The Effect of Fertilized Egg White on Growth, Migration, Differentiation and Genes Expression Profile in SW480 Colon Cancer Cell Line


 Background: Embryonic microenvironments influence cancer stem cells properties, which leads to anti-cancer effects. Therefore, the current study investigates the effects of fertilized egg white, as an embryonic/fetal microenvironment, on survival, apoptosis, self-renewal characteristic, stemness properties, and migration capacity of SW480 colon cancer cells and 5-fluorouracil (5FU) resistant subgroup.Methods: MTT and Flow cytometry was used to study the cell viability and cell cycle analysis. Clonogenic, spheroid formation, and wound healing assays were used to evaluate cancer cells' self-renewal, stemness properties, and migration capacity. RT-PCR was performed to analyze NANOG, c-MYC, E-cadherin, and NDRG1 mRNA expression.Results: The SW480 colon carcinoma cell line and SW480-5FU chemo-resistant subpopulation cells were subjected to Fertilized Egg White (FEW). FEW decreased cell viability and increased the percentage of the sub-G1 stage in both cell lines. In addition, colony and spheroid formations were decreased in both cells, and the FEW inhibited the migration. Expression of NANOG and c-MYC were reduced in both cells. E-cadherin and NDRG1 expression increased in SW480 cells.Conclusion: FEW decreased the SW480 colon cancer cell line and the SW480-5FU chemo-resistant subpopulation growth and migration. Also, by the changes observed in gene expression and spheroid formation, we suggest the possibility of decreased stemness properties and induction of differentiation following fertilized egg white treatment.


Introduction
Despite huge attempts to nd cancer initiation and progression mechanisms, it remains the second leading cause of death globally [1]. Conventional cancer therapies, including chemo and radiotherapy, have adverse side effects and tumor cells resist them [2]. Evidence shows a compensatory mechanism that promotes proliferation in neighboring cells, enforced by apoptotic cells [3]. Even in successful therapies, remission may occur aggressively after an apparent cancer-free period [4,5]. The adverse effects of traditional therapies persuaded scientists to search for new molecular targets and design new therapeutic approaches [4,5].
According to the cancer stem cell paradigm, tumor cells arise from a subpopulation of tumor bulk, which drives tumorigenesis and progression [6]. Cells in this category share numerous characteristics with adult and embryonic human stem cells, speci cally self-renewal, stemness, differentiation to other types of tumor cells, and resistance to conventional medications [6]. The Gene expression pattern of aggressive cancer stem cells shows similarities with adult and embryonic stem cells, particularly in stemness and differentiation-related genes expression [7]. Unlike stem cells, cancer cells are uncontrolled in expressing stemness and differentiation factors, such as c-Myc, NANOG, Sox 2, NDRG1, E-cadherin, and developing pathway including Notch, Hedgehog, and Wnt, resulting in the progression of cancer bulk [8,9].
Despite similarities between embryos and cancer development, and between embryonic stem cells and cancer stem cells, cancer incidence in the embryo and fetus is sporadic [10,11]. Some studies also have shown that embryonic niches could reverse the tumorigenic state of cancer cells [10,11], and the malignancy of aggressive melanoma cells declines when transferred to chick embryos [12]. Also, the injection of cancer cells in the different fetal stages of mouse fetus impaired cancer cell growth [13].
Egg whites are well known for providing all the nutrients needed to form and grow normal embryos, including amino acids, lipids, vitamins, elements, and growth factors [14]. The protein within egg white migrates to the developing chick embryos, potentially establishing that eggs have a direct role in embryogenesis, in addition to nourishing and storing protein, and can create an embryonic niche that can be an anticancer [15,16].
This study aimed to investigate the effect of fertilized egg white (FEW) as an in vitro embryonic/fetal microenvironment on human colorectal SW480 cell line growth, stemness and differentiation. Also, we looked at a subpopulation of SW480 that were made resistant to the chemotherapy agent 5-Fluorouracil (5-FU). We exposed cells to the FEW environments and studied the growth rate, cell cycle, colony, spheroid formations, migration, and expression of genes involved in stemness and differentiation.

Preparation of Egg white (EW)
The fertilized 9-day-old chicken egg was purchased from the chicken incubation farm. Egg white was separated in a sterile condition. In all following experiments, EW was dissolved in the DMEM medium to reach a nal concentration of 10% v/v concentration, similar to FBS concentration in media.

Cell culture
The SW480 human colorectal carcinoma line was obtained from the Cell Bank of Pasteur Institute's Tehran. The cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM) (GIBCO, Life Technologies), cell culture medium containing 10% fetal bovine serum (FBS) (GIBCO, Life Technologies), 1% glutamine (Sigma), and 1% penicillin/streptomycin antibiotic (Sigma) at 37°C and 95% humidity in a 5% CO2 humidi ed incubator. The cells were passaged after reaching appropriate con uency (80-90%), and media was changed every other day.

Establishment of 5FU resistant SW480 colon cancer cell line
According to our previous study, the MTT assay determined the IC50 value of 5-uorouracil (5FU) [17]. In brief, SW480 cells were exposed to the increasing doses (0, 10, 50, 100, 150, 200, 300µM) of 5FU for 72h. MTT assay was performed, and the dose-dependent diagram was created by GraphPad prism. The IC50 dose for 5FU was calculated as 20 µM. After that, an almost fully con uent (90%) monolayer of SW480 cells was treated with 20 µM (the IC50 dose) of 5FU. Following 72 h incubation, the medium was discarded, and the plates were cultured under the standard condition for two to three weeks. When cells reached their logarithmic phase, they were used for the experiments.

Flow cytometry cell analysis
To ascertain the effect of fertilized egg white on cell cycle and apoptosis, we used the Flow cytometry cell analysis. The cells were incubated overnight in a 12-well plate and then exposed to fertilized egg white for three days (72h). Then, cells were collected, rinsed with PBS two times, and xed by 70% ethanol at -20˚C overnight. The following day, the cells were rinsed with PBS containing 0.5 µM/ml RNase (Sigma, St. Louis, MO, USA) for 30 min. Then, cells were stained with 50 µg/ml propidium iodide (Sigma, St. Louis, MO, USA) for 30 minutes in the dark. Finally, the FACScan ow cytometer (Becton Dickinson) analyzed the Cell cycle.
2.5 Colonospheres (spheroid) formation 10 4 cells per well were cultured in a 24-well plate covered with poly-HEMA to make it a low attachment. The media used was serum-free DMEM supplemented with 30 ng/mL epidermal growth factor (EGF) (Royan Institute, Iran), 30 ng/mL broblast growth factor (FGF) (Royan Institute, Iran), and pen/strep antibiotic 1% with and without FEW. In addition, EGF and FGF were added every other day for eight days. Finally, the produced colonospheres were counted under the microscope.
2.6 Clonogenic assay 100 cells/well were seeded in a 12-well plate in triplicate and incubated for 24h at 37°C. Cells were treated with 10% fertilized egg white. After two weeks, Acetic acid/methanol (1:7) was added to x colonies, and they were stained using 0.05% crystal violet (Sigma Chemical Company, Louis, MO, USA) at 25°C for 2h.
Plates were washed with water and kept at 25˚C to dry. Finally, Colonies that had greater than fty cells were selected and counted under a microscope. The clonogenic capacity was determined based on the number of colonies formed in proportion to the number of seeded cells.

Scratch wound healing assay
The wound-healing assay was used to evaluate cell migration. The cells were cultured in a six-well plate for two days to reach 95% con uency, and then the plate was incubated overnight with no-FBS to inhibit proliferation. Gently and slowly, the monolayer was using a sterile tip. Cells were rinsed with PBS to eliminate debris, and the medium containing FEW was added. The images were captured at 0, 48 h, and 72 h following treatment and were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) (10). Cell migration that was calculated as the percentage of gap closure: At =0h is the area of gap measured at time 0 after scratching, and At = Δh is the area of gap measured later [18].

RNA extraction and Real-time PCR analysis
According to the manual, the total RNA of cells was isolated with RNX-plus Solution (Sinaclon, Tehran, Iran). Puri cation was evaluated by determining the 260/280 nm absorbance ratio at 260 nm and 260/280 nm. Then, 3 µg extracted RNA was used to generate cDNA by Frist Strand cDNA Synthesis Kit (2step RT-PCR kit, Vivantis, USA). The real-time PCR was performed for NANOG, c-MYC, E-cadherin, NDRG1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (as the endogenous control) mRNA expression using SYBR Green (Bio-Rad). Table 1 lists the sequences of the primers used. 3 Results

Effect of fertilized egg white Treatment on Cell Viability
The colorimetric MTT assay was performed to assess the effect of FEW on SW480 and SW480-5FU cellular viability, using MTT assay after 72h treatment. As demonstrated in Figure 1, fertilized egg white signi cantly decreased SW480 cells (51%) and SW480-5FU cells (46%) viability compared to control.

Effect of fertilized egg white on Cell Cycle
To further investigate the inhibitory effect of fertilized egg white on SW480 and SW480-5FU, we used owcytometry cell cycle analysis using propidium iodide (

Effect of fertilized egg white on colony formation
The colony formation assay was used to semi-quantitatively study the effects of fertilized egg white on self-renewal and prolonged cell proliferation. SW480 and SW480-5FU cells were treated with FEW for two weeks, and colonies greater than fty cells were counted. Fertilized egg white signi cantly reduced the colony formation after 13 days by 76% and % 80.5 in SW480 and SW480-5FU cells, respectively (P<0.05) ( Figure 4).

Effect of Fertilized egg white on cell migration
We performed the scratch wound healing assay to evaluate the cell migration induced with FEW treatments. The percentage of wound area closure was calculated at 48 and 72h. FEW caused approximately 50% gap area after 72 h compared to control in both cells, inhibiting cell migration. The results are presented in Figure 5.

Effect of fertilized egg white treatment on gene expression
Growth and migration inhibition of SW480-5FU cells persuaded us to check the stemness and differentiation genes expression induced by 72h exposure to FEW. c-MYC, highly expressed in the colon cancer stem cells, causes greater tumor sphere-forming e ciency, apoptosis resistance, invasion, migration, and tumorigenesis in cancer cells [19]. NANOG induces stem-like phenotype in colon cancer cells, similar to Nanog in prostate cancer, Oct4 in melanoma, and Sox2 in breast cancer [20]. E-cadherin is one of class of type-1 transmembrane protein that originated from the epithelia and has an key role in the epithelial to mesenchymal transition in cancer progression [21].
As shown in Figure 6, our data demonstrated that FEW reduced c-MYC and NANOG expression in SW480 cells compared to the control cells. At the same time, the expression of E-cadherin and NDRG1 (3.73 fold) increased. In the SW480-5FU cells, NANOG and c-MYC expression were also decreased following FEW treatment. However, E-cadherin and NDRG1 expression did not change after treatment with FEW.

Discussion
The present study provides preliminary data showing that fertilized egg white, as an embryonic/maternal microenvironment, exerted an inhibitory effect on the growth of colorectal carcinoma cell line (SW480), induced apoptosis, and decreased self-renewal and stemness-related colony and spheroid formation, inhibited migration in both SW480 cells and SW480-5FU cells. In addition, the genes involved in the stemness and differentiation of cells were modulated.
The Stemness properties of cancer cells are an essential feature in the prolonged proliferation, invasion, and metastasis of cancer [22]. We demonstrated that FEW inhibited the proliferation of SW480 cells by ceasing cells at the G0/G1 cell cycle and decreasing the stemness-related sphere formation and selfrenewal characteristics of both SW480 and SW480-5FU cells. The analysis of stemness genes of c-MYC and NANOG in colorectal cancer cells also allowed us to suggest the possibility of a decrease in stemness properties by FEW. Some evidence indicated that abnormal expression of stemness genes promotes tumor growth, increases chemo-resistance, and enhances tumor invasion [23]. Previous studies have reported that the unfertilized egg white did not signi cantly affect the survival rate of the breast cancer cells (MCF-7, MDA-MB-231) [24] and TCam-2 seminoma cells [25]. However, similar to the present study, Klf4, c-MYC, and NANOG expression was diminished following egg white treatment [24]. Furthermore, ovomucin and lysozyme, two components of egg white, have been shown to reduce proliferation, metastasis, and angiogenesis of cancer cells [26][27][28][29][30]. Ovotransferrin increased caspase 9, and 6 changed the morphology of cell and mitochondrial membranes resulting in inhibition of proliferation and induction of apoptosis in colon cancer cells (HCT-116), in contrast to breast cancer cells (MCF-7 ) [26]. Therefore, the reduction of proliferation, induction of cell cycle arrest, and reduction of stemness induced by FEW in the present study could be partially due to the components of egg white mentioned above.
Metastasis is a major cause of death from cancer. Metastatic primer cells belong to a subset of stem cells that migrate to other tissues by expressing epithelial to mesenchymal transition (EMT) markers and other super cial markers involved in the invasiveness and metastasis of cancer cells [31]. Embryonic microenvironments can inhibit cell invasion and metastasis by inducing differentiation and increasing the expression of differentiation markers [32]. In the present study, FEW decreased migration in both SW480 and SW480-5FU cells. E-cadherin and NDRG1 levels were also signi cantly higher in SW480 cells, indicating that differentiation was induced in the subpopulation of SW480 cells that escaped the apoptosis that was induced by FEW. The results from this study are similar to ndings in earlier studies using unfertilized egg white in seminoma and breast cancer cells MCF-7 and MDA-MB-231 [24,25], which demonstrated that egg white microenvironment can inhibit EMT [24].

Conclusion
As the embryonic/maternal microenvironment, fertilized egg white might perform a fundamental role in decreasing growth, migration, self-renewal and stemness, downregulation of stemness genes, and upregulation of differentiation genes in SW480 colon cancer cells. However, additional experiments are required to elucidate the exact mechanism of growth and metastatic inhibitory effect of FEW microenvironments. It also needs to be investigated which components of FEW mediated these stemness mitigation and differentiating induction by fertilized egg white.    Effect of fertilized egg white on colony formation in SW480 and SW480-5FU cells. Cells were treated with fertilized egg white for two weeks and colonies greater than 50 cells counted. Normalized values are represented as mean ±SD of three separated test. *p<0.05 compared to control by student t-test.

Figure 5
Effect fertilized egg white treatment on the migration of (A) SW480 cells, and (B) SW480-5FU cells was evaluated by scratch wound healing assay after 72h. Micrographs were captured at 0, 48 and 72h and gap closures were measured by ImageJ software. The results shown are represented as mean ± SD of three separated tests. *p<0.05 compared to control by student t-test.