Abstract
Darbepoetin (DPO), an erythropoietin (EPO) derivative, was licensed in 2002 to treat patients with solid tumors suffering from chemotherapy-dependent anemia, although various tumors express EPO to improve vascularization, thus favoring tumor growth and spreading. Therefore, we wanted to investigate direct effects of DPO on the liver tumor cell lines HepG2, SkHep1, Huh-7, AKN1, HCC-T and HCC-M, as well as on primary human hepatocytes (hHeps). DPO (0–40 ng/ml) did not affect viability of hHeps, HepG2, SkHep1, AKN1, HCC-T and HCC-M cells, as determined by Resazurin conversion. However, Huh-7 cells’ viability dose-dependently decreased from 5 ng/ml DPO on. Lack of LDH release into culture medium and negative DNA laddering excluded apoptosis or necrosis as the cause for the reduced Resazurin conversion. In Huh-7 cells, DPO increased the expression of p53. Interestingly, Huh-7 cells showed the highest basal TGF-β1 expression as compared to the other cell lines. Upon inhibition of TGF-β1 signaling, DPO no longer reduced viability in Huh-7 cells. On the contrary, co-incubation with TGF-β1 made the other cell lines responsive to DPO. Summarizing our data, we show that DPO reduces the growth of Huh-7 cells by up-regulation of the tumor-suppressor gene p53. This mechanism seems to be dependent on a strong TGF-β expression and corresponding signaling in these cells, as other cell lines became responsive to DPO with TGF-β1 supplementation. The knowledge of this mechanism offers great perspectives for the understanding and treatment of solid liver tumors.
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Abbreviations
- DPO:
-
Darbepoetin
- hHeps:
-
Human hepatocytes
- TGF-β:
-
Transforming growth factor β
References
Akhurst RJ, Derynck R (2001) TGF-beta signaling in cancer—a double-edged sword. Trends Cell Biol 11(11):S44–S51
Arcasoy MO (2008) The non-haematopoietic biological effects of erythropoietin. Br J Haematol 141(1):14–31
Bader A, Pavlica S, Deiwick A, Lotkova H, Kucera O, Darsow K, Bartel S, Schulze M, Lange HA, Cervinkova Z (2011) Proteomic analysis to display the effect of low doses of erythropoietin on rat liver regeneration. Life Sci 89(23–24):827–833
Bockhorn M, Fingas CD, Rauen U, Canbay A, Sotiropoulos GC, Frey U, Sheu SY, Wohlschlager J, Broelsch CE, Schlaak JF (2008) Erythropoietin treatment improves liver regeneration and survival in rat models of extended liver resection and living donor liver transplantation. Transplantation 86(11):1578–1585. doi:10.1097/TP.0b013e31818b22b4
Cordenonsi M, Montagner M, Adorno M, Zacchigna L, Martello G, Mamidi A, Soligo S, Dupont S, Piccolo S (2007) Integration of TGF-beta and Ras/MAPK signaling through p53 phosphorylation. Science 315(5813):840–843
Dooley S, Hamzavi J, Breitkopf K, Wiercinska E, Said HM, Lorenzen J, Ten Dijke P, Gressner AM (2003) Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats. Gastroenterology 125(1):178–191
Ewen ME, Oliver CJ, Sluss HK, Miller SJ, Peeper DS (1995) p53-dependent repression of CDK4 translation in TGF-beta-induced G1 cell-cycle arrest. Genes Dev 9(2):204–217
Kausar H, Gull S, Ijaz B, Ahmad W, Sarwar MT, Iqbal Z, Nawaz Z, Riazuddin S, Hassan S (2011) Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO). J Transl Med 9:186
Klemm K, Eipel C, Cantre D, Abshagen K, Menger MD, Vollmar B (2008) Multiple doses of erythropoietin impair liver regeneration by increasing TNF-alpha, the Bax to Bcl-xL ratio and apoptotic cell death. PLoS One 3(12):e3924. doi:10.1371/journal.pone.0003924
Knobeloch D, Ehnert S, Schyschka L, Buchler P, Schoenberg M, Kleeff J, Thasler WE, Nussler NC, Godoy P, Hengstler J, Nussler AK (2012) Human hepatocytes: isolation, culture, and quality procedures. Methods Mol Biol 806:99–120. doi:10.1007/978-1-61779-367-7_8
Le Minh K, Klemm K, Abshagen K, Eipel C, Menger MD, Vollmar B (2007) Attenuation of inflammation and apoptosis by pre- and posttreatment of darbepoetin-alpha in acute liver failure of mice. Am J Pathol 170(6):1954–1963
Lee KH (2010) CaMKII inhibitor KN-62 blunts tumor response to hypoxia by inhibiting HIF-1alpha in hepatoma cells. Korean J Physiol Pharmacol 14(5):331–336. doi:10.4196/kjpp.2010.14.5.331
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275
Luo YH, Li ZD, Liu LX, Dong GH (2009) Pretreatment with erythropoietin reduces hepatic ischemia–reperfusion injury. Hepatobiliary Pancreat Dis Int 8(3):294–299
Mausberg AK, Meyer Zu Horste G, Dehmel T, Stettner M, Lehmann HC, Sheikh KA, Kieseier BC (2011) Erythropoietin ameliorates rat experimental autoimmune neuritis by inducing transforming growth factor-beta in macrophages. PLoS One 6(10):e26280. doi:10.1371/journal.pone.0026280
Muta H, Funakoshi A, Baba T, Uike N, Wakasugi H, Kozuru M, Jimi A (1994) Gene expression of erythropoietin in hepatocellular carcinoma. Intern Med 33(7):427–431
Prade-Houdellier N, Frebet E, Demur C, Gautier EF, Delhommeau F, Bennaceur-Griscelli AL, Gaudin C, Martinel V, Laurent G, Mansat-De Mas V, Beyne-Rauzy O (2007) Human telomerase is regulated by erythropoietin and transforming growth factor-beta in human erythroid progenitor cells. Leukemia 21(11):2304–2310
Ribatti D, Marzullo A, Gentile A, Longo V, Nico B, Vacca A, Dammacco F (2007) Erythropoietin/erythropoietin-receptor system is involved in angiogenesis in human hepatocellular carcinoma. Histopathology 50(5):591–596
Roumier C, Lejeune-Dumoulin S, Renneville A, Goethgeluck AS, Philippe N, Fenaux P, Preudhomme C (2006) Cooperation of activating Ras/rtk signal transduction pathway mutations and inactivating myeloid differentiation gene mutations in M0 AML: a study of 45 patients. Leukemia 20(3):433–436
Rupertus K, Sperling J, Corsten M, Scheuer C, Nickels RM, Schilling MK, Menger MD, Kollmar O (2010) Darbepoetin-alpha enhances hepatectomy-associated stimulation of colorectal liver metastatic growth. Ann Surg 252(1):131–141. doi:10.1097/SLA.0b013e3181e33915
Rupertus K, Senger S, Menger MD, Schilling MK, Kollmar O (2012) Darbepoetin-alpha promotes neovascularization and cell proliferation in established colorectal liver metastases. J Surg Res 176(2):517–523
Sanchez-Elsner T, Ramirez JR, Sanz-Rodriguez F, Varela E, Bernabeu C, Botella LM (2004) A cross-talk between hypoxia and TGF-beta orchestrates erythropoietin gene regulation through SP1 and Smads. J Mol Biol 336(1):9–24
Schmeding M, Hunold G, Ariyakhagorn V, Rademacher S, Boas-Knoop S, Lippert S, Neuhaus P, Neumann UP (2009) Erythropoietin reduces ischemia–reperfusion injury after liver transplantation in rats. Transpl Int 22(7):738–746
Seehofer D, Neumann UP, Schirmeier A, Carter J, Cho SY, Lederer A, Rayes N, Menger MD, Nussler AK, Neuhaus P (2008) Synergistic effect of erythropoietin but not G-CSF in combination with curcumin on impaired liver regeneration in rats. Langenbecks Arch Surg 393(3):325–332. doi:10.1007/s00423-008-0290-x
Siebert N, Xu W, Grambow E, Zechner D, Vollmar B (2011) Erythropoietin improves skin wound healing and activates the TGF-beta signaling pathway. Lab Invest 91(12):1753–1765
Sigal M, Siebert N, Zechner D, Menschikow E, Abshagen K, Vollmar B, Eipel C (2011) Darbepoetin-alpha inhibits the perpetuation of necro-inflammation and delays the progression of cholestatic fibrosis in mice. Lab Invest 90(10):1447–1456
Tentori L, Graziani G (2007) Doping with growth hormone/IGF-1, anabolic steroids or erythropoietin: is there a cancer risk? Pharmacol Res 55(5):359–369
Tovari J, Pirker R, Timar J, Ostoros G, Kovacs G, Dome B (2008) Erythropoietin in cancer: an update. Curr Mol Med 8(6):481–491
Tsuchiya A, Kubota T, Takizawa K, Yamada K, Wakai T, Matsuda Y, Honma T, Watanabe M, Shirai Y, Maruyama H, Nomoto M, Aoyagi Y (2009) Successful treatment in a case of massive hepatocellular carcinoma with paraneoplastic syndrome. Case Rep Gastroenterol 3(1):105–110. doi:10.1159/000213480
Vassiliou I, Lolis E, Nastos C, Tympa A, Theodosopoulos T, Dafnios N, Fragulidis G, Frangou M, Kondi-Pafiti A, Smyrniotis V (2010) The combined effect of erythropoietin and granulocyte macrophage colony stimulating factor on liver regeneration after major hepatectomy in rats. World J Surg Oncol 8:57
Vik A, Cui G, Isaksen V, Wik T, Hansen JB (2009) Erythropoietin production by a hepatic adenoma in a patient with severe erythrocytosis. Acta Haematol 121(1):52–55
Wang L, Schmitz V, Perez-Mediavilla A, Izal I, Prieto J, Qian C (2003) Suppression of angiogenesis and tumor growth by adenoviral-mediated gene transfer of pigment epithelium-derived factor. Mol Ther 8(1):72–79
Westenbrink BD, Oeseburg H, Kleijn L, van der Harst P, Belonje AM, Voors AA, Schoemaker RG, de Boer RA, van Veldhuisen DJ, van Gilst WH (2008) Erythropoietin stimulates normal endothelial progenitor cell-mediated endothelial turnover, but attributes to neovascularization only in the presence of local ischemia. Cardiovasc Drugs Ther 22(4):265–274. doi:10.1007/s10557-008-6094-y
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We would like to thank Marina Unger for excellent technical assistance.
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The authors declare that they have no conflict of interest.
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Sabrina Ehnert, Thomas Freude, Stefan Pscherer and Andreas K. Nussler have contributed equally.
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Ehnert, S., Freude, T., Eicher, C. et al. Darbepoetin inhibits proliferation of hepatic cancer cells in the presence of TGF-β. Arch Toxicol 88, 89–96 (2014). https://doi.org/10.1007/s00204-013-1094-5
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DOI: https://doi.org/10.1007/s00204-013-1094-5