Bee Product Royal Jelly Suppress EMT and Invasiveness of HCT-116 Cells †

2023


Introduction
As a highly frequent type of cancer diagnosed in both males and females, colorectal cancer (CRC) is also widely recognized as the most common cause of death worldwide.Globally it is one of the cancers whose incidence is increasing, and the prognosis of cancer patients' survival is often poor, due to the acquisition of the invasive and migratory potential of cancer cells which consequently leads to metastasis [1].Firstly, cancer cells succumb to a specific process-the epithelial to mesenchymal transition (EMT)-allowing them to acquire invasive potential.This allows them to detach from the primary cancer site; therefore, cells are able to penetrate the surrounding stiff extracellular matrix.For this purpose, the expressions of certain markers that enable this transition are necessary to assess, such as Snail, nuclear protein, or the cytoskeletal protein vimentin.It is known that Snail expression starts in the first stage of colorectal carcinogenesis, in which this transcriptional factor acts as a repressor of many epithelial hallmarks, as well as the potentiator of mesenchymal markers [2].Hence, therapy designed to battle cancer from this point of cancer progression involves targeting the aforementioned markers.
We aimed to investigate the effects of royal jelly, a natural bee product originating from Serbia, on the invasive potential of the colorectal cancer cell line HCT-116 and the expression of these two invasive EMT proteins.

Materials and Methods
HCT-116 cells, isolated from the rectal region of human colon carcinomas, were purchased as an immortalized cell line from the American Type Culture Collection (ATCC, Manassas, VA, USA).Cells were cultured in a complete culturing medium (Dulbecco's modified Eagle medium-DMEM, supplemented with 10% of fetal bovine serum-FBS, and penicillin/streptomycin), in a humified atmosphere, at 37 • C with 5% of CO 2 , and were seeded for assays at 80-90% of confluency.The royal jelly (RJ) sample (produced by the Apis mellifera L. species) was collected in the central region of Serbia and diluted fresh in a phosphate buffer solution (PBS) and a complete cell culturing medium.In this way, two working concentrations, 10 and 100 µg/mL, were obtained and applied for all the following assays, while the effects of RJ were analyzed after 24 h.
The invasive potential of control (untreated) and HCT-116 cells treated with RJ was investigated using an assay with Transwell inserts coated with a thin collagen layer, which was appied as an extracellular matrix.This assay was performed as described earlier [3].Absorbances were read using a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA) (595 nm wavelength) and the results from three independent experiments, performed in triplicate, are presented, in the form of an invasive index.
In order to assess the protein expression of Snail and vimentin, an immunofluorescent assay was performed in three independent experiments and in triplicate, as described earlier (Jovanovic et al., 2022).An inverted fluorescent microscope Eclipse Ti (Nikon Instruments Inc., Tokyo, Japan) was used to obtain micrographs (at 600× magnification), which were further analyzed to quantify the relative fluorescence intensity of the targeted proteins.For this purpose, the ImageJ software package was applied, following the procedure described earlier [4].
The statistical comparison of the obtained results was performed using the IBM SPSS statistical software package (ver.17, 2008, Armon, NY, USA) and a one-way ANOVA.The results from all the performed assays are presented as mean ± standard error, where * p < 0.05 designates a statistically significant difference between the treatments and the control, and # p < 0.05 designates a statistically significant difference between the effects of the treatment concentrations.

Results
According to our results, as shown in Figure 1, the suppression of the invasiveness of the tested colorectal cancer cell line, HCT-116, was observed 24 h after treatment with two applied RJ concentrations (10 and 100 µg/mL).This suppression is obvious and statistically significant, when compared to the control values.
Biol.Life Sci.Forum 2023, 26, x 2 of 5 (Dulbecco's modified Eagle medium-DMEM, supplemented with 10% of fetal bovine serum-FBS, and penicillin/streptomycin), in a humified atmosphere, at 37 °C with 5% of CO2, and were seeded for assays at 80-90% of confluency.The royal jelly (RJ) sample (produced by the Apis mellifera L. species) was collected in the central region of Serbia and diluted fresh in a phosphate buffer solution (PBS) and a complete cell culturing medium.In this way, two working concentrations, 10 and 100 µg/mL, were obtained and applied for all the following assays, while the effects of RJ were analyzed after 24 h.The invasive potential of control (untreated) and HCT-116 cells treated with RJ was investigated using an assay with Transwell inserts coated with a thin collagen layer, which was appied as an extracellular matrix.This assay was performed as described earlier [3].Absorbances were read using a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA) (595 nm wavelength) and the results from three independent experiments, performed in triplicate, are presented, in the form of an invasive index.
In order to assess the protein expression of Snail and vimentin, an immunofluorescent assay was performed in three independent experiments and in triplicate, as described earlier (Jovanovic et al., 2022).An inverted fluorescent microscope Eclipse Ti (Nikon Instruments Inc., Tokyo, Japan) was used to obtain micrographs (at 600× magnification), which were further analyzed to quantify the relative fluorescence intensity of the targeted proteins.For this purpose, the ImageJ software package was applied, following the procedure described earlier [4].
The statistical comparison of the obtained results was performed using the IBM SPSS statistical software package (ver.17, 2008, Armon, NY, USA) and a one-way ANOVA.The results from all the performed assays are presented as mean ± standard error, where * p < 0.05 designates a statistically significant difference between the treatments and the control, and # p < 0.05 designates a statistically significant difference between the effects of the treatment concentrations.

Results
According to our results, as shown in Figure 1, the suppression of the invasiveness of the tested colorectal cancer cell line, HCT-116, was observed 24 h after treatment with two applied RJ concentrations (10 and 100 µg/mL).This suppression is obvious and statistically significant, when compared to the control values.In order to assess the possible mechanism of RJ's anti-invasive potential, the protein expressions of the regulatory factor Snail and the effector marker vimentin were investigated, using an immunofluorescent method, and the results can be observed on representative micrographs, as well as in graphs (Figure 2).Firstly, the expression intensity of Snail in the control (untreated) HCT-116 cells was at a very high level, and the localization of this protein was in both cell nuclei and cytoplasm, indicating its abundance In order to assess the possible mechanism of RJ's anti-invasive potential, the protein expressions of the regulatory factor Snail and the effector marker vimentin were investigated, using an immunofluorescent method, and the results can be observed on representative micrographs, as well as in graphs (Figure 2).Firstly, the expression intensity of Snail in the control (untreated) HCT-116 cells was at a very high level, and the localization of this protein was in both cell nuclei and cytoplasm, indicating its abundance in cells (Figure 2a).Meanwhile, the treatment was able to suppress this significantly and restrict its localization only to cell nuclei.In this suppression, the lower RJ concentration exerted a more significant effect than when a higher concentration was applied, which correlates with its anti-invasive potential (Figure 1).levels of vimentin, found in both cell nuclei and cytoplasm.When the treatment with RJ was applied, a significantly lower level of this protein expression was observed, which was mainly localized in cell nuclei (Figure 2b).The lower RJ concentration had a slightly stronger suppressive effect on this protein expression when compared to a higher RJ concentration.These results regarding vimentin protein expression also correlate with the antiinvasive properties exerted by RJ, as shown earlier (Figure 1).

Discussion
So far, the common treatments for colon cancer in clinics are chemoradiotherapy, surgery and immunotherapy.However, many disadvantages have been noticed, which is why scientists are turning to the development of alternative types of therapeutic approaches to this disease.Natural products have been used in folk medicine for centuries, and many of them have proved to be effective in treating cancer [5].Royal jelly, as a natural product, has already been shown to possess anticancer properties by suppressing cancer growth and aggressiveness [6].Therefore, we aimed to assess the antiinvasive potential of royal jelly sampled in Serbia, on the colorectal cancer cell line HCT-116, which has already been described as highly invasive and aggressive [7].Therefore, On the other hand, the control (untreated) HCT-116 cells contained relatively high levels of vimentin, found in both cell nuclei and cytoplasm.When the treatment with RJ was applied, a significantly lower level of this protein expression was observed, which was mainly localized in cell nuclei (Figure 2b).The lower RJ concentration had a slightly stronger suppressive effect on this protein expression when compared to a higher RJ concentration.These results regarding vimentin protein expression also correlate with the antiinvasive properties exerted by RJ, as shown earlier (Figure 1).

Discussion
So far, the common treatments for colon cancer in clinics are chemoradiotherapy, surgery and immunotherapy.However, many disadvantages have been noticed, which is why scientists are turning to the development of alternative types of therapeutic approaches to this disease.Natural products have been used in folk medicine for centuries, and many of them have proved to be effective in treating cancer [5].Royal jelly, as a natural product, has already been shown to possess anticancer properties by suppressing cancer growth and aggressiveness [6].Therefore, we aimed to assess the anti-invasive potential of royal jelly sampled in Serbia, on the colorectal cancer cell line HCT-116, which has already been described as highly invasive and aggressive [7].Therefore, the suppression of this cell behavior is a highly desirable approach for designing anticancer therapeutics.
The present study reports on the suppression of the invasiveness of the tested colorectal cancer cells by treatment with RJ, which is obviously induced by lowered protein levels of Snail and vimentin.Previous investigations confirm that natural products possess the ability to repress the levels of these two invasive markers in vitro, as well in vivo [8].
It is generally accepted that a fundamental characteristic responsible for the formation of metastasis is the acquisition of invasive potential, which is associated with Snail and vimentin expression [2].The overexpression of Snail, as a zing-fingered transcriptional factor, is responsible for resistance to chemotherapeutics, lymph node metastasis, activation of the EMT program, the stemness of cancer and the poor prognoses of CRC patients [9,10].The activation of vimentin expression by Snail has already been reported as resulting in the increased invasive and migratory potential of cancer cells, enabling their dissemination (metastasis).Also, the occurrence of this nuclear factor has not been detected in normal (healthy) epithelial cells, however, it was found to be present in the invasive front of cancer tissues [11].The tight connection between the two investigated EMT markers with significant roles in the acquisition of invasiveness in cancer cells resulted in the suppression of this very undesirable trait of the HCT-116 cell line.

Conclusions
RJ exerted significant anti-invasive activity against the very aggressive HCT-116 colorectal cell line by attenuating the Snail and vimentin invasive markers, which is a significant result of the present study, suggesting that this natural product is a valuable source of anticancer effects.We anticipate that these findings will be the focus of increasing attention in both the scientific and clinical fields of research.

Figure 1 .
Figure 1.Invasive potential of control and HCT-116 cells treated with RJ in two selected concentrations (10 and 100 µg/mL) presented as mean values ± SE; * p < 0.05 is considered as statistically significant difference between treatments and control values.

Figure 1 .
Figure 1.Invasive potential of control and HCT-116 cells treated with RJ in two selected concentrations (10 and 100 µg/mL) presented as mean values ± SE; * p < 0.05 is considered as statistically significant difference between treatments and control values.

Figure 2 .
Figure 2. Representative micrographs showing Snail (a) and vimentin (b) fluorescence intensity in control and treated HCT-116 cells.Micrographs were obtained 24 h after treatment with RJ at two selected concentrations (10 and 100 µg/mL).Results of relative protein fluorescence intensity are presented as changes compared to control values (mean ± SE), where * p < 0.05 is considered as statistically significant difference between treatments and control values, and # p < 0.05 is considered as statistically significant difference between treatment concentrations.

Figure 2 .
Figure 2. Representative micrographs showing Snail (a) and vimentin (b) fluorescence intensity in control and treated HCT-116 cells.Micrographs were obtained 24 h after treatment with RJ at two selected concentrations (10 and 100 µg/mL).Results of relative protein fluorescence intensity are presented as changes compared to control values (mean ± SE), where * p < 0.05 is considered as statistically significant difference between treatments and control values, and # p < 0.05 is considered as statistically significant difference between treatment concentrations.