1. Introduction
Around 75% of breast cancer cases are estrogen receptor-positive (ER+), making them the most prevalent subtype [1]. The death rate of ER+ patients was at least four times higher among AAW than CAW, and this higher rate in AAW was not related to differences in tumor stage, grade, or therapy initiation in AAW patients [2], but may be influenced by a combination of genetic, physiological, and social factors [3-6]. Particularly, less fruit and vegetable consumption among AAW over the past three decades [7] may contribute to the higher prevalence and mortality of ER+ breast cancer, and this disparity continues with only 5.5% of African-Americans meeting the federal vegetable intake recommendations, while 9.5% of the white-Americans reached the recommendation [8].
Phytochemicals, natural metabolites from plants, including fruits and vegetables, have strong anti-cancer potential and breast cancer prevention [9]. For instance, luteolin (3,4,5,7-tetrahydroxyflavone, LUT), a flavonoid in a variety of commonly vegetables such as thyme, Chinese celery, radicchio, and peppers and fruits such as lemons, oranges, grapes and apples [10, 11] and indole-3-carbinol (I3C), a biologically active compound found in cruciferous vegetables like Brussels sprouts, cabbage, kale, cauliflower, and broccoli [12] have shown anti-breast cancer effects. However, the required concentrations of LUT and I3C for anti-breast cancer effects are approximately 50-80 µM [13, 14] and 200–490 µM [15, 16], respectively, which are significantly higher than what can be physiologically achieved in vivo. For instance, the peak plasma levels of LUT and I3C (or their metabolites) reach only about 1 µM [17]-15 µM [18] and 27.9 µM (mice) [19]-278.5 µM (rats) [20], respectively, with oral administration of the pure LUT and I3C. To bridge the gap between effective in vitro concentrations and achievable in vivo levels, we combined LUT and I3C at relatively lower levels, which synergistically suppressed ER+ breast cancer in CAW cells and xenograft mice [21]. In contrast, the individual LUT and I3C at the relevant levels did not exhibit an anti-breast cancer effect [21].
In the present study, we investigated whether the combined LUT and I3C exerts similar anti-breast cancer effects and mechanisms both in AAW (HCC1500 and ZR-75-30) and CAW (MCF-7 and T47D)-derived ER+ breast cancer cells. We found that the combinations of LUT and I3C exhibited a synergistic anti-proliferative effect and enhanced apoptosis both in AAW and CAW-derived ER+ breast cancer cells. Interestingly, the combinations specifically reduced the expression of the anti-apoptotic protein BCL-XL in cells derived from CAW. The combinations also significantly inhibited cell migration and invasion in both CAW and AAW-derived cells. These results suggest that the combination of LUT and I3C is an efficient approach to treat ER+ breast cancer in both AAW and CAW.
2. Materials and methods
2.1. Cells and reagents
The ER+ breast cancer cell lines ZR-7530, MCF-7, T-47D, and HCC1500 were purchased from the American Tissue Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured with RPMI-1640 (ZR-75-30 and HCC1500) or DMEM (MCF-7 and T-47D) medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) in an incubator with 5% CO2 at 37°C. Cells were seeded and treated with different concentrations of I3C and luteolin or a combination of LUT and I3C in the relevant assays below. I3C and luteolin were obtained from Sigma Aldrich Inc. (Bellefonte, PA, USA) and dissolved in Dimethyl sulfoxide at 100 mM, aliquoted, and stored at -20°C.
2.2. Cell proliferation
Human cells from AAW (ZR-75-30 and HCC1500) and cells from CAW (MCF-7 and T-47D) were seeded in a 12-well plate at 10%~20% confluency with the relevant medium as described above. Cells were then treated with LUT 30μM, I3C 40μM, I3C 60μM, LUT 30μM + I3C 40μM, or LUT 30μM+ I3C 60μM for 72hrs at 37°C and 5% CO2. The cell proliferation assay was performed using the WST-1 assay kit according to the manufacturer’s instructions and our report [21]. In brief, the medium was aspirated and replaced with 300μL/well of fresh medium, and 40μL/well of WST-1 mixed solution (an equal amount of electro mediator and developer) was added to maintain for 2 hrs in the CO2 incubator. Absorbance was read at 450 nm using a Synergy Hybrid plate reader (BioTek Instruments Inc., Winooski, VT, USA). This proliferation assay in each cell line was repeated five times.
2.3. Cell apoptosis
Cell apoptosis was quantified with the annexin V apoptosis detection kit (eBioscience, San Diego, CA, USA) as we reported [21]. After treatment with the desired concentrations of individual or combinations of LUT and I3C for 24-48 hours, cells were harvested, suspended in a binding buffer, and stained with fluorochrome-labeled annexin and propidium iodide for 30 minutes at room temperature. The portion of late apoptotic cells was analyzed by flow cytometry. This assay was repeated three times in each cell line.
2.4. Immunoblotting
Target proteins such as BCL-XL and GAPDH from cells were measured by immunoblotting, as we reported [21]. Briefly, treated cells were added with mammalian protein extraction buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS. Thermo-scientific, Waltham, MA, USA) to harvest cells, and then sonicated thrice for 5 seconds on ice at intervals. After centrifuging 5 min at 12 RPM, the supernatant was collected to measure protein concentration using the Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Based on the protein concentration of the sample, different volumes of the sample were added to a relevant volume of sample buffer to ensure each sample had the same protein concentration, and then heated at 95 °C for 5 min to prepare the sample for immunoblotting. The exact amount (15 μl) of sample was loaded onto the SDS-PAGE gel to separate proteins, and then the separated proteins were transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Piscataway, NJ, USA). The membranes were blocked with 5% non-fat dry milk at room temperature for 1 hr. Antibodies against BCL-XL, BAX, and GAPDH (D16H11) (purchased from Cell Signaling Technology, Danvers, MA, USA) were incubated overnight, all at a dilution factor of 1:1000, respectively. After three washes, membranes were incubated with secondary antibodies at room temperature for 1 hr, and the intensities of the X-ray films were quantified using ImageJ software. BCL-XL or BAX protein in the experiment was normalized with GAPDH as an internal control to confirm that protein loading is equal across the gel.
2.5. Wound healing
The capacity of cells to migrate was evaluated using a wound-healing experiment, as we reported [22]. The monolayer of cells was allowed to develop to around 80% confluency, and then linear wounds were created using a P20 pipette tip. After rinsing floating cells, cells were treated for 2 hrs with 10 μg/mL mitomycin C to prevent the cells from going through mitosis and make it possible to differentiate between migration and proliferation. Cells were treated with LUT 30μM, I3C 40μM, I3C 60μM, LUT 30μM + I3C 40μM, or LUT 30μM+ I3C 60μM for 72 hrs. Images (4× magnification) of the scrape were captured under the microscope at 0 and 72 hrs. The extent of migration in each well was examined by using ImageJ to determine the closed wound area.
2.6. Statistical analysis
All experiments were repeated three to five times. The data were presented as mean ± standard error. Statistical analysis was performed by one-way ANOVA followed by a two-tailed Students’ t-test. A significant difference was set at * p < 0.05, **p < 0.01, and ***p < 0.001.
3. Results
3.1. Combinations of luteolin and I3C synergistically inhibited human breast cancer cell proliferation both in AAW (ZR-75-30 and HCC1500) and CAW (MCF-7 and T47D)-derived cells
Based on our previous study, the combination of I3C and LUT synergistically inhibited CAW (T47D and MCF-7 cells) ER+ breast cancer cell proliferation [15]. In the present study, we investigated whether combining I3C and luteolin also synergistically inhibits cell proliferation in AAW-derived ER+ breast cancer cells (ZR-75-30 and HCC1500). These four cell lines were selected based on ER+ expression, cell type, tissue, and ethnicity of the original donor, as reported [23, 24]. As shown in Fig. 1 A & 1 B, the combinations L30I40 (LUT 30 μM + I3C 40 μM) and L30I60 (LUT 30 μM + I3C 60 μM) significantly reduced (23% and 18% of control, respectively, P < 0.001) cell numbers compared to the individual chemicals LUT 30μM (L30, 78% of control), 13C 40 μM (I40, 82% of control), or I3C 60 uM (I60, 65% of control, P < 0.05) in MCF-7 cells. These results were also confirmed in another CAW-derived breast cancer T47D cells (Fig. 1 C & 1 D) in which L30I40 and L30I60 synergistically inhibited cell proliferation (27 and 21% respectively, p < 0.001), which is significantly lower than the individual chemicals L30 (43% of control, P < 0.001), I40 (95% of control), or I60 (82% of control). The inhibitory rates of L30I40 in the present study are very similar to those in our previous report (23±4% vs. 27±3%) [15]. To compare the inhibitory effects of the combination treatments between CAW and AAW-derived cells, we also performed a cell proliferation assay using two AAW-derived cell lines, ZR-75-30 and HCC1500. As shown in Fig. 1 E & 1 F, the combinations L30I40 and L30I60 has the best inhibitory effect (reduced cell numbers to 35 and 28% of control respectively, p < 0.001) in ZR-7530 cell which is significantly lower than the individual chemicals L30 (64% of control), I40 (91% of control), I60 (66% of control). These results were also confirmed on another AAW-derived breast cancer HCC1500 cells (Fig. 1 G & 1 H) in which L30I40 and L30I60 synergistically inhibit cell proliferation (23 and 18%, respectively, p < 0.001), is significantly lower than the individual chemicals L30 (64% of control), I40 (93% of control), or I60 (80% of control). Therefore, the combinations of L30I40 and L30I60 synergistically inhibited ER+ breast cancer both in AAW and CAW-derived cells. The synergistic inhibitory effect of the combinations was determined by the combination index (CI): CI > 1, antagonistic effect, and CI < 1, synergistic effect as described by the Chou-Talalay plot [25]. The CIs of L30I40 in MCF-7, T47D, ZR-75-30, and HCC1500 cells are 0.68, 0.66, 0.73, and 0.68, respectively. Moreover, there is no significant difference between L30I40 and L30I60, although L30I60 exhibits more pronounced anti-breast cancer effects, but has higher CIs (0.75, 0.76, 0.79, and 0.76 in MCF-7, T47D, ZR-75-30, and HCC1500 cells, respectively) in all four cell lines.
3.2. Combined luteolin and I3C exerts a similar anti-proliferative effect between AAW and CAW-derived ER+ breast cancer cells
To compare the anti-proliferative effects of combinations between AAW-derived cells and CAW-derived cells, we com bined the data of two cell lines from AAW (ZR-75-30 and HCC1500) and CAW (MCF-7 and T47D) at each treatment and conducted the statistical analysis. As shown in Fig. 2, there is no significant difference (p>0.05) between AAW-derived cells and CAW-derived cells by the two combinations L30I40 and L30I60, although there are more inhibitory effects in CAW (25% of control and 19% of control, respectively) than in AAW (30% of control and 24% of control, respectively). These results indicate that the combinations of luteolin and I3C have a similar anti-ER+ breast cancer effect between AAW and CAW.
3.3. The combination of luteolin and I3C induces apoptosis in AAW (ZR-75-30 and HCC1500) and CAW (MCF-7 and T47D)-derived cells
To further understand how the combination of LUT and I3C suppresses cancer cell growth, we carried out apoptosis analysis in cells stained with Annexin V. We found that the combination L30I60 induced late cell apoptosis in all four cell lines, MCF-7, T47D, ZR-75-30, and HCC1500. As shown in Fig. 3A and 3B, the combinations L30I40 and L30I60 significantly promoted apoptosis (late apoptotic cells up to 40% and 48%, respectively, P < 0.05 and < 0.01, respectively) in MCF-7 cells compared with the control. L30I60 significantly exerted a greater apoptotic effect compared to I60 (P < 0.001). These results were also confirmed in another CAW-derived breast cancer T47D cells (Fig. 3C) in which L30I40 and L30I60 synergistically induced cell apoptosis (P <0.05 and P < 0.01, respectively), and L30 alone also significantly induced apoptosis compared to the control (P < 0.01). To compare the apoptotic effects of the combination treatment between CAW and AAW-derived cells, we also performed apoptotic analysis in AAW-derived ZR-75-30 and HCC1500 cells. As shown in Fig.3D, the combination L30I60 has the best apoptotic effect (late apoptotic cells up 52%, P < 0.01) compared to the control in ZR-75-30 cells. These results were also confirmed in HCC1500 cells (Fig. 3E), in which L30I40 and L30I60 synergistically induced cell apoptosis (P <0.05) compared to the control; however, the single treatment did not show a significant pro-apoptotic effect. Therefore, the combinations L30I40 and L30I60 synergistically induced apoptosis in both AAW and CAW-derived ER+ breast cancer cells.
3.4. Combined luteolin and I3C exert a similar pro-apop totic effect between AAW and CAW-derived ER+ breast cancer cells
To compare the apoptotic effect of the combination of LUT and I3C between AAW-derived cells and CAW-derived cells, we combined the data of two cell lines from CAW (MCF-7 and T47D) and AAW (ZR-75-30 and HCC1500) at each treatment and ran statistical analysis. As shown in Fig. 4, there is no significant difference (p>0.05) between AAW-derived cells and CAW-derived cells by the two combinations L30I40 and L30I60, although there are more pro-apoptotic effects in CAW (45% and 55%, respectively) than in AAW (44% and 51%, respectively).
3.5. Combined luteolin and I3C significantly reduced BCL-XL protein in CAW ER+ breast cancer cells
To test how the combinations induce cell apoptosis in T47D and HCC1500 (CAW and AAW, respectively), we measured the anti-apoptotic effector BCL-XL protein expression by immunoblotting in cells after 48 hrs of treatment. The combinations (L30I40 and L30I60) reduced BCL-XL protein level significantly (67%, 63% to the control, respectively, P < 0.01) in T47D (CAW) cells (Fig. 5A), but there was no significant reduction (89% to the control, p > 0.05) in HCC1500 (AAW) cells (Fig. 5 B). Therefore, the combinations L30I40 and L30I60 enhance cell apoptosis by down-regulating the BCL-XL protein level specifically in T47D (CAW) cells. This different mitigating effect of BCL-XL by the combinations between T47D and HCC1500 (Fig. 5E) indicates that the combinations of LUT and I3C promoted apoptosis via a different mechanism in HCC1500 (AAW) cells compared to T47D cells.
We also checked the pro-apoptotic protein BAX level; the combinations (L30I40 and L30I60) increased BAX protein level significantly (133%, 135% to the control, respectively, P < 0.05) in T47D (CAW) cells (Figure 5c), but there was no significant change in HCC1500 (AAW) cells (Fig. 5D). Therefore, the pro-apoptotic effects of the combinations L30I40 and L30I60 may also be achieved by increasing the BAX protein level only in T47D (CAW) cells (Fig. 5F). Thus, the combinations LUT and I3C enhance T47D cell apoptosis by the dual process of suppressing BCL-XL and increasing BAX protein levels.
3.6. Combination of luteolin and I3C prevented the closure of the induced wound in both T47D and HCC1500 cells
One typical experiment to examine the ability of cancer cells to invade and migrate is the wound-healing assay. After growing to 80-100% confluency, cells were treated with 10 μg/mL mitomycin C for two hours to prevent cell proliferation. A P20 pipette tip was used to make the wounds on the plate, and then various treatments were applied for 72 hrs. The area of the wound was measured after images were captured at 0 and 72 hrs. As demonstrated in Fig. 6A and 6B, the combinations L30I40 and L30I60 significantly (P < 0.001 and P < 0.001) decreased the area closure in comparison to the control in MCF-7 cells, which is also significantly different from the individual L30, I40, and I60. Moreover, combination L30I60 (29%) is better than L30I40 (34%) in inhibiting wound healing. A similar result was also observed in AAW-derived HCC1500 cells (Fig. 6C), and there is no significant difference in the inhibitory effect of the combinations between AAW and CAW-derived cells (Fig. 6D). The findings suggest that luteolin and I3C work synergistically to suppress cancer cell invasion and migration, two essential stages of cancer development, in both CAW and AAW patients.
4. Discussion
The novelty of this study lies in confirming that the synergistic inhibition of ER+ breast cancer, achieved through the combination of luteolin and I3C in CAW-derived cells and xenograft mice, also works in AAW-derived cells. The importance of this finding is that the higher rates of ER+ breast cancer prevalence and mortality in AAW patients [2] may be resulted from the long-term lower vegetable and fruit consumption among black Americans [7, 8], and common vegetables and fruits such as peppers and fruits such as lemons, oranges, cabbage, kale, cauliflower, and broccoli contain high contents of luteolin [10, 11] and I3C [12]. Therefore, consuming more of these fruits and vegetables may be a safe, convenient, customized, and cost-effective potential approach to prevent or treat ER+ breast cancer both in AAW and CAW, although further clinical studies are needed.
The results of this study further confirmed our novel conception that combining two or more phytochemicals at relatively lower levels to synergistically suppress chronic diseases, while the individual chemicals do not have the inhibitory effect at the selected concentrations. The rationale of this conception is to bridge the gap between effective concentrations (~μM) in vitro and achievable in vivo levels (~nM) of phytochemicals, particularly via dietary intakes of foods and supplements [26]. For instance, the required concentrations of LUT and I3C for anti-breast cancer effects are approximately 50 µM [13, 14] and 200–490 µM [15, 16], respectively, but the peak plasma levels of LUT and I3C (or their metabolites) reach only about 1 µM [17]-15 µM [18] and 27.9 µM (mice) [19]-278.5 µM (rats) [20], respectively, with oral administration of the pure LUT and I3C. In the present study, our combination L30I40 significantly inhibited ER+ breast cancer cell growth from both AAW and CAW, whereas the individual chemicals L30 and I40 did not exhibit such an inhibitory effect, consistent with our previous report [21]. These two studies represent significant progress in overcoming the paradox between the higher required levels and the lower physiological levels of the phytochemicals, and our concentrations (LUT at 30 µM and I3C at 40 µM) are very close to the peak physiological levels, LUT at 15 µM [18] and I3C at 27.9 µM [19] in animals. In addition, both LUT and I3C are metabolized into several different forms in the body, such as luteolin-3´-O-β-D-glucuronide and luteolin-3´-O-sulfate [18] and 3,3′-diindolylmethane, [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane, indole[3,2b]carbazole and 1-(3-hydroxymethyl)-indolyl-3-indolylmethan[19], respectively, and most of these metabolites have similar or higher effects with its original form. Notably, the liver contains 6-fold more I3C in plasma [19], and oral intake generates 5-fold more I3C in plasma than intravenous administration [20]. In fact, we have reported the synergistic effects of combining phytochemicals in different diseases. For instance, combined curcumin and luteolin synergistically inhibited triple-negative breast cancer [27] and colon cancer [22] both in cells and xenograft mice. This combination of curcumin and luteolin also synergistically suppressed vascular inflammation [28], and this anti-vascular inflammation effect was also observed by the combination of curcumin and resveratrol [29]. In addition, we reported that the combination of resveratrol, genistein, and epigallocatechin gallate (EGCG) synergistically mitigated preadipocyte differentiation, a critical step of obesity development [30]. Indeed, these synergistic effects were also reported by others, such as the combination of curcumin and EGCG [31] and the combination of resveratrol and quercetin [32] in breast cancer cells.
The next highlight of this study is that the synergistic anti-ER+ breast cancer effects both in AAW and CAW-derived cells through regulating three anti-cancer approaches: 1) inhibiting cell proliferation. Given that uncontrolled cell growth/proliferation is the initial and continued steps of cancer development, our combination of luteolin and I3C may inhibit ER+ breast cancer cell proliferation via disrupting cyclin D1-CDK4/6 complex activities and then inducing G1 cell cycle arrest and modulating ERα level [19]. 2) promoting cell apoptosis via regulating protein levels of Bcl-xL and Bax [19]. BCL-XL is an essential target for cancer treatments because of its ability to inhibit apoptosis, improve cell survival, and interact with the tumor microenvironment, all of which contribute to the proliferation and spread of breast cancer cells [33]. Therefore, the combination of luteolin and I3C synergistically down-regulated this anti-apoptotic protein BCL-XL while increasing pro-apoptotic protein BAX, and then promoted cancer cell apoptosis: 3) mitigating cell migration and invasion, key steps of cancer development, which is in line with our previous study that a combination of luteolin and curcumin suppressed colon cancer cell invasion and migration [22]. It is reasonable to assume that the synergistic anti-breast cancer effect of the combined luteolin and I3C may be the result of regulating all three pathways simultaneously.
While the combination of luteolin and I3C exerts a synergistic anti-breast cancer both in AAW and CAW-derived cells via regulating several pathways in the present study, there are several limitations of this study: 1) The concentration of LUT and I3C are still high compared to the physiological levels of the chemicals in animals, particularly, the blood levels of luteolin is 99.90 nM in humans with ordinary diets [34] and I3C and its metabolites are 132.10 nM in humans with uncooked broccoli consumption (200g) [35]; 2) only in vitro studies; 3) only apoptotic molecules BCL-XL and BAX for the mechanism investigation. Therefore, we will conduct these experiments to understand more about the combination how suppress ER+ breast cancer in the future: (1) chemicals will be fed to xenograft or chemically induced breast cancer mice to test the synergistic anti-breast cancer effects of luteolin and 13C; and (2) whole-genome RNA-sequencing and metabolomic analysis of tumors will determine detailed mechanisms on how luteolin and I3C together synergistically inhibit estrogen receptor positive breast cancer in both CAW and AAW.
5. Conclusion
In summary, we show here that the combination of luteolin and I3C, at relatively low concentrations, exerts a synergistic anti- ER+ breast cancer both in AAW and CAW-derived cells via suppressing proliferation, inducing apoptosis, and inhibiting migration/wound healing. The results from this study provide the notion that increasing consumption of phytochemical-rich foods may lower the chance of developing ER+ breast cancer, particularly among AAW, who have the higher rates of ER+ breast cancer and long-term lower vegetable and fruit consumption.
Credit authorship contribution statement
Hongwei Si: Conceptualization, writing –review & editing, funding acquisition, and project administration; Divine Boka: investigation and data collection and analyze, writing—original draft preparation; Fnu Annu: investigation and data collection. All authors have read and agreed to the published version of the manuscript.
Funding
This work was funded by the National Institute of Food and Agriculture/USDA, grant number 2022-38821-37352, and the National Institute of Food and Agriculture/USDA, grant number TENX-2023-FS to Hongwei Si.
Conflict of interest
The authors declare no conflicts of interest.
LIST OF ABBREVIATIONS
AAW African American women
CAW Caucasian American women
EGCG Epigallocatechin gallate
ER+ Estrogen receptor-positive
FBS Fetal bovine serum
I3C Indole-3-carbinol
P/S Penicillin/streptomycin
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