In vivo silencing of Reptin blocks the progression of human hepatocellular carcinoma in xenografts and is associated with replicative senescence

J Hepatol. 2010 May;52(5):681-9. doi: 10.1016/j.jhep.2009.12.029. Epub 2010 Mar 4.

Abstract

Background & aims: We previously showed that Reptin is overexpressed in hepatocellular carcinoma (HCC), and that in vitro depletion of Reptin with siRNAs led to HCC cell growth arrest and apoptosis. Here, we asked whether in vivo targeting of Reptin in established tumours had a therapeutic effect.

Methods: We used lentiviral vectors to construct HuH7 and Hep3B cell lines with doxycycline (Dox)-dependent expression of Reptin (R2) or control shRNA (GL2). Cells were injected subcutaneously into immunodeficient mice, and Dox was given when tumours reached a volume of 250 mm(3).

Results: In vitro, the growth of GL2-Dox, GL2+Dox, and R2-Dox cells was undistinguishable whereas that of R2+Dox cells stopped 4 days after Dox treatment. The growth decrease was associated with increased apoptosis, and evidence of replicative senescence, as shown by staining for acid beta-galactosidase and the presence of senescence-associated heterochromatin foci. In xenografted mice, R2+Dox tumour growth stagnated or even regressed with prolonged treatment in contrast with the GL2-Dox, GL2+Dox, and R2-Dox tumours that progressed steadily. The blockage of tumour progression was associated with the induction of senescence and reduced cell proliferation.

Conclusions: In vivo Reptin depletion leads to tumour growth arrest. Reptin may prove a valuable target in HCC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATPases Associated with Diverse Cellular Activities
  • Animals
  • Apoptosis / drug effects
  • Carcinoma, Hepatocellular / pathology
  • Carcinoma, Hepatocellular / prevention & control
  • Carrier Proteins / drug effects
  • Carrier Proteins / genetics*
  • Cell Division / drug effects
  • Cell Division / genetics
  • Cell Line, Tumor
  • Cellular Senescence / drug effects
  • DNA Helicases / drug effects
  • DNA Helicases / genetics*
  • DNA Primers
  • Doxycycline / pharmacology
  • Gene Expression Regulation, Neoplastic / drug effects
  • Gene Silencing*
  • Genes, Reporter / drug effects
  • Humans
  • Liver Neoplasms / pathology
  • Liver Neoplasms / prevention & control*
  • Luciferases / genetics
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • RNA, Neoplasm / genetics
  • RNA, Neoplasm / isolation & purification
  • RNA, Small Interfering / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transplantation, Heterologous

Substances

  • Carrier Proteins
  • DNA Primers
  • RNA, Neoplasm
  • RNA, Small Interfering
  • Luciferases
  • ATPases Associated with Diverse Cellular Activities
  • DNA Helicases
  • RUVBL2 protein, human
  • Doxycycline