Telomerase inhibition decreases alpha-fetoprotein expression and secretion by hepatocellular carcinoma cell lines: in vitro and in vivo study

PLoS One. 2015 Mar 30;10(3):e0119512. doi: 10.1371/journal.pone.0119512. eCollection 2015.

Abstract

Alpha-fetoprotein (AFP) is a diagnostic marker for hepatocellular carcinoma (HCC). A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. Apoptosis was determined through the Annexin V assay using flow cytometry. The concentrations of AFP were measured using ELISA kits. The AFP mRNA expression was evaluated using RT-PCR, and cell migration was evaluated using a Boyden chamber assay. The in vivo effect of costunolide on AFP production was tested in NSG mice. Telomerase inhibition by costunolide and BIBR 1532 at 5 and 10 μM decreased AFP mRNA expression and protein secretion by HepG2/C3A cells. The same pattern was obtained with cells treated with hTERT siRNA. This treatment exhibited no apoptotic effect. The AFP mRNA expression and protein secretion by PLC/PRF/5 was decreased after treatment with BIBR1532 at 10 μM. In contrast, no effect was obtained for PLC/PRF/5 cells treated with costunolide at 5 or 10 μM. Inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP concentration. In contrast, the MAPK/ERK pathway appeared to not be involved in HepG2/C3A cells, whereas ERK inhibition decreased the AFP concentration in PLC/PRF/5 cells. Modulation of the AFP concentration was also obtained after the inhibition or activation of PKC. Costunolide (30 mg/kg) significantly decreased the AFP serum concentration of NSG mice bearing HepG2/C3A cells. Both the inhibition of telomerase and the inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP production of HepG2/C3A and PLC/PRF/5 cells, suggesting a relationship between telomerase and AFP expression through the PI3K/Akt/mTOR pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminobenzoates / pharmacology
  • Animals
  • Apoptosis / drug effects
  • Carcinoma, Hepatocellular / drug therapy*
  • Carcinoma, Hepatocellular / metabolism*
  • Carcinoma, Hepatocellular / pathology
  • Cell Line, Tumor
  • Cell Movement / drug effects
  • Enzyme Inhibitors / pharmacology*
  • Hep G2 Cells
  • Humans
  • Liver Neoplasms / drug therapy*
  • Liver Neoplasms / metabolism*
  • Liver Neoplasms / pathology
  • Male
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • Naphthalenes / pharmacology
  • Neoplasm Invasiveness / pathology
  • Neoplasm Invasiveness / prevention & control
  • Phosphatidylinositol 3-Kinases / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Neoplasm / genetics
  • RNA, Neoplasm / metabolism
  • RNA, Small Interfering / genetics
  • Sesquiterpenes / pharmacology
  • Signal Transduction / drug effects
  • TOR Serine-Threonine Kinases / metabolism
  • Telomerase / antagonists & inhibitors*
  • Telomerase / genetics
  • Xenograft Model Antitumor Assays
  • alpha-Fetoproteins / genetics
  • alpha-Fetoproteins / metabolism*

Substances

  • AFP protein, human
  • Aminobenzoates
  • BIBR 1532
  • Enzyme Inhibitors
  • Naphthalenes
  • RNA, Messenger
  • RNA, Neoplasm
  • RNA, Small Interfering
  • Sesquiterpenes
  • alpha-Fetoproteins
  • costunolide
  • MTOR protein, human
  • Proto-Oncogene Proteins c-akt
  • TOR Serine-Threonine Kinases
  • TERT protein, human
  • Telomerase

Grant support

This work was supported, in whole or in part, by the Research Council of Saint-Joseph University and the Lebanese National Council for Scientific Research (CNRS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.