1002/cncr.10632

    Article  PubMed  Google Scholar 

  3. Ortega JA, Douglass EC, Feusner JH, Reynolds M, Quinn JJ, Finegold MJ, Haas JE, King DR, Liu-Mares W, Sensel MG, Krailo MD (2000) Randomized comparison of cisplatin/vincristine/fluorouracil and cisplatin/continuous infusion doxorubicin for treatment of pediatric hepatoblastoma: a report from the Children’s Cancer Group and the Pediatric Oncology Group. J Clin Oncol 18(14):2665–2675

    PubMed  CAS  Google Scholar 

  4. Perilongo G, Maibach R, Shafford E, Brugieres L, Brock P, Morland B, de Camargo B, Zsiros J, Roebuck D, Zimmermann A, Aronson D, Childs M, Widing E, Laithier V, Plaschkes J, Pritchard J, Scopinaro M, MacKinlay G, Czauderna P (2009) Cisplatin versus cisplatin plus doxorubicin for standard-risk hepatoblastoma. N Engl J Med 361(17):1662–1670. doi:10.1056/NEJMoa0810613

    Article  PubMed  CAS  Google Scholar 

  5. Perilongo G, Shafford E, Maibach R, Aronson D, Brugieres L, Brock P, Childs M, Czauderna P, MacKinlay G, Otte JB, Pritchard J, Rondelli R, Scopinaro M, Staalman C, Plaschkes J (2004) Risk-adapted treatment for childhood hepatoblastoma. final report of the second study of the International Society of Paediatric Oncology—SIOPEL 2. Eur J Cancer 40(3):411–421 (S0959804903009328)

    Article  PubMed  CAS  Google Scholar 

  6. Warmann S, Gohring G, Teichmann B, Geerlings H, Fuchs J (2002) MDR1 modulators improve the chemotherapy response of human hepatoblastoma to doxorubicin in vitro. J Pediatr Surg 37(11):1579–1584 (S0022346802001653)

    Article  PubMed  Google Scholar 

  7. Zsiros J, Maibach R, Shafford E, Brugieres L, Brock P, Czauderna P, Roebuck D, Childs M, Zimmermann A, Laithier V, Otte JB, de Camargo B, MacKinlay G, Scopinaro M, Aronson D, Plaschkes J, Perilongo G (2009) Successful treatment of childhood high-risk hepatoblastoma with dose-intensive multiagent chemotherapy and surgery: final results of the SIOPEL-3HR study. J Clin Oncol 28(15):2584–2590. doi:10.1200/JCO.2009.22.4857

    Article  Google Scholar 

  8. Kang MH, Reynolds CP (2009) Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res 15(4):1126–1132. doi:10.1158/1078-0432.CCR-08-0144

    Article  PubMed  CAS  Google Scholar 

  9. Chonghaile TN, Letai A (2008) Mimicking the BH3 domain to kill cancer cells. Oncogene 27(Suppl 1):S149–S157. doi:10.1038/onc.2009.52

    Article  PubMed  CAS  Google Scholar 

  10. Adesina AM, Lopez-Terrada D, Wong KK, Gunaratne P, Nguyen Y, Pulliam J, Margolin J, Finegold MJ (2009) Gene expression profiling reveals signatures characterizing histologic subtypes of hepatoblastoma and global deregulation in cell growth and survival pathways. Hum Pathol 40(6):843–853. doi:10.1016/j.humpath.2008.10.022

    Article  PubMed  CAS  Google Scholar 

  11. Lieber J, Kirchner B, Eicher C, Warmann SW, Seitz G, Fuchs J, Armeanu-Ebinger S (2010) Inhibition of Bcl-2 and Bcl-X enhances chemotherapy sensitivity in hepatoblastoma cells. Pediatr Blood Cancer 55(6):1089–1095. doi:10.1002/pbc.22740

    Article  PubMed  Google Scholar 

  12. Pietsch T, Fonatsch C, Albrecht S, Maschek H, Wolf HK, von Schweinitz D (1996) Characterization of the continuous cell line HepT1 derived from a human hepatoblastoma. Lab Invest 74(4):809–818

    PubMed  CAS  Google Scholar 

  13. Doi I (1976) Establishment of a cell line and its clonal sublines from a patient with hepatoblastoma. Gann 67(1):1–10

    PubMed  CAS  Google Scholar 

  14. Eicher C, Dewerth A, Kirchner B, Warmann SW, Fuchs J, Armeanu-Ebinger S (2011) Development of a drug resistance model for hepatoblastoma. Int J Oncol 38(2):447–454. doi:10.3892/ijo.2010.860

    PubMed  CAS  Google Scholar 

  15. Chou TC (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58(3):621–681. doi:10.1124/pr.58.3.10

    Article  PubMed  CAS  Google Scholar 

  16. Yip KW, Reed JC (2008) Bcl-2 family proteins and cancer. Oncogene 27(50):6398–6406. doi:10.1038/onc.2008.307

    Article  PubMed  CAS  Google Scholar 

  17. Wang JL, Liu D, Zhang ZJ, Shan S, Han X, Srinivasula SM, Croce CM, Alnemri ES, Huang Z (2000) Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci USA 97(13):7124–7129 (97/13/7124)

    Article  PubMed  CAS  Google Scholar 

  18. Arisan ED, Kutuk O, Tezil T, Bodur C, Telci D, Basaga H (2010) Small inhibitor of Bcl-2, HA14–1, selectively enhanced the apoptotic effect of cisplatin by modulating Bcl-2 family members in MDA-MB-231 breast cancer cells. Breast Cancer Res Treat 119(2):271–281. doi:10.1007/s10549-009-0343-z

    Article  PubMed  CAS  Google Scholar 

  19. Oliver L, Mahe B, Gree R, Vallette FM, Juin P (2007) HA14-1, a small molecule inhibitor of Bcl-2, bypasses chemoresistance in leukaemia cells. Leuk Res 31(6):859–863. doi:10.1016/j.leukres.2006.11.010

    Article  PubMed  CAS  Google Scholar 

  20. Simonin K, Brotin E, Dufort S, Dutoit S, Goux D, N’Diaye M, Denoyelle C, Gauduchon P, Poulain L (2009) Mcl-1 is an important determinant of the apoptotic response to the BH3-mimetic molecule HA14-1 in cisplatin-resistant ovarian carcinoma cells. Mol Cancer Ther 8(11):3162–3170. doi:10.1158/1535-7163.MCT-09-0493

    Article  PubMed  CAS  Google Scholar 

  21. Hermanson D, Addo SN, Bajer AA, Marchant JS, Das SG, Srinivasan B, Al-Mousa F, Michelangeli F, Thomas DD, Lebien TW, Xing C (2009) Dual mechanisms of sHA 14-1 in inducing cell death through endoplasmic reticulum and mitochondria. Mol Pharmacol 76(3):667–678. doi:10.1124/mol.109.055830

    Article  PubMed  CAS  Google Scholar 

  22. Lickliter JD, Wood NJ, Johnson L, McHugh G, Tan J, Wood F, Cox J, Wickham NW (2003) HA14-1 selectively induces apoptosis in Bcl-2-overexpressing leukemia/lymphoma cells, and enhances cytarabine-induced cell death. Leukemia 17(11):2074–2080. doi:10.1038/sj.leu.2403102

    Article  PubMed  CAS  Google Scholar 

  23. Manero F, Gautier F, Gallenne T, Cauquil N, Gree D, Cartron PF, Geneste O, Gree R, Vallette FM, Juin P (2006) The small organic compound HA14-1 prevents Bcl-2 interaction with Bax to sensitize malignant glioma cells to induction of cell death. Cancer Res 66(5):2757–2764. doi:10.1158/0008-5472.CAN-05-2097

    Article  PubMed  CAS  Google Scholar 

  24. Niizuma H, Nakamura Y, Ozaki T, Nakanishi H, Ohira M, Isogai E, Kageyama H, Imaizumi M, Nakagawara A (2006) Bcl-2 is a key regulator for the retinoic acid-induced apoptotic cell death in neuroblastoma. Oncogene 25(36):5046–5055. doi:10.1038/sj.onc.1209515

    Article  PubMed  CAS  Google Scholar 

  25. Pei XY, Dai Y, Grant S (2004) The small-molecule Bcl-2 inhibitor HA14-1 interacts synergistically with flavopiridol to induce mitochondrial injury and apoptosis in human myeloma cells through a free radical-dependent and Jun NH2-terminal kinase-dependent mechanism. Mol Cancer Ther 3(12):1513–1524 (3/12/1513)

    PubMed  CAS  Google Scholar 

  26. Doshi JM, Tian D, Xing C (2007) Ethyl-2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H- chromene-3-carboxylate (HA 14-1), a prototype small-molecule antagonist against antiapoptotic Bcl-2 proteins, decomposes to generate reactive oxygen species that induce apoptosis. Mol Pharm 4(6):919–928. doi:10.1021/mp7000846

    Article  PubMed  CAS  Google Scholar 

  27. Tian D, Das SG, Doshi JM, Peng J, Lin J, Xing C (2008) sHA 14-1, a stable and ROS-free antagonist against anti-apoptotic Bcl-2 proteins, bypasses drug resistances and synergizes cancer therapies in human leukemia cell. Cancer Lett 259(2):198–208. doi:10.1016/j.canlet.2007.10.012

    Article  PubMed  CAS  Google Scholar 

  28. Wang G, Nikolovska-Coleska Z, Yang CY, Wang R, Tang G, Guo J, Shangary S, Qiu S, Gao W, Yang D, Meagher J, Stuckey J, Krajewski K, Jiang S, Roller PP, Abaan HO, Tomita Y, Wang S (2006) Structure-based design of potent small-molecule inhibitors of anti-apoptotic Bcl-2 proteins. J Med Chem 49(21):6139–6142. doi:10.1021/jm060460o

    Article  PubMed  CAS  Google Scholar 

  29. Ashimori N, Zeitlin BD, Zhang Z, Warner K, Turkienicz IM, Spalding AC, Teknos TN, Wang S, Nor JE (2009) TW-37, a small-molecule inhibitor of Bcl-2, mediates S-phase cell cycle arrest and suppresses head and neck tumor angiogenesis. Mol Cancer Ther 8(4):893–903. doi:10.1158/1535-7163.MCT-08-1078

    Article  PubMed  CAS  Google Scholar 

  30. Zeitlin BD, Joo E, Dong Z, Warner K, Wang G, Nikolovska-Coleska Z, Wang S, Nor JE (2006) Antiangiogenic effect of TW37, a small-molecule inhibitor of Bcl-2. Cancer Res 66(17):8698–8706. doi:10.1158/0008-5472.CAN-05-3691

    Article  PubMed  CAS  Google Scholar 

  31. Mohammad RM, Goustin AS, Aboukameel A, Chen B, Banerjee S, Wang G, Nikolovska-Coleska Z, Wang S, Al-Katib A (2007) Preclinical studies of TW-37, a new nonpeptidic small-molecule inhibitor of Bcl-2, in diffuse large cell lymphoma xenograft model reveal drug action on both Bcl-2 and Mcl-1. Clin Cancer Res 13(7):2226–2235. doi:10.1158/1078-0432.CCR-06-1574

    Article  PubMed  CAS  Google Scholar 

  32. Wang Z, Song W, Aboukameel A, Mohammad M, Wang G, Banerjee S, Kong D, Wang S, Sarkar FH, Mohammad RM (2008) TW-37, a small-molecule inhibitor of Bcl-2, inhibits cell growth and invasion in pancreatic cancer. Int J Cancer 123(4):958–966. doi:10.1002/ijc.23610

    Article  PubMed  CAS  Google Scholar 

  33. Forastiere AA (2008) Chemotherapy in the treatment of locally advanced head and neck cancer. J Surg Oncol 97(8):701–707. doi:10.1002/jso.21012

    Article  PubMed  CAS  Google Scholar 

  34. High LM, Szymanska B, Wilczynska-Kalak U, Barber N, O’Brien R, Khaw SL, Vikstrom IB, Roberts AW, Lock RB (2010) The Bcl-2 homology domain 3 mimetic ABT-737 targets the apoptotic machinery in acute lymphoblastic leukemia resulting in synergistic in vitro and in vivo interactions with established drugs. Mol Pharmacol 77(3):483–494. doi:10.1124/mol.109.060780

    Article  PubMed  CAS  Google Scholar 

  35. Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S, Deng X, Zhai D, Shi YX, Sneed T, Verhaegen M, Soengas M, Ruvolo VR, McQueen T, Schober WD, Watt JC, Jiffar T, Ling X, Marini FC, Harris D, Dietrich M, Estrov Z, McCubrey J, May WS, Reed JC, Andreeff M (2006) Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell 10(5):375–388. doi:10.1016/j.ccr.2006.10.006

    Article  PubMed  CAS  Google Scholar 

  36. Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, Bruncko M, Deckwerth TL, Dinges J, Hajduk PJ, Joseph MK, Kitada S, Korsmeyer SJ, Kunzer AR, Letai A, Li C, Mitten MJ, Nettesheim DG, Ng S, Nimmer PM, O’Connor JM, Oleksijew A, Petros AM, Reed JC, Shen W, Tahir SK, Thompson CB, Tomaselli KJ, Wang B, Wendt MD, Zhang H, Fesik SW, Rosenberg SH (2005) An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435(7042):677–681. doi:10.1038/nature03579

    Article  PubMed  CAS  Google Scholar 

  37. Trudel S, Stewart AK, Li Z, Shu Y, Liang SB, Trieu Y, Reece D, Paterson J, Wang D, Wen XY (2007) The Bcl-2 family protein inhibitor, ABT-737, has substantial antimyeloma activity and shows synergistic effect with dexamethasone and melphalan. Clin Cancer Res 13(2 Pt 1):621–629. doi:10.1158/1078-0432.CCR-06-1526

    Article  PubMed  CAS  Google Scholar 

  38. van Delft MF, Wei AH, Mason KD, Vandenberg CJ, Chen L, Czabotar PE, Willis SN, Scott CL, Day CL, Cory S, Adams JM, Roberts AW, Huang DC (2006) The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell 10(5):389–399. doi:10.1016/j.ccr.2006.08.027

    Article  PubMed  Google Scholar 

  39. Griffiths GJ, Dubrez L, Morgan CP, Jones NA, Whitehouse J, Corfe BM, Dive C, Hickman JA (1999) Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J Cell Biol 144(5):903–914

    Article  PubMed  CAS  Google Scholar 

  40. E-MEXP-1851 D (http://www.ebi.ac.uk/arrayexpress)

  41. Analysis RRDfGE (http://157.82.78.238/refexa/main_search.jsp)

  42. Konopleva M, Watt J, Contractor R, Tsao T, Harris D, Estrov Z, Bornmann W, Kantarjian H, Viallet J, Samudio I, Andreeff M (2008) Mechanisms of antileukemic activity of the novel Bcl-2 homology domain-3 mimetic GX15-070 (obatoclax). Cancer Res 68(9):3413–3420. doi:10.1158/0008-5472.CAN-07-1919

    Article  PubMed  CAS  Google Scholar 

  43. Siu WY, Arooz T, Poon RY (1999) Differential responses of proliferating versus quiescent cells to adriamycin. Exp Cell Res 250(1):131–141. doi:10.1006/excr.1999.4551

    Article  PubMed  CAS  Google Scholar 

  44. Langer CJ, Albert I, Kovacs P, Blakely LJ, Pajkos G, Petrov P, Somfay A, Szczesna A, Zatloukal P, Kazarnowicz A, Moezi MM, Schreeder MT, Schnyder J, Berger MS (2011) A randomized phase II study of carboplatin (C) and etoposide (E) with or without pan-BCL-2 antagonist obatoclax (Ob) in extensive-stage small cell lung cancer (ES-SCLC). J Clin Oncol 29 (suppl; abstr 7001)

    Google Scholar 

  45. Cvitkovic E (1998) Cumulative toxicities from cisplatin therapy and current cytoprotective measures. Cancer Treat Rev 24(4):265–281

    Article  PubMed  CAS  Google Scholar 

  46. von Schweinitz D, Byrd DJ, Hecker H, Weinel P, Bode U, Burger D, Erttmann R, Harms D, Mildenberger H (1997) Efficiency and toxicity of ifosfamide, cisplatin and doxorubicin in the treatment of childhood hepatoblastoma. Study Committee of the Cooperative Paediatric Liver Tumour Study HB89 of the German Society for Paediatric Oncology and Haematology. Eur J Cancer 33(8):1243–1249 (S0959804997000956)

    Article  Google Scholar 

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Acknowledgments

The authors wish to acknowledge ABBOTT Laboratories for providing ABT-737.

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Authors and Affiliations

  1. Department of Pediatric Surgery and Pediatric Urology, University Children’s Hospital, Hoppe-Seyler-Strasse 1, 72076, Tübingen, Germany

    Justus Lieber, Verena Ellerkamp, Julia Wenz, Bettina Kirchner, Steven W. Warmann, Jörg Fuchs & Sorin Armeanu-Ebinger

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  1. Justus Lieber
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  2. Verena Ellerkamp
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  3. Julia Wenz
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  7. Sorin Armeanu-Ebinger
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Correspondence to Justus Lieber.

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Lieber, J., Ellerkamp, V., Wenz, J. et al. Apoptosis sensitizers enhance cytotoxicity in hepatoblastoma cells. Pediatr Surg Int 28, 149–159 (2012). https://doi.org/10.1007/s00383-011-2988-z

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