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.2013 Apr 16;8(4):e61484.
doi: 10.1371/journal.pone.0061484. Print 2013.

Mechanisms underlying the anti-tumoral effects of Citrus Bergamia juice

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Mechanisms underlying the anti-tumoral effects of Citrus Bergamia juice

Simona Delle Monache et al. PLoS One..

Erratum in

Abstract

Based on the growing deal of data concerning the biological activity of flavonoid-rich natural products, the aim of the present study was to explore in vitro the potential anti-tumoral activity of Citrus Bergamia (bergamot) juice (BJ), determining its molecular interaction with cancer cells. Here we show that BJ reduced growth rate of different cancer cell lines, with the maximal growth inhibition observed in neuroblastoma cells (SH-SY5Y) after 72 hs of exposure to 5% BJ. The SH-SY5Y antiproliferative effect elicited by BJ was not due to a cytotoxic action and it did not induce apoptosis. Instead, BJ stimulated the arrest in the G1 phase of cell cycle and determined a modification in cellular morphology, causing a marked increase of detached cells. The inhibition of adhesive capacity on different physiologic substrates and on endothelial cells monolayer were correlated with an impairment of actin filaments, a reduction in the expression of the active form of focal adhesion kinase (FAK) that in turn caused inhibition of cell migration. In parallel, BJ seemed to hinder the association between the neural cell adhesion molecule (NCAM) and FAK. Our data suggest a mechanisms through which BJ can inhibit important molecular pathways related to cancer-associated aggressive phenotype and offer new suggestions for further studies on the role of BJ in cancer treatment.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. RP-HPLC chromatograms of BJ.
The analysis was performed at two different wavelengths, 283 (A) and 325 (B) nm. The sample was examined for five times and a representative chromatogram is shown. For peak identification see Table 1.
Figure 2
Figure 2. Effect of BJ on cell proliferation.
PC-12 (A), MDA-MB231 (B), PC3 (C), WI-38 (D) and SH-SY5Y (E) cells were incubated with bergamot juice (from 0.5 to 5%) for 24, 48 and 72 hs and assayed by MTT test. Results are expressed as percentage of absorbance respect to control cells (100%). Analysis of the SH-SY5Y proliferation was performed also by cell count assays (F). Experimental data showed that, although with different extent, BJ reduced growth rate of several cell lines, with the maximal effect in the SH-SY5Y. The results are expressed as means ± SEM from at least three independent experiments performed in eightplicate (MTT test) or in triplicate (cell counting). *P<0.05vs ctrl; **P<0.01vs ctrl; ***P<0.001vs ctrl, BJ 0.5 and 1%; °P<0.05vs BJ 2.5%;∧P<0.05vs BJ 1%.
Figure 3
Figure 3. Cytotoxic effect of BJ.
(A) Cytotoxic action of increasing concentrations of BJ (1–5%) was determined in SH-SY5Y, PC12 and WI-38 cells by trypan blue dye exclusion test. The assays were performed for 24, 48 and 72 hs and expressed as % of cell death. Data are the mean ± SEM of three independent experiments. Results display that BJ did not induced cytotoxicity neither in normal nor in tumoral cells. (B) Assessment of DNA damage in SH-SY5Y cells exposed to BJ performed by comet assay. In the panel are reported the images captured by fluorescence microscopy after 24 (on the left), 48 (in the middle) and 72 hs (on the right) of treatment. A representative experiment that was replicated three times with similar results is shown. The images display the round and intact nucleus observed in both BJ-treated and untreated cells, suggesting the lack of genotoxicity by BJ. HDNA: % head DNA. Nuclei were visualized by fluorescence microscopy at a magnification of 400x.
Figure 4
Figure 4. Evaluation of apoptosis on the SH-SY5Y cells exposed to BJ.
(A) Cytofluorimetric evaluation of apoptosis; representative Annexin Vversus PI dot plot analyses of SH-SY5Y cells treated with BJ for 72 hs. (B) Mean percentage of events in each quarter (Q) of cytofluorimetric analyses: Q3 contains the vital cells, Q4 the cells in early apoptosis, Q2 the cells in late apoptosis and Q1 the necrotic cells. The FACS analysis presented is representative of three different experiments. (C) Analysis of caspase-3 and caspase-9 expression in SH-SY5Y cells treated for 72 hs with BJ or etoposide (eto). Lower molecular weight bands of caspases represent the active form. Data show the lack of apoptosis after 72 hs of exposure to BJ.
Figure 5
Figure 5. Analysis of p53 and cyclin D1 expression in SH-SY5Y treated with BJ.
On the left of the panel are display the immunoblot of SH-SY5Y cells exposed to 1–5% BJ for 24–72 hs: a representative of three separate experiments is shown. On the right of the panel are presented the quantification ± SEM of both cyclin D1 and p53 expressions from three independent blots performed after 24–72 hs of BJ incubation. Autoradiographic bands were quantified by ImageJ software and normalized for β-actin levels. Data are extrapolated in reference of the values detected in control cells, which are arbitrarily assigned as 1. Results of Western blot documented that BJ decreases the expression of cyclin D1, unchanging the level of p53. *P<0.05vs ctrl.
Figure 6
Figure 6. Adhesion inhibition by BJ treatment.
(A) Representative images showing the decreased adhesive capacity of SH-SY5Y cells following 2 hs of BJ treatment. Cells were visualized by optical microscopy at a magnification of 200x. (B) Adhesion assay performed by plating SH-SY5Y and PC12 cells on non-coated plastic surface for 24 hs. Data were expressed as percentage of the total cells present in the well and are the mean ± SEM of three different experiments. (C) Number of adhered cells obtained after plating SH-SY5Y cells on three different physiological substrates (Collagen I, Fibronectin and Matrigel) in presence of BJ for 24 hs. The values are expressed as mean percentage with respect to untreated cultures of three different experiments (± SEM). (D) Adhesive skill of SH-SY5Y cells treated with BJ at 0.5 and 1% in comparison with untreated cells when seeded on HUVEC monolayers. Representative images show the decrease capability of SH-SY5Y cell (see the number of round cells) to join to HUVEC cells. Values obtained by counting attached SH-SY5Y cells are shown in the histogram. Data are expressed as mean ± SEM counts of five different optical fields. *P<0.05; **P<0.01; ***P<0.001vs control. Collectively the results demonstrated that BJ impaired cells adhesiveness.
Figure 7
Figure 7. BJ impairs SH-SY5Y migration interfering with actin polymerization.
(A) Effect of BJ on SH-SY5Y migration through gelatine coated filters after 24 hs of treatment. In the upper panel representative images of coated-filters are shown. The graphic in the lower ones represents the mean ± SEM of the migrated cells, as described in the material and methods. **P<0.01 and ***P<0.001vs control. (B) Fluorescence F-actin labelling of SH-SY5Y cells treated with 1% BJ. Representatives images show the loss of the normal actin morphology characterized by stress fibres (arrows)vs a morphology characterized by shorter and contorted actin fibres. White bar = 20 µm.
Figure 8
Figure 8. FAK and pFAK expression in SH-SY5Y and PC12 cells treated with BJ.
Western blotting analysis of FAK and pFAK expression in SH-SY5Y and PC12 cells treated for 24 hs with increasing concentration of BJ (0.1%, 0.5% and 1%). Relative densitometric analyses of pFAK and FAK immunoreactive bands are presented in the histograms. Data (mean ± SEM of three experiments) were normalized to the values yielded for β-actin. *P<0.05, **P<0.01 and ***P<0.001vs untreated cells. Note the significant reduction of pFAK by BJ.
Figure 9
Figure 9. BJ effects on association between FAK and NCAM.
(A) Western blotting analysis of NCAM basal expression in PC3, MDA-MB231, WI-38, PC-12 and SH-SY5Y cells. Note that NCAM is expressed only in both SH-SY5Y and PC12 cells. (B) Western blotting analysis of FAK, pFAK, and NCAM expression in SH-SY5Y cells treated with 1% BJ for different times (from 1 h to 24 hs). Relative densitometric analyses of immunoreactive bands normalized to the values for β-actin are presented in the lateral histogram (mean ± SEM of three experiments). *P<0.05, **P<0.01 and ***P<0.001vs untreated cells. A slight reduction of NCAM within 6 hs of BJ incubation accompanied to a significant decrease of pFAK is shown. (C) Protein extracts from SH-SY5Y cells treated for 24 hs with 0.1–5% BJ were subjected to immunoprecipitation with FAK antibody (IP:FAK, left panel), or with NCAM antibody (IP:NCAM; right panel) and then analysed for the expression of both NCAM and FAK.
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Research was supported by a grant from Sicily Region (PO FESR Sicilia 2007/2013, CUP G73F11000050004 to MN, project “MEPRA”, N. 133 of Linea d’Intervento 4.1.1.1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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