Royal Jelly Suppresses Invasive Potential of Colorectal Cancer Cells by Attenuating Vimentin and Snail ^†

Abstract

Royal jelly (RJ), as an exclusive bee product, showed beneficial effects on various human pathological conditions, including cancer. Therefore, we examined the effects of RJ on the suppression of invasiveness of SW-480 cells and protein expression of Vimentin and Snail, markers of cancer cell invasive properties. This natural treatment suppressed the aggressive behavior of tested cells by inhibiting the expression of transcription factor Snail, and consequently the expression of cytoskeletal protein Vimentin with a key role in supporting invasive potential. In conclusion, our report indicates the possible molecular mechanism of anti-invasive activity of RJ on the colorectal carcinoma cell line via the inhibition of Vimentin and Snail protein expression.

1. Introduction

Colorectal cancer (CRC) is a malignant type of tumor of the gastrointestinal tract with a very high incidence. This disease is ranked as the second type of all malignant tumors in male and third in females [1].

An important property of cancer, including CRC, is the acquisition of invasive potential. Invasive or metastatic phenotypes are initiated during the epithelial–mesenchymal transition (EMT). This complicated cellular process implies loss of epithelial characteristics, gaining the mesenchymal properties and reorganization of the cytoskeletal architecture which enable the motility of cancer cells [2]. Furthermore, these transformed cancer cells secrete proteolytic enzymes which digest the extracellular matrix and lead to the destruction of colon wall layers [2,3]. Snail has been recognized as a core regulator orchestrating the EMT in CRC, and overexpression of this transcription factor is driving the upregulation of EMT effectors, such as proteolytic enzymes and Vimentin. Vimentin is involved in cytoskeletal interaction, cell adhesion, and is a prominent EMT marker highly expressed in invasive cancer cells [4].

Therefore, when invasion occurs, the metastatic process begins, and when this happens, cancer treatment becomes particularly problematic. Hence, science is turning to natural products that can ameliorate standard chemotherapeutical approaches in combating aggressive behavior of CRC [3].

It is known that dietary habits play one of the most important roles in elevating risk of CRC initiation and progression [5]. Almost half of colorectal cancer cases can be prevented by altering dietary patterns; therefore, it is necessary to pay attention to the food quality and food consumption behavior [6].

Royal jelly is well known bee product that has been traditionally used for centuries because of its valuable nutritive characteristics and is considered nowadays as an effective dietary supplement. It possesses significant pharmacological properties exerting beneficial effects on human well-being [5]. Moreover, it already showed remarkable anti-cancer activity in vitro and in vivo [7,8], and proved to affect the invasion of cancer cells and modulate the metastasis of various cancer types [8,9]. Nevertheless, understanding the molecular mechanisms underlying these notable actions of RJ, especially how this natural product inhibit invasive properties of colorectal cancer cells, is important for the development of optimized therapeutic strategies necessary for combating CRC.

Hence, we aimed to investigate royal jelly’s effects on the invasiveness of colorectal carcinoma cell line SW-480 and to elucidate the molecular mechanism of this action.

2. Materials and Methods

Royal jelly sample (RJ) was obtained from beekeepers located in Serbia (Central region) produced by Apis mellifera L. bee species. RJ was diluted in Phosphate buffer solution to obtain stock solution, and further diluted in complete cell culturing medium (Dulbecco’s Modified Eagle Medium supplemented with 10% of Fetal Bovine Serum and penicillin/streptomycin). Two working concentrations 10 and 100 µg/mL were applied and all the following analyses were done 24 h after treatment. Colorectal carcinoma cell line SW-480 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and propagated according to a standard culturing procedure (5% CO₂/37 °C/humified atmosphere).

The invasive potential of RJ on SW-480 cells was determined by applying a Transwell assay with collagen layer, simulating extracellular matrix, and protocol for this assay as described in detail previously in Jovanović et al. [5]. Resulting absorbances were read at 595 nm wavelength on Multiskan SkyHigh Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Results are presented from three independent experiments performed in triplicates as invasive index (%).

Furthermore, the immunofluorescent technique was used to evaluate the protein expression of proinvasive markers Vimentin and Snail, according to the protocol already described in detail [5]. Micrographs were taken on an inverted fluorescent microscope Eclipse Ti (Nikon Instruments Inc., Tokyo, Japan) at 600× magnification. Obtained micrographs were used for the quantification of relative fluorescence intensity following the method described earlier by Schneider et al. [10], using the ImageJ software package. Results were acquired from three independent experiments performed in triplicate and represented as relative fluorescence per cell (%).

Statistical analysis was conducted using a one-way Anova test within IBM SPSS statistical software package (ver. 20.0, Armonk, NY, USA).

3. Results

3.1. RJ Suppresses Invasiveness

The invasive potential of control and treated SW-480 cells was investigated 24 h after treatment with RJ using Transwell technique. We observed responsiveness of this tested carcinoma cell line to applied treatment. Namely, as it can be observed on Figure 1, both applied RJ concentrations showed an inhibitory effect on the invasiveness of SW-480 cells. Moreover, this anti-invasive activity of RJ was showed to be dose-dependent, meaning that higher RJ dose (100 µg/mL) showed slightly stronger suppressive effect (Figure 1).

3.2. RJ Inhibited Expression of Vimentin and Snail

The protein expression of cytoskeletal protein Vimentin and transcription factor Snail was investigated using the immunofluorescent method. As it can be observed from Figure 1, RJ treatment was able to lower the protein expression of Vimentin and Snail in SW-480 cells after 24 h. The observed decrease was more notable regarding Vimentin expression, than expression of Snail. However, this inhibitory effect was dose-dependent regarding both investigated proteins.

The SW-480 cell line showed sensitivity to applied treatment and the higher RJ concentration showed the most prominent suppressive activity regarding both investigated markers. This significant decrease of Vimentin and Snail expression is in correlation with RJ’s anti-invasive property, as it was shown previously (Figure 1 and Figure 2).

Considering these suppressive effects of RJ on protein expression of these two different proteins, we aimed to determine the possible change of Vimentin and Snail location within SW-480 cells.

Obtained micrographs, presented on Figure 3a, show obvious changes in localization of Vimentin in cells treated with RJ, when compared to control cells. Namely, we observed this protein in both cell nuclei and cytoplasm, while in treated cells, its expression was restricted to cell cytoplasm area only (Figure 3a).

When it comes to Snail localization, this transcription factor was present in cell nuclei and cytoplasm in untreated SW-480 cells indicating abundance of this transcription factor. Meanwhile, when cells were treated with RJ, the expression of Snail was gradually inhibited in cytoplasm, and was eventually limited to cell nuclei only in treatment with higher RJ concentration (Figure 3b).

4. Discussion

Many studies proved RJ as an effective anti-cancer agent, exerting anti-proliferative, pro-apoptotic, anti-metastatic activity, as well as affecting the expression of cancer-related molecules in patients with cancer [8]. Also, RJ has been used for a long time as a dietary supplement because of its multiple beneficial effects on human well-being [5]. The analysis of RJ’s chemical composition has shown that this natural product consists of many bioactive components, including unsaturated fatty acid 10H2DA, to which the majority of RJ’s properties has been attributed to [5,8]. However, only few studies provided scientific explanations for some of these RJ’s properties. However, although RJ showed prominent anti-invasive and anti-metastatic activity, the mechanisms underlying these activities still remain unknown. Therefore, the present study aimed to provide more detailed experimentally proved data on its anti-invasive regulatory mechanism of action.

According to our presented results, it can be concluded that the exerted effects of RJ on suppression of invasive properties of SW-480 cells were obviously due to reduced expression of Vimentin and Snail.

Since Vimentin, as part of cell cytoskeletal structure, regulates cell movement, it is obvious that the insufficient presence of this protein in the tested colorectal carcinoma cell line induced by the presence of RJ caused weaker cell movement through the collagen layer. It is known that in cells with induced EMT, the transcription factor Snail is overexpressed and its regulatory role implies concomitant upregulation of Vimentin [11]. Hence, the reduction of Snail caused by applied RJ treatment in SW-480 cells was followed by the downregulation of Vimentin, and reduced invasive potential of these cells. We already showed in Jovanovic et al. [5] the inhibitory effect of RJ’s unsaturated fatty acid 10H2DA on Snail and Vimentin expression at the gene and protein level, which is the explanation for this significant RJ’s effect.

However, more detailed investigations are needed in order to consider the efficacy and safety, as well as clinical utility, of RJ, or various therapies that include RJ, as a complementary bioactive substance for application in patients with CRC.

5. Conclusions

Our report indicates the possible molecular mechanism of the anti-invasive activity of RJ on colorectal carcinoma cell line. This bee product showed pronounced and promising effects on carcinoma cells in vitro; however, our future research will focus on more detailed studies regarding effects of this natural product on invasion of CRC cells.