The Combination of Zerumbone and 5-FU: A Significant Therapeutic Strategy in Sensitizing Colorectal Cancer Cells to Treatment

Objectives. Chemotherapy is considered to be essential in the treatment of patients with colorectal cancer (CRC), but drug resistance reduces its efficacy. Many patients with advanced CRC eventually show resistance to 5-fluorouracil (5-FU) therapy. Synergistic and potentiating effects of combination therapy, using herbal and chemical drugs, can improve patients’ response. Zerumbone (ZER), which is derived from ginger, has been studied for its growth inhibitory function in various types of cancer.Methods. The cytotoxic effects of ZER and 5-FU alone and their combination, on the SW48 and HCT-116 cells, were examined, using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The mRNA and protein levels of β-catenin, survivin, and vimentin were measured in treated CRC cells, using qRT-PCR and western blot. Colony formation assay, scratch test, and flow cytometry were performed to detect the changes of proliferation, migration, and apoptosis. Key Findings. In HCT-116and SW48-treated cells, the proliferation, the gene and protein expression levels of the markers, the migration, the colony formation, and the survival rates were all significantly reduced compared to the control groups, and the sharpest decline was observed in the 5-FU+ZER treatment groups. Conclusions. Combination therapy has shown promising results in CRC cells, especially in drug-resistant cells.


Introduction
CRC is one of the most common diseases in industrialized countries and is currently the third most common cause of cancer-related deaths in males and the second most in females worldwide [1]. 5-FU is commonly used as a chemotherapeutic drug in cancer treatments and in combination with other drugs to treat many types of cancers including breast, anal, stomach, head, and neck cancer [2]. However, the response rate of 5-FU for advanced CRC is only 20%; whereas, combining 5-FU with other chemotherapeutic drugs has improved the response rates in these patients to 40-50% [3]. As drug resistance remains a major clinical problem for the clinical application of 5-FU and related chemotherapeutic drugs, investigating the molecular pathways and genes, responsible for therapeutic resistance to 5-FU in CRC, offers insights into mechanisms of cell survival, thus developing more responsive therapeutic targets [2,4].
Epithelial-mesenchymal transition (EMT) is a very complex yet well-known process in cancer cells that is an essential stage in tumor metastasis and invasion [5,6]. A critical step of this transition is the lack of expression of epithelial markers such as E-cadherin, claudin, occludin, desmoplakin, type IV collagen, and laminin 1 and upregulation of mesenchymal markers such as N-cadherin, intregrin, vimentin, type I collagen, laminin 5, and fibronectin [7]. EMT is associated with several signaling pathways, including the transforming growth factor-β (TGF-β), Wnt, Hedgehog, and Notch pathways, and it can affect the involved genes such as β-catenin which is activated in the Wnt pathway [8]. The Wnt/β-catenin signaling pathway, also called the canonical Wnt pathway, is a major regulating signaling pathway in several cancers due to its effect on the transcription of the targeted genes [9]. Inhibition of Wnt/β-catenin signaling could increase sensitivity to chemotherapeutic agents in cancers [10]. β-Catenin is the main mediator of this pathway that is widely expressed in many tissues.
Vimentin is a widely expressed and highly conserved gene. Vimentin is constitutively expressed in normal mesenchymal cells and is the cytoskeletal component, responsible for maintaining cell integrity, supporting and anchoring the organelles, and providing resistance to avoid cell damage [11]. A previous study has shown the overexpression of vimentin in a wide range of epithelial cancers including prostate cancer, gastrointestinal tumors, CNS tumors, breast cancer, lung cancer, and malignant melanoma [12]. Upregulation of vimentin in EMT and the signaling pathways, contributing to the metastasis, invasion, tumorigenesis, and chemoresistance of various tumors, plays an important role in the progression and prognosis of cancer [13].
Survivin is the smallest bifunctional protein and a member of the inhibitor of apoptosis (IAP) family of proteins that can inhibit apoptosis and promote cell division [14]. Survivin is expressed at low levels in normal cells, but it has also been found to be prominently expressed in many solid and aggressive tumors [15]. In various tumors, high expression of survivin is associated with resistance to chemotherapy, poor prognosis, and increased angiogenesis. Therefore, survivin is an important target in cancer treatment [14].
Overcoming the resistance to intrinsic and therapeutic agents is one of the most important challenges in the treatment of cancer patients, as chemoresistance causes disease relapse and metastasis and remains the main barrier in cancer therapy. Therefore, it is very important to identify the molecular mechanisms of chemoresistance [16]. In chemotherapy, the use of the combination of nontoxic or less toxic phytochemicals with chemotherapy agents may reduce the toxicity, especially toxicity to normal tissues. Moreover, the lower dose of drugs, used in combination therapy, reduces the drug resistance in cancer cells. Therefore, the use of less-toxic agents, such as those used in herbal therapy, could be a promising therapeutic approach in cancer treatment [17].
Most therapeutic approaches in cancer treatment are associated with toxicity, side effects, lack of selectivity, high cost, and chemoresistance. Herbs, plants, and plant-based compounds that are commonly referred as safe compounds have been demonstrated to exert chemopreventive features and mediate anticancer roles in diverse cells [18]. One such herbal compound is ZER with the chemical name (2E,6E,10E)-2,6,9,9-tetramethylcycloundeca-2,6,10-trien-1-one that is extracted from the rhizomes of traditional plant, Zingiber zerumbet Smith [19]. ZER is known for its biomedical properties such as having antioxidant, antibacterial, antihypersensitive, and anti-inflammatory activities and exhibiting its diverse effects on proliferation, angiogenesis, and apoptosis against a wide variety of tumor cells including colon, liver, myeloid, breast, and gastric cancer [20]. Recent research has shown that ZER also mediates antiproliferative properties against various cancers such as skin, lung, liver, brain, breast, pancreas, and colon cancer [18,20].
The current study suggests, for the first time, the synergistic and potentiating effects of the combination of both ZER and 5-FU in increasing the sensitivity of CRC cell lines to 5-FU treatment. As EMT, metastasis, and chemoresistance are closely related to tumor progression, we attempted to establish that ZER treatment may mediate 5-FU resistance by targeting the important genes involved in cancer chemoresistance such as vimentin, survivin, and β-catenin. Consequently, combination treatment of ZER with 5-FU is expected to prove more beneficial for CRC patients.

Material and Method
2.1. Reagents. Zerumbone (z3902-50M),HPLC grade ≥ 98%purity , and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; ZER was prepared in stock solution of 1 mM (MW 218.23 g/mol) using DMSO; the final concentration of DMSO for in vitro study was less than 0.01%; the different concentrations of ZER ranging from 0 to 100 μM were prepared from 1 mM stock. Trypan blue was purchased from Sigma Chemical (St. Louis, MO, USA). 5-FU was purchased from Sigma-Aldrich. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (molecular weight: 335.43) was purchased from Sigma-Aldrich. High Pure RNA Isolation Kit was obtained from Roche Applied Science (Germany), and SYBR Green QPCR Master Mix and DNA ladder 1 kb and 50 bp were products of Thermo Fisher Scientific (USA). The first-strand cDNA synthesis kit was obtained from Thermo Scientific (USA). Acrylamide and bis-acrylamide were purchased from Sigma-Aldrich. TEMED, APS, and isopropanol were purchased from Merck. Tris base was purchased from Sigma-Aldrich; anti-rabbit secondary antibody was purchased from Santa Cruz; anti-β-catenin antibody (ab223075), anti-vimentin antibody (ab20346), and anti-survivin antibody (ab76424) were purchased from Abcam; ECL Kit was purchased from Amersham; protein extraction kit was purchased from Bio Basic.

Cell
Culture. The SW48 and HCT-116 (human colon cancer) cell lines were purchased from the National Cell Bank of Pasteur Institute (Tehran, Iran). Cells were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco), containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco), and were maintained at 37°in a humidified incubator containing 5% CO 2 . Cells were treated with ZER, 5-FU, and 5-FU+ZER. Untreated cells were used as the control groups.
2.3. Cell Viability Assay. The inhibitory effects of ZER and 5-FU alone and together on cell viability were determined by the analysis of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cells were seeded at 7000 cells per well (triplicates) in 96-well tissue culture plates. After 24 h, the cells were treated with the increasing concentrations of ZER (0-100 μM) and 5-FU (0-700 μM) and cotreated with 5-FU and ZER based on their concentration and treatment time. After 24, 48, and 72 h of incubation, 10 μl of MTT solution was added to each well of the plate and the mixture was incubated for 3-4 h at 37°C. Then, the medium was removed, and 100 μl of dimethyl sulfoxide Real-time PCR was performed with 1 μl cDNA, 3.6 μl H 2 O, 5 μl SYBR Green QPCR Master Mix, and 0.2 μl of specific primers in the Roche LightCycler machine (Roche Diagnostics). 18srRNA was applied as an internal control gene. Duplicate reactions were run for each cDNA sample, and relative expression of genes was determined by using the 2 −ΔΔCT method. Sequences for gene-specific primers are provided in Table 1. 2.6. Protein Extraction and Western Blot Analysis. The HCT-116 and SW48 cells with different treatment groups (5-FU, ZER, and 5-FU+ZER) were trypsinized and then washed twice with cold PBS followed by centrifugation at 1500 RPM for 5 minutes. Then, the extraction of protein was performed using the Extraction Kit. Finally, the total protein concentration was measured by Bradford assay.
SDS-PAGE was then performed to resolve the equivalent proteins which were then transferred to nitrocellulose membrane. After that, blocking was done using 5% nonfat dry milk. The membrane was incubated with primary antibodies which include anti-β-catenin antibody (ab223075), antivimentin antibody (ab20346), and anti-survivin antibody (ab76424) overnight at 4°C. Membranes were then incubated with secondary antibody (anti-rabbit secondary antibody conjugated with horseradish peroxidase) for 1 h at 4°C. The proteins of interest were visualized using the chemiluminescence detection kit, and bands were quantified by ImageJ software.

Colony Formation Assay.
Clonogenic assay is the method of choice to determine cell reproductive death after treatment with cytotoxic agents. HCT-116 and SW48 cells were seeded in a 6-well plate at a density of 3 × 10 5 for each group and kept in the incubator. After 24 h, the medium was pulled out from a 6-well plate and cells were treated with optimum concentrations of ZER, 5-FU alone, and together. Then, conditioned media of cells was removed and replaced with complete media. The plates were incubated again overnight. After a specified time (the optimum time that was chosen for HCT-116 was 24 h and for SW48 was 72 h), the cells were resuspended with trypsin and counted. 450 cells were double-replicated in another 6-well plate, and the plates were incubated for 3-4 days without shaking. After 8-10 days, and observing the colonies (with at least 50 cells) on the plates, the colonies were washed with PBS and then crystal violet was used to stain them. The formed colonies were counted, and then, their photos were taken for further analysis.

Migration
Assay. Migration assay of HCT-116 and SW48 cells was performed by scratch assay. First, 2 × 10 5 HCT-116 and SW48 cells were seeded into a 24-well tissue culture plate. Then, a scratch was made in the monolayer using a yellow pipette tip across the center of the well. Detached cells were washed twice with medium, and then, cells were treated with optimum concentrations of ZER, 5-FU, and 5-FU+ZER for 24 and 48 h. Finally, cell migration to the gap was examined by recording images at the beginning of treatment and at intervals of 24 and 48 h during cell transfer to close the scratch.
2.9. Flow Cytometry Analysis of Apoptosis. HCT-116 and SW48 cells (1 × 10 6 ) were seeded into a 12-well culture plate and incubated with 5-FU, ZER, and 5-FU+ZER, in optimum concentration for prime time. A total of cells were trypsinized, washed, and resuspended in 1 ml PBS with 5% fetal bovine serum. After centrifugation, cells were stained with Annexin V-FITC/propidium iodide (PI) detection kit (Mab-Tag, Germany) that was used to explore several phases of apoptosis and cell death in SW48 and HCT-116 cell lines. Finally, the apoptosis ratio was analyzed using Attune NxT acoustic focusing cytometer (Life Technology, USA).

Statistical
Analysis. The data were analyzed using GraphPad Prism 5.0 software using the one-way analysis of variance test for comparison between three groups. Data were presented as the mean ± standard error of the mean. The p value of <0.05 was considered statistically significant.

ZER Augments Cytotoxicity Effects of 5-FU in HCT-116
and SW48 Cells. The effect of different concentrations of ZER, 5-FU treatment, and a combination of both on the viability of HCT-116 and SW48 cells compared to untreated cells at 24, 48, and 72 h was evaluated, using MTT assay.

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The cytotoxic effect of ZER treatment on CRC cells was also determined by calculating 50% of cell death, which was about 14 μM and 19 μM in HCT-116 and SW48 cells, respectively ( Figure 1). The cytotoxic effect of 5-FU in HCT-116 cells was calculated to be 20 μM, and in SW48 cells was about 158 μM ( Figure 2). Moreover, the cytotoxic effects of combination treatments were 9.5 μM in HCT-116 cells and 100 μM in SW48 cells ( Figure 3) (in combination treatment, first cells were treated with ZER in 14 μM and 19 μM concentration in HCT-116 and SW48 cells, respectively, and then, 5-FU with different concentrations was added (0-700 μM)). In this study, the dose-dependent effect of ZER, 5-FU, and the combination of these two agents was observed in both cell lines at 24, 48, and 72 h. As a precaution, a concentration lower than and above the IC50 concentration was chosen for treating the cells. The best result was observed in the cells that were treated with IC50 concentrations; hence, the IC50 concentrations were used for further experiments.

Treatment with ZER+5-FU Reduced the β-Catenin
Gene Expression in CRC Cell Lines. As shown in Figure 8, the substantial decrease in expression of β-catenin mRNA level was detected in HCT-116 cells treated with ZER+5-FU for 24 h. This significant reduction in β-catenin gene expression in ZER+5-FU treatment at 24 h is stemmed from both inhibitory effects of ZER and 5-FU (0.48-fold for 24 h) (Figure 8(a)).
Treatment of SW48 cells revealed that there was no significant change in the mRNA level of β-catenin in all treatments in 24 h. Moreover, the combination of both ZER+5-FU treatment could significantly decrease the expression of β-catenin mRNA level at 48 and 72 h, which was due to the potentiating effect of ZER (0.46-fold for 48 h and 0.16-fold for 72 h) (Figure 8(b)).

Decreased Expression of Vimentin Protein in Cells
Treated with ZER, 5-FU, and 5-FU+ZER. The protein expression of vimentin was assessed using western blot analysis. Results of western blot showed that the level of protein was , and 40% reduction in ZER-, 5-FU-, and 5-FU +ZER-treated cells, respectively) ( Figure 9(a)). No significant changes were observed in the protein levels of vimentin in SW48 cells treated with 5-FU, while considerable reduction was observed in other treated groups including 5-FU+ZER and ZER. The optimum time was 72 h for all treatments in SW48 cells (70% and 73.3% reduction in ZER-and 5-FU+ZER-treated cells, respectively) ( Figure 9(b)). Treatment with 5-FU+ZER caused the sharpest decline in all groups in both cell lines.
In the SW48 cell line with the exception of 5-FU treatment, in other treatments, significant decline was observed in the optimum time (72 h) (46.6% and 58.5% reduction in ZERand 5-FU+ZER-treated cells, respectively) ( Figure 10(b)).
Taken together, the results showed that the 5-FU+ZER treatment groups demonstrated a sharper decline than each treatment alone, in both cell lines.

Proliferation of CRC Cells Was
Inhibited by the Effect of 5-FU, ZER, and 5-FU+ZER Treatment. The clonogenic assay was performed to compare the effects of ZER and 5-FU treatment alone or together on colorectal cancer cell lines. Cells were counted by ImageJ software after staining. Figure 12 shows a significant decrease in the number of HCT-116 and SW48 cells. All three groups showed a significant reduction in the number of cells in HCT-116 cells; however, the combination therapy exerted the most inhibitory effect, followed by cells treated with ZER. Treatment with 5-FU alone showed the least inhibitory effect (5-FU: 63%, ZER: 23%, and 5-FU+ZER: 82% reduction in the number of colonies). The reduction in the number of colonies in SW48 cells was due to the effect of ZER and 5-FU+ZER treatments, and no inhibition was observed in the 5-FU-treated group (ZER: 19% and 5-FU+ZER: 39% reduction in the number of colonies).

Migration of HCT-116 and SW48 Cells Was Significantly
Reduced by the Effect of 5-FU, ZER, and 5-FU+ZER Treatment. The wound healing assay was applied to evaluate the potential role of 5-FU, ZER, and 5-FU+ZER treatment on migration in SW48 and HCT-116 cells. The results showed that the wound widths were significantly reduced after 24 and 48 h in the control groups, while 5-FU-, ZER-, and 5-FU+ZER-treated cells demonstrated less migration in these time points (Figure 13

Discussion
CRC is a major cause of morbidity and mortality all over the world. CRC is generally diagnosed as a malignant disease in both men and women [21]. CRC is most commonly diagnosed in patients of 50 years of age and older. Earlier onset is observed in hereditary and familial CRC. In patients with stage III CRC, common therapies such as surgery, chemotherapy, and radiation therapy could result in complete remission or increase life expectancy [21].
Chemotherapy resistance is a major barrier to cancer treatment that is primarily associated with EMT process and leads to cancer progression. Oncogenes and tumor   BioMed Research International suppressors, which also play a role in inducing EMT, are major contributing factors to chemoresistance. In chemotherapy, often, a combination of two anticancer agents is more effective than each agent alone [17]. 5-FU, a fluoropyrimidine analog, is a chemotherapeutic agent widely used for treating colorectal cancer, but resistance remains a major obstacle to 5-FU clinical efficacy. Recent studies have suggested that EMT is associated with chemoresistance in animal models of lung and pancreatic cancers [22,23]. Zhang et al. showed that downregulation of snail, an EMT marker, might be a potential therapeutic approach to solve chemoresistance and prevent metastasis during 5-FU chemotherapy in breast cancer [24]. Ginger, a key component in functional foods, has been used for thousands of years as a medicinal herb to treat a variety of chronic diseases [25]. ZER is a cyclic sesquiterpene from the rhizomes of ginger plant (Zingiber zerumbet Smith)

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which is known for its biomedical properties such as having antioxidant, antibacterial, anti-inflammatory, and immunomodulatory activities [26].
There are a number of mechanisms, such as the Wnt signaling pathway, that are suggested to be responsible for drug resistance. As high as 50% of metastatic CRC, patients are resistant to 5-FU-based chemotherapy [27]. Studies suggest that 5-FU combination therapies may be more beneficial [18,28]. Some studies found that the 5-FU-resistant CRC cells demonstrate high expression of TCF4 and β-catenin, indicating an upregulated Wnt pathway. Subsequently, β-catenin-silenced CRC cells were relatively more sensitive to chemotherapy reagents [2,10]. Furthermore, survivin has been proven to play an important role in colorectal carcinogenesis. Virrey et al. have shown that chemoresistance in tumor cells might be correlated with an overexpression of the inhibitor of bifunctional proteins, like survivin which inhibits apoptosis [15]. Moreover, in 2018, Chung and colleagues revealed that the 5-FU treatment could decrease the gene and protein expression of vimentin as an EMT marker in HCT-116 and DLD1 cells [20].
Here, we investigated the treatment effect of ZER alone and with 5-FU on the proliferation, gene and protein   Figure 6: Synergic effect of ZER and 5-FU on mRNA level of vimentin in HCT-116 (a) and SW48 (b) cells. CRC cells were treated with ZER and 5-FU alone and together for 24, 48 h, and 72 h followed by total RNA extraction for reverse transcription to cDNA. The cDNAs were used to assess expression levels of selected genes by SYBR green-based real-time quantitative PCR. The fold changes were derived using the comparative 2 −ΔΔCT method. Each data point is presented as the mean ± SD (n = 3). All data were normalized to levels of 18srRNA ( * p < 0:05, * * p < 0:01, and * * * p < 0:001 versus nontreated cells).   Figure 7: Synergic effect of ZER and 5-FU on mRNA level of survivin in HCT-116 (a) and SW48 (b) cells. CRC cells were treated with ZER and 5-FU alone and together for 24, 48 h, and 72 h followed by total RNA extraction for reverse transcription to cDNA. The cDNAs were used to assess expression levels of selected genes by SYBR green-based real-time quantitative PCR. The fold changes were derived using the comparative 2 −ΔΔCT method. Each data point is presented as the mean ± SD (n = 3). All data were normalized to levels of 18srRNA ( * p < 0:05, * * p < 0:01, and * * * p < 0:001 versus nontreated cells).  Figure 8: Synergic effect of ZER and 5-FU on mRNA level of β-catenin in HCT-116 (a) and SW48 (b) cells. CRC cells were treated with ZER and 5-FU alone and together for 24, 48 h, and 72 h followed by total RNA extraction for reverse transcription to cDNA. The cDNAs were used to assess expression levels of selected genes by SYBR green-based real-time quantitative PCR. The expression fold changes were derived using the comparative 2 −ΔΔCT method. Each data point is presented as the mean ± SD (n = 3). All data were normalized to levels of 18srRNA. ( * p < 0:05, * * p < 0:01, and * * * p < 0:001 versus nontreated cells). . In accordance to our findings, other studies have previously reported that ZER can reduce the gene and protein expression of survivin, vimentin, and β-catenin genes [29,30]. In contrast, the results of another study in 2009 by Yodkeeree et al. showed that ZER has little or no effect on survivin gene expression [31].
We found no significant inhibition of the interested genes in SW48 cells treated with 5-FU in all time points. However, in HCT-116 cells, a significant decrease was observed in all three time points, except for β-catenin which inhibitory effect was only seen in the  [20]. In another study in 2017, an aptamer containing the survivin RNAi was used, in order to increase sensitivity of HT-29 CRC cells to 5-FU and oxaliplatin [33].
Here for the first time, we evaluated the synergistic effect of ZER and 5-FU treatment in CRC cell lines (HCT-116 and SW48). We found a significant drop in expression of vimentin and survivin mRNA level in HCT-116 cells that were treated with ZER+5-FU in 24, 48, and 72 h, while treatment of cells with ZER alone could markedly inhibit the expression of vimentin and survivin mRNA level in 24 h. Therefore, it could be suggested that in combination therapy, the potentiating effect of 5-FU could make the CRC cells more sensitive to the combination of 5-FU and ZER compared to using each alone (Figures 6 and 7(a)). In

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BioMed Research International namely, berberine (a chemical found in several plants), curcumin (another member of the ginger family), and quercetin (a plant pigment) alone and in combination with 5-FU on gastric cancer cells. They reported that the synergistic effect of each of these substances with 5-FU decreased the expression of survivin and STAT3 levels resulting in an increase in cell death in gastric cancer cells [35].
Similarly, we found that in SW48 cells at some time points, 5-FU and ZER alone did not have a significant inhibitory effect on the markers of interest, and when used in combination, they can be more effective by having a complementary effect. As shown in Figures 6 and 7, a significant drop in the expression of genes in ZER+5-FU-treated cells was due to the potential effect of ZER at 72 h ( Figures 6 and 7(b)).
As illustrated in Figure 8(a), a significant decrease in expression of β-catenin mRNA level was detected in HCT-116 cells which were treated with ZER+5-FU for 24 h. This significant decrease in the ZER+5-FU treatment group at 24 h is related to both inhibitory effects of ZER and 5-FU (Figure 8(a)).
Similar to our study, the synergic effects of 5-FU with EPLE (from salvia plant) on HCT-116 and SW480 CRC cells have been demonstrated by Ye et al. in 2015. Their results revealed that EPLE alone and along with 5-FU suppressed the Wnt/β-catenin pathway, thus reducing the gene expression of survivin [36].
Regarding SW48 cells, we found that the combination of ZER and 5-FU treatment could significantly decrease the expression of β-catenin mRNA level in 48 and 72 h which was due to the potentiating effect of ZER treatment (Figure 8(b)).
Metastasis is the leading cause of cancer mortality and accounts for about 11% of cancer-related deaths. Metastasis consists of separation, migration, invasion, and adhesion. It is regulated by different signaling pathways and affected by the surrounding extracellular matrix (ECM) [37]. Studying cell migration and the involved factors provides valuable insights into cancer diagnosis, prognosis, treatment, and drug development. The inhibition of migration in HCT-116  Figure 12: The inhibitory effect of ZER, 5-FU, and 5-FU+ZER on colony formation of (a) HCT-116 and (b) SW48 cells. In both cell lines, the number of colonies in the treated group showed a significant decrease compared to the control group with the exception of the 5-FU-treated group in SW48 cells. * p < 0:05, * * p < 0:01, and * * * p < 0:001 compared to the control group (n = 2).     15 BioMed Research International cells was due to the effect of ZER, 5-FU, and 5-FU+ZER treatment, while in SW48 cells, no inhibition was seen in the 5-FU treatment group. Similar to our findings, Manmuan et al. in 2018 reported the combinatory effects of oxymatrine (alkaloid compound derived from Sophora flavescens root) along with 5-FU markedly reduced migration of CRC cells in 24 and 48 h [38]. Colony formation is a method used to assess the independent cell proliferation of a cancer cell, during which a single tumor cell with a high proliferation rate forms a colony in the plate within a few weeks [39]. Our results demonstrated a significant reduction in all three groups with a further inhibitory effect of combination in HCT-116 cells, while in 5-FU-treated SW48 cells, no significant inhibitory effect was observed. Liu et al. in 2018 examined the effect of HQGGT (a Chinese herbal compound) along with 5-FU in H630R1 and MC38 cells of CRC. Their result revealed that the combination of HQGGT and 5-FU reduced the number of colonies more effectively than 5-FU alone [40].
Apoptosis is a physiological process in which cell death is caused by a cascade of events. It leads to the programmed removal of specific cells, without harming neighbor cells. Any changes in this process could result in a variety of diseases. In a study by Fang et al. in 2019, ethanolic extract of Spica Prunellae (EESP) (a Chinese drug) along with 5-FU increased the sensitivity of 5-FU-resistant cells (HCT-8/5-FU) and increased the apoptosis rate compared to the individual use of each component [41].
EGFR is a transmembrane protein receptor that is involved in the pathogenesis and progression of many malignancies. They are generally activated once ligands bind to the extracellular domain. After binding to their ligands, intracellular cascade reactions occur, which mainly cause cell proliferation [42]. Several studies have found various genetic changes, for example, the mutations in EGFR family in many types of tumors, including colon cancer. The mutations of EGFR, the low expression of EGFR, and the changes in its ligands have been shown to be related to drug resistance [43]. In this regard Gu et al. in 2019 demonstrated that EGFR contributed to 5-FU resistance in colon cancer cells through autophagy induction. Therefore, their results highlight the potential clinical utility of targeting autophagy genes [44]. Dysregulation of the autophagy pathway and the various signaling pathways involved in this process in cancer cells is closely related to drug resistance of tumors. The PI3K/AKT/mTOR and mitogen-activated protein kinase (MAPK) pathways are the main regulators of autophagy [45]. As mentioned earlier, mutations in the EGFR pathway could be one of the factors that contribute to drug resistance. As HCT-116 and SW48 cell lines have different molecular profiles of EGFR, SW48 cells are bearing a mutation of EGFR, but the HCT-116 cells have wild-type EGFR; it could be assumed that the observed drug resistance in SW48 cells is associated with the EGFR pathway.
Therefore, the main purpose of this study was to compare the effects of the combination of ZER and the chemotherapeutic agent, 5-FU alone, and together on the expression of important markers involved in progression, migration, proliferation, and apoptosis of CRC cells. Altogether, our findings suggest that ZER may be a promising compound to be used in combination treatment regimens to induce chemosensitization to 5-FU in CRC cell lines through downregulation of EMT marker (vimentin), apoptosis marker (survivin), and the Wnt/β-catenin pathway in CRC cells.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that there is no conflict of interests.