Advertisement

Molecular and Cellular Biochemistry

, Volume 383, Issue 1–2, pp 213–222 | Cite as

Hepatitis B virus X (HBx) play an anti-apoptosis role in hepatic progenitor cells by activating Wnt/β-catenin pathway

  • Lihong Shen
  • Xifeng Zhang
  • Daixi Hu
  • Tao Feng
  • Hongli Li
  • Yongliang Lu
  • Jiayi Huang
Article

Abstract

Increasing evidence has shown that normal stem cells may act as cancer-initiating cells and contribute to the development and progression of cancer. HBx has a close relationship with hepatocellular carcinoma, however, the role of HBx in hepatic progenitor cells (HPCs) is poorly understood. In this study, we sought to determine the role of HBx in regulating HPCs apoptosis and the underlying molecular mechanism(s) using HPCs derived from mouse fetal liver. The apoptotic ratio of HPCs infected with adenovirus-expressing HBx (Ad-HBx) was examined using flow cytometry. Results showed that the Ad-HBx treatment led to substantially decreased apoptotic ratio of HPCs, as confirmed by the Hoechst 33342 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Possible alterations of relative proteins were examined using Western blot and real-time PCR assays. The HBx expression in HPCs increased the expression levels of Bcl2 and Mcl1 while decreasing the expression levels of Bax and cleaved caspase-9 and -3. In addition, the mRNA and protein expression levels of β-catenin were both increased. The β-catenin protein were mainly accumulated in cytoplasm and tended to transfer into cell nucleus after Ad-HBx treatment. The over-expression of β-catenin decreased the apoptotic ratio of HPCs and inhibited the expression of cleaved caspase-9 and -3 while blocking β-catenin expression resulted in the opposite results. Taken together, our results strongly suggested that the HBx protein may inhibits apoptosis of hepatic progenitor cells, at least in part by activating the WNT/β-catenin pathway. This provided a new insight into the molecular mechanism of HBx-mediated live carcinogenesis.

Keywords

Hepatitis B virus X Hepatic progenitor cells Apoptosis WNT/β-catenin pathway Cancer stem cells 

Notes

Acknowledgments

We thank Dr. T. -C. He of The University of Chicago Medical Center for providing the cell lines, vectors and technical support. The reported work was supported by Research Grants from the Natural Science Foundation of China (Grant# 81071770, TF and Grant# 81201679, J Y H) .

Conflict of interest

The authors report no conflicts of interest.

References

  1. 1.
    Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM (2006) Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 66(19):9339–9344. doi: 10.1158/0008-5472.CAN-06-3126 PubMedCrossRefGoogle Scholar
  2. 2.
    Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367(6464):645–648. doi: 10.1038/367645a0 PubMedCrossRefGoogle Scholar
  3. 3.
    Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3(7):730–737PubMedCrossRefGoogle Scholar
  4. 4.
    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100(7):3983–3988. doi: 10.1073/pnas.0530291100 PubMedCrossRefGoogle Scholar
  5. 5.
    Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63(18):5821–5828PubMedGoogle Scholar
  6. 6.
    Matsui W, Huff CA, Wang Q, Malehorn MT, Barber J, Tanhehco Y, Smith BD, Civin CI, Jones RJ (2004) Characterization of clonogenic multiple myeloma cells. Blood 103(6):2332–2336. doi: 10.1182/blood-2003-09-3064 PubMedCrossRefGoogle Scholar
  7. 7.
    Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445(7123):111–115. doi: 10.1038/nature05384 PubMedCrossRefGoogle Scholar
  8. 8.
    Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ (2005) Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65(23):10946–10951. doi: 10.1158/0008-5472.CAN-05-2018 PubMedCrossRefGoogle Scholar
  9. 9.
    Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, Weissman IL, Clarke MF, Ailles LE (2007) Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA 104(3):973–978. doi: 10.1073/pnas.0610117104 PubMedCrossRefGoogle Scholar
  10. 10.
    Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, Hotz S, Van Belle PA, Xu X, Elder DE, Herlyn M (2005) A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 65(20):9328–9337. doi: 10.1158/0008-5472.CAN-05-1343 PubMedCrossRefGoogle Scholar
  11. 11.
    Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, Zheng BJ, Guan XY (2007) Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 132(7):2542–2556. doi: 10.1053/j.gastro.2007.04.025 PubMedCrossRefGoogle Scholar
  12. 12.
    Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM (2007) Identification of pancreatic cancer stem cells. Cancer Res 67(3):1030–1037. doi: 10.1158/0008-5472.CAN-06-2030 PubMedCrossRefGoogle Scholar
  13. 13.
    Eramo A, Lotti F, Sette G, Pilozzi E, Biffoni M, Di Virgilio A, Conticello C, Ruco L, Peschle C, De Maria R (2008) Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ 15(3):504–514. doi: 10.1038/sj.cdd.4402283 PubMedCrossRefGoogle Scholar
  14. 14.
    Fan X, Matsui W, Khaki L, Stearns D, Chun J, Li YM, Eberhart CG (2006) Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors. Cancer Res 66(15):7445–7452. doi: 10.1158/0008-5472.CAN-06-0858 PubMedCrossRefGoogle Scholar
  15. 15.
    Chiba T, Kita K, Zheng YW, Yokosuka O, Saisho H, Iwama A, Nakauchi H, Taniguchi H (2006) Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 44(1):240–251. doi: 10.1002/hep.21227 PubMedCrossRefGoogle Scholar
  16. 16.
    Peacock CD, Wang Q, Gesell GS, Corcoran-Schwartz IM, Jones E, Kim J, Devereux WL, Rhodes JT, Huff CA, Beachy PA, Watkins DN, Matsui W (2007) Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc Natl Acad Sci USA 104(10):4048–4053. doi: 10.1073/pnas.0611682104 PubMedCrossRefGoogle Scholar
  17. 17.
    Krivtsov AV, Twomey D, Feng Z, Stubbs MC, Wang Y, Faber J, Levine JE, Wang J, Hahn WC, Gilliland DG, Golub TR, Armstrong SA (2006) Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 442(7104):818–822. doi: 10.1038/nature04980 PubMedCrossRefGoogle Scholar
  18. 18.
    Bjerkvig R, Tysnes BB, Aboody KS, Najbauer J, Terzis AJ (2005) Opinion: the origin of the cancer stem cell: current controversies and new insights. Nat Rev Cancer 5(11):899–904. doi: 10.1038/nrc1740 PubMedCrossRefGoogle Scholar
  19. 19.
    Ganem D, Prince AM (2004) Hepatitis B virus infection–natural history and clinical consequences. N Engl J Med 350(11):1118–1129. doi: 10.1056/NEJMra031087 PubMedCrossRefGoogle Scholar
  20. 20.
    Neuveut C, Wei Y, Buendia MA (2010) Mechanisms of HBV-related hepatocarcinogenesis. J Hepatol 52(4):594–604. doi: 10.1016/j.jhep.2009.10.033 PubMedCrossRefGoogle Scholar
  21. 21.
    Tang H, Delgermaa L, Huang F, Oishi N, Liu L, He F, Zhao L, Murakami S (2005) The transcriptional transactivation function of HBx protein is important for its augmentation role in hepatitis B virus replication. J Virol 79(9):5548–5556. doi: 10.1128/JVI.79.9.5548-5556.2005 PubMedCrossRefGoogle Scholar
  22. 22.
    Benhenda S, Cougot D, Buendia MA, Neuveut C (2009) Hepatitis B virus X protein molecular functions and its role in virus life cycle and pathogenesis. Adv Cancer Res 103:75–109. doi: 10.1016/S0065-230X(09)03004-8 PubMedCrossRefGoogle Scholar
  23. 23.
    Sell S (1993) The role of determined stem-cells in the cellular lineage of hepatocellular carcinoma. Int J Dev Biol 37(1):189–201PubMedGoogle Scholar
  24. 24.
    Arzumanyan A, Friedman T, Ng IO, Clayton MM, Lian Z, Feitelson MA (2011) Does the hepatitis B antigen HBx promote the appearance of liver cancer stem cells? Cancer Res 71(10):3701–3708. doi: 10.1158/0008-5472.CAN-10-3951 PubMedCrossRefGoogle Scholar
  25. 25.
    Zhang WY, Cai N, Ye LH, Zhang XD (2009) Transformation of human liver L-O2 cells mediated by stable HBx transfection. Acta Pharmacol Sin 30(8):1153–1161. doi: 10.1038/aps.2009.99 PubMedCrossRefGoogle Scholar
  26. 26.
    Wang C, Yang W, Yan HX, Luo T, Zhang J, Tang L, Wu FQ, Zhang HL, Yu LX, Zheng LY, Li YQ, Dong W, He YQ, Liu Q, Zou SS, Lin Y, Hu L, Li Z, Wu MC, Wang HY (2011) HBx induces tumorigenicity of hepatic progenitor cells in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treated HBx transgenic mice. Hepatology. doi: 10.1002/hep.24675 Google Scholar
  27. 27.
    Huang J, Shen L, Lu Y, Li H, Zhang X, Hu D, Feng T, Song F (2012) Parallel induction of cell proliferation and inhibition of cell differentiation in hepatic progenitor cells by hepatitis B virus X gene. Int J Mol Med 30(4):842–848. doi: 10.3892/ijmm.2012.1060 PubMedGoogle Scholar
  28. 28.
    Huang J, Bi Y, Zhu GH, He Y, Su Y, He BC, Wang Y, Kang Q, Chen L, Zuo GW, Luo Q, Shi Q, Zhang BQ, Huang A, Zhou L, Feng T, Luu HH, Haydon RC, He TC, Tang N (2009) Retinoic acid signalling induces the differentiation of mouse fetal liver-derived hepatic progenitor cells. Liver Int 29(10):1569–1581. doi: 10.1111/j.1478-3231.2009.02111.x PubMedCrossRefGoogle Scholar
  29. 29.
    Bi Y, Huang J, He Y, Zhu GH, Su Y, He BC, Luo J, Wang Y, Kang Q, Luo Q, Chen L, Zuo GW, Jiang W, Liu B, Shi Q, Tang M, Zhang BQ, Weng Y, Huang A, Zhou L, Feng T, Luu HH, Haydon RC, He TC, Tang N (2009) Wnt antagonist SFRP3 inhibits the differentiation of mouse hepatic progenitor cells. J Cell Biochem 108(1):295–303. doi: 10.1002/jcb.22254 PubMedCrossRefGoogle Scholar
  30. 30.
    Shih WL, Kuo ML, Chuang SE, Cheng AL, Doong SL (2003) Hepatitis B virus X protein activates a survival signaling by linking SRC to phosphatidylinositol 3-kinase. J Biol Chem 278(34):31807–31813. doi: 10.1074/jbc.M302580200 PubMedCrossRefGoogle Scholar
  31. 31.
    Takada S, Kaneniwa N, Tsuchida N, Koike K (1997) Cytoplasmic retention of the p53 tumor suppressor gene product is observed in the hepatitis B virus X gene-transfected cells. Oncogene 15(16):1895–1901. doi: 10.1038/sj.onc.1201369 PubMedCrossRefGoogle Scholar
  32. 32.
    Diao J, Khine AA, Sarangi F, Hsu E, Iorio C, Tibbles LA, Woodgett JR, Penninger J, Richardson CD (2001) X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway. J Biol Chem 276(11):8328–8340. doi: 10.1074/jbc.M006026200 PubMedCrossRefGoogle Scholar
  33. 33.
    Kim KH, Seong BL (2003) Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal. EMBO J 22(9):2104–2116. doi: 10.1093/emboj/cdg210 PubMedCrossRefGoogle Scholar
  34. 34.
    Su F, Schneider RJ (1997) Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor alpha. Proc Natl Acad Sci USA 94(16):8744–8749PubMedCrossRefGoogle Scholar
  35. 35.
    Hu M, Kurobe M, Jeong YJ, Fuerer C, Ghole S, Nusse R, Sylvester KG (2007) Wnt/beta-catenin signaling in murine hepatic transit amplifying progenitor cells. Gastroenterology 133(5):1579–1591. doi: 10.1053/j.gastro.2007.08.036 PubMedCrossRefGoogle Scholar
  36. 36.
    Apte U, Thompson MD, Cui S, Liu B, Cieply B, Monga SP (2008) Wnt/beta-catenin signaling mediates oval cell response in rodents. Hepatology 47(1):288–295. doi: 10.1002/hep.21973 PubMedCrossRefGoogle Scholar
  37. 37.
    Yang W, Yan HX, Chen L, Liu Q, He YQ, Yu LX, Zhang SH, Huang DD, Tang L, Kong XN, Chen C, Liu SQ, Wu MC, Wang HY (2008) Wnt/beta-catenin signaling contributes to activation of normal and tumorigenic liver progenitor cells. Cancer Res 68(11):4287–4295. doi: 10.1158/0008-5472.CAN-07-6691 PubMedCrossRefGoogle Scholar
  38. 38.
    Shih WL, Kuo ML, Chuang SE, Cheng AL, Doong SL (2000) Hepatitis B virus X protein inhibits transforming growth factor-beta -induced apoptosis through the activation of phosphatidylinositol 3-kinase pathway. J Biol Chem 275(33):25858–25864. doi: 10.1074/jbc.M003578200 PubMedCrossRefGoogle Scholar
  39. 39.
    Huo TI, Wang XW, Forgues M, Wu CG, Spillare EA, Giannini C, Brechot C, Harris CC (2001) Hepatitis B virus X mutants derived from human hepatocellular carcinoma retain the ability to abrogate p53-induced apoptosis. Oncogene 20(28):3620–3628. doi: 10.1038/sj.onc.1204495 PubMedCrossRefGoogle Scholar
  40. 40.
    Gottlob K, Fulco M, Levrero M, Graessmann A (1998) The hepatitis B virus HBx protein inhibits caspase 3 activity. J Biol Chem 273(50):33347–33353PubMedCrossRefGoogle Scholar
  41. 41.
    Marusawa H, Matsuzawa S, Welsh K, Zou H, Armstrong R, Tamm I, Reed JC (2003) HBXIP functions as a cofactor of survivin in apoptosis suppression. EMBO J 22(11):2729–2740. doi: 10.1093/emboj/cdg263 PubMedCrossRefGoogle Scholar
  42. 42.
    Lee YI, Hwang JM, Im JH, Kim NS, Kim DG, Yu DY, Moon HB, Park SK (2004) Human hepatitis B virus-X protein alters mitochondrial function and physiology in human liver cells. J Biol Chem 279(15):15460–15471. doi: 10.1074/jbc.M309280200 PubMedCrossRefGoogle Scholar
  43. 43.
    Waris G, Huh KW, Siddiqui A (2001) Mitochondrially associated hepatitis B virus X protein constitutively activates transcription factors STAT-3 and NF-kappa B via oxidative stress. Mol Cell Biol 21(22):7721–7730. doi: 10.1128/MCB.21.22.7721-7730.2001 PubMedCrossRefGoogle Scholar
  44. 44.
    Kim S, Kim HY, Lee S, Kim SW, Sohn S, Kim K, Cho H (2007) Hepatitis B virus x protein induces perinuclear mitochondrial clustering in microtubule- and dynein-dependent manners. J Virol 81(4):1714–1726. doi: 10.1128/JVI.01863-06 PubMedCrossRefGoogle Scholar
  45. 45.
    Shirakata Y, Koike K (2003) Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential. J Biol Chem 278(24):22071–22078. doi: 10.1074/jbc.M301606200 PubMedCrossRefGoogle Scholar
  46. 46.
    Pan J, Duan LX, Sun BS, Feitelson MA (2001) Hepatitis B virus X protein protects against anti-Fas-mediated apoptosis in human liver cells by inducing NF-kappa B. J Gen Virol 82(Pt 1):171–182PubMedGoogle Scholar
  47. 47.
    Protzer U, Seyfried S, Quasdorff M, Sass G, Svorcova M, Webb D, Bohne F, Hosel M, Schirmacher P, Tiegs G (2007) Antiviral activity and hepatoprotection by heme oxygenase-1 in hepatitis B virus infection. Gastroenterology 133(4):1156–1165. doi: 10.1053/j.gastro.2007.07.021 PubMedCrossRefGoogle Scholar
  48. 48.
    Lee YI, Kang-Park S, Do SI (2001) The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade. J Biol Chem 276(20):16969–16977. doi: 10.1074/jbc.M011263200 PubMedCrossRefGoogle Scholar
  49. 49.
    Liu Q, Chen J, Liu L, Zhang J, Wang D, Ma L, He Y, Liu Y, Liu Z, Wu J (2011) The X protein of hepatitis B virus inhibits apoptosis in hepatoma cells through enhancing the methionine adenosyltransferase 2A gene expression and reducing S-adenosylmethionine production. J Biol Chem 286(19):17168–17180. doi: 10.1074/jbc.M110.167783 PubMedCrossRefGoogle Scholar
  50. 50.
    Liu H, Yuan Y, Guo H, Mitchelson K, Zhang K, Xie L, Qin W, Lu Y, Wang J, Guo Y, Zhou Y, He F (2012) Hepatitis B virus encoded X protein suppresses apoptosis by inhibition of the caspase-independent pathway. J Proteome Res 11(10):4803–4813. doi: 10.1021/pr2012297 PubMedCrossRefGoogle Scholar
  51. 51.
    Nejak-Bowen KN, Monga SP (2011) Beta-catenin signaling, liver regeneration and hepatocellular cancer: sorting the good from the bad. Semin Cancer Biol 21(1):44–58. doi: 10.1016/j.semcancer.2010.12.010 PubMedCrossRefGoogle Scholar
  52. 52.
    Cavard C, Colnot S, Audard V, Benhamouche S, Finzi L, Torre C, Grimber G, Godard C, Terris B, Perret C (2008) Wnt/beta-catenin pathway in hepatocellular carcinoma pathogenesis and liver physiology. Future Oncol 4(5):647–660. doi: 10.2217/14796694.4.5.647 PubMedCrossRefGoogle Scholar
  53. 53.
    Gheorghiade M, Palazzuoli A, Ronco C (2010) Acute heart failure treatment: traditional and new drugs. Contrib Nephrol 165:112–128 Retraction in Contrib Nephrol 167:VIPubMedCrossRefGoogle Scholar
  54. 54.
    Tetsu O, McCormick F (1999) Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398(6726):422–426. doi: 10.1038/18884 PubMedCrossRefGoogle Scholar
  55. 55.
    Orford K, Orford CC, Byers SW (1999) Exogenous expression of beta-catenin regulates contact inhibition, anchorage-independent growth, anoikis, and radiation-induced cell cycle arrest. J Cell Biol 146(4):855–868PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Lihong Shen
    • 1
    • 3
  • Xifeng Zhang
    • 1
    • 3
  • Daixi Hu
    • 1
    • 3
  • Tao Feng
    • 1
    • 3
  • Hongli Li
    • 1
    • 3
  • Yongliang Lu
    • 1
    • 3
  • Jiayi Huang
    • 1
    • 2
  1. 1.Molecular Medicine and Cancer Research CenterChongqing Medical UniversityChongqingPeople’s Republic of China
  2. 2.Departments of PathophysiologyChongqing Medical UniversityChongqingPeople’s Republic of China
  3. 3.Departments of Biochemistry and Molecular BiologyChongqing Medical UniversityChongqingPeople’s Republic of China

Personalised recommendations