Açaí Extract and Anticancer Drug Combination Promotes Synergistic Toxicity and Apoptosis in MCF-10A Cells of Breast Cancer Model

Abstract

Ethnopharmacological Relevance:

Euterpe Oleracea Mart., açaí, is a species that has been used for centuries by many Central and South American natives as a staple food as well as providing numerous health benefits, such as anti-tumor and anti-proliferative effects. There has been a recent increase in the global consumption of açaí and açaí food products because its various benefits.

Aim of the Study:

To evaluate the toxicological potential between açaí extracts and two widely used anticancer drugs – methotrexate and tamoxifen.

Materials and methods:

Euterpe Oleracea Mart. fruit powder extracts obtained from a name brand company in Brazil and two açaí dietary supplement brands were tested on two cancer cell lines, MCF-7 and MDA-MB-231, and one normal epithelial breast cell line, MCF-10A. Cell viability was measured using MTT assay. The data was analyzed using SynergyFinder Plus and Compusyn to determine the potential synergism, antagonism, or additive effect of the açaí extract when combined with the selected anticancer drugs, tamoxifen and methotrexate.

Results:

When combined with methotrexate, the methanol extract of the açaí powder and the acidic methanol extract of the açaí dietary supplements caused significant synergy (potentiation), thus increasing toxicity, of the combination to the normal cell line, MCF-10A. The combination of the acidic methanol extract of the açaí dietary supplements also induced apoptosis with the MCF-10A cells. Methotrexate was potentiated by the aqueous extract of the açaí powder when tested with the MCF-7 cells. Tamoxifen toxicity was increased only with the MCF-7 cells. Pre-exposure of the MCF-10A cells to the methanol extract of the açaí powder caused increased tamoxifen toxicity for the normal cell line but decreased toxicity for methotrexate combination. There were no significant indications of the açaí extracts causing an antagonistic relationship with either anticancer drug.

Conclusion:

The açaí extracts evaluated here potentiated both anticancer drugs, increasing their toxicity for all cell lines with the most significant increase in toxicity occurring with normal cell line, MCF-10A. The toxicity was further confirmed to be through induction of apoptosis. This calls for further investigation of the chemical constituents of açaí that are the cause of the toxicity of the combination of anticancer drug and açaí extract.

Graphical Abstract

1. INTRODUCTION

Breast cancer is a life-threatening disease that affects millions of people worldwide (Sedeta et al., 2023). It is the most prevalent cancer in females and contributes to the highest percentage of mortality due to cancer-related deaths in women (Sedeta et al., 2023). In recent years, there has been a growing interest in the use of alternative approaches for cancer prevention including botanical dietary supplements (BDS) (Marian, 2017). These natural compounds, derived from various plants and herbs, have been evaluated for their potential roles in reducing cancer progression. After receiving a cancer diagnosis, anywhere from 20% to 80% of individuals reportedly consume dietary supplements (Marian, 2017). Individuals with prostate, colorectal, and lung cancers are among many individuals who utilize dietary supplements. But the most prevalent use of BDS is by survivors of breast cancer diagnoses (Marian, 2017). The likely rationale, reported by Marian (2017), underlying this widespread adoption of dietary supplements involves aspirations for enhancing one’s quality of life, alleviating treatment-related symptoms, and addressing the disease progression itself. Additionally, recommendations from healthcare providers and the influence of family and friends play a role in this decision. The question of whether these supplements impact the effectiveness of cancer treatment remains unresolved (Hudson et al., 2018). The FDA defines dietary supplements as a product intended for ingestion that contains a dietary ingredient to supplement the diet. (Food and Drug Administration, 2024). Some studies have shown that antioxidant-rich foods can lessen the effects of cancer; however, it is possible that botanical dietary supplements (BDS), can interfere with the efficacy of anticancer drugs as well(Dietz et al, 2016).

Euterpe oleracea Mart. (Arecaceae), commonly known as açaí, is a palm tree native to the Amazon region of Central and South America, well known for producing antioxidant rich fruit that has various anti-inflammatory and anti-proliferative effects (Laurindo et al., 2023). For centuries, the açaí fruit has played a significant role in the daily lives of Amazonian tribes and is traditionally consumed as a staple food, providing essential nutrients and antioxidants to maintain health. Açaí has also been traditionally used for endocrine disorders, (Silva et al., 2020) which are known to include certain breast cancers (Berstein, 2011). In Columbia and Ecuador, Euterpe oleracea Mart. has traditional uses related to general cancer and tumors (Macía, M. et al., 2011). Despite its long history as a versatile dietary plant in its native habitat, açaí’s introduction to new regions has led to a rapid increase in global demand for its fruit, with Brazil emerging as the primary producer and exporter to meet this growing consumption (Laurindo et al., 2023). Açaí use in the United States is in alignment with the traditional use as a supplementation to the diet as well as for antioxidant and antiproliferative benefits of açaí. Açaí has antitumoral properties due to its anti-inflammatory, antiproliferative, and pro-apoptotic mechanisms (Laurindo et al., 2023). In vitro studies have demonstrated that açaí, when administered alone, decreased cell viability through apoptosis induction in glioma cells and murine melanoma cells (Deaconeasa et al., 2015). However, the pharmacodynamics of concomitant use of açaí and anticancer drugs in the context of off-target toxicity have yet to be evaluated even though there is a large prevalence of possible toxicities from this combination.

Researchers analyzed data from the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS) to investigate the risk of adverse events associated with açaí botanical dietary supplement use and anticancer drugs metabolized and non-metabolized by cytochrome P450 3A4 (CYP3A4), the major xenobiotic metabolizing enzyme in the liver (Fahim et al., 2019). Amongst açaí users, a 50% increase in adverse events was reported in the cohort of patients prescribed anticancer agents that were not interactive with CYP3A4 as compared to patients using agents that did interact with the enzyme system. Methotrexate was one of the non-CYP3A4 interactive drugs in the analysis which supported its selection in this study. The adverse events were of cardiovascular nature and ranged in severity, with hospitalization required in some settings (Fahim et al., 2019). Further investigation using the FDA Center for Food Safety and Applied Nutrition Event Reporting System (CAERS) indicated that when acai-containing BDS were used concomitantly with anticancer drugs, there were hospitalizations with symptoms that included chest pain and pulmonary thrombosis (Fahim et al., 2019). The reports of adverse events in both FDA databases show a urgent need for an assessment of the safety of concomitant açaí BDS use with anticancer drugs.

The studies on Euterpe oleracea (açaí) provide valuable insights into its biological effects, including its antioxidant properties and potential cardiovascular benefits. However, none of these studies address the critical gap of how açaí interacts with anticancer drugs, particularly in the context of concomitant use. For instance, while one study evaluates the vascular effects of açaí extract in vivo, showing increased blood flow (Pontes, V. et al., 2021), it does not explore how açaí may influence the metabolism or efficacy of chemotherapeutic agents. Similarly, research on açaí’s antioxidant activity focuses on its potential to reduce oxidative stress but overlooks the possibility that açaí could alter the interactions with cytochrome P450 enzymes, which are crucial for drug metabolism. Additionally, studies on açaí’s cardioprotective effects during chemotherapy and its potential to reduce doxorubicin-induced cardiotoxicity (Polegato, B.F et al., 2019) do not address whether açaí co-administration could impact the effectiveness or toxicity of anticancer treatments more broadly. They also do not assess the impact on cancer cells. Although there is evidence that açaí supplementation may protect against chemotherapyinduced damage in specific tissues, the lack of research on how açaí interacts with a range of chemotherapy drugs leaves an important gap in understanding.

Although many studies evaluate the pharmacokinetic and pharmacodynamic implications of concomitant BDS and anticancer medication uses, no studies assessed the putative pharmacodynamic interactions between açaí extracts and anticancer drugs that cause off-target toxicity in breast cancer. This work aims to attenuate this gap in the current literature through an analysis of both açaí fruit material and BDS extracts in combination with two anticancer drugs: methotrexate (MTX), a non-CYP3A4 interactive drug, and tamoxifen (TAM), a CYP3A4 interactive drug as comparison. The viability of two breast cancer cell lines (MCF-7, MDA-MB-231) and one typical breast tissue cell line (MCF-10A) will be assessed following exposure of açaí extracts as a monotherapy and in combination with anticancer drugs via MTT assay. To further represent potential synergistic, additive, or antagonistic effects of these combinations, GraphPad, SynergyFinder Plus and CompuSyn were utilized to create 2D and 3D visuals of combinatorial analysis of the treatments. An analysis of apoptosis will be assessed via measurements of Annexin V CF488A fluorescence signal.

2. MATERIALS AND METHODS

2.1. Chemicals

MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (≥97.5%) and dimethyl sulfoxide (DMSO) (≥99.7%) were obtained from Sigma. Puromycin (≥98%) and tamoxifen (≥95%) were obtained from Cayman Chemical Company. Methotrexate (≥99.14%) and camptothecin (≥99.34%) were obtained from ApexBio. Vectashield Vibrance Antifade Mounting Medium with 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Vector Laboratories. Annexin V CF488A Dye Conjugate and Live-or-Dye NucFix Red Staining Kit was purchased from Biotium. Paraformaldehyde (8%) (PFA) was purchased from Electron Microscopy Sciences.

2.2. Preparation and Analysis of Açaí Extracts

Açaí extracts were prepared as previously described in Heck et al., (2023). In short, four LC-MS grade solvents, acidic methanol (70% methanol, 0.1% HCl v/v), methanol, 95% ethanol and water were used separately to extract certified organic açaí fruit powder (Catalog no. AÇAÍ4, Lot#26579) from Mountain Rose Herbs. (Heck et al., 2024).. Formulations from two different botanical dietary supplement companies were extracted using only acidic methanol and methanol. Nature’s Way (NW) (Green Bay, WI, USA, 2022 batch #20137327) acidic methanol and Natrol (Chatsworth, CA, USA, 2022 lot#2086344) acidic methanol and methanol extracts were used in this study. The BDS capsule extracts will be referred to as F3 (NW 2022) and F4 (Natrol 2022). All açaí extracts used in this study have similarities to ways botanical extracts are traditionally prepared, (Handa, S.S., 2008) with the alcohol-based extracts having similarity to the tinctures that are prepared from plants in traditional medicine and the aqueous extracts having similarity to decoctions or cold aqueous percolation preparation. The extracts were subjected to additional solvent removal via rotary evaporation and subsequent lyophilization to remove any residual solvent. The plant names have been checked with http://www.worldfloraonline.org; accessed on June 1, 2024. Total anthocyanins were quantified for each extract via liquid-chromatography electrospray ionization mass spectrometry (LC-ES-IMS). The extracts were standardized to cyanidin-3-O-glucoside (C3G) content using the method in Heck et al., (2023). The aqueous, methanol, acidic methanol, and ethanol extracts of the Brazilian açaí powder used in this study were abbreviated MRAQ, MRME, MRAC, and MRET, respectively. The acidic methanol extracts for two brand dietary supplement formulations, abbreviated F3AC and F4AC, previously extracted by Heck et al., 2023, were used along with the methanol extracts of the second brand formulation abbreviated F4ME.

2.3. Cell Culture

MCF-7 (lot#0507779) and MDA-MB-231 (lot#0509796) cells were obtained from the NCI repository for cancer cells (NCI, 2021). Both cell lines were cultured using the suggested basal Roswell Park Memorial Institute (RPMI) 1640 medium without L-glutamine obtained from Fisher Scientific. L-glutamine (2mM), penicillin/gentamycin, and 10% fetal bovine serum from Hyclone were supplemented to the basal medium (Coussens, et al., 2025). Non-tumorigenic mammary epithelial, MCF-10A, cells were obtained from the American Type Culture Collection (ATCC) and were cultured using suggested conditions. The medium for the MCF-10A cells used was the Mammary Epithelial Cell Growth Medium (MEGM) Bulletkit from Lonza that included the basal Mammary Epithelial Cell Basal Medium (MEBM) with SingleQuot aliquots for supplementation. All aliquots were used along with an additional supplementation of cholera toxin from Sigma to form the fully supplemented MEBM media. All cells were incubated at 37 °C and 5% CO₂. The cells were passaged using Dulbecco’s Phosphate Buffered Saline (D-PBS) and trypsin from Life Technologies Corporation. The two cancer cell lines, MCF-7 and MDA-MB-231, were passaged using 0.25% trypsin and the non-tumorigenic mammary epithelial cell line, MCF-10A, was passaged using 0.05% trypsin.

2.4. Optimization Experiments

Since the two cancer cell lines, MCF-7 and MDA-MB-231, were obtained from the NCI Cancer Cell Repository, the cell seeding density that the repository used was implemented in this study. To validate the proposed cell seeding density and replicability for the assays to be performed, a range of cell densities, including densities used by the NCI, for each cell line was seeded in 96 well plates for the time points of the assays that would be performed. (Close, D.A., et al., 2019) The cell seeding densities were also tested for the non-malignant breast cell line, MCF-10A under supplemented media conditions and non-supplemented media conditions. Previously reported cell seeding densities were used in this optimization experiment. (Hafner, M. et al., 2016). All three cell lines were subjected to cell viability assays of puromycin and dimethyl sulfoxide (DMSO) treatment. DMSO served as the vehicle for all the treatments and puromycin was implemented as a positive control for complete cell death or 0% cell viability. To optimize puromycin concentration, all three cell lines were seeded at the cell seeding density that was confirmed for each cell at the respective time point. After 24 hours, the cells were treated with a range of puromycin concentrations from 1 μg/ml to 10 μg/ml per Sigma guidelines. The puromycin solution was made using a 1 mg/ml stock solution and a 1:100 dilution factor to obtain a 10 μg/ml solution. The 10 μg/ml solution diluted further to create the treatment range. To ensure the vehicle, DMSO, did not cause any decrease in cell viability, the cells were seeded at the optimized cell seeding density for respective time points. After 24 hours, DMSO concentrations ranging from 0.05% to 10% were added to the cells. For both DMSO and puromycin treatments, an MTT assay was performed after the exposure time to determine the concentration of vehicle tolerable to the cells and the concentration of puromycin to use for the positive control, respectively.

2.5. Cell Viability Assays

Cells were plated into 96 well plates at optimized cell densities depending on the time frame and cell type. MCF-7 cells were seeded at 10,000 cells per well (1.0 × 10⁵ cells/ml), MDA-MB-231 cells were seeded at 20,000 cells per well (2.0 × 10⁵ cells/ml), and MCF-10A cells were seeded at 10,000 cells per well (1.0 × 10⁵ cells/ml) for the 48-hour treatment period. For the 72-hour treatment for MDA-MB-231 and MCF-10A cells, the seeding density was 10,000 (1.0 × 10⁵ cells/ml) and 5,000 cells per well (5.0 × 10⁴ cells/ml), respectively. After 24 hours, treatment was added to the wells of the 96 well plate that contains cells. Prior to the end of the treatment window, 10% of the well volume, (either 20 μL or 30 μL) of MTT reagent (1 mg/ml) was added to each treatment well of the 96 well plate to create a final MTT reagent concentration of 0.1 mg/ml. The MCF-7 and MDA-MB-231 cells were incubated for 3 hours, when purple precipitate was visible, while the MCF-10A cell line was incubated for 4 hours. After the incubatory period, the solution was removed from the wells and any purple formazan crystals that formed were dissolved using 100 μL of DMSO per well. (Razak et al, 2019). The absorbance was measured at 570 nm using a ThermoSkanit Varioskan Flash (ThermoFisher Scientific) spectrophotometer. The cell viability was determined using the vehicle as the negative control (100% cell viability). (Razak et al, 2019).

2.6. Anticancer Drug Cytotoxicity Studies

Stock solutions of MTX and TAM were made in DMSO which served as the vehicle for all experiments. The stock solutions were added to cell culture medium to produce the desired concentration while not exceeding the optimized amount of DMSO for the cells. Each cell line (MCF-7, MDA-MB-231, and MCF-10A) was seeded into 96 well plates for 24 hours prior to the addition of the anticancer drug. For MTX, MCF-7 and MCF-10A cells were treated with a dose range of 1.0 × 10⁻⁵ μM to 1 μM. MDA-MB-231 cells were treated with MTX in a dose range of 1.0 × 10⁻⁴ μM to 10 μM. The two cancer cell lines and non-tumorigenic mammary epithelial breast cell line were treated with TAM at an optimized dose range of 1 to 100 μM and 0.6 to 60 μM, respectively. The doses were chosen using steady-state concentrations as a reference (Christophidis, N et al., 1979 and Hutson P.R. et al., 2005). The cells were incubated at the respective times that were optimized per cell line per anticancer drug. Cell viability was measured using an MTT assay. Experiments were completed in triplicates per 96 well plate and with at least three biological replicates.

2.7. Cytotoxicity Evaluation of Açaí Extracts

Cells were seeded into 96 well plates and after 24 hours, all three cell lines were treated with the respective açaí extract. The extract treatment range was formed by using the concentration of the compound that all extracts were standardized to, cyanidin-3-glucoside (C3G). The treatment range was from 1.0 × 10⁻⁵ ng/ml to 1000 ng/ml of C3G depending on the solubility of the extract in DMSO. The treatment range included the pre-determined human-relevant concentration (HRC) of C3G (2.321 ng/ml). (Mertens-Talcott et al, 2008). The calculation of HRC is located in the supplementary data (Supplementary Calculation). Cell viability was determined using an MTT assay. Experiments were conducted in triplicates and with at least three biological replicates.

2.8. Human Relevant Concentration (HRC) Screening of Açaí Extracts for Combinatorial Experiments

Cells were plated as described above. The same range of anticancer drug utilized in the anticancer drug assays was implemented for this screening but the açaí extract dose remained constant at a concentration of 2.321 ng/ml C3G per extract. Both treatments were initially created at a concentration double that of the final required concentration with consecutive dilution across wells to reach the desired final concentration for each treatment. Prior to the end of the treatment window, MTT reagent was added to each well. After the MTT assay time frame, the 96-well plate was processed, and absorbance was measured. Initially, to determine differences in the dose-response curves of the screening combinations, GraphPad Prism analysis using the non-linear regression comparison of fit with the logIC₅₀ as the parameter was used. The data was subsequently analyzed in Excel using a t-test for comparison of each dose of the anticancer drug alone and the combination of the anticancer drug and HRC of the respective extracts to determine any statistically significant differences between anticancer drug alone and anticancer drug with HRC of açaí extracts.

2.9. Combinatorial Experiments of Açaí Extracts and Anticancer Drugs

The açaí extracts that exhibited the most statistically significant differences in cell viability per dose of combination in the HRC screening of the açaí extracts were used to perform a combinatorial assay using a range of anticancer drugs and a range of açaí extracts. The optimized treatment range of the individual anticancer drug and açaí extract in the single-agent testing were used for the combinatorial assays. Similar to the HRC screening, both solutions of anticancer drug or respective açaí extract were made in double concentrations, and 90 μL of each solution was added to the wells for the 48-hour assays, and 135 μL of each solution was added to the wells for the 72-hour assays. This gave a final concentration of the respective doses and a total volume of 180 μL and 270 μL for the 48-hour and 72-hour treatment times. Additional wells of each plate contained the açaí extract and anticancer drug at respective concentrations to monitor the solubility of the extract. Cell viability was measured using MTT. The data obtained was analyzed via GraphPad Prism, SynergyFinder Plus and CompuSyn. Experiments were completed in triplicates and with at least three biological replicates.

2.10. Pre-Exposure of Açaí Botanical Dietary Supplement Extracts and Combination with Anticancer Drugs

To simulate the condition where individuals are consuming açaí containing substances prior to receiving anticancer medication, pre-exposure experiments were conducted. Cells were plated at the optimized density for the respective time points followed by incubation for 12 hours and then subsequent treatment with the range of extract concentrations used for combinatorial assays. After an additional 12 hours of incubation, the cells were administered a combination of açaí extract and anticancer drug and incubated for the length of the respective assay. MTT reagent was added to the treatment wells, and after an incubatory period, plates were processed, and absorbance was measured. Cell viability was calculated using the vehicle well as 100% cell viability. Experiments were completed in triplicates and with at least three biological replicates.

2.11. Fluorescence Microscopy for Determination of Cytotoxicity

An exploratory assessment of apoptosis was completed to determine whether the combination of açaí caused cytotoxicity through induction of apoptosis measured via fluorescence microscopy. Cells were seeded into an 8-well glass bottom chamber slide at the optimized seeding densities for the respective anticancer drug. After 24 hours, the cells were treated with F4AC açaí BDS extract, an anticancer drug, or a combination of both treatments for 48 and/or 72 hours. The açaí and respective anticancer drug were dosed at a single concentration that was chosen using the Synergy Plots obtained from SynergyFinder Plus. The dose of MTX used for MCF-7 cells fluorescence assay was 1.0 × 10⁻⁴ μM. The dose of F4AC extract used was 10 ng/mL of C3G (1.67 ug/mL extract). For the MCF-10A cells, the dose of MTX was 1.0 × 10⁻³ μM. The dose of F4AC extract was 1.0 × 10⁻⁴ ng/mL C3G (0.2 ng/mL extract), respectively. In a designated chamber slide well, Camptothecin at 5 μM, per assay guidelines, was added four hours prior to the end of the treatment window as a positive control for apoptosis. At the end of the treatment period, the cells were washed with D-PBS and then incubated with NucFix Red viability dye for 30 minutes at room temperature, protected from light. The cells were then washed with D-PBS and the Annexin V CF488A conjugate dye dissolved in media was added to the chamber wells for 15 minutes protected from light at 37°C. After the incubatory period, the cells were washed with 1X Annexin V CF488A binding buffer. The cells were fixed with 4% PFA for 10 minutes and subsequently washed with 1X Annexin V CF488A binding buffer. Excess buffer was removed, if necessary, and 25 μL of Vectashield with 4’,6-diamidino-2-phenylindole (DAPI) with mounting media was placed on each well of the chamber slide. The coverslip was applied and the slide was allowed to cure for 24 hours prior to imaging. Fluorescence imaging was conducted using a Keyence BZ-810X All-in-One Fluorescent Microscope.

3. THEORY AND CALCULATION

Cell viability from the MTT analysis was calculated by using the Equation 1 below.

$$\text{Cell Viability}\%=(\text{absorbance of treatment wells}/\text{absorbance of control wells})\times 100\%$$ Eq. 1.

Synergy plots of the combinations using CompuSyn software involved Supplementary Equation 1 (SE.1), the median effect plot equation and Supplementary Equation 2 (SE.2), the combination index (CI).

To measure the synergy from SynergyFinder Plus program using the Loewe Additivity Model, Highest Single Agent, Bliss Model and Zip Model, respectively, Supplementary Equations 3, 4, 5, and 6 were used (Zheng et al, 2022).

RESULTS

4.1. Açaí Extracts Standardized by Cyanidin-3-O-Glucoside (C3G) Content

Using LC-ESI-MS quantitation, the extracts used in this study were standardized based on C3G content. The individual and combination assays were conducted using the açaí extract in concentrations of standardized compound. The human-relevant concentration (HRC) was determined to be 2.321 ng/ml from the standardized compounds using previously reported Cmax values from a human pharmacokinetic study (Mertens-Talcott, 2008). In Figure 1, the total ion chromatogram (TIC) of F4AC, the botanical dietary supplement extract, and extracted ion chromatogram of C3G of F4AC is shown. Additional chromatograms can be seen in Heck et al., (2023).

Figure 1.

The total ion chromatogram (TIC) of F4AC açaí BDS extract and the extracted ion chromatogram (EIC) of cyanidin-3-O-glucoside (CG3) at 5.476 min retention time.

4.2. Optimization Experiments

The optimized parameters, shown in Table 1, for each cell line, were determined and used for the subsequent experiments, with the exception of vehicle concentration which was maintained at 0.1% DMSO. These conditions include assay time frame, puromycin (positive control) concentration optimization, and negative control/vehicle concentration optimization. The vehicle concentration was considered to alter the assay when the cell viability was less than 95%. As MTX was optimized for a 48-hour treatment window for MCF-7 cells, the cell seeding density for 72 hours was not needed to be confirmed for that cell line.

Table 1.

The optimized parameters for the individual and combinatorial assays for each cell line.

Optimized Conditions	MCF-7	MDA-MB-231	MCF-10A
48 HR Cell Seeding Density	1.0 × 105 cells/ml	2.0 × 105 cells/ml	1.0 × 105 cells/ml
72 HR Cell Seeding Density	N/A	1.0 × 105 cells/ml	5.0 × 104 cells/ml
Puromycin Concentration	3 μg/ml	1 μg/ml	1 μg/ml
DMSO (Vehicle) Max Concentration	0.3%	0.5%	0.3%

4.3. Cytotoxicity of Anticancer Drugs and Açaí Extracts as Single Treatments

MCF-7 cells were treated with MTX and TAM for the optimized time period of 48 hours. MDA-MB-231 and MCF-10A cells were treated at the optimized time periods of 48 hours for TAM and 72 hours for MTX. The data was analyzed using GraphPad Prism using the non-linear regression model with Hill slope analysis. Table 2 depicts the IC₅₀ values for each cell line with respective anticancer drugs.

Table 2.

The IC₅₀ values in micromolar of MTX and TAM, respectively, for the MCF-7, MDA-MB-231, and MCF-10A cell lines.

Cell Line	MTX IC50 (μM)	TAM IC50 (μM)
MCF-7 (ER+)	0.002 ± 0.002	26.97 ± 2.1
MDA-MB-231 (ER-)	0.141 ± 0.004	27.35 ± 1.5
MCF-10A (normal)	0.011 ± 0.01	7.914 ± 0.7

The açaí extracts in the single treatments were not toxic for the two cancer cell lines, MCF-7 and MDA-MB-231 cells; therefore, no IC₅₀ value was determined. This was expected as the extract concentrations were within human-relevant ranges, which may not have been high enough to cause significant toxicity to the cancer cells. As for the normal breast cell model, MCF-10A, one of the acidic methanol açaí extracts, F3AC, was the only extract to show cytotoxicity for the 48-hour exposure time with an IC₅₀ of 12.9 ng/ml of C3G. Additionally, at the 72-hour time point, for the normal cells, all açaí extracts caused at least a 20% decrease in cell viability, and the IC₅₀ of F3AC decreased to 5.013 ng/mL of C3G with the increase in the treatment period.

4.4. Açaí Botanical Dietary Supplement Extracts at Human Relevant Concentration (HRC) Causes Dose-Dependent Modulation of Anticancer Drug Toxicity

For the HRC screening, the comparison of fit analysis using GraphPad Prism showed no significant differences in dose-response curves between the anticancer drug alone and the HRC combination. It was decided to further analyze the cell viability by dose due to the low concentration of extract used to create the HRC (2.321 ng/ml C3G). The individual dose analysis using a t-test with unequal variances provided more insight into the screening results. Anticancer drug dose-response and HRC combination dose-response were compared, p-values lower than 0.05 were considered significant, and two extracts per cell line were chosen to move forward for a full combinatorial assessment. Overall, the two açaí extracts that had the most significant p-values were the two acidic methanol extracts of the BDS formulations of açaí, F3AC and F4AC. Per cell line, the most significant doses in the HRC screening differed depending on the extract and drug combination. For MCF-7 cells, the HRC screening combinations with the highest number of significant p-values were MRAQ and F4AC with both anticancer drugs, respectively. For the MDA-MB-231 cells, the screening combinations that had the most significant p-values were F3AC and F4ME across both anticancer drugs, respectively, and F4AC with TAM. There were two dose concentrations of significance for both F4AC and F4ME extracts, but F4AC only had significant dose concentrations with TAM and not with MTX. As for F4ME, the formulation açaí extract, there was a significant difference in dose concentration response in combination with MTX and TAM, which is why it was chosen for the combinatorial assay for these cell lines. For the MCF-10A cell line, there were various extracts that showed significance from the parameters previously discussed, but F3AC and F4AC were chosen for full combinatorial assays because they were common with either cancer cell line, which was used for comparison. The açaí extract MRME was included for the 7 by 7 combinatorial assays because it is the methanol extract of açaí powder obtained from Brazil, and it has been shown in the literature that the methanol extracts of botanicals contain the most bioactive compounds (Onyebuchi and Kavaz, 2020). Table 3 highlights the differences in the cell viability found between each dose of the IC₅₀ curve of the anticancer drug alone and each dose of the IC₅₀ curve of the anticancer drug with the HRC of the açaí extract.

Table 3.

The number of significant changes in dose for human-relevant concentration screening.

	1MCF-7 (ER+)		2MDA-MB-231 (ER−)		3MCF-10A (normal)
Açaí Extract	MTX	TAM	MTX	TAM	MTX	TAM
MRAQ 1	1	1	-	-	-	-
MRAC	-	1	-	-	1	1
MRET	-	-	-	1	-	-
MRME*	-	-	1	-	1	-
F3AC 2,3	-	-	3	2	1	1
F4AC 1,3	2	2	-	2	1	-
F4ME 2	-	1	1	1	-	-

The numbers on the açaí extracts correspond to the cell line that was used for the future combinatorial assays. The asterisk (*) indicates the extract that was used in combination on all cell lines. The hyphen indicates no significant changes (p-values>0.05) in dose-response found in the combination.

4.5. Zero Interaction Potency (ZIP) Score Provides the Most Comprehensive Preliminary Combinatorial Information for Açaí Extract Combinations with Anticancer Drugs

As we were unable to establish an IC₅₀ value for the açaí extracts alone with the two cancer cell lines, we chose to perform a non-constant dose ratio combinatorial assay using the same optimized dose range of the individual anticancer drug assays and the individual açaí extract assays in a 7 by 7 combination index, instead of using concentrations based on IC₅₀ values (Zhang et al., 2016). SynergyFinder Plus and CompuSyn were used to determine synergism, antagonism, or additive effect in combination with either TAM or MTX. Any synergy calculated from the combinations is referred to as potentiation due to the açaí extracts not showing any toxicity when administered alone on the cancer cells.

Cell viability data values were entered into CompuSyn for analysis of synergy or antagonism. The synergy information from CompuSyn such as isobologram, dose reduction index, and combination index are included in the supplementary data (Supplementary Figures 25-31). Additional data can be made upon request.

The data used for CompuSyn was entered into SynergyFinder Plus which provides synergy plots based on four different drug combination equations that were created depending on how the two drugs may interact (Zheng et al., 2022). The types of drug combination analyses include zero interaction potency (ZIP), SE. 6., which calculates the expected effect of two drugs under the assumption that they do not interact with each other (Zheng et al, 2022). The Loewe synergy score is calculated using the Loewe additivity model based on SE.3., which defines the expected effect as if the drug was combined with itself and considers the dose-response curves of individual drugs. The highest single agent (HSA) synergy plot is determined using SE. 4., and it states that the expected combination effect equals to the higher effect of individual drugs. The Bliss model, SE. 5., provides synergy data under the assumption that the two drugs exert their effects independently. The ZIP model was chosen as the synergy plot analysis to use for the comparison of drug-botanical interactions due to the number of lower p-values provided by SynergyFinder Plus (Figure 2.) in comparison to the other models across the experiments. Since the exact mechanism of the interaction between the açaí extracts and the anticancer drugs is unknown, there was also an assumption that the two treatments would not interact with each other. Although the Bliss model contains similar assumptions, the ZIP model was ultimately chosen. For simplicity, the ZIP model will be used for comparisons of synergy throughout the entirety of this paper. The ZIP synergy scores for all tested combinations are in Table 4. All four plots of synergy for each combination are in the supplementary data (Supplementary Figures 1-24).

Figure 2.

Synergy plots of all four drug combination analyses from SynergyFinder Plus of the combination of MTX and the aqueous extract of açaí on MCF-7 cells.

Table 4.

The ZIP synergy scores of concomitant açaí extract and anticancer drug combinations provided by SynergyFinder Plus.

	MCF-7 (ER+)		MDA-MB-231 (ER−)		MCF-10A (normal)
EXTRACT	MTX	TAM	MTX	TAM	MTX	TAM
Methanol Extract of Fruit Powder from Brazil (MRME)	2.22 Additive	7.68 Additive	7.22 Additive	−1.79 Additive	16.75 Synergistic	−1.36 Additive
Aqueous Extract of Fruit Powder from Brazil (MRAQ)	8.98 Additive	9.44 Additive	-	-	-	-
Acidic Methanol Extract of Brand #2 Supplement (F4AC)	8.95 Additive	6.31 Additive	-	-	23.04 Synergistic	10.35 Synergistic
Acidic Methanol Extract of Brand #1 Supplement (F3AC)	-	-	7.08 Additive	0.37 Additive	10.2 Synergistic	6.26 Additive
Methanol Extract of Brand #2 Supplement (F4ME)	-	-	4.64 Additive	−0.14 Additive	-	-

Values greater than 10 are considered synergistic, less than −10 are antagonistic, and between −10 and 10 are considered additive. The hyphen indicates combinations that were not tested for those cell lines.

4.6. Açaí BDS Extracts Cause an Additive Effect in Combination with Anticancer Drugs for Breast Cancer Cells

There was an additive effect produced for all concomitant combinations of the tested açaí extracts and either anticancer drug for the two breast cancer cell lines. There was no synergy in any of the combinations for the breast cancer cells by the definition of SynergyFinder Plus. It is important to note that since the extracts did not cause any toxicity alone, the interaction between the anticancer drugs and the açaí extracts is termed potentiation. There were a few combinations that did get close to reaching the threshold, a score of 10 or higher, set by SynergyFinder Plus as the definition of synergy. As shown in Table 4, The two highest synergy scores for MCF-7 cells were from the same extract, MRAQ. The synergy score was higher with TAM at 9.44. In the combinations, the synergy scores were generally higher for TAM for the MCF-7 cells than with MTX with the exception of MRME. The two highest synergy scores for the MDA-MB-231 cells were for the MRME and F3AC açaí extracts and MTX. In the case of MDA-MB-231 cells, all synergy scores were higher in combinations with MTX.

4.7. Açaí Extracts and MTX Have a Synergistic Relationship in Concomitant Exposure for MCF-10A Cells

The non-tumorigenic mammary epithelial breast cell model, MCF-10A, was analyzed for synergy via cell viability. MRME, F3AC, and F4AC all exhibited synergy with MTX, according to SynergyFinder Plus analysis. The combination of MRME and MTX produced a synergistic interaction, whereas the same açaí extract and TAM produced a low signal in the additive effect range. This result was similar to the acidic methanol açaí extracts, F3AC and F4AC. F4AC had synergy with MTX for the normal cells across the majority of the dose range, as shown in Figure 3. The MDA-MB-231 cells were used as a comparison in this combination due to the HRC significance points. Although the same general results were seen in the MDA-MB-231 cell line, the overall potentiation signal was lower and in the additive effect range provided by SynergyFinder Plus. It should be noted that F3AC caused significant cytotoxicity towards MCF-10A cells when administered alone but at high doses of extract.

Figure 3.

The 2D (top right) and 3D (top left) ZIP synergy plots of MCF-10A cells treated with F4AC and MTX combination. The Compusyn isobologram of the combination (bottom).

For all the açaí BDS extracts, there was a common effect where more cytotoxicity was seen on the normal cell model in combination with methotrexate. There is a possibility that compounds present in these extracts increase methotrexate toxicity or directly cause toxicity. Further studies are required to validate the mechanism of toxicity. The methanol extract from the açaí powder from Brazil was decided for a pre-exposure evaluation because of the chemical complexity of methanol extracts as well as the effects across all cell lines as it was the only extract tested among all 3 cell lines with both anticancer drugs.

4.8. Pre-Exposure of Methanol Extract of Açaí Powder before Combination of Anticancer Drug and Açaí Extract Combination Suggests Different Toxicity Profiles Depending on Anticancer Drug

In the concomitant exposure of MRME and MTX, the açaí extract slightly potentiated MTX for the two cancer cells causing an overall additive effect, shown in Figure 4.

Figure 4.

The 3D ZIP synergy plots of the concomitant combination of MTX and MRME on the MCF-7 (left), MDA-MB-231 (middle), and MCF-10A (right) cells.

When the MCF-7 cells are exposed to MRME prior to an administration of MRME and MTX, the synergy score increases from 2.22 to 5.63, shown in Table 5 and Figure 5, indicating an increased toxicity of the combination. For the MDA-MB-231 cells, the synergy score decreased from 7.22, concomitant exposure, to 6.33 in the pre-exposure conditions indicating a slight decrease in toxicity when the cells were exposed to the extract before the combination. As for the normal cell line, MCF-10A, the combination treatment of MTX and MRME caused a synergistic effect with a score of 16.75 but when the cells were treated with MRME prior to the combination, there was a dramatic decrease in synergy score to 0.86. This is indicative of a dramatic reduction in MTX cytotoxicity when the cells are treated with MRME before the combination of MTX and MRME.

Table 5.

The ZIP Synergy scores of the concomitant vs. pre-exposure MRME and MTX combinations.

	MCF-7 (ER+)		MDA-MB-231 (ER−)		MCF-10A (normal)
EXTRACT
	MTX	TAM	MTX	TAM	MTX	TAM
MRME	2.22 Additive	7.68 Additive	7.22 Additive	−1.79 Additive	16.75 Synergistic	−1.36 Additive
Pre-Treatment w/MRME	5.63 Additive ⬆	3.15 Additive ⬇	6.33 Additive ⬇	2.64 Additive ⬆	0.86 Additive ⬇	6.94 Additive ⬆

The red arrows indicate increases in synergy score and the green arrows indicate decrease in synergy score.

Figure 5.

The 3D ZIP synergy plots of the pre-exposure combination of MTX and MRME on the MCF-7 (left), MDA-MB-231 (middle), and MCF-10A (right) cells.

In the concomitant exposure of MRME and TAM, potentiation of TAM was noted exclusively in the MCF-7 cells, shown in Figure 6.

Figure 6.

The 3D ZIP synergy plots of the concomitant combination of TAM and the MRME on MCF-7 (left), MDA-MB-231 (middle), and MCF-10A (right) cell lines.

However, in the pre-exposure combination assay, Figure 7, the potentiation of TAM decreased by half for the MCF-7 cells, making this combination scenario less toxic to that cell line. This is the only case where the MRME pre-treatment caused a reduction in synergy for TAM combinations. The ZIP synergy score in the concomitant combination for MDA-MB-231 cells was trending towards antagonism with a score of −1.79, but the pre-treatment with MRME caused an increase in synergy score, which supports increased toxicity.

Figure 7.

The 3D ZIP synergy plots of the pre-exposure combination of TAM and MRME on the MCF-7 cells (left), MDA-MB-231 (middle), and MCF-10A(right) cells.

The MCF-10A cells experienced the most significant change in synergy scores from the combination scenarios of TAM. In the concomitant exposure, the cells were in the additive range at −1.79. When the cells were treated with MRME and then exposed to the combination, there was an increase in synergy score to 6.94, indicating an increase in toxicity. Although there are significant changes between the synergy scores of the concomitant and the pre-exposure combinations, the scores remained in the additive range. No scores moved from one level to another for the TAM combinations.

When comparing the two anticancer drugs used for this study, there is an indication of an increase in cytotoxicity only for MCF-7 with the pre-exposure conditions in the MTX combinations, and the other two cell lines showed a decrease in toxicity. The effect of the pre-exposure is the opposite for TAM, where there is a decrease in toxicity for the MCF-7 cells but an increase in toxicity for the other two cell lines, MDA-MB-231 and MCF-10A. There is also a significant possibility of cytoprotective characteristics of MRME when treated before the cells are exposed to the combination.

4.9. Acidic Methanol Açaí BDS Supplement (F4AC) Extract Shows Most Toxicity through Synergy with MTX in MCF-10A Cells

As the focus of this study was determining increased toxicity in the concomitant exposure of açaí and anticancer drugs, the highest synergy score for the MCF-10A cells was further analyzed. The acidic methanol extract of one brand name BDS formulation, F4AC, showed the most synergy with MTX overall and with MCF-10A cells (Figure 3).

Synergistic concomitant exposure is exhibited at nearly all doses of extract and MTX combinations on the MCF-10A cells. MCF-7 cells were also tested with this MTX and F4AC açaí BDS extract combination with potentiation of MTX seen at higher doses of F4AC; particularly, these doses were above that of the HRC of C3G, as shown in Figure 8.

Figure 8.

The 2D (top right) and 3D (top left) ZIP synergy plots of MCF-7 cells treated with F4AC and methotrexate combination.

This indicates that the F4AC extract may cause significant cytotoxic effects on normal breast cells while only slightly increasing cytotoxic impact on the breast cancer cells. It is imperative to distinguish the fact that F4AC showed little cytotoxicity at high doses when administered alone but was able to cause a dramatic effect of toxicity in the combination.

4.10. Combination of Açaí and Methotrexate Increases Apoptosis Signal for MCF-10A Cells

In an exploratory study of apoptosis induction, F4AC, one of the açaí BDS extracts, was chosen as it produced the highest synergy score of all drug combinations and cell lines. Also, it was the extract that showed a possible indication of methotrexate-induced toxicity for MCF-10A cells, as it did not significantly reduce cell viability at high concentrations when administered alone. The cells were stained with DAPI, Annexin V CF488A, and NucFix Red for assessment of apoptosis and necrosis. As shown in Figure 9, there was a significant reduction in the number of cells on the slide of the combination of F4AC and MTX shown with the nuclei staining via DAPI.

Figure 9.

DAPI, Annexin V, and NucFix Red of MCF-10A cells treated with MTX, F4AC, and the combination of both. The images were taken at 200x magnification. (a)Vehicle; (b)MTX at 1 nM (c)F4AC at 1 pg/mL C3G (d)Combination of MTX and F4AC (e)Positive Control for Apoptosis

There was also a slight increase in Annexin V CF488A signal, which indicates early apoptosis in the combination. It seems that there may have also been slight apoptosis induction with the F4AC single treatment due to the increase in fluorescence of Annexin V CF488A. There was also a significant increase in necrotic cells with the combination higher than any other treatment.

The MCF-7 cells showed no apparent signs of apoptosis with any of the single treatments or combinations, Figure 10. This result was somewhat expected due to the low concentration of both anticancer drug and açaí BDS extracts used. These results corroborated with the results from the cell viability values and synergy plots of enhanced cytotoxicity in combinatorial exposure of açaí BDS extracts and non-CYP3A4 interactive anticancer agent, methotrexate, for the normal breast cells, MCF-10A.

Figure 10.

DAPI, Annexin V, and NucFix Red of MCF-7 cells treated with MTX, F4AC, and the combination of both. The images were taken at 200x magnification. (a)Vehicle; (b)MTX at 0.1 nM (c)F4AC at 10 ng/mL C3G (d)Combination of MTX and F4AC (e)Positive Control for Apoptosis

5. DISCUSSION

This study examines the pharmacodynamic interactions between anticancer drugs and açaí with a particular focus on the implications of induced toxicity of non-CYP3A4 interactive drugs from açaí in breast cancer with a preliminary mechanistic assessment. Açaí, a supplement commonly used in South America for its antioxidant effects, has been previously studied for its health benefits, but limited information on the toxicity profile of açaí, especially in combined use with medication. The cytotoxicity of the various combinations of açaí extracts with MTX and TAM were investigated in three in vitro breast cell lines to determine safety and to evaluate potential anticancer drug efficacy changes induced by concomitant açaí BDS use.

Açaí BDS extracts were standardized based on cyanidin-3-O-glucoside content and tested in the in vitro analyses using the concentration of the standard compound which, to the authors’ knowledge, has not been done before for açaí or for cyanidin-3-O-glucoside. The cancer cells had no reduction in cell viability with the single treatment of the açaí extracts. The MCF-10A cell viability decreased by at least 20% for all of the açaí extracts in single treatments. This is the first observation of açaí toxicity in the non-tumorigenic mammary epithelial breast cell model.

Using the standardized extracts, a screening was conducted to determine which extracts should be moved forward for the full combinatorial assay. The human-relevant concentration was based on a previously reported amount of cyanidin-3-O-glucoside (C3G) that was found in the blood after oral administration of açaí (Mertens-Talcott et al., 2008). The screening narrowed down the extracts for testing from 7 extracts to 3 extracts using p-values from t-test unequal variances analyses. The screening was integrative to the research as it showed that low concentrations of açaí can cause significant differences in response to anticancer drugs even if there is not a drastic change in dose-response curves. Some cases have addressed açaí or an anticancer drug in vitro as a single treatment but not in combination. (da Silva, et al., 2022). In particular, the MCF-7 cell line has been tested with açaí seed extract, and cell death and other issues were reported, but the antiproliferative activity was reported at concentrations beyond the human achievable of extract (da Silva, et al., 2022). This is the first report on the concept of using human-relevant doses for in vitro assessment of açaí and the response of low concentrations of açaí in combination with anticancer drugs in vitro. Lower concentrations of açaí causing synergistic interactions with methotrexate, in this study, indicated that there is a higher risk of adverse events occurring from this combination since only a minimal quantity of açaí constituents is needed to produce this synergistic interaction contributing to possible toxicity. Further combinatorial analyses were needed to determine the exact doses of synergy.

To analyze the potential synergy, it was decided to use SynergyFinder Plus and CompuSyn to gather information from two types of synergy analysis tools. This work is the first to report on the use of SynergyFinder Plus for the assessment of anticancer drug and botanical extract combinations via cell viability measurements. The use of SynergyFinder Plus in this work allows for multiple analyses of various types of interactions based on mathematical drug interaction equations such as Loewe and Bliss (Zhang et al., 2016). SynergyFinder Plus also provided 3D graphs and 2D heatmaps that allowed for better visualization and understanding of the data. CompuSyn was also used to validate the information provided from SynergyFinder Plus and the data from SynergyFinder Plus was more informative and palatable than the isobolograms from CompuSyn. In this study, SynergyFinder Plus assisted with determining whether the interactions between açaí and the anticancer drugs were synergistic, additive, or antagonistic.

Once extracts, F3AC, F4AC, and MRME, were confirmed via human-relevant concentration screening, they were used for the combinatorial assays. It was determined that, for the breast cancer cells, MCF-7 and MDA-MB-231, most of the açaí extracts enhanced the effects of the anticancer drugs through potentiation, which is where there was no effect of the açaí extract alone, but when combined with the anticancer drug, the effect increased. Although SynergyFinder Plus calculated the interactions between the açaí extracts and the anticancer drugs as an additive effect, it is considered potentiation by definition. This is the first account, to the authors’ knowledge, of the pharmacodynamic interactions of açaí and anticancer drugs, so it is imperative to make this distinction. It is also important to note that the extract was from the açaí pulp and not the seed, as in other studies (da Silva, M.A.C.N. et al., 2022) where cytotoxicity to cancer cells was found. This could also explain why the açaí extracts had no antiproliferative activity for the cancer cells when treated alone. For the MCF-10A cells, the combinations produced mostly synergistic interactions except for TAM with MRME or F3AC, where there was an additive effect. This marks the first occurrence of açaí and anticancer drug toxicity for MCF-10A cells.

Potentiation and synergy have been demonstrated amongst various combinations of natural products and anticancer drugs, such as echinacea, ginkgo biloba, and green tea (Sridhar, 2020). These interactions can reduce drug efficacy or, conversely, increase toxicity (Sridhar, 2020). Many of these botanical-drug interactions were determined through pharmacokinetic studies, but these interactions can also be evaluated with pharmacodynamic studies (Li, X. et al, 2019). This work provides the foundation for future studies to determine the interaction between açaí botanical extracts and anticancer drugs.

MTX potentiation was low for the MCF-7 cell line and higher with the MDA-MB-231 cell line. Conversely, for TAM, the potentiation was high for the MCF-7 cells and low for the MDA-MB-231 cell line. This same combination was also performed on the non-tumorigenic mammary epithelial cells, MCF-10A, and the results were similar to the MDA-MB-231 cells. This suggests that the effect of the methanol extract and anticancer combination is specific to MCF-7 cells and/or TAM drug mechanisms. TAM generally had the same pattern of potentiation by all extracts with both cancer cell lines. The highest signal of potentiation was produced with the MCF-7 cells which could be due to the mechanism of TAM and the presence of overexpressed estrogen receptors (Mokhtar et al., 2022). There are reports of plant-derived non-steroidal compounds interfering with estrogen receptors (Basu et al., 2018), such as isoflavones, flavonoids, and lignans, which have been reported in açaí (Heck et al., 2024). There is also a possibility of interactions with the MCF-7 cell membrane transporters, in particular, p-glycoprotein, which is also overexpressed in these MCF-7 cells (Mokhtar et al., 2022). The MCF-7 cell line was the only cell line that had more potentiation or toxicity from the extracts with TAM than with MTX. The MDA-MB-231 and MCF-10A cell lines showed higher synergy scores, with the MCF-10A cell line experiencing the highest synergy in the combination of all açaí extracts and MTX.

Pre-exposure of the açaí extract with subsequent addition of the combined anticancer drug and açaí extract showed varying results. Regarding MTX, there was a slight increase in potentiation following açaí extract pre-exposure for MCF-7 cells when compared to the concomitant exposure. For the MDA-MB-231 cells, there was a slight decrease in potentiation. As for the MCF-10A cells, there was a significant reduction in synergy score with the pre-treatment of MRME in combination with MTX. The concomitant interaction was regarded as 16.75, which is synergistic by SynergyFinder Plus conditions, but when the cells were exposed to MRME before the combination, the synergy score dropped to 0.86, which is in the additive effect range, but the number is so close to zero, it is indicative of no response. Açaí has been previously shown to exhibit cytoprotective activity with normal cells, specifically with cardiovascular cells in an in vivo model (Alessandra-Perini et al., 2001 and Polegato, B.F et al., 2019) in the subject of attenuating anticancer-induced toxicity. In Polegato, B.F et al., there were reports of in vivo activity that could not be verified in vitro. However, in this study, the cytoprotective nature of açaí was produced with pre-administration. Although the mechanism is currently unknown, this is the first report of cytoprotective activity of açaí fruit extract in MCF-10A cells.

In the case of TAM, there was a slight decrease in potentiation following açaí extract pre-exposure for the cancer cell lines. However, there was a contrasting increase in potentiation for the normal cells under these same conditions. This could potentially be due to the aforementioned cell membrane protein interactions (Basu et al., 2018) with the numerous compounds in the extracts, although this remains unclear.

The original focus of this study was to determine the implications of açaí and anticancer drug toxicity for non-tumorigenic mammary epithelial cells in a breast cancer model, so it was decided to move forward with apoptosis studies of F4AC and MTX combinations in MCF-10A and MCF-7 cells. The FAERS database analysis indicated possible interaction between açaí containing supplements and non-CYP3A4 interactive anticancer drugs. This analysis is supported by the in vitro testing of the combination in this study. It is shown that the non-CYP3A4 interactive anticancer drug, MTX, caused the most potentiation overall with all açaí extracts with the estrogen receptor-negative cell line, MDA-MB-231, and normal breast cell model, MCF-10A. The strongest synergy was with the MCF-10A cells, which provided strong support for further investigation into the mechanisms of toxicity.

The preliminary fluorescence imaging for assessment of apoptosis using DAPI, Annexin V CF488A, and NucFix Red confirmed that there was a mechanism of increased toxicity in the combination of açaí BDS extracts and non-CYP3A4 interactive anticancer drug, methotrexate, via apoptosis induction. The MCF-10A cells were the most impacted by the combination of F4AC and methotrexate as indicated by the synergy scores. There was a significant decrease in the number of cells and there was an increase of apoptosis and necrosis signals. It is relevant to note that the toxicity of the combinations was at low doses of both the açaí extracts and the anticancer drugs, which indicates a possibility for more interactions as the dose is within the range of human relevancy. There is evidence of flavonoid-related compounds from botanical origins that have been shown to cause oxidative DNA damage, leading to cancer in estrogen-sensitive organs like the vulva and uterus. (Murata, M. et al., 2001). Specifically, the isoflavones genistein and daidzein were shown to be the inducers of oxidative DNA damage in MCF-10A cells. (Murata, M. et al., 2001). This is a possible explanation apart from the known cytotoxic effects of MTX on the MCF-10A toxicity from the F4AC and MTX combination since isoflavones have been reported in the açaí extracts used in this study (Heck et al., 2024). Another explanation for the observed toxicity to the normal breast cells is that there are previous reports of other bioactive natural compounds that cause disruptions of E-cadherin which is responsible for cell adhesion. (Lee, J., et al., 2015). The fluorescence images showed a significant decrease in the number of MCF-10A cells which may have been due to a disruption of E-cadherin which is required for MCF-10A cell survival. (Martin, S. S., et al., 2001). This could also explain the cardiovascular injury reported in the FDA database for the concomitant use of açaí and non-CYP3A4 interactive drug (Nachtigal, P., et al., 2001). Further investigation into the chemical composition of açaí and the specific compounds that lead to toxicity is needed to evaluate the cause of the observed interaction.

One limitation of this study is the limited clinical relevance due to the study being conducted on in vitro models and it may not fully reflect the complex interactions in the human body. Another limitation of this study is that the açaí phytochemical composition may vary widely and there were only two botanical dietary supplements chosen which may not represent all commercial products consumed by patients. There are also limitations with the SynergyFinder Plus analysis in the context of botanicals and the mechanisms of the toxicity towards normal cells in the combination remain unclear.

In this study, it was ultimately determined that low doses of both anticancer drugs and açaí botanical extract caused significant toxicity towards normal cells but little to no toxicity for cancer cells. There is strong evidence for an increase in incidences of adverse events from the concomitant use of açaí and anticancer drugs for breast cancer. It was also confirmed that the combination of MTX and açaí leads to apoptotic signals in normal cells. Future studies should be conducted on the mechanism of the toxicity of the combination of açaí and methotrexate and other non-CYP3A4 interactive drugs.

6. CONCLUSION

To the best of the authors’ knowledge, this is the first report of the pharmacodynamic interactions between açaí extracts and the anticancer drugs, MTX and TAM on MCF-7, MDA-MB-231, and MCF-10A cell lines. Additionally, this is the first report of SynergyFinder Plus utilization to investigate the synergy of natural products and anticancer drug combinations through a 3D visualization. This is also the first report of MCF-10A toxicity from the combination of MTX and açaí extracts with measurements of apoptosis. The synergy that is exhibited by the combination of açaí extracts and anticancer drugs is shown here to be dependent on the drug and its dose, in addition to the açaí extraction solvent, and açaí source. There was significant potentiation of TAM and the açaí extracts for the MCF-7 cell line and with MTX and açaí extracts for the other two cell lines, MDA-MB-231 and MCF-10A.

The potentiation that was shown from the combinations on the cancer cell lines was not strong enough to make any conclusions on improving drug efficacy or drug outcome. It should be noted, however, that no significant antagonism was determined from these experiments, so it can be stated that there was no significant decrease in in vitro drug efficacy on the breast cancer cells. The strongest potentiation was observed by the acidic methanol extract of one of the açaí BDS, F4AC, on the MCF-10A cells. By the definitions of SynergyFinder Plus, almost all combinations were in the range of additive effect, apart from MTX with the methanol extract of açaí powder (MRME) and MTX and TAM with the acidic methanol extract of one of the BDS (F4AC). The normal cell line was significantly affected by the combination of the anticancer drug and açaí extract combination via apoptosis induction, which calls for further evaluation of the toxicity of the combination of açaí and anticancer drugs.

Future studies will be conducted to evaluate the toxicity mechanisms of these combinations with an assessment of the 3D synergy plots to best visualize the exact interaction between açaí and anticancer drugs. Additional assessments will be completed for C3G compound alone with the non-CYP3A4 interactive anticancer drugs. Future studies will also include analysis of key proteins related to normal cell toxicity and the exact mechanism of the açaí and anticancer drug combination.

Highlights.

• Anticancer drugs show synergy with açaí extracts from powder from fruit pulp and dietary supplements

• Methotrexate and açaí supplement extract combinations cause an increase in toxicity in MCF-10A breast cells

• Pre-exposure of açaí fruit pulp extract shows cytoprotective activity for MCF-10A

• SynergyFinder Plus provides 2D and 3D botanical-drug combination visualization to help with confirming doses with the highest synergistic interactions

ABBREVIATIONS

ATCC

American Type Culture Collection

BDS

Botanical dietary supplement

C3G

Cyanidin-3-O-Glucoside

CAERS

Center for Food Safety and Applied Nutrition Event Reporting System

CYP3A4

Cytochrome p450 3A4

DAPI

4’,6-diamidino-2-phenylindole

DMSO

Dimethyl sulfoxide

D-PBS

Dulbecco’s Phosphate Buffered Saline

EIC

Extracted Ion Chromatogram

F3AC

Acidic methanol extract of Nature Way’s Açaí Supplement

F4AC

Acidic methanol extract of Natrol’s Açaí Supplement

F4ME

Methanol extract of Natrol’s Açaí supplement

FAERS

Food and Drug Administration Adverse Event Reporting System

FDA

Food and Drug Administration

HRC

Human Relevant Concentration

HSA

Highest single agent

IC₅₀

Half-maximal inhibitory concentration

LC-ESI-MS

Liquid chromatography electrospray ionization mass spectrometry

MEBM

Mammary Epithelial Cell Basal Medium

MEGM

Mammary Epithelial Cell Growth Medium

MRAC

Acidic methanol extract of Mountain Rose Powder

MRAQ

Aqueous extract of Mountain Rose Powder

MRET

Ethanol extract of Mountain Rose Powder

MRME

Methanol extract of Mountain Rose Powder

MTX

Methotrexate

MTT

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

NCI

National Cancer Institute

NW

Nature’s Way

PFA

Paraformaldehyde

RPMI

Roswell Park Memorial Institute

TAM

Tamoxifen

TIC

Total Ion Chromatogram

ZIP

Zero Interaction Potency