Copper (II) and 2,2'-bipyridine complexation improves chemopreventive effects of naringenin against breast tumor cells

Abstract

Cancer is the second leading cause of death worldwide and there is epidemiological evidence that demonstrates this tendency is emerging. Naringenin (NGEN) is a trihydroxyflavanone that shows various biological effects such as antioxidant, anticancer, anti-inflammatory, and antiviral activities. It belongs to flavanone class, which represents flavonoids with a C6-C3-C6 skeleton. Flavonoids do not exhibit sufficient activity to be used for chemotherapy, however they can be chemically modified by complexation with metals such as copper (Cu) (II) for instance, in order to be applied for adjuvant therapy. This study investigated the effects of Cu(II) and 2,2'-bipyridine complexation with naringenin on MDA-MB-231 cells. We demonstrated that naringenin complexed with Cu(II) and 2,2'-bipyridine (NGENCuB) was more efficient inhibiting colony formation, proliferation and migration of MDA-MB-231 tumor cells, than naringenin (NGEN) itself. Furthermore, we verified that NGENCuB was more effective than NGEN inhibiting pro-MMP9 activity by zymography assays. Finally, through flow cytometry, we showed that NGENCuB is more efficient than NGEN inducing apoptosis in MDA-MB-231 cells. These results were confirmed by gene expression analysis in real time PCR. We observed that NGENCuB upregulated the expression of pro-apoptotic gene caspase-9, but did not change the expression of caspase-8 or anti-apoptotic gene Bcl-2. There are only few works investigating the effects of Cu(II) complexation with naringenin on tumor cells. To the best of our knowledge, this is the first work describing the effects of Cu(II) complexation of a flavonoid on MDA-MB-231 breast tumor cells.

Figure 1. Complexation of Naringenin (NGEN) with copper (II) and 2,2′-bipyridine (NGENCuB).

Figure 2. Cellular Morphology of MDA-MB-231 (right panels) and MCF-10A (left panels) control cells, naringenin (NGEN)-treated or naringenin complexed with copper (II) and 2,2′-bipyridine (NGENCuB)-treated cells.

Cells were allowed to grow in a humidified incubator at 37°C in 5% CO₂ overnight and then treated with 1, 10 and 100 µM of NGEN or NGENCuB for 24, 48 and 72 hours. Cell morphology was examined under an inverted microscope with amplification of 100×. White arrows indicate detaching round cells. Scale bar  = 100 µm.

Figure 3. Effects naringenin (NGEN) or naringenin complexed with copper (II) and 2,2′-bipyridine (NGENCuB) on MDA-MB-231 colony formation.

A. Clonogenic assay of untreated MDA-MB-231 cells (control) or treated with NGEN or NGENCuB. A photograph of Petri-dishes in a representative experiment is shown. B. Quantification of colony number. C. Quantification of colony size. D. Plate efficiency. Quantification of colony number and size was performed using Image J public domain software. Data represent means ± SD from three different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey's post-hoc analysis. Asterisks represent *p≤0.05, **p≤0.01, ***p≤0.001 compared to control.

Figure 4. Effects naringenin (NGEN) or naringenin complexed with copper (II) and 2,2′-bipyridine (NGENCuB) on MDA-MB-231 (left) and MCF-10A (right) cell proliferation and IC₅₀ values for each compound and cell line.

Cells were plated in a 96-well plate and incubated with different concentrations (1 to 1000 µM) of NGEN or NGENCuB for 48 hours. Viable cells were estimated by MTT assay. Results are expressed as percent viability relative to control (untreated) cells. Data represent mean ± SD of three independent assays in triplicate. Results were compared using ANOVA, followed by a Tukey's post-hoc analysis. Asterisks represent *p≤0.05, **p≤0.01, compared to NGEN.

Figure 5. Effects of naringenin (NGEN) or naringenin complexed with copper (II) and 2,2′-bipyridine (NGENCuB) on MDA-MB-231 cell migration.

A. Cells were scratched using a pipette tip to make gaps between cells before NGEN and NGENCuB treatment. At 0, 6, 12, 24 and 48 hours of treatment, the plates were photographed under a light microscope (40 x). B. Percentage of wound closure of control cells or cells treated with 100 µM of NGEN or NGENCuB. Results are expressed as percent of wound closure relative to control (untreated) cells. Data represent mean ± SD of three independent assays in triplicate. Results were compared using ANOVA, followed by a Tukey's post-hoc analysis. Asterisks represent *p≤0.05, **p≤0.01, ***p≤0.001 compared to control.

Figure 6. Effects of naringenin (NGEN) or naringenin complexed with copper (II) and 2,2′-bipyridine (NGENCuB) on MDA-MB-231 cell migration in Boyden chambers.

Control cells or compounds-treated cells were allowed to migrate towards lower insert chambers containing FBS as chemoattractant, for 22 hours. The morphology of cells in the different treatments migrating toward FBS is in the bottom panels. Positive control (C+) represents migrating cells without any treatment, negative control (C-) represents untreated cells migrating toward an FBS-free medium. Data represent mean ± SD of three independent assays in triplicate. The results were compared using ANOVA, followed by a Tukey's post-hoc analysis. Asterisks represent *p≤0.05, **p≤0.001 compared to control (C+).

Figure 7. Effects of naringenin (NGEN) or naringenin complexed with copper (II) and 2,2′-bipyridine (NGENCuB) on activity of pro-MMP9 in MDA-MB-231 breast tumor cells.

A. Zymography in 1% gelatin-SDS-PAGE. Lane 1: molecular mass marker (M); lane 2: control cells; lanes 3–5: cells treated with NGEN; lane 6: control cells and lanes 7–9: cells treated with NGENCuB (n = 3; 10 µg of total protein was loaded in each lane). A photograph of a representative zymography gel is shown. B. pro-MMP9 concentrations were determined by the integrated optical density (IOD) obtained from the bands. Gels were analyzed by densitometry, and activity was expressed as arbitrary units (AU) and data were normalized in percentage compared to untreated control cell lysate. Data represent mean ± SD of three independent assays in triplicate. The results were compared using ANOVA, followed by a Bonferroni's multiple comparison test. Asterisk represents *p≤0.05.

Figure 8. Effects of naringenin (NGEN) or naringenin complexed with copper (II) and 2,2′-bipyridine (NGENCuB) on apoptosis in MDA-MB-231 breast tumor cells.

A. Nuclear 4', 6-diamidino-2-phenylindole (DAPI) staining. Cells treated or not (control) with different concentrations of NGEN or NGENCuB were observed under a fluorescence microscope. Representative phase-contrast and DAPI staining images were taken 24 h post-treatment. B. The number of cells with apoptotic nuclei was counted and plotted in a graphic. Scale bar  = 100 µm. C. Cytometry analysis of MDA-MB-231 cells treated or not (control) with 100 µM of NGEN or NGENCuB for 24 hours. After treatment, cells were harvested by trypsinization, centrifuged, washed twice with cold PBS and incubated with PE Annexin V and 7AAD (upper right panel) for 15 minutes in the dark at room temperature and then analyzed by cytometry. D. The percentage of apoptotic and necrotic cells was plotted in a graph. Data represent mean ± SD of three independent assays in triplicate. The results were compared using ANOVA, followed by a Tukey's post-hoc analysis. Asterisks represent *p≤0.05, **p≤0.01 (compared to control), #p≤0.05 (compared to NGEN).

Figure 9. Effects of naringenin (NGEN) or naringenin complexed with copper (II) and 2,2′-bipyridine (NGENCuB) on the expression of apoptotic or anti-apoptotic genes.

MDA-MB-231 cells were incubated or not (control) with 1 µM of NGEN or NGENCuB for 24 h. After, total RNA was extracted with Trizol reagent. cDNAs were synthesized and amplification of endogen control (GAPDH) and target genes was run in a Rotor-Gene 6000 real-time rotary analyzer using the gene-specific primer pairs described in MM section. Data represent mean ± SD of three independent assays in triplicate. The results were compared using ANOVA, followed by a Tukey's post-hoc analysis. Asterisk represents *p≤0.05.