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. 2015 Feb 24;20(3):3730-43.
doi: 10.3390/molecules20033730.

Dose-dependent cytotoxic effects of boldine in HepG-2 cells-telomerase inhibition and apoptosis induction

Sakineh Kazemi Noureini  1 , Michael Wink  2
Affiliations

Dose-dependent cytotoxic effects of boldine in HepG-2 cells-telomerase inhibition and apoptosis induction

Sakineh Kazemi Noureini et al. Molecules. .

Abstract

Plant metabolites are valuable sources of novel therapeutic compounds. In an anti-telomerase screening study of plant secondary metabolites, the aporphine alkaloid boldine (1,10-dimethoxy-2,9-dihydroxyaporphine) exhibited a dose and time dependent cytotoxicity against hepatocarcinoma HepG-2 cells. Here we focus on the modes and mechanisms of the growth-limiting effects of this compound. Telomerase activity and expression level of some related genes were estimated by real-time PCR. Modes of cell death also were examined by microscopic inspection, staining methods and by evaluating the expression level of some critically relevant genes. The growth inhibition was correlated with down-regulation of the catalytic subunit of telomerase (hTERT) gene (p < 0.01) and the corresponding reduction of telomerase activity in sub-cytotoxic concentrations of boldine (p < 0.002). However, various modes of cell death were stimulated, depending on the concentration of boldine. Very low concentrations of boldine over a few passages resulted in an accumulation of senescent cells so that HepG-2 cells lost their immortality. Moreover, boldine induced apoptosis concomitantly with increasing the expression of bax/bcl2 (p < 0.02) and p21 (p < 0.01) genes. Boldine might thus be an interesting candidate as a potential natural compound that suppresses telomerase activity in non-toxic concentrations.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Boldine.
Figure 2
Figure 2
Dose responding viability of HepG-2 cells after 24 (light gray), 48 (gray) and 72 (black) h treatment with boldine (A). Cell viability of HEK293 (B) and HFF3 cells (C) after 48 h treatment with boldine. HepG-2 viability after 48 h treatment with berberine using MTT (D).
Figure 3
Figure 3
Dose-dependent inhibition of telomerase activity and hTERT mRNA levels in HepG-2 cells 48 h treated with boldine (P values are < 0.002 and 0.01 respectively).
Figure 4
Figure 4
HepG-2 and HFF cells seeded on cover slip (A and D respectively) and treated with 50 µg/mL boldine for 48 h (B and E respectively), fixed and viewed with inverted phase microscope. Fluorescence microscopy after staining with acridine orange and ethidium bromide (C and F respectively). The magnification is 200× in A and D, E, F and 400× in B and C. Relative expression of p21 p < 0.01, and bax/bxl2 in HepG-2 cells treated with boldine for 48 h; p < 0.02 (G). Apoptotic DNA fragmentation in HepG-2 cells after boldine treatment (from right to left: 0 (untreated control), 3, 30, 50 µg/mL, respectively) separated in agarose gel electrophoresis. HFF3 treated with 0, 5, 50 and 95 µg/mL showed no DNA fragmentation. DNA sample from HepG2 cells treated with staurosporin as a known apoptosis inducing compound and untreated cells, respectively (H).
Figure 4
Figure 4
HepG-2 and HFF cells seeded on cover slip (A and D respectively) and treated with 50 µg/mL boldine for 48 h (B and E respectively), fixed and viewed with inverted phase microscope. Fluorescence microscopy after staining with acridine orange and ethidium bromide (C and F respectively). The magnification is 200× in A and D, E, F and 400× in B and C. Relative expression of p21 p < 0.01, and bax/bxl2 in HepG-2 cells treated with boldine for 48 h; p < 0.02 (G). Apoptotic DNA fragmentation in HepG-2 cells after boldine treatment (from right to left: 0 (untreated control), 3, 30, 50 µg/mL, respectively) separated in agarose gel electrophoresis. HFF3 treated with 0, 5, 50 and 95 µg/mL showed no DNA fragmentation. DNA sample from HepG2 cells treated with staurosporin as a known apoptosis inducing compound and untreated cells, respectively (H).
Figure 5
Figure 5
Morphology of un-treated HepG-2 cells after β-galactosidase staining (A). Treated HepG-2 cells after four treatments with 3 µg/mL boldine 48 h in each of the consecutive passage (B) and after β-galactosidase staining (C). The magnification is 200× in all pictures. Percent of positively stained HepG-2 cells for β-galactosidase activity (D) and HepG2 cell doubling time in long-term treating with 3 µg/mL boldine 48 h per passage as described in text (E); each bar shows the average of data collected from two independent experiments each in duplicates.
Figure 5
Figure 5
Morphology of un-treated HepG-2 cells after β-galactosidase staining (A). Treated HepG-2 cells after four treatments with 3 µg/mL boldine 48 h in each of the consecutive passage (B) and after β-galactosidase staining (C). The magnification is 200× in all pictures. Percent of positively stained HepG-2 cells for β-galactosidase activity (D) and HepG2 cell doubling time in long-term treating with 3 µg/mL boldine 48 h per passage as described in text (E); each bar shows the average of data collected from two independent experiments each in duplicates.
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