Bergamottin and 5-Geranyloxy-7-methoxycoumarin Cooperate in the Cytotoxic Effect of Citrus bergamia (Bergamot) Essential Oil in Human Neuroblastoma SH-SY5Y Cell Line

Abstract

The plant kingdom has always been a treasure trove for valuable bioactive compounds, and Citrus fruits stand out among the others. Bergamottin (BRG) and 5-geranyloxy-7-methoxycoumarin (5-G-7-MOC) are two coumarins found in different Citrus species with well-acknowledged pharmacological properties. Previously, they have been claimed to be relevant in the anti-proliferative effects exerted by bergamot essential oil (BEO) in the SH-SY5Y human neuroblastoma cells. This study was designed to verify this assumption and to assess the mechanisms underlying the anti-proliferative effect of both compounds. Our results demonstrate that BRG and 5-G-7-MOC are able to reduce the proliferation of SH-SY5Y cells, inducing apoptosis and increasing cell population in sub-G0/G1 phase. Moreover, we demonstrated the pro-oxidant activity of the two coumarins that increased reactive oxygen species and impaired mitochondrial membrane potential. From a molecular point of view, BRG and 5-G-7-MOC were able to modulate apoptosis related factors at both protein and gene levels. Lastly, we evaluated the synergistic effect of their combination, finding that the highest synergy was observed at a concentration ratio similar to that occurring in the BEO, supporting our initial hypothesis. Taken together, our results deepen the knowledge regarding the effect of BRG and 5-G-7-MOC in SH-SY5Y cells, emphasizing the relevance of their cooperation in achieving this effect.

Keywords: 5-geranyloxy-7-methoxycoumarin; Citrus bergamia; bergamot; bergamottin; cancer; coumarin; pharmacological combination; psoralen.

Figure 1

Effects of BRG and 5-G-7-MOC (3.125 to 100 µM) on SH-SY5Y neuroblastoma cell proliferation for 24–72 h. Viability rate was assessed by MTT (A) and BrdU incorporation (B) tests. Results of both assays are expressed as percentages ± standard error of the means (SEM) of the absorbance values detected in the control (CTRL) cells. Each concentration was tested eight-fold and three independent experiments were carried out (n = 24). ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. CTRL.

Figure 2

Fluorescence-activated cell sorting (FACS) analysis of apoptosis in SH-SY5Y cells exposed to BRG and 5-G-7-MOC. The detection of apoptosis was performed through the Annexin V-FITC/PI test. Representative Annexin V vs PI dot plots of the SH-SY5Y cells treated with 25 and 50 μM of both compounds for the indicated periods are displayed. Q3 contains the viable cells (Annexin V −/PI −), Q4 contains the cells in early apoptosis (Annexin V +/PI −), Q2 contains the cells in late apoptosis (Annexin V +/PI +), while Q1 contains the necrotic ones (Annexin V −/PI +). Histograms depict the percentages of cells present in the corresponding quadrants ± SEM of three experiments separately performed in triplicate (n = 9).

Figure 3

Impact of BRG and 5-G-7-MOC on cell cycle progression of SH-SY5Y cells. The outcomes of the treatment for 48h with BRG and 5-G-7-MOC at 25 and 50 µM on cell cycle of SH-SY5Y cells were appreciated by the propidium iodide assay through flow cytometry. The plots are representative of three different experimental sessions performed in triplicate (n = 9). Percentages of cells present in each phase of the cell cycle are reported in the donut charts (sub G0/G1: white; G0/G1: green; S: yellow; G2/M: blue).

Figure 4

Generation of ROS and fall of ΔΨm in SH-SY5Y after treatment with BRG and 5-G-7-MOC. (A) ROS levels were assessed through the fluorescent probe DCFH-DA. (B) Variations of ΔΨm were evaluated through the cationic fluorochrome R123. Results of both assays are expressed as percentages ± SEM of the fluorescence values detected in the control cells of three different experiments for eight replicates (n = 24). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. CTRL.

Figure 5

Modulation of apoptosis-related gene and protein levels in SH-SY5Y cells treated with BRG and 5-G-7-MOC. Cells exposed to BRG and 5-G-7-MOC at 25 and 50 µM for 24 h were processed for mRNA and protein expression studies. (A) Relative quantities of mRNA, obtained through real-time PCR made in triplicate, were calculated by the 2^–∆∆Ct method, with β-actin as housekeeping gene. (B) Immunoblots of proteins, obtained from Western blotting studies, are shown along with their densitometric analyses, on the right. The expression of BAX, p53, Bcl-2 and Bcl-XL was normalized against β-actin (β-act), while that of both caspases is expressed as ratio of the cleaved form with respect to the relative zymogen. Results are expressed as fold change respect to untreated cells and expressed as mean ± SEM of three different sets of experiments performed in triplicate (n = 9). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. CTRL.

Figure 6

Synergistic effect of BRG and 5-G-7-MOC treatment in SH-SY5Y cells. Cell viability was assessed by MTT assay and the effect of the drug combination was calculated and visualized using SynergyFinder 2.0 software through the ZIP reference model. The synergy score is expressed as the mean of all δ scores for the dose-response analysis, and the maps shown are representative of three different experimental sessions. The red, white and green regions of the graph indicate the synergy, additivity and antagonism, respectively. The white square represents the highest synergistic area.