Antitumor Effects of Freeze-Dried Robusta Coffee (Coffea canephora) Extracts on Breast Cancer Cell Lines

Abstract

Coffee consumption is believed to have chemopreventive and chemotherapeutic effects and to contribute to preventing the development and progression of cancer. However, there is still controversy around these claims. As indicated in our previous works, diet can influence the risk of breast cancer. Intake of coffee is hypothesized to reduce this risk, but current scientific evidence is not conclusive. This work is aimed at studying the effects of Robusta coffee bean extract on cell viability, proliferation, and apoptosis of different human cancers, especially breast cancer cell lines. To this end, cell viability was evaluated by Alamar Blue in 2D and 3D models, the cell cycle by PI, apoptosis by annexin V, mitochondrial morphology, and functionality by mitoTracker, and colony formation capacity by the clonogenic assay. Green and dark coffee extract significantly reduced viability in human breast, colorectal, brain, and bone cancer cells. Coffee anticancer activity was clearly evidenced in MDA-MB-231 (ER^-) and MCF-7 (ER⁺) breast cancer cells but not in the normal breast cell line. In addition, coffee extract induces an increase S phase and a decrease G2/M population in breast cancer cells, affected the mitochondrial morphology, and triggered apoptosis. MDA-MB-231 breast cancer cells lost their clonogenic capacity after treatment. The antitumor activity was demonstrated in both 2D and 3D culture cell models.

Figure 1

Coffee extract effect on cell cancer viability. Human cancer cell lines (MCF7 and MDA-MB-231) and human epithelial breast cell lines (MCF 10A) were treated for 24 h with increasing concentrations of (a) green coffee or (b) dark coffee (0-5000 μg/mL). (c) Inhibitory concentration 50% (IC50) on breast cell line treated with green and dark coffee was determined and expressed as μg/mL. ^∗ represents statistically significant difference in the column indicated with respect to treated MCF 10A, as determined by ANOVA (p < 0.05), with Tukey's multiple comparison posttest. (d) Cell morphology was analyzed by light microscopy. Scale bar = 50 μm. Values are represented as mean ± SEM from three independent experiments.

Figure 2

Coffee extract effect on cell morphology and cell cycle progression. MCF 10A, MDA-MB-231, and MCF7 cells were treated during 24 h with green coffee extract at 0 (control), 500, or 1000 μg/mL. (a) DNA content profiles using propidium iodide stain to compare the cell cycle progression. (b) Quantification of cell population proportion in different cell cycle phases. Values represent the mean ± SEM from three independent experiments. Cell cycle were determined using FlowJo V10 software (10.7.1) and analyzed by the Watson Pragmatic algorithm model. Each concentration of coffee extract used was compared with its respective cell line control (^∗p < 0.005; ^∗∗p < 0.001; ^∗∗∗p = 0.0001) as determined by ANOVA, with Tukey's multiple comparison posttest. (c) Nuclear fragmentation was analyzed using Höestch 33342 staining. Staurosporine (STS) was used as a positive control for nuclear fragmentation. The white arrows indicate fragmented nuclei. Scale bar = 50 μm.

Figure 3

Apoptosis induction by green coffee extract. MCF 10A, MDA-MB-231, and MCF7 cells were treated for 24 h with green coffee at 0, 500, or 1000 μg/mL. (a) Apoptosis flow cytometry analysis by Annexin V/propidium iodide staining was performed, and representative plots are presented. (b) Quantification of apoptosis induction (relative to control). Values are mean ± SEM from three independent experiments (^∗∗∗p < 0.0001) as determined by ANOVA, with Tukey's multiple comparison posttest.

Figure 4

Green coffee effect on mitochondrial morphology. MDA-MB-231, MCF7, and MCF 10A cells were treated for 0, 2, and 4 h with green coffee extracts at 1000 μg/mL or STS 6 nM for 4 h. Representative microphotographs show the mitochondrial (green-MitoTracker Red CMXRos) and nuclear (blue-DAPI) stain. Scale bar = 50 μm (upper row), zoom scale bar = 10 μm (lower row).

Figure 5

Green coffee effect on mitochondrial membrane potential. MDA-MB-231, MCF7, and MCF 10A cells were treated for 0, 4, and 24 h with green coffee extracts at 500 and 1000 μg/mL. Cells were collected and incubated with 100 nM MitoTracker Red CMXRos for 30 min and then analyzed by flow cytometry. Representative histograms of each treatment are presented.

Figure 6

Green coffee effect on MDA-MB-231 and MCF 10A cell clonogenic capacity. Cells were treated for 24 h with green coffee extracts at 0, 500, and 1000 μg/mL. Then, the surviving cells were seeded at low density for a clonogenic assay. (a) Representative results are shown for each condition stained with crystal violet. Scale bar = 0.5 cm. (b) Quantification of the viable clones. Values are the mean ± SEM of three independent experiments (^∗∗∗p = 0.0001 with respect to control and ^^p < 0.001) between cases as determined by ANOVA, with Tukey's multiple comparison posttest.

Figure 7

Green coffee extract effect on a 3D model of spheroids. 6 × 10³ MDA-MB-231, MCF7, or MCF 10A cells were seeded (40 μL) and cultured for 7, 6, and 4 days, respectively. Spheroids were treated with green coffee extracts at 0, 2000, 3000, 3500, and 4000 μg/mL for 24 h. (a) Representative microphotographs showing spheroid morphology are presented. Scale bar = 100 μm. (b) Spheroids viability was analyzed for the AlamarBlue ® assay and quantified. Values are the mean ± SEM from three independent experiments (^∗p < 0.005; ^∗∗p < 0.001; ^∗∗∗p = 0.0001) between cases as determined by ANOVA, with Tukey's multiple comparison posttest.