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Proceeding Paper

Unsaturated 10H2DA Queen Bee Acid from Royal Jelly Modulates Epithelial-to-Mesenchymal Transition in SW-480 Colorectal Cancer Cells †

by
Milena M. Jovanović
1,* and
Dragana S. Šeklić
2
1
Department for Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34000 Kragujevac, Serbia
2
Institute for Information Technologies Kragujevac, Department of Natural Sciences, University of Kragujevac, Jovana Cvijića bb, 34000 Kragujevac, Serbia
*
Author to whom correspondence should be addressed.
†
Presented at the 2nd International Electronic Conference on Genes, 11–13 December 2024; Available online: https://sciforum.net/event/IECGE2024.
Biol. Life Sci. Forum 2025, 43(1), 3; https://doi.org/10.3390/blsf2025043003
Published: 26 May 2025
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Abstract

:
Cancer research largely focuses on epithelial-to-mesenchymal transition (EMT) as a critical mechanism required for the formation of metastases. This process involves the transformation of epithelial cells into mesenchymal cells by acquiring suppressed levels of anti-EMT and elevated expression of pro-EMT markers. Unsaturated fatty acid 10H2DA has not been investigated hitherto regarding its potential to target specific EMT markers in colorectal cancer (CRC). In our study, this substance showed successful upregulation of the expression of the anti-EMT marker E-cadherin and downregulation of the expression of pro-EMT markers SNAIL, N-cadherin, and Vimentin at the gene and protein levels. This prominent effect of 10H2DA in modulating the expression of specific and significant EMT markers in CRC should not be neglected in future studies regarding anticancer therapeutic approaches.

1. Introduction

Cancer presents a global health problem; therefore, it is the focus of many scientific studies. Colorectal cancer (CRC) is one of the most common types of cancer worldwide, affecting both sexes. This second most deadly type of cancer has high global incidence and mortality rates, which have been increasing recently, especially in high-income countries [1].
The treatment of CRC mostly consists of standard procedures such as surgery, radiation, and chemotherapy. In most cases, these approaches induce severe toxicity of numerous organs, which is why medicine is turning to the application of alternative natural products [2]. The use of natural agents is preferable, especially in combination with chemotherapeutics used as standard, mostly due to their cell-selective mechanism of action, which distinguishes them from cytostatics. Namely, the specific effects of natural agents have already been intensively reported, and many of them target specific signal pathways in cancer cells, or specific proteins, regulatory or effective [3,4].
It is known that epithelial-to-mesenchymal transition (EMT) is a critical molecular mechanism responsible for the acquisition of the invasive and migratory properties of cancer cells and can ultimately lead to the formation of metastases. This key step in the transdifferentiation process of immotile epithelial cells into mobile mesenchymal cells primarily involves deregulation and/or suppression of strong intercellular junctions consisting of E-cadherin and β-catenin. Consequently, cancer cells dissociate from the cancer mass, and, after invasion into surrounding tissue, they reach blood vessels, which allows them to disseminate and colonize secondary tissues [5]. Therefore, studies using treatments that are able to elevate the level of anti-EMT marker E-cadherin, as well as being able to reestablish intercellular junctions, have the potential to improve therapeutic approaches in the field of cancer treatment. Furthermore, EMT is tightly regulated by a pro-EMT protein, the transcription marker SNAIL, which primarily downregulates the expression of E-cadherin and upregulates the expression of other pro-EMT markers, such as N-cadherin and effector intermediate filament protein Vimentin [5]. Therefore, a growing need has arisen for bioactive substances able to target and modulate EMT markers and reverse this process.
Bee products are highly appreciated in medicine worldwide and are used for the treatment of various pathological conditions, among which is cancer. Royal jelly is considered a bee product with many bioactive substances: proteins, lipids, carbohydrates, amino acids, etc. [6,7]. Unsaturated fatty acid 10H2DA is a qualitative marker of royal jelly, present only in this natural product with already proven anticancer potential [6]. Previous studies have shown its remarkable potential to inhibit the formation of tumors in vivo and suppress tumor growth and the occurrence of metastatic lesions [7,8,9,10]. However, its potential to target specific EMT markers in the colorectal cancer (CRC) cell line SW-480 has not been investigated so far, which was the aim of this study.

2. Materials and Methods

Colorectal cancer cell line SW-480, isolated from stage II CRC, was obtained from the American Type Culture Collection (ATCC, Manassas, VI, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Basel, Switzerland) supplemented with 100 U/mL penicillin/100 μg/mL streptomycin (Gibco, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and 10% fetal bovine serum (FBS, Capricorn Scientific, Ebsdorfergrund, Germany). Maintenance of cells was completed in a humidified atmosphere at 37 °C and 5% CO2. After reaching 70 to 80% confluence in 75 cm2 cell culture flasks, the cells were harvested through trypsinization and seeded for the following assays.
Queen bee acid 10H2DA was purchased from TCI Chemicals (Tokyo, Japan) as powder, which was diluted in DMEM and dimethyl sulfoxide (SERVA, Heidelberg, Germany) The cytotoxic effects of 10H2DA on the SW-480 cell line were assessed using the MTT test, whereby cells were seeded in 96-well plate (1 × 104 cells per well). MTT was performed in triplicate in two independent experiments, 24 and 72 h after treating the cells with the following concentrations: 0.1, 1, 10, 50, 100, and 500 µM. Two concentrations, 10 and 100 µM, were selected for further experiments based on their non-cytotoxic effect. For the purpose of the analysis of the relative gene expression of E-cadherin, SNAIL, N-cadherin, and Vimentin markers, the cells were seeded in 25 cm2 cell culture flasks, wherein, after reaching 80% confluence, the treatment was applied. After 24 h, the quantitative polymerase chain reaction (qPCR) method was utilized according to the protocol already described [5], and the results were compared to the expression of housekeeping gene β-actin. This experiment was performed using a Mic qPCR Cycler (Biomolecular Systems, Yatala, Australia), and the obtained results were calculated according to the 2−ΔΔCt formula [5].
The changes in protein concentration of these markers between the control (untreated) and cells treated with two selected 10H2DA concentrations were evaluated using an immunofluorescent assay. The SW-480 cells were propagated in 6-well culture dishes, and when 80% confluency was reached, treatment was applied in two selected concentrations. After 24 h, the protocol described in Šeklić et al.’s study [11] was followed and E-cadherin, SNAIL, N-cadherin, and Vimentin protein expression was determined by using an inverted fluorescent microscope Eclipse Ti (Nikon Instruments Inc., Melville, NY, USA) to obtain micrographs at 600× magnification, while the changes in relative fluorescent intensity were quantified using the ImageJ software package (ver. 1.52a) [12].

Statistical Analysis

Statistical analyses were performed using the IBM SPSS Statistics software package (v. 17, IBM Corp., Armonk, NY, USA), whereby Student’s t-test and one-way ANOVA were applied for multiple comparisons.

3. Results

3.1. Effects of 10H2DA on Cell Viability

We assessed the effects of unsaturated fatty acid 10H2DA on the viability of SW-480 colorectal cancer cells (Figure 1a), as well as its effects on a normal (healthy) cell line, MRC-5 (Figure 1b). As can be observed, this chemical compound exerted no significant cytotoxicity on both tested cell lines after both 24 and 72 h of treatment. The treatment was able to reduce the cell viability of SW-480 cells by 20% only when the highest concentration was applied (500 µM) (Figure 1a), with no detected IC50 values (Table 1). On the other hand, 10H2DA had a proliferative effect on the healthy MRC-5 cell line when applied in lower concentrations (0.1 and 1 µM), while even higher concentrations had no impact on cell viability (with IC50 > 500 µM) when compared to the control (untreated) cells (Figure 1b, Table 1).

3.2. Gene Expression of Targeted Markers

According to our results regarding the levels of the investigated EMT markers, E-cadherin, SNAIL, N-cadherin, and Vimentin, the tested substance 10H2DA was found to be an effective treatment. As can be observed in Figure 2, queen bee acid successfully upregulated the expression of anti-EMT marker E-cadherin at the gene level 24 h after treatment, while our results point to the downregulated expression of the pro-EMT regulatory marker SNAIL, as well as the effector markers N-cadherin and Vimentin. Among the two tested 10H2DA concentrations applied, the higher concentration, 100 µM, exerted a significantly stronger effect. Namely, this concentration was more powerful in the elevation of E-cadherin expression and in lowering the expression of pro-EMT markers at the same time.

3.3. Protein Expression of Targeted Markers

The expression of the investigated markers, E-cadherin, SNAIL, N-cadherin, and Vimentin, at the protein level was also investigated using the immunofluorescent method, and the results are presented in Figure 3a,b. According to the obtained results, the expression of these markers at the gene level was accompanied by protein expression. Treatment successively led to significant elevation of E-cadherin at the protein level, when compared to the control values (Figure 3a). 10H2DA was able to simultaneously inhibit pro-EMT proteins, the regulatory marker SNAIL, and the effector markers N-cadherin and Vimentin. Their expression levels were significantly below the control values. Moreover, representative micrographs show the distribution of targeted parameters within the SW-480 cells (Figure 3b). The localization of E-cadherin in control cells was mainly in the cytoplasmic membrane area, while in treated cells, this protein was observed both in the membrane and the nucleus area, indicating its higher concentration in cells. Meanwhile, transcription factor SNAIL was located in both the cytoplasmic and nuclear compartments in control SW-480 cells, and its expression was evidently intense. However, when 10H2DA treatment was applied, the intensity of SNAIL expression was lowered (Figure 3a) and was limited mostly to the nuclear area (Figure 3b). Effector marker N-cadherin was observed mostly in the cytoplasmic compartment of control SW-480 cells; however, a small portion of this protein was also found in the nuclear area (Figure 3b). The location of N-cadherin remained unchanged after treatment with 10H2DA, however, with it being limited to the cytoplasmic area. The intensity of this protein expression was evidently lower, especially after treatment with higher treatment concentrations (Figure 3a,b). Vimentin, an effector pro-EMT protein, was found to be highly expressed in the control SW-480 cells. This intermediate filament, responsible for the advanced EMT and increased cell migratory and invasive capacity of cancer cells, was present in all cell compartments, cell nuclei, the cytoplasm, and the membrane area (Figure 3b). 10H2DA significantly and successfully suppressed this protein’s expression and limited it to mostly the cytoplasmic area (Figure 3a,b).

4. Discussion

Many studies have confirmed the significant suppressive effect of royal jelly on tumor growth and progression in vivo [2,8,13]. It has also been demonstrated through several studies that royal jelly significantly inhibits tumor formation, slows the growth of some tumor types, prevents the formation of metastases, and positively affects the successful survival of these tested model systems [7,9,10,14,15]. The reduction in tumor size under the influence of royal jelly was found to be approximately 50%, and the lifespan of the in vivo model system was extended by about 20% [7]. These effects of royal jelly are mainly attributed to its active component 10H2DA. However, the effects of this unsaturated fatty acid on the expression of specific EMT markers have not been investigated hitherto. Our study shows, for the first time, the significant effects of 10H2DA on the expression of anti- and pro-EMT markers present in the SW-480 immortalized colorectal carcinoma cell line.
Analysis of EMT markers’ expression rate in untreated (control) SW-480 cells, originating from early stage–stage II CRC [11,16], revealed a strong presence of pro-EMT markers, SNAIL, N-cadherin, and Vimentin, indicating an aggressive potential. The migratory capacity of these cells has already been reported, among moderate differentiation and cluster growth [11,17,18,19,20]. According to our research, SNAIL was found to be an important regulator of EMT in SW-480 cells, considering that this transcription factor regulates the expression of effector markers N-cadherin and Vimentin, which concords with the strong expression of these proteins in the investigated SW-480 cells in this study. It is known that SNAIL also inhibits the expression of E-cadherin in cancer cells [5], which is confirmed by our study, wherein we observed the effects of 10H2DA on SNAIL inhibition at the gene and protein levels, as well as the consequent rise in E-cadherin levels. Moreover, when 10H2DA inhibited SNAIL, the expression level of N-cadherin and Vimentin dropped. This indicates 10H2DA as a very promising natural agent in inhibiting the EMT via a very specific target, the regulation of SNAIL expression.
Additionally, our previous study showed a suppressive effect of 10H2DA acid on the migratory and invasive potential of this cell line [5], and considering the results of the present study, we can conclude that the mechanism of action of this treatment in SW-480 cells was through the reduction in pro-EMT markers, primarily the regulatory marker SNAIL. The detailed molecular mechanism in colorectal cancer cells during treatment with 10H2DA has not been investigated so far; thus, this is the first study showing the targeted effects of this bioactive substance on this type of cancer. Similar effects of 10H2DA have been shown in previous studies on lung cancer [21]. The basis of the suppression of migratory potential and the intensity of EMT in a study conducted by Lin et al. [21] was the increase in E-cadherin levels and the suppression of N-cadherin, Vimentin, and SNAIL at the gene and protein levels.
The effects of this unsaturated fatty acid on the SW-480 cell line can be explained by the very strong affinity of 10H2DA for binding to estrogen receptor β (ERβ) within cells that express it. The binding of this acid to the receptor causes its activation, which initiates its movement to the nucleus where it binds to the promoter regions of genes designated as estrogen-responsive sequences (EREs) within the DNA molecule [22]. Several studies have confirmed that this acid, 10H2DA, activates ERβ [22,23,24], through which the expression of various genes is regulated, including E-cadherin, N-cadherin, β-catenin, Vimentin, and SNAIL [22]. The presence and predominance of this type of receptor (ERβ) are responsible for regulating the maintenance of the epithelial phenotype, and its presence has been confirmed in CRC and in this tested cell line [25,26,27].

5. Conclusions

The results of the present study reveal that unsaturated fatty acid 10H2DA has remarkable effects on the EMT process in colorectal cancer cell line SW-480, wherein this unique chemical compound significantly elevated anti-EMT marker E-cadherin and inhibited pro-EMT markers SNAIL, N-cadherin, and Vimentin. The potential of unsaturated acid 10H2DA to modulate the expression of specific and significant EMT markers in CRC is obvious and prominent and should not be neglected in future studies regarding anticancer therapeutic approaches.

Author Contributions

Conceptualization, D.S.Š. and M.M.J.; methodology, M.M.J.; software, M.M.J.; validation, D.S.Š.; formal analysis, M.M.J.; investigation, M.M.J.; resources, D.S.Š.; data curation, D.S.Š.; writing—original draft preparation, M.M.J.; writing—review and editing, D.S.Š.; visualization, M.M.J.; supervision, D.S.Š.; project administration, D.S.Š.; funding acquisition, D.S.Š. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia, grant numbers 451-03-66/2024-03/200378 and 451-03-136/2025-03.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effect of 10H2DA on the viability of SW-480 (a) and MRC-5 (b) cell lines after 24 h and 72 h. The results are presented as the mean ± standard error from two independent experiments performed in triplicate.
Figure 1. Effect of 10H2DA on the viability of SW-480 (a) and MRC-5 (b) cell lines after 24 h and 72 h. The results are presented as the mean ± standard error from two independent experiments performed in triplicate.
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Figure 2. Results of relative gene expression in the SW-480 cell line treated with 10H2DA applied at two selected concentrations, 10 and 100 µM, after 24 h. Values are presented as the fold change in mRNA expression in a target sample, normalized to a reference gene, and relative to the control sample. Control values are designated as a line. * p < 0.05 is designated as statistically significant difference between treatments and control values, while the # p < 0.05 is statistically significant difference between treatment concentrations.
Figure 2. Results of relative gene expression in the SW-480 cell line treated with 10H2DA applied at two selected concentrations, 10 and 100 µM, after 24 h. Values are presented as the fold change in mRNA expression in a target sample, normalized to a reference gene, and relative to the control sample. Control values are designated as a line. * p < 0.05 is designated as statistically significant difference between treatments and control values, while the # p < 0.05 is statistically significant difference between treatment concentrations.
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Figure 3. Representative micrographs showing the protein expression of the targeted markers in SW-480 cells 24 h after treatment with 10H2DA. Cell nuclei were stained blue (DAPI); E-cadherin, SNAIL and vimentin proteins were stained red (secondary antibody conjugated with Cy3), while the N-cadherin proteins were stained green (secondary antibody conjugated with Alexa). Merged Scale bar: 50 μm (b) Fluorescence intensity of analyzed proteins presented as changes relative to control cells (values of the control designated as a line) in % (a), where * p < 0.05 is a statistically significant difference between the control and treatment, and # p < 0.05 is a statistically significant difference between the treatment concentrations. The results are presented from two independent experiments performed in triplicate.
Figure 3. Representative micrographs showing the protein expression of the targeted markers in SW-480 cells 24 h after treatment with 10H2DA. Cell nuclei were stained blue (DAPI); E-cadherin, SNAIL and vimentin proteins were stained red (secondary antibody conjugated with Cy3), while the N-cadherin proteins were stained green (secondary antibody conjugated with Alexa). Merged Scale bar: 50 μm (b) Fluorescence intensity of analyzed proteins presented as changes relative to control cells (values of the control designated as a line) in % (a), where * p < 0.05 is a statistically significant difference between the control and treatment, and # p < 0.05 is a statistically significant difference between the treatment concentrations. The results are presented from two independent experiments performed in triplicate.
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Table 1. Cytotoxic effects—IC50 values (μM) of 10H2DA on the SW-480 and MRC-5 cell lines.
Table 1. Cytotoxic effects—IC50 values (μM) of 10H2DA on the SW-480 and MRC-5 cell lines.
10H2DASW-480MRC-5
24 h>500>500
72 h>500>500
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MDPI and ACS Style

Jovanović, M.M.; Šeklić, D.S. Unsaturated 10H2DA Queen Bee Acid from Royal Jelly Modulates Epithelial-to-Mesenchymal Transition in SW-480 Colorectal Cancer Cells. Biol. Life Sci. Forum 2025, 43, 3. https://doi.org/10.3390/blsf2025043003

AMA Style

Jovanović MM, Šeklić DS. Unsaturated 10H2DA Queen Bee Acid from Royal Jelly Modulates Epithelial-to-Mesenchymal Transition in SW-480 Colorectal Cancer Cells. Biology and Life Sciences Forum. 2025; 43(1):3. https://doi.org/10.3390/blsf2025043003

Chicago/Turabian Style

Jovanović, Milena M., and Dragana S. Šeklić. 2025. "Unsaturated 10H2DA Queen Bee Acid from Royal Jelly Modulates Epithelial-to-Mesenchymal Transition in SW-480 Colorectal Cancer Cells" Biology and Life Sciences Forum 43, no. 1: 3. https://doi.org/10.3390/blsf2025043003

APA Style

Jovanović, M. M., & Šeklić, D. S. (2025). Unsaturated 10H2DA Queen Bee Acid from Royal Jelly Modulates Epithelial-to-Mesenchymal Transition in SW-480 Colorectal Cancer Cells. Biology and Life Sciences Forum, 43(1), 3. https://doi.org/10.3390/blsf2025043003

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