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.
Cancer is currently a major public health problem, and the available therapeutic strategies are not fully effective in several tumor types. The American Cancer Society estimates that by 2019, approximately 17,624,50 new cancer cases will be diagnosed, which is equivalent to more than 4,800 new cases every day [
Breast cancer etiology is considered multifactorial, and it includes interactions between genetic, behavioral, and environmental factors. Breast cancer is a heterogeneous disease, but cancer subtypes are hormone-related. Breast tumors that express the ER (ER+ tumors) are more strongly associated with hormone-related factors than tumors that do not express the ER (ER− tumors).
According to an analysis that verified the antitumor molecules approved by drug entities, such as FDA and similar organizations, 49% of anticancer molecules approved between 1940 and 2014 were natural products or chemical derivatives worldwide [
As stated in previous work, the diet may influence breast cancer risk. Coffee intake has been hypothesized to reduce the risk of breast cancer, but the current evidence is inconclusive [
This drink contains caffeine, chlorogenic acid (CGA) (which is caffeic bound to quinic acid), p-coumaroylquinic, and feruloylquinic acids [
Green coffee consumption as a dietary supplement or as a beverage is increasing due to its reported antioxidant benefits. Furthermore, the chemopreventive and anticancer potential of bioactive molecules present in a standard cup of coffee has been described not only in green coffee but also in the beverage made from coffee, the black coffee. Indeed, many retrospective meta-analysis and several human studies have shown the benefits of coffee consumption for reduced the incidence and risk of breast cancer (without showing possible mechanisms) ([
The coffee used in this study was
Human breast carcinoma (MCF7 and MDA-MB-231), human breast (MCF 10A), human bone carcinoma (U2OS), human colorectal carcinoma (HCT116), and human brain glioblastoma multiform (T98G) cell lines were obtained from the ATCC-Bethesda, MD, USA. Cell lines were plated and maintained routinely in Dulbecco’s Modified Eagle’s Medium high glucose (DMEM) supplemented with 10% fetal bovine serum (FBS) plus antibiotics (100 U/mL penicillin/0.1 mg/mL streptomycin), and pH 7.4, under 5% CO2. Culture medium for MCF 10A cell line was supplemented with 20 ng/mL epidermal growth factor, 100 ng/mL cholera toxin, 0.01 mg/mL human insulin, and 500 ng/mL hydrocortisone. Once the cells reached 80% confluence, they were dissociated using 0.05% trypsin-EDTA and subcultured. Culture medium was replaced every 2 days. Cells were seeded in 96-well plates, and after 24 h, the medium was changed to fresh supplemented DMEM medium. Cells were treated for 24 h with increasing concentrations of green or dark coffee extracts dissolved in supplemented DMEM (25 to 5000
Cell lines were plated in 96-wells plates and cultured for 24 h. Then, cells were treated for 24 h, and the culture medium replaced with alamarBlue® 10% v/v dissolved in DMEM supplemented with 10% FBS and antibiotics. Three hours later, fluorescence (590 nm) was monitored using a Biotek microplate reader, as recommended by the manufacturer.
After treatment, cells were rinsed briefly with phosphate buffered saline (PBS) and detached using trypsin at room temperature. After centrifugation, the cells were washed twice with PBS, resuspended in cold 70% v/v ethanol solution, incubated for 24 h at 4°C, and treated with RNAse (200
To measure the apoptosis rate, the cells were stained using FITC-conjugated Annexin V and PI. The nonadherent cells were collected, and the adherent cells were quickly washed with PBS and detached using trypsin/EDTA 0.125% (Sigma chemical Co., St. Louis, USA) at room temperature. Subsequently, cells were stained with Annexin V-FITC/propidium iodide (PI) (BD Pharmingen, New Jersey, USA) according to the manufacturer’s instructions, quantified by flow cytometer using a Beckton Dickinson FACSCanto II and analyzed using FlowJo V10 software [
Exponentially growing MDA-MB-231 and MCF 10A cells were harvested, counted, and seeded (1.106 cells/plate) in Petri dishes. Cells were allowed to grow at 37°C in 5% CO2 overnight. Then, cells were incubated with different coffee concentrations for 24 h. Next, the cells were harvested, counted, and reseeded at low density (about 50-250 cell/well) in 24 multiwell plates (Corning Costar USA). After incubation for additional 15 days, the colonies were stained with crystal violet 5% v/v solution for 20 min. The number of clones (
MCF7 is a breast ductal carcinoma cell line that corresponds to a relatively less aggressive hormone-responsive breast cancer (better prognosis). MCF-7 cells express lower levels of VEGF than MDA-MB-231 cells, which have high invasive and migration capacities. Due to this, we believe that MCF7 cell lines were not suitable for the colony formation assay [
MDA-MB-231, MCF7, and MCF 10A cells were grown on 12 mm glass coverslips and treated using coffee extracts (0 and 1000
MDA-MB-231, MCF7, and MCF 10A cells were grown on 24 multiwell plates for 48 h and treated using coffee extracts (5000 and 1000
The spheres were formed from cells using the hanging drop system: Perfecta3D® 96-well plates (3D Biomatrix; Michigan, USES)
Results are expressed as mean values and the corresponding standard deviation of experiments done in triplicate. The data were analyzed with the statistical software GraphPad Prism (version 8.0, GraphPad software, San Diego, CA). The significant differences are indicated by letters as determined by one-way analysis of variance (ANOVA) followed by Tukey’s posttest (
The coffee extracts used in this study were obtained by standardized processes by the Brazilian national company of “Pesquisa Agropecuária” from green
Experiments were carried out to evaluate the effects of coffee extracts on cancer cells. For this purpose, cell viability was analyzed after incubation with different concentrations of coffee extracts dissolved in DMEM medium for 24 h. As observed in Figures
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
The chemical composition of coffee varies because the preparation process may change the content of bioactive compounds, resulting in different types of coffee beverages around the world, as widely reported in the literature. During roasting, the chlorogenic acid content decreases, while melanoidins are formed as complexes of sugars, amino acids, and chlorogenic acid through the Maillard reaction, or vitamin B niacin from the trigonelline alkaloid. Instant coffee or a paper filter on the coffee beans leads to almost complete removal of the diterpenes of cafestol and kahweol. The caffeine content varies according to the type of coffee and can also be modified by the preparation process [
Caffeine and the polyphenol content have been suggested to contribute to coffee anticancer activities. Dried green coffee beans contain carbohydrates (59–62%), CGAs (7–10%), aliphatic acids (2%), caffeine (1–2%), trigonelline (1%), and free amino acids (<1%), but roasting coffee reduces the contents of carbohydrates, CGAs, and free amino acids and increases those of alkaloids (mostly caffeine) and aliphatic acids [
To investigate the effects on cell morphology, breast cancer cell lines were incubated with green coffee extracts at concentrations of 500 and 1000
The results of the cell cycle analysis indicate that the treatment of cells with green coffee extract at 1000
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
Our results show that cell incubation with coffee extracts induces cell death. Consequently, we next evaluated cell death type triggered by nuclear fragmentation analysis. After treatment, cells were stained using Höechst 33342, and nuclear morphology was analyzed by fluorescent microscopy. A chromatin condensation distinctive pattern and nuclear fragmentation (apoptotic bodies) were observed in the MDA-MB-231 and MCF7 cells treated, but not in MCF 10A cells (Figure
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
The antitumor effect of coffee is thought to be based on several mechanisms. The antioxidant effect is conditioned not only by the content of direct antioxidants but also by the ability to activate endogenous antioxidant enzymes like superoxide dismutase (SOD) and
Mitochondria are critical organelles that ensure the normal function of the cells. These organelles play a crucial role in generating cellular energy and regulating cell fate through their participation in cell-death regulation [
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
Since changes in mitochondrial membrane potential (MMP) are associated with cell death and given the results of the microscopy analysis (Figure
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
In agreement with our results, several studies proposed that caffeine treatment significantly suppressed cell growth and viability and induced apoptosis by activating the caspase pathway in gastric cancer cells [
To further investigate the effect of coffee on breast cancer cells, we conducted a colony formation assay (in vitro cell survival assay, based on the single-cell ability to grow in a colony). This assay allowed us to determine the cell clonogenic capacity after exposure to cytotoxic agents [
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
Our results are similar to those found in prostate tumor cells (DU-145) treated with coffee CGAs, which showed a reduced colony formation capacity after treatment [
In addition, we investigated the therapeutic potential of green coffee extract on a 3D cell culture model, generating spheroids from MCF 10A, MCF7, and MDA-MB-231 cells. On days 4, 6, or 7 of culture, respectively, these spheroids were treated with different concentrations of green coffee extracts for 24 h and then analyzed by microscopy. MDA-MB-231 and MCF 10A spheroid morphology was not modified by the treatment, but MCF7 spheroids lost their cellular compaction level until forming very lax aggregates (Figure
Green coffee extract effect on a 3D model of spheroids.
The ability of dietary substances to modulate the immune response and suppress the proinflammatory environment in the body is considered to be one of the important mechanisms of tumor chemoprevention [
In previous work, the antioxidant activity of extracts with different degrees of roasting was analyzed. We demonstrated, through several tests, that Robusta green coffee and lightly roasted extracts had a higher antioxidant potential [
A reduction or even elimination of coffee consumption has been traditionally recommended in view of a global risk profile, but its consumption has progressively been considered in a less negative light due to its better-known phytochemistry [
Recent epidemiological and prospective (meta-analysis) studies demonstrate that consumption of healthy foods, such as coffee, especially rich in polyphenol content, might have antitumor activity against several cancer types, such as colorectal [
Especially in reference to breast cancer, numerous authors provided evidence on a strong and significant inverse association between cancer risk and coffee consumption [
In the present work, we showed that coffee extracts have antiproliferative and cytotoxic effects on breast cancer cells in 2D and 3D culture models. Surprisingly, coffee extracts do not affect viability on human epithelial breast cell lines (noncarcinogens). Our results suggest that green and roasted coffee bean extracts showed a strong bioactive capacity, promoting cell viability decrease, cell cycle alteration, cytostatic effect, mitochondrial morphology, and MMP alterations and clonogenic capacity loss.
These extracts of
The results presented herein have far-reaching health relevance since coffee compounds could be used as chemopreventive and chemotherapeutic medicine which, in addition to their antioxidant activities and capacities, can also provide nutrition and contribute to preventing cancer development and progression. As suggested by other authors, a cancer diagnosis could be a stimulus for patients to make protective changes in health, and health professionals should consider this as a window of opportunity to educate patients about a healthy lifestyle [
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper.
Ayelén Denise Nigra and Deborah de Almeida Bauer Guimarães contributed equally to this work. They are first and co-first authors respectively. Ayelén D. Nigra performed the conceptualization and acquisition of data, methodology, validation, Formal analysis, investigation, and writing-original draft, writing-review and editing, visualization, and final approval of the manuscript. Deborah de A. Bauer performed the conceptualization and acquisition of data, methodology, formal analysis, investigation, original draft, writing-review and editing and final approval of the manuscript. Cesar G. Prucca performed the conceptualization, methodology, resources, writing-original draft, writing-review and editing, project administration, funding acquisition, and final approval of the manuscript. Otniel Freitas-Silva performed the analysis and interpretation of data, methodology, original draft, writing-review and editing, resources, and final approval of the manuscript. Anderson Junger Teodoro performed the resources, original draft, writing-review and editing, supervision, project administration, funding acquisition, and final approval of the manuscript. German A. Gil performed the conceptualization and design of the study, formal analysis and interpretation of data, investigation, methodology, resources, writing-original draft, writing-review and editing, visualization, supervision, project administration, funding acquisition, and final approval of the manuscript.
We thank CIQUIBIC (CONICET, Universidad Nacional de Córdoba, Córdoba, Argentina). This work was supported by the Secretaría de Ciencia y Técnica, Universidad Nacional de Córdoba; Fondo para la Investigación Científica y Tecnológica, Secretaría de Ciencia, Tecnología e Innovación Productiva, Argentina, and CONICET (Consejo Nacional de Investigaciones Científicas y Tecnológicas), Argentina. We acknowledge the excellent technical and imaging assistance provided by Dr. Carlos Mas and Dr. Cecilia Sampedro from Centro de Micro y Nanoscopía de Córdoba, CEMINCO-CONICET. We appreciate cytometry assistance by Dr. Pilar Crespo and Paula Abadie and to Maria Alejandra Scotti for English editing of the manuscript. Deborah de A. Bauer Guimarães thanks the Programa de Movilidad Universitaria de la Red De Macrouniversidades de América Latina y El Caribe for a short-term fellowship. Ayelén D. Nigra acknowledges the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET Argentina) for a postdoctoral fellowship; GAG and CGP are career members of CONICET. This work was supported by the Ministerio de Ciencia, Tecnología e Innovación Productiva, Argentina (PICT 2016-0986), PIP-CONICET 2015, and SeCyT 2016 and 2018 to GG. This study was partially funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001 and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro–FAPERJ (225031).
Supplementary Figure 1: coffee extract effect on cell cancer viability. Human cancer cell lines (U2OS, HCT116, and T98G) were treated for 24 h with increasing concentrations of (a) green coffee or (b) dark coffee (0-5000