Aged garlic extract and its constituent, S-allyl-L-cysteine, induce the apoptosis of neuroblastoma cancer cells due to mitochondrial membrane depolarization

Abstract

Aged garlic extract (AGE) has been demonstrated to have therapeutic properties in tumors; however its mechanisms of action have not yet been fully elucidated. A previous study revealed that AGE exerts an anti-proliferative effect on a panel of both sensitive [wild-type (WT)] and multidrug-resistant (MDR) human cancer cells. Following treatment of the cells with AGE, cytofluorimetric analysis revealed the occurrence of dose-dependent mitochondrial membrane depolarization (MMD). In this study, in order to further clarify the mechanisms of action of AGE, the effects of AGE on mitochondria isolated from rat liver mitochondria (RLM) were also examined. AGE induced an effect on the components of the electrochemical gradient (Δµ_H ⁺), mitochondrial membrane potential (ΔΨ_m) and mitochondrial electrochemical gradient (ΔpH_m). The mitochondrial membrane dysfunctions of RLM induced by AGE, namely the decrease in both membrane potential and chemical gradient were associated with a higher oxidation of both the endogenous glutathione and pyridine nucleotide content. To confirm the anti-proliferative effects of AGE, experiments were performed on the human neuroblastoma (NB) cancer cells, SJ-N-KP and the MYCN-amplified IMR5 cells, using its derivative S-allyl-L-cysteine (SAC), with the aim of providing evidence of the anticancer activity of this compound and its possible molecular mechanism as regards the induction of cytotoxicity. Following treatment of the cells with SAC at 20 mM, cell viability was determined by MTT assay and apoptosis was detected by flow cytometry, using Annexin V-FITC labeling. The percentages of cells undergoing apoptosis was found to be 48.0% in the SJ-N-KP and 50.1% in the IMR5 cells. By cytofluorimetric analysis, it was suggested that the target of SAC are the mitochondria. Mitochondrial activity was examined by labeling the cells with the probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylimidacarbocyanine iodide (JC-1). Following treatment with SAC at 50 mM, both NB cell lines exhibited a marked increase in MMD. On the whole, the findings of this study indicate that both natural products, AGE and SAC, cause cytotoxicity to tumor cells via the induction of mitochondrial permeability transition (MPT).

Keywords: S-allyl-L-cysteine; aged garlic extract; anti-proliferative activity; human neuroblastoma cancer cells; mitochondria; mitochondrial membrane depolarization.

Figure 1.

Effect of AGE on the components of the Δµ_H+, ΔΨ_m and ΔpH_m. RLM were incubated in standard medium as described in the Materials and methods section supplemented with 2 µM TPP+ for detecting ΔΨ_m and with 400 µM [14C]DMO (1 µCi/mmol) plus 5 mM [3H]glycerol (100 µCi/mmol) for detecting 58ΔpH_m. Where indicated, 2 mg/ml AGE was present. Both measurements of ΔΨ_m and ΔpH_m were performed on the same sample. A representative experiment is shown. Five other experiments yielded almost identical results. AGE, aged garlic extract; Δµ_H+, electrochemical gradient; ΔΨ_m, mitochondrial membrane potential; ΔpH_m, mitochondrial electrochemical gradient.

Figure 2.

Redox state of mitochondrial glutathione in the presence of AGE. RLM were incubated for 30 min in standard medium as described in the Materials and methods section. AGE concentrations are indicated in the figure. When present, 1 µM CsA. Results are the mean values ± SD of 5 experiments. Data were analyzed by one-way ANOVA, followed by Dunnett's post hoc test. Statistically significant differences (*P<0.05) are indicated by asterisks. AGE, aged garlic extract; RLM, rat liver mitochondria.

Figure 3.

Changes in the redox state of mitochondrial pyridine nucleotides in the presence of AGE. RLM were incubated for 30 min in standard medium as described in the Materials and methods section. AGE concentrations are indicated in the figure. When present, 1 µM CsA. A typical experiment is reported, 4 others yielded identical results. AGE, aged garlic extract; RLM, rat liver mitochondria.

Figure 4.

GC-MS chromatogram of SAC. SAC and internal standard were analyzed as TBDMS-derivatives. The inset presents the electron impact mass spectrum of SAC bearing two TBDMS-groups. GC-MS, gas chromatography-mass spectrometry; SAC, S-allyl-L-cysteine; TBDMS, tert-butyldimethylsilyl.

Figure 5.

GC-MS chromatogram of AGE. GC-MS, gas chromatography-mass spectrometry; AGE, aged garlic extract.

Figure 6.

(A and B) Anti-proliferative effects of SAC on human neuroblastoma cell lines. (A) SJ-N-KP and (B) IMR5 cells were treated with 0, 1, 5, 10, 20 and 30 mM of SAC alone for 48 h. The effects were determined by MTT assay. Each point represents the mean ± SEM of 2 independent experiments, with 3 wells per experiment. Where not shown, error bars lie within symbols. Data represent the means ± SD. SAC, S-allyl-L-cysteine.

Figure 7.

Annexin V/PI assay: Effect of SAC on SJ-N-KP and IMR5 cells. Flow cytometric analysis of apoptosis of neuroblastoma cells following double labeling with Annexin V-FITC and PI. Neuroblastoma cells were treated with increasing concentrations (0, 10 and 20 mM) of SAC. At 48 h after the end of the treatment, and incubation at 37°C, cells were analyzed by flow cytometry. (A) Representative Annexin V-FITC and PI flow cytometry dot plots of SJ-N-KP and IMR5 cells are shown. The x-axis represents FITC staining, and the y-axis represents PI staining. The percentage of cells displaying Annexin V-FITC positive/PI-negative (early apoptosis), Annexin V-FITC positive/PI-positive (late apoptotic or dead), Annexin V-FITC negative/PI-positive (necrotic) and double negative cells (viable cells) is indicated. The dot plots profile of cells were obtained from 1 out of 2 independent experiments, performed in the same experimental conditions, which yielded similar results. (B) Each bar represents the mean ± SD of normal or total apoptotic cells of 2 independent experiments. Data were analyzed by one-way ANOVA, followed by a Dunnett's post hoc test. (A) *P<0.05 vs. control SJ-N-KP cells; and (B) *P<0.05 and ***P<0.001 vs. control IMR5 cells. SAC, S-allyl-L-cysteine.

Figure 7.

Annexin V/PI assay: Effect of SAC on SJ-N-KP and IMR5 cells. Flow cytometric analysis of apoptosis of neuroblastoma cells following double labeling with Annexin V-FITC and PI. Neuroblastoma cells were treated with increasing concentrations (0, 10 and 20 mM) of SAC. At 48 h after the end of the treatment, and incubation at 37°C, cells were analyzed by flow cytometry. (A) Representative Annexin V-FITC and PI flow cytometry dot plots of SJ-N-KP and IMR5 cells are shown. The x-axis represents FITC staining, and the y-axis represents PI staining. The percentage of cells displaying Annexin V-FITC positive/PI-negative (early apoptosis), Annexin V-FITC positive/PI-positive (late apoptotic or dead), Annexin V-FITC negative/PI-positive (necrotic) and double negative cells (viable cells) is indicated. The dot plots profile of cells were obtained from 1 out of 2 independent experiments, performed in the same experimental conditions, which yielded similar results. (B) Each bar represents the mean ± SD of normal or total apoptotic cells of 2 independent experiments. Data were analyzed by one-way ANOVA, followed by a Dunnett's post hoc test. (A) *P<0.05 vs. control SJ-N-KP cells; and (B) *P<0.05 and ***P<0.001 vs. control IMR5 cells. SAC, S-allyl-L-cysteine.

Figure 8.

Cell cycle assay: Effect of SAC on SJ-N-KP and IMR5 cells. Cell cycle analysis carried out by flow cytometry on neuroblastoma cells. SJ-N-KP and IMR5 cells were treated with increasing concentrations (0, 10 and 20 mM) of SAC. At 48 h after the end of the treatment, and incubation at 37°C, followed by the staining with PI and RNase, the cells were analyzed by flow cytometry. (A) Representative histograms of sub-G1 analysis performed on SJ-N-KP and IMR5 cells using PI staining are shown. The percentage of the sub-G1 cell population is indicated. The histograms have been obtained from 1 out of 2 experiments carried out in the same experimental conditions, which gave similar results. (B) Each bar represents the mean ± SD of sub-G1 cell population of 2 independent experiments. Data were analyzed by one-way ANOVA, followed by Dunnett's post hoc test. ***P<0.001 vs. control SJ-N-KP or IMR5 cells. SAC, S-allyl-L-cysteine.

Figure 8.

Cell cycle assay: Effect of SAC on SJ-N-KP and IMR5 cells. Cell cycle analysis carried out by flow cytometry on neuroblastoma cells. SJ-N-KP and IMR5 cells were treated with increasing concentrations (0, 10 and 20 mM) of SAC. At 48 h after the end of the treatment, and incubation at 37°C, followed by the staining with PI and RNase, the cells were analyzed by flow cytometry. (A) Representative histograms of sub-G1 analysis performed on SJ-N-KP and IMR5 cells using PI staining are shown. The percentage of the sub-G1 cell population is indicated. The histograms have been obtained from 1 out of 2 experiments carried out in the same experimental conditions, which gave similar results. (B) Each bar represents the mean ± SD of sub-G1 cell population of 2 independent experiments. Data were analyzed by one-way ANOVA, followed by Dunnett's post hoc test. ***P<0.001 vs. control SJ-N-KP or IMR5 cells. SAC, S-allyl-L-cysteine.

Figure 9.

Effects of the treatment with SAC on mitochondrial membrane potential on SJ-N-KP and IMR5 cells. The neuroblastoma cells, SJ-N-KP and IMR5, were incubated with increasing concentrations (0, 30 and 50 mM) of SAC for 48 h at 37°C. Subsequently, mitochondrial membrane depolarization was assayed using JC-1 and was examined by flow cytometry. The ratio of red (JC-1 aggregate)/green (JC-1 monomer) fluorescence intensity was used to represent the mitochondrial membrane potential. Results represent the mean values ± SD from 3 independent experiments. Data were analyzed by one-way ANOVA, followed by Dunnett's post hoc test. *P<0.05 vs. control SJ-N-KP or IMR5 cells. SAC, S-allyl-L-cysteine.