, Volume 13, Issue 9, pp 1135–1147 | Cite as

Downregulation of hepatoma-derived growth factor activates the Bad-mediated apoptotic pathway in human cancer cells

  • Tsun Yee Tsang
  • Wan Yee Tang
  • Wing Pui Tsang
  • Ngai Na Co
  • Siu Kai Kong
  • Tim Tak Kwok
Original Paper


Hepatoma-derived growth factor (HDGF) is highly expressed in human cancer and its expression is correlated with poor prognosis of cancer. The growth factor is known to stimulate cell growth while the underlying mechanism is however not clear. Transfection with HDGF cDNA stimulated while its specific antisense oligonucleotides repressed the growth of human hepatocellular carcinoma HepG2 cells. Furthermore, knock-down of HDGF by antisense oligos also induced apoptosis in HepG2 cells and in other human cancer cells, e.g. human squamous carcinoma A431 cells. HDGF knock-down was found to induce the expression of the pro-apoptotic protein Bad and also inactivate ERK and Akt, which in turn led to dephosphorylation of Bad at Ser-112, Ser-136, and activation of the intrinsic apoptotic pathway, i.e. depolarization of the mitochondrial membrane, release of mitochondrial cytochrome c, increase in the processing of caspase 9 and 3. As HDGF knock-down not only suppresses the growth but also induces apoptosis in human cancer cells, HDGF may therefore serve as a survival factor for human cancer cells and a potential target for cancer therapy.


HDGF Apoptosis Bad 



This work was supported by a Grant from the Shanghai-Hong Kong Anson Research Foundation, Hong Kong.

Supplementary material

10495_2008_241_MOESM1_ESM.tif (170 kb)
Supplementary Fig. 1 Apoptosis induction in HepG2 cells after knock-down of HDGF with HDGF specific siRNA. The cells were transfected with HDGF siRNA (Hi) and scramble siRNA (S) for 24h. The antisense strand sequence for the HDGF siRNA is GUAUUUGUUGGCUGUUGAU-dTdT. (A) After transfection, the cells were allowed to grow for 72h followed by DNA fragmentation assay. (B) The cells were allowed to grow for 48h after transfection followed by annexin V/propidium iodide staining assay. (C) The expression level of HDGF after transfection with HDGF siRNA was determined by Western blot analysis. The experiments have been repeated at least 3 times with similar results. The one shown is the representative one (TIF 170 kb)
10495_2008_241_MOESM2_ESM.tif (141 kb)
Supplementary Fig. 2 Apoptosis in HepG2 cells with knock-down of HDGF and/or Bad. The cells were transfected with HDGF antisense oligos with or without the Bad antisense oligos. After transfection, the cells were incubated for 48h followed by Annexin V binding assay. The % increase in apoptotic cells is calculated by subtracting the % of apoptotic cells in the group transfected with HDGF antisense oligos from that of the corresponding group transfected with the sense oligos. H-AS: transfection with HDGF antisense oligos only. H+B-AS: co-transfection with HDGF and Bad antisense oligos. The data are average from three independent experiments. Mean±SEM. *p<0.05, significantly different from those transfected with H-AS (TIF 141 kb)


  1. 1.
    Nakamura H, Kambe H, Egawa T, Kimura Y, Ito H, Hayashi E et al (1989) Partial purification and characterization of human hepatoma-derived growth factor. Clin Chim Acta 183:273–284. doi: 10.1016/0009-8981(89)90361-6 PubMedCrossRefGoogle Scholar
  2. 2.
    Sue SC, Chen JY, Lee SC, Wu WG, Huang TH (2004) Solution structure and heparin interaction of human hepatoma-derived growth factor. J Mol Biol 343:1365–1377. doi: 10.1016/j.jmb.2004.09.014 PubMedCrossRefGoogle Scholar
  3. 3.
    Abouzied MM, Baader SL, Dietz F, Kappler J, Gieselmann V, Franken S (2004) Expression patterns and different subcellular localization of the growth factors HDGF (hepatoma-derived growth factor) and HRP-3 (HDGF-related protein-3) suggest functions in addition to their mitogenic activity. Biochem J 378:169–176. doi: 10.1042/BJ20030916 PubMedCrossRefGoogle Scholar
  4. 4.
    Nakamura H, Izumoto Y, Kambe H, Kuroda T, Mori T, Kawamura K et al (1994) Molecular cloning of complementary DNA for a novel human hepatoma-derived growth factor. its homology with high mobility group-1 protein. J Biol Chem 269:25143–25149PubMedGoogle Scholar
  5. 5.
    Everett AD, Lobe DR, Matsumura ME, Nakamura H, McNamara CA (2000) Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development. J Clin Invest 105:567–575. doi: 10.1172/JCI7497 PubMedCrossRefGoogle Scholar
  6. 6.
    Oliver JA, Al-Awqati Q (1998) An endothelial growth factor involved in rat renal development. J Clin Invest 102:1208–1219. doi: 10.1172/JCI785 PubMedCrossRefGoogle Scholar
  7. 7.
    Enomoto H, Yoshida K, Kishima Y, Kinoshita T, Yamamoto M, Everett AD et al (2002) Hepatoma-derived growth factor is highly expressed in developing liver and promotes fetal hepatocyte proliferation. Hepatology 36:1519–1527PubMedGoogle Scholar
  8. 8.
    Machuy N, Thiede B, Rajalingam K, Dimmler C, Thieck O, Meyer TF et al (2005) A global approach combining proteome analysis and phenotypic screening with RNA interference yields novel apoptosis regulators. Mol Cell Proteomics 4:44–55. doi: 10.1074/mcp.M400089-MCP200 PubMedGoogle Scholar
  9. 9.
    Zhou Z, Yamamoto Y, Sugai F, Yoshida K, Kishima Y, Sumi H et al (2004) Hepatoma-derived growth factor is a neurotrophic factor harbored in the nucleus. J Biol Chem 279:27320–27326. doi: 10.1074/jbc.M308650200 PubMedCrossRefGoogle Scholar
  10. 10.
    Kishima Y, Yamamoto H, Izumoto Y, Yoshida K, Enomoto H, Yamamoto M et al (2002) Hepatoma-derived growth factor stimulates cell growth after translocation to the nucleus by nuclear localization signals. J Biol Chem 277:10315–10322. doi: 10.1074/jbc.M111122200 PubMedCrossRefGoogle Scholar
  11. 11.
    Nameki N, Tochio N, Koshiba S, Inoue M, Yabuki T, Aoki M et al (2005) Solution structure of the PWWP domain of the hepatoma-derived growth factor family. Protein Sci 14:756–764. doi: 10.1110/ps.04975305 PubMedCrossRefGoogle Scholar
  12. 12.
    Yoshida K, Nakamura H, Okuda Y, Enomoto H, Kishima Y, Uyama H et al (2003) Expression of hepatoma-derived growth factor in hepatocarcinogenesis. J Gastroenterol Hepatol 18:1293–1301. doi: 10.1046/j.1440-1746.2003.03191.x PubMedCrossRefGoogle Scholar
  13. 13.
    Hu TH, Huang CC, Liu LF, Lin PR, Liu SY, Chang HW et al (2003) Expression of hepatoma-derived growth factor in hepatocellular carcinoma. Cancer 98:1444–1456. doi: 10.1002/cncr.11653 PubMedCrossRefGoogle Scholar
  14. 14.
    Yoshida K, Tomita Y, Okuda Y, Yamamoto S, Enomoto H, Uyama H et al (2006) Hepatoma-derived growth factor is a novel prognostic factor for hepatocellular carcinoma. Ann Surg Oncol 13:159–167. doi: 10.1245/ASO.2006.11.035 PubMedCrossRefGoogle Scholar
  15. 15.
    Huang JS, Chao CC, Su TL, Yeh SH, Chen DS, Chen CT et al (2004) Diverse cellular transformation capability of overexpressed genes in human hepatocellular carcinoma. Biochem Biophys Res Commun 315:950–958. doi: 10.1016/j.bbrc.2004.01.151 PubMedCrossRefGoogle Scholar
  16. 16.
    El-Rifai W, Frierson HF Jr, Harper JC, Powell SM, Knuutila S (2001) Expression profiling of gastric adenocarcinoma using cDNA array. Int J Cancer 92:832–838. doi: 10.1002/ijc.1264 PubMedCrossRefGoogle Scholar
  17. 17.
    Lepourcelet M, Tou L, Cai L, Sawada J, Lazar AJ, Glickman JN et al (2005) Insights into developmental mechanisms and cancers in the mammalian intestine derived from serial analysis of gene expression and study of the hepatoma-derived growth factor (HDGF). Development 132:415–427. doi: 10.1242/dev.01579 PubMedCrossRefGoogle Scholar
  18. 18.
    Ren H, Tang X, Lee JJ, Feng L, Everett AD, Hong WK et al (2004) Expression of hepatoma-derived growth factor is a strong prognostic predictor for patients with early-stage non-small-cell lung cancer. J Clin Oncol 22:3230–3237. doi: 10.1200/JCO.2004.02.080 PubMedCrossRefGoogle Scholar
  19. 19.
    Bernard K, Litman E, Fitzpatrick JL, Shellman YG, Argast G, Polvinen K et al (2003) Functional proteomic analysis of melanoma progression. Cancer Res 63:6716–6725PubMedGoogle Scholar
  20. 20.
    Yamamoto S, Tomita Y, Hoshida Y, Morii E, Yasuda T, Doki Y et al (2007) Expression level of hepatoma-derived growth factor correlates with tumor recurrence of esophageal carcinoma. Ann Surg Oncol 14:2141–2149. doi: 10.1245/s10434-007-9369-9 PubMedCrossRefGoogle Scholar
  21. 21.
    Yamamoto S, Tomita Y, Hoshida Y, Takiguchi S, Fujiwara Y, Yasuda T et al (2006) Expression of hepatoma-derived growth factor is correlated with lymph node metastasis and prognosis of gastric carcinoma. Clin Cancer Res 12:117–122. doi: 10.1158/1078-0432.CCR-05-1347 PubMedCrossRefGoogle Scholar
  22. 22.
    Uyama H, Tomita Y, Nakamura H, Nakamori S, Zhang B, Hoshida Y et al (2006) Hepatoma-derived growth factor is a novel prognostic factor for patients with pancreatic cancer. Clin Cancer Res 12:6043–6048. doi: 10.1158/1078-0432.CCR-06-1064 PubMedCrossRefGoogle Scholar
  23. 23.
    Okuda Y, Nakamura H, Yoshida K, Enomoto H, Uyama H, Hirotani T et al (2003) Hepatoma-derived growth factor induces tumorigenesis in vivo through both direct angiogenic activity and induction of vascular endothelial growth factor. Cancer Sci 94:1034–1041. doi: 10.1111/j.1349-7006.2003.tb01397.x PubMedCrossRefGoogle Scholar
  24. 24.
    Kishima Y, Yoshida K, Enomoto H, Yamamoto M, Kuroda T, Okuda Y et al (2002) Antisense oligonucleotides of hepatoma-derived growth factor (HDGF) suppress the proliferation of hepatoma cells. Hepatogastroenterology 49:1639–1644PubMedGoogle Scholar
  25. 25.
    Zhang J, Ren H, Yuan P, Lang W, Zhang L, Mao L (2006) Down-regulation of hepatoma-derived growth factor inhibits anchorage-independent growth and invasion of non-small cell lung cancer cells. Cancer Res 66:18–23. doi: 10.1158/0008-5472.CAN-04-3905 PubMedCrossRefGoogle Scholar
  26. 26.
    Bae J, Hsu SY, Leo CP, Zell K, Hsueh AJ (2001) Underphosphorylated BAD interacts with diverse antiapoptotic bcl-2 family proteins to regulate apoptosis. Apoptosis 6:319–330. doi: 10.1023/A:1011319901057 PubMedCrossRefGoogle Scholar
  27. 27.
    Tan Y, Demeter MR, Ruan H, Comb MJ (2000) BAD ser-155 phosphorylation regulates BAD/Bcl-XL interaction and cell survival. J Biol Chem 275:25865–25869. doi: 10.1074/jbc.M004199200 PubMedCrossRefGoogle Scholar
  28. 28.
    Fang X, Yu S, Eder A, Mao M, Bast RC Jr, Boyd D et al (1999) Regulation of BAD phosphorylation at serine 112 by the ras-mitogen-activated protein kinase pathway. Oncogene 18:6635–6640. doi: 10.1038/sj.onc.1203076 PubMedCrossRefGoogle Scholar
  29. 29.
    Lizcano JM, Morrice N, Cohen P (2000) Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155. Biochem J 349:547–557. doi: 10.1042/0264-6021:3490547 PubMedCrossRefGoogle Scholar
  30. 30.
    Ayllon V, Martinez-A C, Garcia A, Cayla X, Rebollo A (2000) Protein phosphatase 1alpha is a ras-activated bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis. EMBO J 19:2237–2246. doi: 10.1093/emboj/19.10.2237 PubMedCrossRefGoogle Scholar
  31. 31.
    Chiang CW, Harris G, Ellig C, Masters SC, Subramanian R, Shenolikar S et al (2001) Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin- 3-dependent lymphoid cells by a mechanism requiring 14–3-3 dissociation. Blood 97:1289–1297. doi: 10.1182/blood.V97.5.1289 PubMedCrossRefGoogle Scholar
  32. 32.
    Wang Q, Huang Y, Ni Y, Wang H, Hou Y (2007) siRNA targeting midkine inhibits gastric cancer cells growth and induces apoptosis involved caspase-3, 8, 9 activation and mitochondrial depolarization. J Biomed Sci 14:783–795. doi: 10.1007/s11373-007-9192-0 PubMedCrossRefGoogle Scholar
  33. 33.
    Schinzel A, Kaufmann T, Borner C (2004) Bcl-2 family members: Integrators of survival and death signals in physiology and pathology. Biochim Biophys Acta 1644:95–105. corrected. doi: 10.1016/j.bbamcr.2003.09.006 Google Scholar
  34. 34.
    Donovan M, Cotter TG (2004) Control of mitochondrial integrity by bcl-2 family members and caspase-independent cell death. Biochim Biophys Acta 1644:133–147. doi: 10.1016/j.bbamcr.2003.08.011 PubMedCrossRefGoogle Scholar
  35. 35.
    Datta SR, Katsov A, Hu L, Petros A, Fesik SW, Yaffe MB et al (2000) 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell 6:41–51. doi: 10.1016/S1097-2765(00)00006-X PubMedCrossRefGoogle Scholar
  36. 36.
    Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ (1996) Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell 87:619–628. doi: 10.1016/S0092-8674(00)81382-3 PubMedCrossRefGoogle Scholar
  37. 37.
    Yang J, Everett AD (2007) Hepatoma-derived growth factor binds DNA through the N-terminal PWWP domain. BMC Mol Biol 8:101. doi: 10.1186/1471-2199-8-101 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Tsun Yee Tsang
    • 1
  • Wan Yee Tang
    • 1
    • 2
  • Wing Pui Tsang
    • 1
  • Ngai Na Co
    • 1
  • Siu Kai Kong
    • 1
  • Tim Tak Kwok
    • 1
  1. 1.Department of BiochemistryThe Chinese University of Hong KongShatinHong Kong SAR, China
  2. 2.Department of Environmental HealthUniversity of Cincinnati Medical CenterCincinnatiUSA

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