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

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.

Figure 1

The effect of chelidonine on the growth of HepG2 cells after 48 h exposure. Viability of cells at each concentration of chelidonine was evaluated by neutral red uptake test and is expressed as a percentage relative to untreated control cells. The values presented are the mean ± SD of six repeats each one in triplicate.

Figure 2

Morphological changes in HepG2 cells after 48 h treatment with 12 μmol/L chelidonine (LD₅₀) visualized by phase contrast microscopy. A: Apoptotic cells (black arrows) and a few cells undergoing blister cell death (white arrows) are clearly seen; B: Untreated control HepG2 cells.

Figure 3

Induction of DNA fragmentation in HepG2 cells after 48 h treatment with different concentrations of chelidonine. The concentration of chelidonine is indicated at the top of each well.

Figure 4

Telomerase activity using TRAP assay in HepG2 control and 48 h treated cells with different concentration of chelidonine up to LD₅₀. A: The relative activity of treated cells against untreated control cells are indicated as mean ± SD; B: TRAP products resolved on non-denaturing PAGE and stained with SYBR Green. The concentration of chelidonine is indicated on top of each well in μmol/L. C+: Positive control; C-: Negative control.

Figure 5

HepG2 cells were treated for 48 h with medium supplemented with solvent (ethanol, not exceeding 0.1%) or 0.1, 10 and 50 μmol/L chelidonine. Relative hTERT gene expression as measured by real-time RT-PCR which was calibrated with the constitutive expression of β2-microglobulin gene. hTERT level in control samples was considered as 100%. The values indicated as mean ± SD (n ≥ 6).

Figure 6

Number of population doublings after long-term treatment with a sub-apoptotic dose of chelidonine (0.1 μmol/L) or recovered cells after 48 h exposure to LD₅₀ concentration of chelidonine in comparison with control HepG2 cells. In re-treatment experiments (long-term treatment) HepG2 cells were exposed to 0.1 μmol/L only 48 h in each passage and followed by recovery with normal medium devoid of chelidonine. Each point indicates the mean value of a duplicated experiment.

Figure 7

The morphological changes and induction of senescence in HepG2 cells in a long-term treatment experiment. A: HepG2 cells after treatment five times with 0.1 μmol/L chelidonine; B: Cells retreated five times stained for β-galactosidase activity; C: Stained control cells.

Figure 8

Relative expression of Bcl-2 gene in HepG2 cells after 48 h treatment with different concentrations of chelidonine in relation to untreated control cells assessed by real-time RT-PCR and calibrated with β2-microglobulin gene expression. The expression level of Bcl-2 in control cells was considered as 100%. mean ± SD are indicated (n = 3).

Figure 9

Relative expression level of P-glycoprotein in HepG2 cells 48 h after treatment with different concentrations of chelidonine. Each bar indicates the mean ± SD (n = 3).