Alpha-Hederin, the Active Saponin of Nigella sativa, as an Anticancer Agent Inducing Apoptosis in the SKOV-3 Cell Line

Abstract

Alpha-hederin (α-HN), a pentacyclic triterpene saponin, has recently been identified as one of the active compounds of Nigella sativa, as a potential anticancer agent. However, no extensive studies on α-HN have been done as yet, as it was in the case of thymoquinone-the main ingredient of the N. sativa essential oil. To our knowledge, there are also no data available on how α-HN acts on the human cancer ovarian cell line SKOV-3. In this study we attempt to present the cytotoxic influence of α-HN on the SKOV-3 cell line by means of two methods: Real-Time xCELLigence and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The obtained IC₅₀ values are 2.62 ± 0.04 μg/mL and 2.48 ± 0.32 μg/mL, respectively. An induction of apoptosis in SKOV-3 cells was confirmed by staining cellular nuclei with Hoechst 33342 dye and by flow cytometry analysis by binding annexin V to the cell membranes. We found that α-HN induces apoptosis in a dose-dependent manner. In the first stages of apoptosis, the mitochondrial membrane potential was found to decrease. Also, inactivation of anti-apoptotic protein Bcl-2 was observed, as well as the caspase-9 and then caspase-3/7 activation. In addition, the treatment of SKOV-3 cells with α-HN induced the cell cycle arrest of cancer cells in G0/G1 phase. The results of our investigations indicate that α-HN induces apoptosis in the SKOV-3 cell line and that the intrinsic mitochondrial pathway is involved in the programmed cancer cell death.

Keywords: MMP; MTT assay; RTCA; annexin; caspases; cell cycle; flow cytometry; triterpene saponin.

Figure 1

The structure of alpha-hederin (α-HN) [1].

Figure 2

The SKOV-3 cells’ viability declines after the treatment with α-HN. The SKOV-3 cells were incubated with α-HN for 24 h. The curves are labeled with numbers that represent increasing concentrations of α-HN (1–70 µg/mL, respectively).

Figure 3

The dose-response curves obtained from the RTCA system and MTT assay after the SKOV-3 cells’ treatment with α-HN. α-HN was used in concentrations of 1–70 and 0.5–50 µg/mL, respectively. IC₅₀ values of α-HN were measured on the grounds of the dose-response curves by the Real-Time xCELLigence system (A) and MTT assay (B). Standard deviations are represented by error bars.

Figure 4

Apoptotic changes induced in SKOV-3 cells by α-HN. The state of SKOV-3 nuclear chromatin after treating with 0.1% DMSO (A) or α-HN at concentration of 0.5, 2 and 10 µg/mL (B–D) was evaluated by Hoechst 33342 staining. The cells after treatment with α-HN depict condensed chromatin (contrary to the DMSO control cells). Arrows point out apoptotic cells.

Figure 5

Apoptosis assay on SKOV-3 cells treated with α-HN. The SKOV-3 cells were examined by flow cytometry using annexin V/7-AAD staining method. The percentage of apoptotic cells was estimated in the control sample of the untreated cells (control), after the cells’ incubation with DMSO (control with DMSO) at concentration of 0.5% (A) or with α-HN at concentrations of 0.5–30 μg/mL for 24 h (particular concentrations are represented in pictures: 0.5 (B), 2 (C), 10 (D), 17 (E), and 30 μg/mL (F)). The mean values of three independent trials of the total apoptotic rate were calculated in comparison to the DMSO control (G). Standard deviation is represented by error bars. Significant differences relative to the DMSO control are marked with an * (p < 0.05).

Figure 6

α-HN triggered alterations in the inner transmembrane mitochondrial potential in SKOV-3 cells. The loss of potential was determined after 3 h in untreated SKOV-3 cells (control), in the cells treated with 0.5% DMSO (control with DMSO) (A), and within the cells treated with increasing concentrations of α-HN in the range 0.5–30 μg/mL (particular concentrations are represented in pictures: 0.5 (B), 2 (C), 10 (D), and 30 μg/mL (E)). The measurement of SKOV-3 mitochondrial depolarization was evaluated in relation to the 0.3% DMSO control (F). Each sample was run three times, independently. Standard deviation is represented by error bars. Significant differences relative to the control are marked with an * (p < 0.05).

Figure 7

Estimation of Bcl-2 inactivation in SKOV-3 cells treated with α-HN. The diagrams depict the mean values of three independent experiments with the levels of anti-apoptotic protein, Bcl-2, which was measured in SKOV-3 cells that were previously treated with 0.3% DMSO (control with DMSO) (A) and the increasing concentrations of α-HN in the range 0.5–30 µg/mL for 24 h (particular concentrations are represented in pictures: 0.5 (B), 2 (C), 10 (D), 17 (E), and 30 μg/mL (F)). The results are presented as percentages of non-expressing, activated (via phosphorylation), and inactivated cells (G). Control: The untreated cells.

Figure 8

Caspase-8 and caspase-9 activity after the SKOV-3 cells’ treatment with α-HN. A significant increase was observed in caspase-9 activity in the SKOV-3 cell line after 5 h of incubation with different concentrations of α-HN in comparison to the control (0.3% DMSO-treated cells). Each sample was run three times, independently. Error bars represent standard deviations. Significant differences relative to the DMSO control are marked with * (p < 0.05).

Figure 9

Caspase-3/7 activation in SKOV-3 cells treated with α-HN. Diagrams depict the mean values of early, late, and totally apoptotic cells of three independent experiments after incubation of the SKOV-3 cells with 0.3% DMSO (control with DMSO) (A) and increasing concentrations of α-HN in the range 0.5–30 µg/mL for 24 h (particular concentrations are represented in pictures: 0.5 (B), 2 (C), 10 (D), 17 (E), and 30 μg/mL (F)). Significant differences relative to DMSO control are marked with * (p < 0.05) (G). Error bars represent standard deviation. Control: the untreated cells.

Figure 10

Cell cycle analysis of the SKOV-3 cells treated for 24 h with increasing concentrations of α-HN. A dose-dependent increase of the cells in G0/G1 populations was compared with that of the untreated cells (control) and the DMSO control sample (control with DMSO). Diagram depicts the mean values of percentages of three independent analysis of the cells accumulated in particular cell cycle phase.