Transcriptional down regulation of hTERT and senescence induction in HepG2 cells by chelidonine

World J Gastroenterol. 2009 Aug 7;15(29):3603-10. doi: 10.3748/wjg.15.3603.

Abstract

Aim: To investigate the potential effects of chelidonine, the main alkaloid of Chelidonium majus, on telomerase activity and its regulation in HepG2 cells.

Methods: Cytotoxicity of chelidonine for HepG2 cells was determined by neutral red assay. A modified polymerase chain reaction (PCR)-based telomerase repeat amplification protocol was used to estimate relative telomerase activity in chelidonine-treated cells in comparison with the untreated control cells. Relative expression level of the catalytic subunit of telomerase (hTERT) gene and P-glycoprotein (pgp) were estimated using semi-quantitative real-time reverse transcription-PCR (RT-PCR). Cell senescence in treated cells was demonstrated using a beta-galactosidase test.

Results: Cytotoxicity of chelidonine in HepG2 cells was not dose-dependent and tended to reach plateau immediately after the living cells were reduced in number to slightly higher than 50%. However, 12 micromol/L concentration of chelidonine was considered as LD(50), where the maximal attainable effects were realized. Real-time RT-PCR data showed that the expression of pgp increased three-fold in chelidonine treated HepG2 cells in comparison with the untreated controls. Morphologically, treated HepG2 cells showed apoptotic features after 24 h and a small fraction of cells appeared with single blister cell death. The relative expression level of Bcl-2 dropped to less than 50% of control cells at a sub-apoptotic concentration of chelidonine and subsequently increased to higher than 120% at LD(50). Telomerase activity was reduced considerably after administration of very low doses of chelidonine, whereas higher concentrations of chelidonine did not remarkably enhance the effect. Real-time RT-PCR experiments indicated a drastic decrease in expression level of hTERT subunit of telomerase under treatment with chelidonine. Repeated treatment of cells with very low doses of chelidonine caused a decline in growth rate by 4 wk and many of the cells appeared to be aged with large volume and dark staining in the beta-galactosidase assay.

Conclusion: Chelidonine reduces telomerase activity through down-regulation of hTERT expression. Senescence induction might not be directly caused by reducing telomerase activity as it occurs after a few population doublings.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • Antineoplastic Agents, Phytogenic / analysis
  • Apoptosis / drug effects
  • Benzophenanthridines / pharmacology*
  • Cell Line, Tumor
  • Cellular Senescence / drug effects
  • Chelidonium / chemistry
  • Down-Regulation / drug effects
  • Genes, bcl-2 / drug effects
  • Humans
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Telomerase / metabolism*
  • beta-Galactosidase / metabolism

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Antineoplastic Agents, Phytogenic
  • Benzophenanthridines
  • Proto-Oncogene Proteins c-bcl-2
  • chelidonine
  • TERT protein, human
  • Telomerase
  • beta-Galactosidase