Abstract
BACKGROUND/OBJECTIVES
Colorectal cancer (CRC) is a common malignancy worldwide and continues to account for significant cancer-associated morbidity and mortality. This study aimed to explore the anti-cancer effects of estradiol (E2), a female hormone, and β-carotene (BC), a dietary antioxidant, focusing on their roles in promoting apoptosis and regulating the expression of BC-related metabolic enzymes in human colon cancer cells.
MATERIALS/METHODS
E2, BC, and a combination of both were administered to 2 human colon cancer cell lines, HCT116 and HT29. Cell survival was analyzed using the MTT assay. Expression of estrogen receptor beta (ERβ), β,β-carotene-15′,15′-monooxygenase 1 (BCMO1), β,β-carotene-9′,10′-oxygenase 2 (BCO2), and apoptosis-related markers such as Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) was analyzed using polymerase chain reaction or Western blotting.
RESULTS
E2 treatment decreased cell viability in both cell lines, indicating that E2 promotes anti-cancer effects. BC treatment modestly increased E2 secretion, while the combined E2 and BC treatment enhanced this effect. Treatment with E2, BC, and their combination suppressed Bcl-2 expression and upregulated Bax. The combination treatment was more effective than each treatment administered individually. Furthermore, treatment with E2, BC, and their combination upregulated the expression of ERβ, BCMO1, and BCO2.
CONCLUSION
The anti-cancer effects of E2, BC, and their combination are mediated through apoptosis induction. These findings highlight the potential for utilizing dietary antioxidants and hormonal regulation as complementary strategies in CRC treatment.
Keywords: Beta carotene, estradiol, colorectal neoplasms, apoptosis
INTRODUCTION
Globally, colorectal cancer (CRC) accounted for approximately 10% of all cancer cases in 2020. CRC also accounted for 9.4% of cancer-related deaths, underscoring the significant associated incidence and mortality rates [1]. Meanwhile, CRC ranked among the top five most commonly diagnosed cancers in Korean in 2020. Moreover, CRC represents the fourth most prevalent cancer in men and the third in women [2]. Therefore, the development and equitable implementation of treatment strategies are crucial in addressing disparities in cancer outcomes across different regions [3].
CRC exhibits notable gender differences in both incidence and mortality rates [4]. These disparities can be attributed to a range of factors, including hormonal influences [5]. Estrogen, a sex steroid hormone, contributes to reproductive system masculinization, organ formation during embryonic development, and regulation of reproductive functions [6]. Differences in the estrogen receptor beta (ERβ) expression have been observed between malignant and normal colon tissues, with a significant reduction in ERβ expression observed in malignant tissues in both men and women, indicating a relationship between ERβ levels and cancer progression [7]. Moreover, the structure known as helix 12 (H12) in the estrogen receptor (ER) undergoes distinct conformational changes depending on ligand binding; in the estradiol (E2) structure, H12 blocks ligand binding, whereas, in the RAL structure, H12 shifts to create a binding site, thus demonstrating how ER antagonists can inhibit ER function by preventing ligand binding [8].
Vitamin A is crucial for immune and visual functions, as well as for regulating cell growth and differentiation [9]. Additionally, β-carotene (BC), a precursor of vitamin A, has demonstrated anti-cancer potential, notably inhibiting the proliferation of specific CRC cell lines. Carotenoids, including BC, undergo oxidative cleavage and modifications through the carotenoid cleavage oxygenase (CCO) family [10]. Specifically, BC is cleaved by enzymes including β,β-carotene-15′,15′-monooxygenase 1 (BCMO1) and β,β-carotene-9′,10′-oxygenase 2 (BCO2). Studies involving human colon cancer cell lines treated with BC have shown that BC demonstrates anti-cancer properties through inducing apoptosis by arresting the cell cycle at the G2/M phase and reducing the expression of Bcl-2, an anti-apoptotic protein [11].
Cancer develops due to an imbalance between cell division and apoptosis [12]. The process of apoptosis allows cells to die safely without harming neighboring cells, which is critical for cancer suppression [13]. Although the individual anti-cancer activities of BC and E2 have been reported, the potential for their synergistic interaction in colorectal cancer remains largely unexplored. The rationale for combining these agents lies in the potential crosstalk between retinoid-activated retinoic acid receptor (RAR)/retinoid X receptor (RXR) and ER signaling. BC-derived retinoids activate RAR/RXR pathways [14], while E2 primarily exerts anti-cancer effects through ERβ [15]. Given that both paths are known to intersect in the co-regulation of genes related to cell growth and survival, their simultaneous activation may offer a more robust anti-proliferative response [16]. Furthermore, as both BC and E2 target the intrinsic apoptotic pathway by modulating the Bcl-2/Bax ratio, a combined approach could potentially exert a synergistic effect on apoptosis induction, providing a more effective therapeutic strategy for colorectal cancer [11]. Thus, this study explored the potential of BC and E2 in modulating apoptosis and BC metabolism in CRC cells.
MATERIALS AND METHODS
Cell culture
Two human colon cell lines (HCT116 and HT29) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in medium obtained from Welgene (Daegu, Korea). Fetal bovine serum (Gibco, Gaithersburg, MD, USA), penicillin (100 U/mL; Invitrogen, Carlsbad, CA, USA), and streptomycin (100 μg/mL; Invitrogen) were added to the medium. The cell lines were maintained under conditions of 37°C and 5% CO2 during the entire experiment. BC (catalog number: C9750; Sigma Aldrich, St. Louis, MO, USA) was dissolved in tetrahydrofuran (Sigma Aldrich), using red light conditions to protect BC since the pigment is light sensitive. E2 (Sigma Aldrich) was dissolved in ethanol (Sigma Aldrich).
Cell viability assay
Cell viability was evaluated using the thiazolyl blue tetrazolium bromide (catalog number: M5655, MTT; Sigma Aldrich) assay. Each cell line was seeded into a 96-well plate. After 24-h incubation, various doses of E2, BC, and their combination treatment were applied for an additional 24 h. Then, the medium was removed, and MTT solution was added to each well. After 3 h of incubation, the supernatant was discarded, and 100 μL dimethyl sulfoxide solution (Sigma Aldrich) was added to solubilize the formazan crystals. The absorbance of each well was analyzed using a microplate reader (Molecular Device, Sunnyvale, CA, USA). The absolute values for each treatment group were calculated relative to the control group, representing 100% cell viability.
RNA extraction and quantitative real-time polymerase chain reaction (PCR)
RNA was extracted using TRIzol reagent (Invitrogen). The cDNA was converted using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time PCR was performed with 1 μg of RNA and processed SYBR green mix (Qiagen, Hilden, Germany). The extracted cDNA samples were analyzed using specific primers (Table 1).
Table 1. Primer sequences used in quantitative real-time PCR.
| Gene name | Gene ID | Primer sequence (5′→3′) |
|---|---|---|
| ERβ | 2100 | Forward: GCA CCT TTC TCC TTT AGT GG |
| Reverse: ATA GTG ATA TCC CGA TGC GT | ||
| BCMO1 | 53630 | Forward: GGG AGT CCA GAT ACA ACC AT |
| Reverse: GGT GTT GTA GGT ATC GCT TC | ||
| BCO2 | 83875 | Forward: GAG GAC CAG GGC TGT GTT AT |
| Reverse: CTC AGG TTG TCT CCC TCA GG | ||
| GAPDH | 2597 | Forward: GTT CCA ATA TGA TTC CAC CCA |
| Reverse: GAC TCC ACG ACG TAC TCA G |
PCR, polymerase chain reaction; ERβ, estrogen receptor beta; BCMO1, β,β-carotene-15,15′-monooxygenase 1; BCO2, β,β-carotene-9′,10′-dioxygenase 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Western blot analysis
Protein lysates for Western blot analysis were obtained from the cells. The cell plates were washed twice with 1× phosphate-buffered saline, and proteins were extracted using PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) supplemented with a phosphatase inhibitor (Sigma Aldrich). Protein concentration was determined using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA). Extracted protein samples were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, and the proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with bovine serum albumin and skimmed milk at room temperature for at least 1 h. After blocking, the membrane was incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study are as follows: ERβ (catalog number: GTX70174; GeneTex, Irvin, CA, USA), BCMO1 (catalog number: A15848; ABclonal, Woburn, MA, USA), BCO2 (catalog number: 14324-1-AP; Proteintech Group Inc., Resmont, IL, USA), Bcl-2 (catalog number: SC-7382; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (catalog number: SC526; Santa Cruz Biotechnology), and β-actin (catalog number: ab6276; Abcam, Cambridge, England).
Statistical analysis
All results are presented as the mean ± SE of the mean from at least 3 independent experiments, with a P-value lower than 0.05 considered statistically significant. Data were analyzed using GraphPad PRISM (9.0 version; GraphPad Software Inc., San Diego, CA, USA). Differences between groups were assessed using a one-way analysis of variance followed by the Newman-Keuls post hoc test.
RESULTS
The effects of E2, BC, a combination treatment on cell survival
E2 treatments of 100 and 1,000 nM significantly suppressed cell viability in HCT116 cells by 15.8% (P < 0.01) and 22.9% (P < 0.001), respectively, compared to the control (Fig. 1A). Similarly, E2 treatments of 100 and 1000 nM reduced cell viability in HT29 cells by 12.0% (P < 0.001) and 19.7% (P < 0.0001), respectively, compared to the control group, demonstrating a dose-dependent inhibitory effect on cancer cell proliferation (Fig. 1B). BC treatment at 20 and 40 µM significantly reduced cell viability in both cell lines, with reductions of 21.9% and 41.6%, respectively, in HCT116 cells (Fig, 1C, P < 0.0001 for both) and 19.5% and 32.3%, respectively, in HT29 cells, compared to the control (Fig. 1D, P < 0.0001 for both). Despite treatment with BC or E2 alone significantly reducing cell viability in both HCT116 and HT29 cells, the combination treatment exhibited the strongest inhibitory effect by 26.2% in HCT116 cells (Fig. 1E) and 31.8% in HT29 cells (Fig. 1F) compared to the non-treated control (P < 0.0001 for both).
Fig. 1. The effects of E2, BC, and the combination treatment on cell survival.
The MTT assay was performed to assess cell viability. HCT116 (A) and HT-29 (B) cells were administered E2 (0, 1, 10, 100, 1,000 nM) for 24 h. HCT116 (C) and HT-29 (D) cells were administered BC (20, 40 µM) for 24 h. HCT116 (E) and HT-29 (F) cells were administered a combination of E2 (100 nM) and BC (20 µM) for 24 h. Each value represents the mean ± SE of the mean. One-way analysis of variance was used to analyze differences between treatments with the Newman-Keuls post hoc test. A P-value ≤ 0.05 was considered to indicate statistical significance.
E2, estradiol; BC, β-carotene; NC, normal control; Combi, combination treatment.
a-dThe letters represent statistically distinct groups determined by post hoc analysis. Groups sharing the same letter are not significantly different, whereas groups with different letters are significantly different (P < 0.05).
E2, BC, and a combination treatment induce apoptosis and enhance antioxidant defense in human CRC cells
The effects of BC, E2, and a combination treatment on apoptosis-related markers Bcl-2 and Bax were assessed in CRC cell lines. The combination treatment significantly downregulated the expression of Bcl-2, an anti-apoptotic marker, by 48.4% in HCT116 cells (P < 0.05) and a 73.5% reduction in HT29 cells (P < 0.01) compared to the control group (Fig. 2A). In contrast, Bax, a pro-apoptotic marker, was significantly upregulated by the combination treatment in both cell lines, with a 6.2-fold increase in HCT116 cells (Fig. 2D, P < 0.01) and a 3.4-fold increase in HT29 cells compared to the control group (Fig. 2E, P < 0.05). Both the singular E2 and BC treatments promoted effects on each marker, whereas the combination treatment demonstrated a more enhanced effect, indicating that the combination promotes apoptosis more effectively than an individual treatment.
Fig. 2. E2, BC, and the combination treatment induce apoptosis and enhance antioxidant defense processes in human CRC cells.
The relative protein expression of (A-C) Bcl-2 and (D-F) Bax was analyzed by Western blotting. HCT 116 and HT-29 cells were administered a combination of E2 (100 nM) and BC (20 µM) for 24 h. β-actin was used as a loading control. Each value represents the mean ± SE of the mean. One-way analysis of variance was used to analyze differences between treatments with the Newman-Keuls post hoc test. A P-value ≤ 0.05 was considered to indicate statistical significance.
E2, estradiol; BC, β-carotene; CRC, colorectal cancer; NC, normal control; Combi, combination treatment.
a-cThe letters represent statistically distinct groups determined by post hoc analysis. Groups sharing the same letter are not significantly different, whereas groups with different letters are significantly different (P < 0.05).
E2, BC, and the combination treatment increase endogenous E2 concentration and ERβ expression
E2 treatment increased the E2 concentration in both cell lines. Comparatively, the BC treatment alone did not increase E2 concentration; however, the combination treatment of E2 and BC promoted an additional increase in E2 concentration compared to the E2 treatment alone (Fig. 3A and B, P < 0.0001 for both). Furthermore, the ERβ mRNA expression was significantly upregulated in the BC group, showing a 9.3-fold increase in HCT 116 cells (Fig. 3C) and a 2.9-fold increase (P < 0.01) in the HT29 cells compared to the control group (Fig. 3D). The most prominent upregulation was observed in the combination group, with a 17.6-fold increase (P < 0.0001) in HCT 116 cells and a 3.9-fold increase in HT-29 cells (P < 0.001) compared to the control group. ERβ expression was strongly upregulated by the combination treatment, with a 1.8-fold increase (P < 0.001) in HCT 116 cells (Fig. 3E, P < 0.05) and a 2.5-fold increase in the HT29 cells compared to the control group (Fig. 3F, P < 0.05).
Fig. 3. E2, BC, and the combination treatment increase endogenous E2 concentration and ERβ expression.
(A, B) The E2 concentration was measured using ELISA. (C, D) The relative mRNA expression of ERβ was analyzed using quantitative real-time PCR. GAPDH was used as a loading control. (E, F) The relative protein expression of ERβ was analyzed by Western blotting. HCT116 and HT-29 cells were administered a combination of E2 (100 nM) and BC (20 µM) for 24 h. β-actin was used as a loading control. Each value represents the mean ± SE of the mean. One-way analysis of variance was used to analyze differences between treatments with the Newman-Keuls post hoc test. A P-value ≤ 0.05 was considered to indicate statistical significance.
E2, estradiol; BC, β-carotene; ERβ, estrogen receptor beta; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, normal control; Combi, combination treatment.
a-cThe letters represent statistically distinct groups determined by post hoc analysis. Groups sharing the same letter are not significantly different, whereas groups with different letters are significantly different (P < 0.05).
E2, BC, and the combination treatment upregulate BCMO1 and BCO2 expression
E2, BC, and the combination treatments significantly upregulated the BCMO1 and BCO2 mRNA expression. In particular, the combination treatment promoted a 7.9-fold (P < 0.001) increase in BCMO1 mRNA expression in HCT116 cells (Fig. 4A) and a 3.7-fold (P < 0.0001) increase in HT29 cells (Fig. 4B) compared to the control group. Meanwhile, the BCO2 mRNA levels were increased by 15.4-fold (P < 0.0001) in HCT 116 cells (Fig. 4C) and 2.3-fold (P < 0.001) in HT29 cells (Fig. 4D) compared to the control group, indicating a strong transcriptional response in both cell lines. The combination treatment promoted in the highest BCMO1 protein expression levels, with a 2.3-fold increase in HCT116 cells and a 1.9-fold increase in HT29 cells compared to the control (Fig. 4E-G, P < 0.05 for both cell lines). Similarly, the BCO2 expression increased by 3.3-fold (P < 0.01) in HCT 116 and by 1.2-fold in HT-29 cells (P < 0.05) following the combination treatment (Fig. 4H-J), indicating that both treatments markedly elevated key enzymes in BC metabolism.
Fig. 4. E2, BC, and the combination treatment upregulate BCMO1 and BCO2 expression.
The relative mRNA expression of (A, B) BCMO1, and (C, D) BCO2 was confirmed using quantitative real-time PCR. GAPDH was used as a loading control. The relative protein expression of (E-G) BCMO1 and (H-J) BCO2 was analyzed by Western blotting. β-actin was used as a loading control. HCT116 and HT-29 cells were administered a combination of E2 (100 nM) and BC (20 µM) for 24 h. Each value represents the mean ± SE of the mean. One-way analysis of variance was used to analyze differences between treatments with the Newman-Keuls post hoc test. A P-value ≤ 0.05 was considered to indicate statistical significance.
E2, estradiol; BC, β-carotene; BCMO1, β,β-carotene-15′,15′-monooxygenase 1; BCO2, β,β-carotene-9′,10′-oxygenase 2; PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, normal control; Combi, combination treatment.
a-dThe letters represent statistically distinct groups determined by post hoc analysis. Groups sharing the same letter are not significantly different, whereas groups with different letters are significantly different (P < 0.05).
DISCUSSION
This study provides compelling evidence for the anti-cancer effects of E2 and BC treatments in human CRC cells, specifically the HCT116 and HT-29 cell lines. Moreover, these findings demonstrate that these compounds, particularly in combination, effectively reduce cell viability and modulate apoptotic and metabolic pathways related to BC that are involved in tumor suppression.
Both E2 and BC treatments individually reduced CRC cell viability; however, the combination treatment showed a more pronounced inhibitory effect, suggesting a potential synergistic interaction. This enhanced efficacy may be attributed to the convergence of hormone-mediated effects, wherein E2 exerts pro-apoptotic effects via ERβ, meanwhile, BC is metabolized into bioactive derivatives that interfere with cancer-promoting pathways [15]. Consistent with these mechanistic insights, the MTT assay results demonstrated that both E2 and BC individually reduced cell viability in HCT116 and HT-29 cells, with the combination treatment exhibiting the most pronounced inhibitory effect.
In this study, the combined treatment of E2 and BC induced robust apoptosis signaling, which is a desirable outcome for anti-cancer efficacy. However, the high apoptotic activity also raises questions regarding its potential toxicity. While our data—specifically, the regulated expression of Bcl-2 family proteins—suggest a programmed apoptotic process rather than nonspecific necrotic damage, the safety of this combination in nonmalignant cells remains to be fully elucidated. Further studies utilizing non-cancerous cell lines and cytotoxicity assays, such as lactate dehydrogenase release, are necessary to establish a comprehensive safety profile and determine the therapeutic index of this combination.
To determine whether the reduction in cell viability was associated with apoptosis, the expression levels of apoptosis-related proteins Bcl-2 and Bax were analyzed. Bcl-2 and Bax were selected as markers due to the critical roles of these proteins in the intrinsic apoptotic pathway [17]. Bcl-2 functions as an anti-apoptotic protein that preserves mitochondrial membrane integrity, whereas Bax promotes mitochondrial outer membrane permeabilization, facilitating cytochrome c release and caspase activation [18]. This study found that E2 and BC treatments downregulated Bcl-2 expression and upregulated Bax expression, with the combination treatment inducing the strongest effects on expression. This shift in the balance toward pro-apoptotic signaling supports the hypothesis that E2 and BC collaboratively promote programmed cell death in CRC cells. Indeed, previous studies have shown that ERβ activation can suppress Bcl-2 expression and promote Bax-mediated apoptosis [19].
ERβ acts a key mediator of estrogen signaling in CRC, and plays a tumor-suppressive role by regulating genes involved in apoptosis and proliferation [15,20]. Moreover, the upregulation of ERβ following the E2 and BC treatments emphasizes the functional relevance of this receptor, as ERβ signaling is known to modulate apoptotic pathways and suppress oncogenic processes across various cancer types, including CRC [21]. This study demonstrated that ERβ expression was significantly upregulated by the E2 and BC treatments, particularly in the combination group. This suggests that ERβ activation may serve as a central mechanism through which both agents exert anti-tumor activity. Notably, ERβ is known to engage in crosstalk with apoptotic regulators, enhancing the transcription of Bax while repressing Bcl-2 [15,17].
BCMO1 and BCO2, 2 key enzymes involved in BC metabolism, were also analyzed. BCMO1 catalyzes the symmetric cleavage of BC into retinal (vitamin A aldehyde), whereas BCO2 mediates asymmetric cleavage, generating various apocarotenoids with known anti-inflammatory and anti-proliferative properties [22,23]. These enzymes facilitate the oxidative cleavage of BC into bioactive metabolites, which contribute to the observed anti-cancer effects [24]. These results present a strong upregulation in mRNA expression of BCMO1 and BCO2 in both cell lines upon BC and combination treatment. While this study focused on the mRNA expression of BC cleavage enzymes, the identification and quantification of their downstream metabolites, such as retinal and apocarotenoids, would provide deeper insight into the functional contributions of the combined treatment of E2 and BC in BC metabolism [25]. Specifically, apocarotenoids, have been shown to exert anti-cancer effects by modulating oxidative stress responses, immune signaling, and epigenetic regulation [26]. Therefore, incorporating metabolite profiling in future studies could substantiate the metabolic axis of BC-mediated tumor suppression. Indeed, prior studies have shown that apocarotenoids produced by BCO2 can modulate oxidative stress responses and transcriptional programs involved in tumor suppression [25]. This indicates that the anti-cancer effects of BC may be mediated by its metabolites, which interact with nuclear receptors such as ERβ and RARs, further influencing apoptotic and metabolic gene networks [27].
Similar trends to those observed in the expressions of metabolic enzymes associated with ERβ and BC in this study support the hypothesis that ERβ may transcriptionally regulate carotenoid cleavage enzymes, thereby influencing the local production of BC-derived bioactives. While this regulatory axis requires further validation, previous research suggests that nuclear receptors, such as ERβ and RARs co-regulate shared gene networks in epithelial tissues [28].
In addition to the enhanced apoptotic response, the present study demonstrated that the combined treatment of E2 and BC selectively increased BCMO1 and BCO2 expression, whereas single treatments exerted little or no effect. One plausible interpretation is that BC-derived retinoids regulate gene transcription through RAR/RXR activation, as RAR/RXR nuclear receptors function as ligand-dependent transcription factors that directly control the expression of retinoid-responsive genes [14]. Furthermore, RAR/RXR signaling has been reported to participate not only in genomic transcription but also in extra-nuclear signaling cascades and to integrate with other nuclear receptor pathways, highlighting its role as a regulatory hub in nuclear receptor crosstalk [29]. Based on these findings, we propose that E2 primes ERβ-dependent transcription, and the presence of BC and its metabolites provides a retinoid signal through RAR/RXR, enabling transcriptional co-regulation and leading to the prominent induction of carotenoid-cleaving enzymes under the combination condition. This model supports the possibility that ERβ may act as a regulatory node linking apoptosis and carotenoid metabolism.
Despite these promising findings, several limitations remain. This study was conducted in vitro and may not fully replicate the complexity of in vivo CRC environments. Therefore, future studies should focus on validating these results using in vivo models and exploring the pharmacokinetics and bioavailability of E2 and BC. Moreover, ERα was not expressed in the CRC human cell lines used in this study [17]; thus the potential role of ERα and subsequent interactions with BC metabolites should be further explored in future in vivo studies to provide a more comprehensive understanding of the pathways involved [30].
The present study demonstrated that the combined treatment of BC and E2 exerted greater anti-cancer effects in CRC cells than either compound alone, suggesting that their actions may extend beyond a simple additive effect. A key finding of this study is that BC and E2 co-treatment more strongly reduced cell viability and more markedly modulated apoptosis-related markers, including suppression of Bcl-2 and upregulation of Bax, compared with single treatments. These results are consistent with previous studies showing that BC-derived retinoids activate RAR/RXR-mediated regulatory networks and promote intrinsic apoptosis by reducing Bcl-2 family signaling [11], while E2 mediates tumor-suppressive effects through ERβ-dependent activation of pro-apoptotic transcriptional programs [15]. Moreover, retinoid and estrogen receptor signaling have been reported to influence one another and co-regulate transcriptional programs associated with cell growth [16], supporting our hypothesis that the two pathways may converge to enhance anti-cancer activity.
Taken together, these results suggest that BC and E2 may activate distinct upstream signals that converge on a shared downstream apoptotic pathway. Although direct assessment of signaling pathway involvement using inhibitors was not performed in the present study, the observed enhancement of apoptosis under the combination treatment—along with the coordinated modulation of Bcl-2 and Bax—provides indirect mechanistic evidence for interplay between ERβ signaling and retinoid-mediated regulatory pathways. Future investigations employing pathway-specific inhibitors such as ERβ antagonists or RAR/RXR inhibitors would be highly valuable to dissect whether E2 and BC converge on a unified upstream axis or operate through complementary signaling mechanisms. Such studies would further validate the molecular basis of the enhanced anti-cancer effect observed in the combination group.
In conclusion, this study highlights the synergistic anti-cancer potential of E2 and BC in CRC cells. The combined effects of these treatments are mediated through ERβ activation, modulation of apoptosis-related genes (Bcl-2, Bax), and upregulation of carotenoid cleavage enzymes (BCMO1, BCO2), suggesting a multi-targeted therapeutic mechanism. These findings pave the way for future exploration of hormone-antioxidant combination therapies in CRC management.
Footnotes
Funding: This work was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Minister of the Ministry of Science and ICT (grant No. 2022R1A2C1009512) and was also supported by the BK21 Fostering Outstanding Universities for Research (FOUR) funded by the Ministry of Education (MOE, Korea) and National Research Foundation of Korea (NRF-5199990614253, Education Research Center for 4IR-Based Health Care).
Conflict of Interest: The authors declare no potential conflicts of interests.
- Conceptualization: Kim Y.
- Formal analysis: Park S; Funding acquisition, Kim Y.
- Investigation: Kim Y.
- Methodology: Park S.
- Supervision: Kim Y.
- Writing - original draft: Park S.
- Writing - review & editing: Park S, Kim Y.
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