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. Author manuscript; available in PMC: 2024 Nov 21.
Published in final edited form as: Phytomed Plus. 2024 May 13;4(3):100578. doi: 10.1016/j.phyplu.2024.100578

Stilbene-rich extract increases the cytotoxic effects of paclitaxel in hormone receptor-positive and triple-negative breast cancer spheroids

Sepideh Mohammadhosseinpour a,b, Alexx Weaver b, Sara V Hernandez-Madrigal b, Gaurav Gajurel a,b, Amit Raj Sharma b,c, Fabricio Medina-Bolivar b,c,*
PMCID: PMC11580422  NIHMSID: NIHMS2018657  PMID: 39574482

Abstract

Background:

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that lacks three receptors commonly found in breast cancer cells. It is associated with high mortality rates, and therefore, investigating therapies to increase survival rates is crucial. Plant-derived compounds are being explored as potential adjuvants for common chemotherapy drugs, such as paclitaxel (Pac).

Purpose:

The study aimed to evaluate the cytotoxic effect of a prenylated stilbene-rich extract (SRE) produced via a sustainable peanut hairy root culture system and observe its potential as an adjuvant for Pac in human triple-negative and hormone receptor-positive (HR+) breast cancer spheroids. The effects were compared to arachidin-1 (A-1), a cytotoxic prenylated stilbene present in the extract.

Methods:

SRE was produced from elicited peanut hairy root cultures. The extract was purified using chromatography techniques to obtain the prenylated stilbene arachidin-1 (A-1) with a purity of over 95 %. TNBC cell lines MDA-MB-231, MDA-MB-436, and HR+ breast cancer cell line MCF-7 were used to evaluate the cytotoxicity and apoptotic activity of SRE in comparison with A-1. Two-dimensional (2D) experiments were performed using cell viability assays and imaging microscopy. Three-dimensional (3D) spheroids cultures were established, and the impact of SRE alone and in combination with Pac on cell viability and caspase 3/7 activity was evaluated.

Results:

SRE (10 μg/mL) inhibited cell proliferation by approximately 50 % in TNBC and MCF-7 cells in a time-dependent manner. Additionally, Annexin V FITC/PI staining revealed that SRE (10 μg/mL) induced more apoptosis than A-1 at the equimolar concentration (5 μM) in MDA-MB-231 cells. Combining SRE with Pac decreased spheroid cell viability and induced apoptosis by activating caspases 3 and 7 in TNBC and HR+ breast cancer spheroids.

Conclusions:

These findings highlight the potential of SRE as a novel adjuvant for Pac chemotherapy in TNBC and HR+ breast cancer treatment.

Keywords: Triple-negative breast cancer, Stilbene-rich extract, Arachidin-1, Paclitaxel, Spheroid cultures

1. Introduction

Plants naturally possess a variety of bioactive specialized metabolites which have been historically used as treatments for numerous diseases in humans, including cancer (Atanasov et al., 2021). Plant extracts are a combination of diverse phytochemicals such as phenolics, terpenes, polysaccharides, alkaloids, and many other compounds (Pradeep et al., 2022). Whole plant extracts are a preferable alternative to purified compounds due to the absence of harmful chemicals in the purification process, availability of abundant raw materials, and low production cost. Furthermore, extracts have shown improved effectiveness when compared to purified compounds due to the beneficial interactions among individual constituents (Pradeep et al., 2022; Caesar and Cech, 2019). However, biotic and abiotic stresses, as well as seasonal variations, affect the yield and composition of metabolites in tissues from plants grown in the field (Li and Zidorn, 2022; Piasecka et al., 2019). To overcome these issues, hairy root cultures from various plants have been established as sustainable and controlled bioproduction platforms for specialized metabolites. Consequently, hairy root culture-derived extracts exhibit high levels of bioactive metabolites and reproducible chemical profiles; therefore, hairy root cultures are amenable and sustainable bioproduction platforms to produce extracts suitable for bioassays, nutraceutical, and pharmaceutical applications (Condori et al., 2010; Nopo-Olazabal et al., 2013; Gajurel et al., 2021, 2022; Wawrosch and Zotchev, 2021).

Stilbenes are polyphenolic phytoalexins that are naturally produced by certain plants, response to biotic and abiotic stresses such as pathogens, UV light, or agricultural chemicals (Donato et al., 2012). These specialized metabolites have garnered research interest due to their intricate structures, including linear and cyclic prenylated derivatives, and diverse biological activities, such as antioxidant, anti-inflammatory, antimicrobial, and anticancer properties (de Bruijn et al., 2018; Abbott et al., 2010; Chen et al., 2018; Chang et al., 2006; Shen et al., 2009). The peanut hairy root culture system has been established as a sustainable platform for producing non-prenylated and prenylated stilbenes when stressed with chemical elicitors (Fang et al., 2020; Yang et al., 2015). The addition of methyl jasmonate (MeJA), methyl-β-cyclodextrin (CD), hydrogen peroxide (H2O2), and magnesium chloride (MgCl2) to peanut hairy root cultures results in elevated production of stilbenes, which are secreted into the culture medium facilitating the production of extracts enriched in these polyphenolic compounds (Fang et al., 2020; Niesen et al., 2013). The Medina-Bolivar laboratory has thoroughly analyzed this extract which includes the non-prenylated stilbenes resveratrol (RES) and piceatannol, as well as prenylated stilbenes arachidin-1 (A-1), arachidin-2, arachidin-3 (A-3), arachidin-5, and arachidin-6 (Fig. 1; Supplementary Fig. S1) (Yang et al., 2015; Gajurel et al., 2021). Earlier research indicates that this extract exhibits strong antioxidant properties in vitro (Gajurel et al., 2021). However, the effect of peanut hairy root stilbene-rich extract (SRE) on cancer cells remains to be studied.

Fig. 1.

Fig. 1.

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Chemical structures of non-prenylated and prenylated stilbenes present in the peanut hairy root culture extract.

Breast cancer is one of the most common cancers and is a leading cause of cancer-related mortality among females (Waks and Winer, 2019). Triple-negative breast cancer (TNBC) accounts for approximately 15–20 % of all breast cancer cases and is associated with a high risk of recurrence and poor prognosis (Zagami and Carey, 2022; Ge et al., 2022). TNBC is characterized by the absence of estrogen, progesterone, and human epidermal growth factor 2 receptors (Ge et al., 2022). The morbidity and mortality rates of TNBC are significantly higher due to its fast growth rate, metastatic potential, and high tendency to acquire treatment resistance (Yang et al., 2020). Anthracycline or taxane-based regimens are the first-line treatment for TNBC patients (Dent et al., 2007; Ge et al., 2022). Paclitaxel (Pac), a taxane natural product originally derived from the Pacific yew tree, Taxus brevifolia, is an FDA-approved microtubule-stabilizing drug that has been used for the treatment of ovarian, breast, and lung cancer (Weaver, 2014). Patients treated with conventional chemotherapy drugs, such as Pac, often experience severe side effects. Therefore, exploring plant extracts as emerging therapies that specifically target metastasis and cause minimal side effects is desirable.

Several types of mammalian cells when cultured in a non-adhesive environment or suspension can cluster together and develop into three-dimensional (3D) multicellular spheroids (Ricci et al., 2013). In contrast to standard monolayer cultures, these spheroids possess structural and functional characteristics that more closely mimic actual tissues (Fennema et al., 2013). 3D spheroid culture possesses advantages over two-dimensional (2D) monolayer culture by facilitating cell-cell and cell-matrix interaction, providing a similar in vivo physicochemical environment, maintaining intrinsic phenotypic properties, and secreting cytokines, chemokines, and angiogenic factors (Ryu et al., 2019; Fennema et al., 2013). Recently, the 3D spheroid culture system has gained popularity in diverse fields, including cancer research and drug screening, due to its ability to mimic features of tumors in vivo and bridge the gap between 2D cell culture systems and in vivo models.

The prenylated stilbene A-1, purified from hairy root cultures of peanut, induces apoptosis in TNBC cells and spheroids (Mohammadhosseinpour et al., 2022, 2023). In addition, this stilbene exhibited a synergistic effect with Pac on inhibiting cell proliferation in TNBC cells and spheroids, and importantly, A-1 was not toxic to non-cancerous breast epithelial cells. However, the effect of stilbene-rich extract (SRE), containing A-1 and other prenylated stilbenes, on cancer cells has not been investigated. The main objectives of the study are to evaluate the cytotoxic effects of SRE and its potential as an adjuvant therapy alongside paclitaxel (Pac) in treating TNBC and HR+ breast cancer. The study aims to assess the efficacy of SRE alone and in combination with Pac, comparing its effects with those of A-1. Therefore, the purpose of this study was to evaluate the potential of the SRE as a novel anticancer agent and adjuvant for Pac in spheroid culture systems of hormone receptor-positive and TNBC cells. The anticancer capacity was compared between SRE and the equimolar concentrations of A-1 in the extract.

2. Materials and methods

2.1. Production of SRE and purification of A-1

Previously established hairy root culture of peanut cv. Hull was used as a bioproduction platform for prenylated stilbenes as described previously (Condori et al., 2010). To produce SRE, ten hairy root cultures of peanut grown, in 250 mL flasks containing 50 mL of culture medium, were co-treated with a combination of four elicitors i.e., MeJA (125 μM), CD (18 g/L), H2O2 (3 mM), and MgCl2 (1 mM) for 192 h, and extraction was performed as previously described (Fang et al., 2020; Gajurel et al., 2021). The extract was analyzed using reversed-phase HPLC in a SunFire™ C18 column (Waters; Milford, MA, USA) (5 μm, 4.6 × 250 mm, UV detection at 340 nm).

For the purification of A-1, a previously established strategy was used (Sharma et al., 2022). Briefly, peanut hairy roots were co-treated with MeJA (125 μM) and CD (18 g/L) for 192 h, and the elicited culture medium was extracted using ethyl acetate. The extract was fractionated using silica gel column chromatography with a step gradient of CHCl3–MeOH. Fraction 10:1 was further fractionated by reversed-phase octadecyl silica (ODS) column chromatography. The final purification was achieved by semi-preparative HPLC of fraction 6:4 obtained from ODS column chromatography with gradient elution of MeOH/0.5 % formic acid in a SunFire C18 OBD™ Prep column (Waters; Milford, MA, USA) (10 × 250 mm, 4 mL/min, UV detection at 340 nm). A-1 with a purity >95 % was obtained.

2.2. Breast cancer and non-cancerous cell lines and reagents

TNBC cell lines MDA-MB-231 (ATCC HTB-26™), MDA-MB-436 (ATCC HTB-130™), and MCF-7 (ATCC HTB-22) were purchased from the American Type and Culture Collection (ATCC; Manassas, VA, USA). MDA-MB-231 and MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC; Manassas, VA, USA) at 37 °C, with a 5 % CO2 humidified environment. MDA-MB-436 cells were cultured in Leibovitz’s l-15 medium (ATCC; Manassas, VA, USA) supplemented with 10 μg/mL of insulin (Gibco; Life Technologies; Grand Island, NY, USA) and 16 μg/mL of glutathione (Sigma-Aldrich; St. Louis, MO, USA) at 37 °C with 0 % CO2. All culture media (DMEM and Leibovitz’s l-15 media) included 10 % fetal bovine serum and 1 % penicillin–streptomycin solution (100 IU/mL of penicillin and 100 μg/mL of streptomycin; ATCC; Manassas, VA, USA). Pac was purchased from Sigma-Aldrich (St. Louis, MO, USA). SRE and Pac were dissolved in dimethyl sulfoxide (DMSO) (ATCC; Manassas, VA, USA). Non-cancerous epithelial cell line MCF-10A (ATCC CRL-10,317™) was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 medium supplemented with a MEGM Kit (Lonza Pharma and Biotech; Basel, Switzerland) at 37 °C, with a 5 % CO2 humidified environment.

2.3. 2D SRE cytotoxicity assay

To determine cell viability, RealTime-Glo™ MT Cell Viability Assays (Promega; Madison, WI, USA) were used following the manufacturer’s instructions. Cells were seeded (2 × 104 cells/well) in triplicate, with 100 μL of media in 96-well plates, and incubated for 24 h at 37 °C with 5 % or 0 % CO2, depending on the cell type. After the initial incubation, cells were treated with various 2-fold concentrations of SRE ranging from 0.7 μg/mL to 180 μg/mL. Cells treated with 0.01 % DMSO were used as a control. Luminescence was measured at 24, 48, and 72 h using a Cytation™ 5 plate reader (BioTek; Winooski, VT, USA). Data were analyzed using GraphPad Prism 9 (San Diego, CA, USA).

2.4. 2D imaging

Cells were seeded (1 × 104 cells/well) into 96-well plates and incubated for 24 h at 37 °C with 5 % or 0 % CO2, depending on the cell type. Cells were treated with A-1 (5 μM) and SRE (10 μg/mL); cells with DMSO (0.01 %) were used as controls. After a 48 h incubation period, media was removed, and wells were washed with cold 1X PBS. Following the Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific; Waltham, MA, USA) imaging microscopy protocol, working dye solutions were made, and cells were stained for 30 min. After staining, cells were washed with 1X Annexin-binding buffer. For total cell count, 100 μL of Hoechst 33,342 solution (1 μg/mL; Thermo Fisher Scientific; Waltham, MA, USA) in 1X Annexin-binding buffer was added to each well and incubated for 15 min. Fluorescence was observed using a Cytation™ 5 plate reader (BioTek; Winooski, VT, USA). The red and green mean fluorescence intensity was determined from the areas of interest in each image using ImageJ software (NIH; Bethesda, MD). Data were analyzed using GraphPad Prism 9 (San Diego, CA, USA).

2.5. 3D culture

Breast cancer cell lines MDA-MB-231, MDA-MB-436, and MCF-7 cells were grown into spheroids as previously described (Sudhakaran et al., 2020). Cells were seeded (2 × 103 cells/well) into V-bottom 96-well plates (Greiner bio-one; #651,101, Frickenhausen, Germany) and centrifuged at 500 g for 8 min for MDA-MB-231 and MCF-7, and 1000 g for 10 min for MDA-MB-436. Each well was supplemented with complete media containing 2.5 % Matrigel (Corning; Glendale, AZ, USA). Spheroids were cultured for 6 days before treatment. Then the spheroids were treated with 1.25, 2.5, 5, 10, 20, or 40 μg/mL SRE or DMSO (0.01 %) for 3 days. For combination treatments, spheroids were treated with SRE (10 μg/mL), A-1 (5 μM), Pac (12.5 nM) or DMSO (0.01 %) for 3 days. Cell viability and death were assessed by staining spheroids with calcein AM (2 μM) (Invitrogen; Carlsbad, CA, USA) and propidium iodide (PI; 2 μg/mL) (Thermo Fisher Scientific; Waltham, MA, USA) for 1 h at 37 °C, as previously described (Sudhakaran et al., 2020). The diameter and PI mean fluorescence intensity were quantified using ImageJ software (NIH; Bethesda, MD). Change in spheroid diameter (Δ diameter) was used to quantify change in spheroid growth. In addition, cell viability was determined using CellTiter-Glo® 3D Cell Viability Assay (Promega; Madison, WI, USA) following the manufacturer’s instructions. Luminescence readings were taken with a Cytation™ 5 plate reader (BioTek; Winooski, VT, USA). Cell viability percentage was determined using the formula: (Ytreatment – Yblank)/(Ycontrol – Yblank) × 100 %, where Y represents the luminescence values.

2.6. 3D caspase 3 and 7 activity apoptosis assay

TNBC cell lines MDA-MB-231 and MDA-MB-436, as well as HR+ MCF-7 spheroids, were produced as described above. The Caspase-Glo® 3/7 3D Assay (Promega; Madison, WI, USA) kit was used to determine caspase 3/7 activity according to the manufacturer’s protocol. Initially, the Caspase-Glo® 3/7 buffer is combined with the substrate and thoroughly mixed. The reagent should then be allowed to reach room temperature before adding an equal amount of Caspase-Glo® 3/7 Reagent to the volume of the medium in each well. Gently mix to homogenize the cells. Incubate at room temperature for 30 min. Measure the luminescence using a Cytation™ 5 plate reader (BioTek; Winooski, VT, USA).

2.7. Statistical analyses

Data is shown as the mean ± standard error of the mean (SEM). When comparing the difference between two groups, students’ t-test analyses were used; one-way analysis of variance (ANOVA) tests were used to compare more than two groups. All statistical analyses were performed using GraphPad Prism 9 (San Diego, CA, USA). Calculations with a p-value of p< 0.05 were deemed statistically significant.

3. Results

3.1. Cytotoxicity of SRE in human breast cancer cells and non-cancerous cells

To observe and quantify the cytotoxic effects of SRE in human TNBC cells and HR+ breast cancer cells, the IC50 values for each cell line were determined. Cells were treated with a 1:2 serial dilution of SRE, with concentrations ranging from 0.7 μg/mL to 180 μg/mL, for 24, 48, and 72 h. At 24 h, the IC50 value of SRE in MDA-MB-231 cells was 14.46 μg/mL, whereas the IC50 values were slightly lower in MDA-MB-436 (11.84 μg/mL) and MCF-7 (8.48 μg/mL) (Fig. 2). In the latter two cell lines, the IC50 values of SRE showed a time-dependent decrease. Specifically, in MDA-MB-436 and MCF-7, the IC50 values of SRE at 24 h were ~0.6-fold and 0.2-fold higher, respectively, when compared to other treatment times (Figs. 2B, C). However, in MDA-MB-231, the IC50 values of SRE at 24 h of incubation showed a modest increase of ~0.03-fold compared to 48 h and 72 h of treatment (Fig. 2A).

Fig. 2.

Fig. 2.

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Effect of SRE on cell proliferation in TNBC cell lines MDA-MB-231 (A) and MDA-MB-436 (B) and hormone receptor-positive MCF-7 (C). Cells were treated with a 1:2 serial dilution of SRE with concentrations ranging from 0.7 μg/mL to 180 μg/mL for 24, 48, and 72 h; Cells treated with 0.01 % DMSO were used as a control. Each point is the average of 3 repetitions ± SEM. Inserts show the IC50 at each time period.

To study if SRE was cytotoxic to non-cancerous human breast epithelial cells, MCF-10A cells were treated with a 1:2 serial dilution of SRE with concentrations ranging from 0.7 μg/mL to 45 μg/mL for 24, 48, and 72 h. No toxic effects were observed in MCF-10A cells treated with SRE at concentrations up to ~11 μg/mL of SRE, however, at this concentration, there were significant decreases in cell viability in all breast cancer cell lines (Fig. 3). These results demonstrated that SRE at concentrations approximately equal to or below the IC50 value in the cancer cell lines did not show cytotoxicity to non-cancerous cells (Figs. 2 and 3). Therefore, the study focused on concentrations lower than 11 μg/mL of SRE (i.e., 10 μg/mL) for the following analysis, as it inhibited cell proliferation in TNBC cells and HR+ breast cancer cells while showing no significant cytotoxicity to non-cancerous breast epithelial MCF-10A cells.

Fig. 3.

Fig. 3.

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Cytotoxicity of SRE in MCF-10A cells compared to other cancer cells. Cell viability was evaluated with SRE concentrations ranging from 0.7 μg/mL to 45 μg/mL for (A) 24, (B) 48, and (C) 72 h. DMSO (0.01 %) was used as diluent control. Data represents mean ± SEM. N= 3. * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001 versus MCF-10A.

3.2. SRE induces higher apoptosis than A-1 in MDA-MB-231 cells

Imaging studies were conducted to confirm the relationship between the growth inhibition effect of SRE and apoptosis. SRE contains several stilbenes, the majority of which are prenylated. Among these, A-1 is present at a high concentration (~16 % w/w; Supplementary Fig. S1) and has been shown to induce apoptosis in TNBC (Mohammadhosseinpour et al., 2022, 2023). To assess if SRE is more effective than A-1 at inducing apoptosis, the cytotoxicity of the SRE was compared to A-1 at its equimolar concentration, i.e. 5 μM, in 10 μg/mL SRE. MDA-MB-231 cells were treated with SRE (10 μg/mL) or A-1 (5 μM) for 48 h which was determined as the optimal time point for this experiment due to the higher fluorescent intensity signal. After treatment with SRE (10 μg/mL) for 48 h, red and green fluorescence was observed in the nucleus and cell membrane of MDA-MB-231 cells, respectively. However, when the cells were treated with A-1 (5 μM) or the control (DMSO 0.01 %), there was less green fluorescence and fewer red dots were detected. These observations indicate that MDA-MB-231 cells treated with SRE (10 μg/mL) induced more apoptosis compared to A-1 (5 μM). SRE with no fluorescent dye was used as a control for the fluorescence of SRE. No fluorescence was detected in the SRE group with the red and green fluorescence dye (Fig. 4A). The results revealed that the cells treated with SRE (10 μg/mL) had significantly higher ~0.7-fold and ~2-fold increases in apoptosis activity compared to cells treated with A-1 and DMSO, respectively (Fig. 4B). Therefore, these results support that SRE has increased apoptosis activity when compared to A-1 alone.

Fig. 4.

Fig. 4.

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Staining and fluorescence imaging of triple negative breast cancer MDA-MB-231 cells using Hoechst 33,342, Annexin V, Alexa Fluor® 488 conjugate and propidium iodide (PI). (A) Cultures were treated for 48 h treatment with A-1 (5.2 μM), SRE (10 μg/mL), or diluent control DMSO (0.01 %); SRE without dye was performed to control for fluorescence of the extract. Blue-fluorescent Hoechst 33,342 was used as a nuclear segmentation, Annexin V, Alexa Fluor® 488 conjugate (Green-fluorescence) was used to visualize apoptotic and dead cells, and red was used to indicate dead cells. (B) the bar graph showed cell death with fluorescence intensity of PI in red and apoptosis with fluorescence intensity of Annexin V, Alexa Fluor® 488 in green. Data represents mean ± SEM. N= 3. * p< 0.05, *** p< 0.001, **** p< 0.0001.

3.3. SRE induces cell death in TNBC spheroids

To assess the effects of SRE on 3D models of human TNBC, MDA-MB-231 and MDA-MB-436 cells were seeded for six days to generate TNBC spheroids. Afterward, the spheroids were treated with increasing concentrations of SRE, ranging from 1.25 to 40 μg/mL, for an additional three days, with DMSO as a control. Our findings reveal that SRE suppressed the growth of TNBC spheroids in a dose-dependent manner, resulting in significant reductions of spheroid diameter by approximately ~30 % and ~60 % at concentrations of 5 and 10 μg/mL, respectively (Fig. 5B). Moreover, the viability of the spheroids remarkably decreased to ~60 % and ~40 % at SRE concentrations of 5 and 10 μg/mL, respectively (Fig. 5C). Furthermore, the results indicated a significant increase in relative fluorescence intensity of PI staining, approximately 1.6-fold and 2-fold for 5 and 10 μg/mL, respectively (Fig. 5D). We also investigated the activity of caspase 3 and 7 in SRE-treated spheroids. The results revealed an approximately 2.5-fold increase in caspase 3 and 7 activity in TNBC spheroids when treated with 10 μg/mL of SRE compared to the control. However, this increase was not statistically significant. In contrast, significant increases in activity were observed at concentrations of 20 μg/mL (~3-fold) and above. Therefore, for the following study, 10 μg/mL of SRE which contains ~5 μM A-1 was used.

Fig. 5.

Fig. 5.

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SRE reduces growth and induces cell death of MDA-MB-231 spheroids. MDA-MB-231 spheroids were treated with 1.25, 2.5 5, 10, 20, 40 μg/mL SRE or diluent DMSO 0.01 % (denoted as 0) for 3 days. (A) Cell death and viability was evaluated by staining spheroids with calcein AM (green) and PI (red). Two representative spheroids, R1 and R2, are shown for each treatment. Scale bar: 500 μm. (B) Growth of spheroids treated with different concentrations of SRE or DMSO was evaluated and represented as change in diameter (Δ diameter) between day 3 and day 0. (C) Percentage of cell viability of spheroids treated for 3 days with different concentrations of SRE or DMSO. (D) Cell death was evaluated in spheroids stained with PI and quantified as relative fluorescence intensity of PI. (D) Caspase 3 and 7 fold increase was measure using Caspase-Glo® 3/7 3D Assay (Promega). Data represent mean ± SEM, N= 4. ##p< 0.05, ##p< 0.01, ### p< 0.001, ####p< 0.0001compared to DMSO.

3.4. Combining SRE with Pac enhances cell death and reduces spheroid growth by activating caspase 3 and 7 in TNBC and HR+ breast cancer spheroids

The effect of SRE combined with Pac treatment on TNBC and HR+ breast cancer spheroids was evaluated and compared with the effect of A-1 combined with Pac. Pac (12.5 nM) was selected because it was the lowest concentration that showed significant results in spheroid viability, according to previous studies (Mohammadhosseinpour et al., 2023). Six-day-old spheroids were treated with Pac (12.5 nM), SRE (10 μg/mL), or A-1 (5 μM) and combinations of Pac with SRE or A-1 for three days. Spheroids treated with DMSO (0.01 %) were used as the control. The combination of Pac and SRE showed a significant reduction in spheroid growth compared with SRE alone or Pac combined with A-1. In comparison to SRE alone, the combination of SRE and Pac yielded approximately 67 %, 40 %, and 18 % reduction of growth in MDA-MB-231, MDA-MB-436, and MCF-7, respectively. Meanwhile, when SRE was compared to Pac combined with A-1, the reductions were approximately 50 %, 10 %, and 26 % in MDA-MB-231, MDA-MB-436, and MCF-7, respectively (Fig. 6B, 7B, 8B). To understand the mechanism underlying the decrease in spheroid growth after the Pac and SRE combination treatment, the study examined the cell viability and cell death effects of the combination. The Pac and SRE combination treatment significantly decreased spheroid viability by about ~20 %, ~26 %, and ~23 % compared with SRE alone and 18 %, ~35 %, and ~41 % compared to Pac combined with A-1 in MDA-MB-231, MDA-MB-436, and MCF-7, respectively (Fig. 6C, 7C, 8C). These findings were confirmed by measuring the relative mean fluorescence intensity of PI staining, which revealed that the combination of Pac with SRE induced cell death in MDA-MB-231, MDA-MB-436, and MCF-7 spheroids. This was indicated by higher relative PI fluorescence intensity of approximately 21 %, ~68 %, and ~74 % in comparison to SRE alone, respectively. Additionally, there was a significant increase of 32 %, ~13 %, and ~155 % in the relative PI fluorescence intensity for the respective cell lines mentioned (MDA-MB-231, MDA-MB-436, and MCF-7) when comparing the combination treatment of Pac with SRE to Pac combined with A-1 (Fig. 6D, 7D, 8D). Lastly, caspase 3 and 7 activity was evaluated for each treatment group; there was ~27 %-fold, ~15 %-fold and ~5 %-fold change of caspase 3/7 activity in spheroids treated with Pac combined with SRE compared to SRE alone ~28 %, ~52 % and ~17 % compared to Pac combined with A-1 in MDA-MB-231, MDA-MB-436, and MCF-7 spheroids, respectively (Figs. 6E, 7E, 8E). These findings demonstrate that SRE (10 μg/mL) increases Pac efficacy by inducing cell death and activating caspase 3 and 7 in human TNBC spheroids.

Fig. 6.

Fig. 6.

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SRE increases the efficacy of Pac in reducing viability in MDA-MB-231 spheroids. Spheroids were treated with 10 μg/mL SRE, 5 μM arachidin-1 (A-1), or 12.5 nM Pac or diluent DMSO 0.01 % (denoted as 0) for 3 days. (A) Cell death and viability were evaluated by staining spheroids with calcein AM (green) and PI (red). Two representative spheroids, R1 and R2, shown for each treatment. Scale bar: 500 μm. (B) Growth of spheroids treated with different concentrations of SRE, A-1, Pac and/or DMSO was evaluated and represented as change in diameter (Δ diameter) between day 3 and day 0. (C) Percentage of cell viability of spheroids treated with SRE, A-1, and/or Pac as well as diluent DMSO. (D) Cell death was observed in spheroids stained PI and was quantified as relative fluorescence intensity of PI. (E) Caspase 3 and 7 activity was measured for treated spheroids using Caspase-Glo® 3/7 3D Assay (Promega; Madison, WI, USA). All data represent mean ± SEM, N= 4. * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001 compared to treatment. #p< 0.05, ##p< 0.01, ### p< 0.001, ####p< 0.0001 versus DMSO.

Fig. 7.

Fig. 7.

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SRE increases the efficacy of Pac in reducing spheroid viability. Spheroids were treated with 10 μg/mL SRE, 5 μM arachidin-1 (A-1), or 12.5 nM Pac or diluent DMSO (0.01 %) (denoted as 0) for 3 days. (A) Cell death and viability were evaluated by staining spheroids with calcein AM (green) and PI (red). Two representative spheroids, R1 and R2, shown for each treatment. Scale bar: 500 μm. (B) Growth of spheroids treated with different concentrations of SRE, A-1, Pac and/or DMSO was evaluated and represented as change in diameter (Δ diameter) between day 3 and day 0. (C) Percentage of cell viability of spheroids treated with SRE, A-1, Pac and/or DMSO. (D) Cell death was evaluated in spheroids stained with PI and quantified as relative fluorescence intensity of PI. (E) Caspase 3 and 7 activity was measured for treated spheroids using Caspase-Glo® 3/7 3D Assay (Promega; Madison, WI, USA). All data represent mean ± SEM, N= 3. * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001 compared to treatment. #p< 0.05, ##p< 0.01, ####p< 0.0001 versus DMSO.

Fig. 8.

Fig. 8.

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SRE increases the efficacy of Pac in reducing viability of HR positive MCF-7 spheroids. Spheroids were treated with 10 μg/mL SRE, 5 μM arachidin-1 (A-1), or 12.5 nM Pac or diluent DMSO (0.01 %) (denoted as 0) for 3 days. (A) Cell death and viability were evaluated by staining spheroids with calcein AM (green) and PI (red). Two representative spheroids, R1 and R2, shown for each treatment. Scale bar: 500 μm.(B) Growth of spheroids treated with different concentrations of SRE, A-1, Pac and/or DMSO was evaluated and represented as change in diameter (Δ diameter) between day 3 and day 0. (C) Percentage of cell viability of spheroids treated with SRE, A-1, Pac and/or DMSO. (D) Cell death was observed in spheroids stained with PI and was quantified as relative fluorescence intensity of PI. Spheroids were treated with SRE (10 μg/mL), A-1 (5 μM), and Pac (12.5 nM) alone and in combination or in the presence of diluent DMSO (denoted as 0). (E) Caspase 3 and 7 activity was measured for treated spheroids using Caspase-Glo® 3/7 3D Assay (Promega; Madison, WI, USA). All data represent mean ± SEM, N= 3. * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001 compared to treatment. #p< 0.05, ##p< 0.01, ###p< 0.001, ####p< 0.0001 versus DMSO.

4. Discussion

TNBC stands apart from hormone receptor-positive breast cancer due to its increased likelihood of tumor invasiveness, higher recurrence rates, and a worse patient prognosis. Moreover, TNBC is considered to be a refractory disease, and thus, there is a lack of effective drugs available to treat patients (Sun et al., 2019). Multicomponent medicines are made up of numerous molecules that interact with different targets. Accordingly, using drugs with multiple components can increase both effectiveness and toxicity. By incorporating the ideal combination of stilbenes, there may be a higher likelihood of inducing death in cancer cells (Balasubramani et al., 2019). SRE, derived from peanut hairy roots, is rich in a combination of prenylated and non-prenylated stilbenoids that exhibit significant capability as antioxidant compounds (Gajurel et al., 2021). Consequently, SRE could function as an effective multicomponent drug, when compared to purified metabolites alone. The combination of stilbenes found in SRE may be more effective at inducing cancer cell death and could exhibit higher bioavailability due to the complex combination (Balasubramani et al., 2019). Certain stilbenes, specifically prenylated derivatives such as A-1 and A-3, have demonstrated antioxidant, anti-inflammatory, and anti-adipogenic effects that could be beneficial for human health (Gajurel et al., 2021). Moreover, these prenylated compounds have been shown to possess enhanced or novel biological activities in vitro compared to their non-prenylated counterparts, such as resveratrol (RES) (Huang et al., 2010).

In addition to the increased pharmacological activity, extracts are easier to produce, more cost-effective, and do not require harsh chemicals for purification compared to purified metabolites. SRE obtained from peanut hairy roots was studied due to its potentially higher bioavailability compared purified stilbenes (Brents et al., 2012). Therefore, further investigation into SRE from peanut hairy roots is needed to fully understand its potential as a nutraceutical resource. In this study, SRE was evaluated as a multicomponent drug to treat TNBC within 2D and 3D spheroid cell culture models.

A previous study showed that A-1 (5 μM) was the most effective concentration to induce cell death in MDA-MB-231 spheroids (Mohammadhosseinpour et al., 2023). Interestingly, the equimolar concentration of A-1 in the 10 μg/mL of extract was ~5 μM, and this concentration (5 μM) was the most effective at inducing apoptosis and cell death (Figs. 2 and 4).

Earlier studies revealed that RES exhibits dose-dependent cytotoxicity on MCF-7 and MDA-MB-231 cells (Gao and Tollefsbol, 2018; Gomes, et al., 2019). These studies demonstrated that concentrations exceeding 50 μM of RES result in a significant reduction of cell viability in both MCF-7 and MDA-MB-231 cells. However, that study showed that MDA-MB-231 cells displayed greater resistance to treatment than MCF-7 cells, indicating MDA-MB-231 has a significant degree of chemotherapeutic drug resistance (Abu Samaan et al., 2019; Gomes et al., 2019). The current study focused on evaluating SRE within TNBC cell lines MDA-MB-231 and MDA-MB-436 as well as HR+ MCF-7. the key difference between these breast cancer subtypes is their hormone receptor status. Due to the lack of receptors, TNBC has no targeted treatment options; in contrast, HR+ breast cancer allows for targeted treatments and better outcomes. Additionally, recent metastatic potential mapping studies demonstrate that MDA-MB-231 has high invasiveness and metastatic potential, while MDA-MB-436 has a much lower rate of metastatic spread and is relatively less aggressive (Jin et al., 2020). Comparing cell lines from these subtypes allows for a comprehensive evaluation of the anticancer capabilities that SRE has on a variety of breast cancer types.

A previous study showed that the cytotoxic effects of A-1, A-3, and RES vary between MDA-MB-231 and MDA-MB-436. Additionally, that study revealed that the IC50 of A-1, A-3, and RES were not time-dependent (Mohammadhosseinpour et al., 2022). Interestingly, SRE showed comparable cytotoxicity in TNBC cell lines MDA-MB-231 and MDA-MB-436 and HR+ MCF-7. Therefore, SRE did not show selective cytotoxicity for TNBC cells. SRE also showed time dependent cytotoxic effects in all three cell lines. When comparing the effect of serial dilution concentrations of SRE on all cell lines, MDA-MB-231 exhibited the highest percentage of cell viability; this could indicate that MDA-MB-231 is more resistant to SRE when compared to other cell lines. Various studies detailed that MDA-MB-231 can demonstrate resistance to stilbenoid compounds, namely, RES (Abu Samaan et al., 2019; Gao and Tollefsbol, 2018). In all three breast cancer cell lines, SRE at 10 μg/mL induced cytotoxicity, despite hormone receptor status (Fig. 2). In addition, concentrations of SRE lower than 10 μg/mL were found to be non-toxic to the normal breast epithelial cell line MCF-10A; cytotoxicity increased with time, suggesting a time-dependent relationship (Fig. 3). This is supported by the previous study that showed A-1 was not cytotoxic to the epithelial cell line MCF-10A at a low micromolar concentration (2 μM) (Mohammadhosseinpour et al., 2023).

Previous studies within 2D models indicate that A-1 halts the cell cycle in G2/M phase triggering intrinsic apoptosis and cell death within TNBC cells (Mohammadhosseinpour et al., 2022, 2023). A-1 promotes apoptosis in MDA-MB-231 and MDA-MB-436 cells in a dose-dependent manner, resulting in an increase in early and late apoptotic cells (Mohammadhosseinpour et al., 2022). Similar studies indicate that the administration of pterostilbene, a methoxylated stilbene, suppresses cellular proliferation of MCF-7 and MDA-MB-231 breast cancer cells in a dose- and time-dependent manner, without significantly affecting the MCF-10A control cells. Moreover, pterostilbene was found to enhance apoptosis in both breast cancer cell lines (Daniel and Tollefsbol, 2017). RES and pterostilbene are monomeric stilbenes. The latter has increased lipophilicity which may improve capacity to pass through cell membranes and increase bioavailability and biological efficiency (Chan et al., 2019). A similar capacity could be expected for prenylated stilbenes present in the SRE due to their lipophilicity when compared to non-prenylated stilbenes. Using caspase 3/7 as indicators of apoptosis induction, this study also revealed that SRE (10 μg/mL) induced apoptosis in MDA-MB-231 cells at a higher rate than A-1 within a 2D assay, suggesting a synergistic effect among the stilbenes in the extract (Fig. 4).

Spheroid cell cultures demonstrate unique advantages over 2D culture systems including the presence of tumor-like scaffolding and allow for complex intercellular interactions. Additionally, when treating 3D cell cultures with chemotherapeutics, spheroids were notably less responsive to these agents than monolayer cell cultures and thereby spheroids may more accurately mimic the tumor microenvironment (Dubois et al., 2017). With this in mind, the cytotoxicity of SRE on TNBC cell line MDA-MB-231 was examined in a 3D spheroid model; a significant decrease in diameter was observed with increasing concentrations of SRE. Importantly, spheroids treated with 10 μg/mL of SRE demonstrated significant decreases in diameter, and the mean PI fluorescence intensity was significantly increased (Fig. 5).

In the current study, HPLC analysis revealed that 10 μg/mL SRE contains 5.2 μM A-1 (Supplementary Fig. S1). Previous study indicates that A-1 inhibited TNBC spheroid development in a dose-dependent manner, with significant reductions of up to 35 % reported at 5 μM, while spheroid cell viability was severely reduced at A-1 concentrations of 5 μM and higher (Mohammadhosseinpour et al., 2023). Given this, the results of the first study were further validated through the significant cytotoxicity results in this study. In earlier studies, the combination of low micromolar concentrations of A-1 (5 μM) with Pac (12.5 nM) successfully decreased cell proliferation, and reduced spheroid formation. These findings imply that A-1 combined with Pac could be developed as a novel plant-derived treatment for TNBC; however, as mentioned before the complex combination of stilbenes in SRE could potentially enhance its effectiveness in inducing cancer cell death and increase its bioavailability (Brents et al., 2012; Mohammadhosseinpour et al., 2023). Thus, the cytotoxic capabilities of A-1, SRE, and Pac were analyzed separately and in combination within the 3D model. The results demonstrated that SRE (10 μg/mL) in combination with Pac (12.5 nM) causes a significant decrease in spheroid viability. This was further confirmed as this combination displayed the highest amount of cell death, measured by relative PI fluorescence intensity, and a significant increase in apoptosis activity as seen in the significant fold change increase of caspase 3/7 activity. These results were seen across TNBC cell line MDA-MB-231 and HR+ cell line MCF-7 (Figs. 6, 7, and 8).

The results from the 3D spheroid model showed similar results as the 2D culture model indicating that SRE is not selectively cytotoxic for TNBC cell lines and it also demonstrated efficacy within the HR+ breast cancer cells. Additionally, this study demonstrated that Pac (12.5 nM) combined with SRE (10 μg/mL) is more toxic to all breast cancer cell lines compared to A-1 (5 μM) combined with Pac (12.5 nM). Therefore, potentially the concentrations 10 μg/mL SRE or lower than 10 μg/mL SRE combined with Pac 12.5 nM could be effective as well.

5. Conclusion

In conclusion, this study demonstrated that SRE derived from peanut hairy roots, which is rich in prenylated stilbenes, could function as a more effective multicomponent drug when compared to purified compounds alone. SRE showed potential for inducing cancer cell death, and the combination of stilbenes in SRE could be more effective and potentially exhibit higher bioavailability than A-1 alone. The results showed that SRE at 10 μg/mL induced cytotoxicity in all three-breast cancer cell lines, including TNBC and HR+ cell lines, as well as a 3D spheroid model. Moreover, the combination of SRE and Pac was found to be effective in inducing cell death and apoptosis activity in TNBC and HR+ cell lines. However, SRE was not selectively cytotoxic for TNBC cell lines and demonstrated potential for HR+ breast cancer cells. These findings suggest that SRE could potentially be a plant-derived therapeutic. Further investigation is needed to fully understand its potential as a novel breast cancer treatment.

Supplementary Material

1

Acknowledgements

Funding for this research was provided by the Arkansas Biosciences Institute (fund no. 200129). This publication was made possible by the Arkansas INBRE program, supported by a grant from the National Institute of General Medical Sciences, (NIGMS), 5P20 GM103429 from the National Institutes of Health. Sepideh Mohammadhosseinpour was supported by a graduate assistantship from the Molecular Biosciences Graduate Program at Arkansas State University.

Abbreviations:

A-1

Arachidin-1

A-3

Arachidin-3

CD

Methyl-β-cyclodextrin

MeJA

Methyl jasmonate

ODS

Octadecyl-silica

Pac

Paclitaxel

PI

Propidium iodide

RES

Resveratrol

SRE

Stilbene-rich extract

TNBC

Triple-negative breast cancer

Footnotes

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Fabricio Medina-Bolivar reports financial support was provided by Arkansas INBRE Program. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Sepideh Mohammadhosseinpour: Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. Alexx Weaver: Writing – original draft, Investigation. Sara V. Hernandez-Madrigal: Writing – original draft, Investigation. Gaurav Gajurel: Writing – original draft, Investigation. Amit Raj Sharma: Writing – original draft, Investigation. Fabricio Medina-Bolivar: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phyplu.2024.100578.

References

  1. Abbott JA, Medina-Bolivar F, Martin EM, Engelberth AS, Villagarcia H, Clausen EC, Carrier DJ, 2010. Purification of resveratrol, arachidin-1, and arachidin-3 from hairy root cultures of peanut (Arachis hypogaea) and determination of their antioxidant activity and cytotoxicity. Biotechnol. Prog. 26 (5), 1344–1351. 10.1002/btpr.454. [DOI] [PubMed] [Google Scholar]
  2. Abu Samaan TM, Samec M, Liskova A, Kubatka P, Büsselberg D, 2019. Paclitaxel’s mechanistic and clinical effects on breast cancer. Biomolecules. 9 (12), 789. 10.3390/biom9120789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Atanasov AG, Zotchev SB, Dirsch VM, the International Natural Product Sciences Taskforce, Supuran CT, 2021. Natural products in drug discovery: advances and opportunities. Nat. Reviews Drug Discov 20, 200–216. 10.1038/s41573-020-0083-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balasubramani SP, Rahman MA, Basha SM, 2019. Synergistic action of stilbenes in muscadine grape berry extract shows better cytotoxic potential against cancer cells than resveratrol alone. Biomedicines. 7 (4), 96. 10.3390/biomedicines7040096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brents LK, Medina-Bolivar F, Seely KA, Nair V, Bratton SM, Ñopo-Olazabal L, Patel RY, Liu H, Doerksen RJ, Prather PL, Radominska-Pandya A, 2012. Natural prenylated resveratrol analogs arachidin-1 and -3 demonstrate improved glucuronidation profiles and have affinity for cannabinoid receptors. Xenobiotica 42 (2), 139–156. 10.3109/00498254.2011.609570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caesar LK, Cech NB, 2019. Synergy and antagonism in natural product extracts: when 1 + 1 does not equal 2. Nat. Prod. Rep. 36, 869–888. 10.1039/C8NP00092K. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chan EWC, Wong CW, Tan YH, Foo JPY, Wong SK, Chan HT, 2019. Resveratrol and pterostilbene: A comparative overview of their chemistry, biosynthesis, plant sources and pharmacological properties. J. Appl. Pharm. Sci 9 (07), 124–129. 10.7324/JAPS.2019.90718. [DOI] [Google Scholar]
  8. Chang JC, Lai YH, Djoko B, Wu PL, Liu CD, Liu YW, Chiou RYY, 2006. Biosynthesis enhancement and antioxidant and anti-inflammatory activities of peanut (Arachis hypogaea L.) arachidin-1, arachidin-3, and isopentadienyl resveratrol. J. Agric. Food Chem. 54, 10281–10287. 10.1021/jf061603f. [DOI] [PubMed] [Google Scholar]
  9. Chen LG, Zhang YQ, Wu ZZ, Hsieh CW, Chu CS, Wung BS, 2018. Peanut arachidin-1 enhances Nrf2-mediated protective mechanisms against TNF-α-induced ICAM-1 expression and NF-κB activation in endothelial cells. Int. J. Mol. Med. 41, 541–547. 10.3892/ijmm.2017.3286. [DOI] [PubMed] [Google Scholar]
  10. Condori J, Sivakumar G, Hubstenberger J, Dolan MC, Sobolev VS, Medina-Bolivar F, 2010. Induced biosynthesis of resveratrol and the prenylated stilbenoids arachidin-1 and arachidin-3 in hairy root cultures of peanut: Effects of culture medium and growth stage. Plant Phys. Biochem. 48, 310–318. 10.1016/j.plaphy.2010.01.018. [DOI] [PubMed] [Google Scholar]
  11. Daniel M, Tollefsbol TO, 2017. Pterostilbene down-regulates hTERT at physiological concentrations in breast cancer cells: Potentially through the inhibition of cMyc. J. Cell Biochem. 119 (4), 3326–3337. 10.1002/jcb.26495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. de Bruijn WJC, Araya-Cloutier C, Bijlsma J, de Swart A, Sanders MG, de Waard P, Gruppen H, Vincken JP, 2018. Antibacterial prenylated stilbenoids from peanut (Arachis hypogaea). Phytochem. Lett. 28, 13–18. 10.1016/j.phytol.2018.04.020. [DOI] [Google Scholar]
  13. Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA, 2007. Triple-negative breast cancer: Clinical features and patterns of recurrence. Clin. Cancer Res. 13, 4429–4434. 10.1158/1078-0432.CCR-06-3045. [DOI] [PubMed] [Google Scholar]
  14. Donato F, Romagnolo CD, Milner JA, 2012. Phytoalexins in cancer prevention. Front Biosci (Landmark Ed.) 17 (6), 2035–2058. 10.2741/4036. [DOI] [PubMed] [Google Scholar]
  15. Dubois C, Dufour R, Daumar P, Aubel C, Szczepaniak C, Blavignac C, Mounetou E, Penault-Llorca F, Bamdad M, 2017. Development and cytotoxic response of two proliferative MDA-MB-231 and non-proliferative sum1315 three-dimensional cell culture models of triple-negative basal-like breast cancer cell lines. Oncotarget. 10.18632/oncotarget.18791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fang L, Yang T, Medina-Bolivar F, 2020. Production of prenylated stilbenoids in hairy root cultures of peanut (Arachis hypogaea) and its wild relatives A. ipaensis and A. duranensis via an optimized elicitation procedure. Molecules. 25, 509. 10.3390/molecules25030509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J, 2013. Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol. 31, 108–115. 10.1016/j.tibtech.2012.12.003. [DOI] [PubMed] [Google Scholar]
  18. Gajurel G, Hasan R, Medina-Bolivar F, 2021. Antioxidant assessment of prenylated stilbenoid-rich extracts from elicited hairy root cultures of three cultivars of peanut (Arachis hypogaea). Molecules. 26, 6778. 10.3390/molecules26226778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gajurel G, Nopo-Olazabal L, Hendrix E, Medina-Bolivar F, 2022. Production and secretion of isowighteone in hairy root cultures of pigeon pea (Cajanus cajan) co-treated with multiple elicitors. Plants 11, 834. 10.3390/plants11050834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gao Y, Tollefsbol TO, 2018. Combinational proanthocyanidins and resveratrol synergistically inhibit human breast cancer cells and impact epigenetic–mediating machinery. Molecules. 23 (8), 2204. 10.3390/molecules23082204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ge J, Zuo W, Chen Y, Shao Z, Yu K, 2022. The advance of adjuvant treatment for triple-negative breast cancer. Cancer Biol. Med. 19, 187–201. 10.20892/j.issn.2095-3941.2020.0752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gomes L, Sorgine M, Passos CLA, Ferreira C, Rosa de Andrade I, Silva JL, Atella GC, Mermelstein CS, Fialho E, 2019. Increase in fatty acids and flotillins upon resveratrol treatment of human breast cancer cells. Sci. Rep. 10.1038/s41598-019-50272-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Huang CP, Au LC, Chiou RYY, Chung PC, Chen SY, Tang WC, Chang CL, Fang WH, Lin SB, 2010. Arachidin-1, a peanut stilbenoid, induces programmed cell death in human leukemia HL-60 cells. J. Agric. Food Chem. 58 (22), 12123–12129. 10.1021/jf103332q. [DOI] [PubMed] [Google Scholar]
  24. Jin X, Demere Z, Nair K, Ali A, Ferraro GB, Natoli T, Deik A, Petronio L, Tang AA, Zhu C, Wang L, Rosenberg D, Mangena V, Roth J, Chung K, Jain RK, Clish CB, Vander Heiden MG, Golub TR, 2020. A metastasis map of human cancer cell lines. Nature 588 (7837), 331–336. 10.1038/s41586-020-2969-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li Y, Zidorn C, 2022. Seasonal variations of natural products in European herbs. Phytochem. Rev. 21, 1549–1575. 10.1007/s11101-021-09797-7. [DOI] [Google Scholar]
  26. Mohammadhosseinpour S, Ho LC, Fang L, Xu J, Medina-Bolivar F, 2022. Arachidin-1, a prenylated stilbenoid from peanut, induces apoptosis in triple-negative breast cancer cells. Int. J. Mol. Sci. 23 (3), 1139. 10.3390/ijms23031139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mohammadhosseinpour S, Weaver A, Sudhakaran M, Ho L-C, Le T, Doseff AI, Medina-Bolivar F, 2023. Arachidin-1, a prenylated stilbenoid from peanut, enhances the anticancer effects of paclitaxel in triple-negative breast cancer cells. Cancers 15, 399. 10.3390/cancers15020399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mohammadhosseinpour S, Weaver A, Sudhakaran M, Ho LC, Le T, Doseff AI, Medina-Bolivar F, 2023. Arachidin-1, a prenylated stilbenoid from peanut, enhances the anticancer effects of paclitaxel in triple-negative breast cancer cells. Cancers 15 (2), 399. 10.3390/cancers15020399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Niesen DB, Hessler C, Seeram NP, 2013. Beyond resveratrol: A review of natural stilbenoids identified from 2009–2013. J. Berry. Res. 3, 181–196. 10.3233/JBR-130047. [DOI] [Google Scholar]
  30. Nopo-Olazabal C, Hubstenberger J, Nopo-Olazabal L, Medina-Bolivar F, 2013. Antioxidant activity of selected stilbenoids and their bioproduction in hairy root cultures of Muscadine grape (Vitis rotundifolia Michx.). J. Agric. Food Chem. 61, 11744–11758. 10.1021/jf404292z. [DOI] [PubMed] [Google Scholar]
  31. Piasecka A, Kachlicki P, Stobiecki M, 2019. Analytical methods for detection of plant metabolomes changes in response to biotic and abiotic stresses. Int. J. Mol. Sci. 20 (2), 379. 10.3390/ijms20020379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pradeep M, Kruszka D, Kachlicki P, Mondal D, Franklin G, 2022. Uncovering the phytochemical basis and the mechanism of plant extract-mediated eco-friendly synthesis of silver nanoparticles using ultra-performance liquid chromatography coupled with a photodiode array and high-resolution mass spectrometry. ACS Sustain. Chem. Eng. 10, 562–571. 10.1021/acssuschemeng.1c07897. [DOI] [Google Scholar]
  33. Ricci C, Moroni L, Danti S, 2013. Cancer tissue engineering: new perspectives in understanding the biology of solid tumors: a critical review. Tissue Eng. Part B: Rev. 19, 1–16. 10.1089/ten.teb.2012.0358.23253031 [DOI] [Google Scholar]
  34. Ryu NE, Lee SH, Park H, 2019. Spheroid culture system methods and applications for mesenchymal stem cells. Cells 8, 1620. 10.3390/cells8121620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sharma AR, Gajurel G, Ahmed I, Roedel K, Medina-Bolivar F, 2022. Induction of the prenylated stilbenoids arachidin-1 and arachidin-3 and their semi-preparative separation and purification from hairy root cultures of peanut (Arachis hypogaea L.). Molecules. 27 (18), 6118. 10.3390/molecules27186118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shen T, Wang XN, Lou HX, 2009. Natural stilbenes: An overview. Nat. Prod. Rep. 26, 916–935. 10.1039/b813414p. [DOI] [PubMed] [Google Scholar]
  37. Sudhakaran M, Parra MR, Stoub H, Gallo KA, Doseff AI, 2020. Apigenin by targeting HnRNPA2 sensitizes triple-negative breast cancer spheroids to doxorubicin-induced apoptosis and regulates expression of ABCC4 and ABCG2 drug efflux transporters. Biochem. Pharmacol. 182, 114259 10.1016/j.bcp.2020.114259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sun Y, Zhou QM, Lu YY, Zhang H, Chen QL, Zhao M, Su SB, 2019. Resveratrol inhibits the migration and metastasis of mda-mb-231 human breast cancer by reversing tgf-β1-induced epithelial-mesenchymal transition. Molecules. 24 (6), 1131. 10.3390/molecules24061131. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  39. Waks AG, Winer EP, 2019. Breast cancer treatment: A Review. JAMa 321, 288–300. 10.1001/jama.2018.19323. [DOI] [PubMed] [Google Scholar]
  40. Wawrosch C, Zotchev SB, 2021. Production of bioactive plant secondary metabolites through in vitro technologies-status and outlook. Appl microbiol and biotechnol 105 (18), 6649–6668. 10.1007/s00253-021-11539-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weaver BA, 2014. How Taxol/paclitaxel kills cancer cells. Mole Biol Cell 25, 2677–2681. 10.1091/mbc.e14-04-0912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yang CQ, Liu J, Zhao SQ, Zhu K, Gong ZQ, Xu R, Lu HM, Zhou RB, Zhao G, Yin DC, Zhang CY, 2020. Recent treatment progress of triple negative breast cancer. Prog. Biophys. Mol. Biol. 151, 40–53. 10.1016/j.pbiomolbio.2019.08.003. [DOI] [PubMed] [Google Scholar]
  43. Yang T, Fang L, Nopo-Olazabal C, Condori J, Nopo-Olazabal L, Balmaceda C, Medina-Bolivar F, 2015. Enhanced production of resveratrol, piceatannol, arachidin-1, and arachidin-3 in hairy root cultures of peanut co-treated with methyl jasmonate and cyclodextrin. J. Agric. Food Chem. 63, 3942–3950. 10.1021/acs.jafc.5b00922. [DOI] [PubMed] [Google Scholar]
  44. Zagami P, Carey LA, 2022. Triple negative breast cancer: Pitfalls and progress. NPJ. Breast. Cancer 8, 95. 10.1038/s41523-022-00382-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

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