Promising Modulatory Effects of Cenicriviroc on the Progression of Mouse Colorectal Cancer through Inhibition of CCR2_CCL2 Signaling Pathway

The study was designed to assay the efficacy of cenicriviroc (CVC) on the progression of mouse colorectal cancer by downregulation of CCR2_CCL2. In this study, CVC was used to inhibit the CCR2 receptor. Next, an MTT assay was performed to evaluate the cytotoxic effects of CVC on the CT26 cell line. CT26 cells were implanted subcutaneously in BALB/c mice. After tumor implantation, one group of animals received 20 mg/kg of CVC several times. The mRNA levels of CCR2, CCL2, VEGF, NF-κB, c-Myc, vimentin, and IL33 were determined in the CT26 cell line and then tumor tissues (after 21 days), by qRT-PCR. Protein levels of the above-mentioned targets were determined by western blot and ELISA. Flow cytometry was performed to assess the changes in apoptosis. Tumor growth inhibition was measured on the 1st, 7th, and 21st days after the first treatment. In both cell line and tumor cells treated with CVC, expression levels of the markers of our interest in mRNA and protein levels were significantly reduced compared to controls. A significantly higher apoptotic index was observed in CVC-treated groups. The rates of tumor growth were significantly decreased on the 7th and 21st days after the first injection. To our knowledge, this was the first time that we demonstrated the promising effect of CVC on the development of CRC through inhibition of the CCR2_CCL2 signaling and its downstream biomarkers.


Introduction
Colorectal cancer (CRC) is known with a high prevalence rate. Many risk factors such as age, family history, and lifestyle including diet, physical activity, and smoking afect its occurrence [1]. CRC tumors can be surgically removed, but for the tumor in advanced stages, tumor metastasis and progression are important issues [2]. Several fundamental factors in the tumor microenvironment contribute to the development of cancer [3]. Te cellular interactions in the tumor microenvironment can facilitate metastasis cascades which are often possible through the signaling of chemokines [4]. CCL2 is over-expressed in CRC, along with its main receptor CCR2 boosts the progression of cancer by promoting proliferation, survival, angiogenesis, migration, and invasion [5] and contributes to tumor metastasis [6].
Studies have shown that MDSCs (myeloid-derived suppressor cells) [7] and TAMs (tumor-associated macrophages) are attracted and accumulated in the tumor site by CCL2 [8]. TAMs and MDSCs are important cells in the development of cancer [5]. Studies have shown that TAMs promote cancer progression by increasing invasiveness and angiogenesis [9]. TAMs produce EGF (epidermal growth factor) which enhances the invasion and motility of cancer cells through increasing matrix degradation [10]. In addition, TAMs inhibit T cell proliferation and suppress the immune system by producing immunosuppressive molecules, which ultimately lead to the development of cancer [11,12]. MDSCs expand strongly in some pathological conditions, including cancer. In addition, the activity of immune cells is regulated by these cells, and evidence has shown that MDSCs can cause tumor growth and cancer progression by inhibiting T cells that have antitumor activity [13].
In addition to the cases mentioned, CCL2 is produced by cancer cells, and TAMs and CAFs (cancer-associated fbroblast) can react with the CCR2 receptor expressed on the surface of cancer cells and cause its activation, which subsequently increases the proliferation, migration, and resistance of cancerous cells to apoptosis. In addition, CCL2 by binding to the CCR2 receptor on the surface of vascular endothelial cells can increase angiogenesis. Tis chemokine increases the attraction and recruitment of Tregs (T regulatory cells) in the tumor microenvironment, which, in turn, suppresses immunity and increases cancer progression [7].
According to these documents, the CCR2_CCL2 signaling pathway plays a key role in the development of cancer, which makes the CCR2 receptor an important and potential therapeutic target in cancer treatment [5].
Cenicriviroc (CVC) is a dual inhibitor of CCR2 and CCR5 receptors. Tis compound was initially used to treat individuals with HIV [14]. It has also recently shown promising results in limiting infammation and fbrosis in liver fbrosis disease [15].
Considering the signifcant role of the CCR2 receptor in cancer, in this study, for the frst time, we studied the efect of this compound in preventing the development of CRC in mouse models and cell lines through inhibition of the CCR2_CCL2 signaling pathway.
Herein, we examined the efects of CVC on the mRNA and protein levels of CCR2, CCL2, VEGF, NF-κB, c-Myc, IL33, and vimentin, which are involved in the progression of cancer. VEGF is the key mediator of angiogenesis in cancer. Angiogenesis is necessary for the development of cancer, and therefore, VEGF can promote the development of cancer [16].
Te expression of many genes efective in the proliferation, infammation, migration, and survival of cancer cells is regulated by nuclear factor kappa B (NF-κB), which is an important transcription factor. Tis factor can cause the development of CRC [17].
Many genes that are efective in cellular processes are regulated by c-Myc, which is an important transcription factor [18].
High expression of this factor has been observed in CRC. Similarly, some studies have shown a strong correlation between the positive expression of c-Myc and poor prognosis in patients with CRC [19].
IL33 is a member of the super family of interleukin 1 cytokines, which plays an important role in infammation [20]. It can cause tumor progression, and other research has proven that IL33 can increase the invasion and migration of glioma cancer cells by up-regulating MMP2 and MMP9 through the ST2_NF-κB signaling pathway [20]. Abnormal expression of IL33 has been reported in human CRC tissues. Also, studies have shown that IL33 can facilitate the proliferation of CRC cells through the ST2 receptor and NF-κB signaling, which up-regulates the expression of COX2 and PGE2 [21].
Vimentin is one of the main components of the IF (intermediate flament) family that preserves cell integrity.
Vimentin over-expression increases tumor growth, invasion, and poor prognosis in cancer. It is also considered an important marker for EMT [22]. Initiation and progression of metastasis are enhanced by EMT [23]. Over-expression of vimentin in some cancers including CRC has been reported [22].

Treatment of CT26 Cells with CVC.
After the determination of IC 50 (40 μM), this concentration of the compound was used to treat the cells, and then, the conditioned media were discarded, and the complete cell culture medium was added. After 24, 48, and 72 hours of incubation, trypsin was used to separate the cells, and subsequent analyses were performed.
2.5. RNA Isolation and QRT-PCR. RNA extraction was performed from CT26 cells using the RNA extraction kit. Ten, RNA quality was determined by using gel electrophoresis, and ribosomal RNA bands (18 S and 28 S) were observed on the gel. Ten, RNA concentration and purity were measured by NanoDrop (Termo Fisher Scientifc, Waltham, MA) spectrophotometer. After the synthesis of cDNA from RNA, the expression of the target genes was evaluated using the real-time PCR technique. Real-time PCR was performed with 1 μl cDNA, 3.6 μl H2O, 5 μl SYBR Green qPCR Master Mix, and 0.2 μl of specifc primers in the Roche Light Cycler machine (Roche Diagnostics). At this stage, the 18 s rRNA gene was used as a reference gene. Finally, the relative expression of the genes was obtained by using the 2 −ΔΔCT method. In Table 1, the primer sequences are shown.

ELISA.
To quantitate the soluble form of CCL2 and VEGF proteins in the conditioned medium of the CT26 cells, a commercial ELISA kit (R&D Systems) was used, and according to the kit protocol, we assessed the level of these proteins. At an absorbance of 450 nm, the light absorption of the standard and samples were read.

Western Blot Analysis.
First, by using trypsin, a cell suspension with a concentration of 1 × 10 7 CT26 cells was prepared. Ten, the cells were washed using PBS and then lysed using RIPA bufer (Santa Cruz, USA). Te lysates were removed by centrifugation at 14,000 rpm for 20 min at 4°C. After that, the Bradford method was used to determine the protein concentration. Te lysates were mixed with an equal volume of 2X Laemmli sample bufer, and then, the lysates were exposed to SDS-PAGE (50 μg) and subsequently transferred to the PVDF membranes. After transferring, 5% BSA (Sigma-Aldrich) was used for membrane blocking. After blocking, incubation with primary antibodies (1 : 1000) was done, and then, the membranes were washed with TBST. Subsequently, incubation with goat anti-rabbit IgG H&L (HRP) secondary antibody (1 : 4000) was done, and after that, chemiluminescence detection kit (ECL, Amersham, USA) was used to visualize protein bands, and fnally, the bands were quantifed by using Image J software.

Flow Cytometry.
To evaluate the number of apoptotic cells, the fow cytometry technique was used. After seeding CT26 cells (1 × 10 6 ) into a 6-well plate and treating the cells with CVC, the cells were collected and washed, and then, according to the steps of the kit (Mab-Tag, Germany), the cells were stained with Annexin V and propidium iodide (PI). Finally, the amount of apoptosis in the treated and control cells was determined by using Attune NxT acoustic focusing cytometer (Life Technology, USA).

Animal Experiment.
For animal experiments, BALB/c mice, male, 8 weeks old (25 gr), were bought from Pastor Institute of Iran (n � 10). Mice were fed with a normal mice pellet and water and maintained in suitable conditions including temperature (23°C ± 3°C), humidity (50 ± 10%), and 12 h light and dark cycle [24]. CT26 cells (400000) were injected subcutaneously to induce colorectal cancer tumors in mice. After 10 days, the mice were ready to receive treatment. Tese mice were categorized into two groups: the treated group (n � 5) and the control group (n � 5), and then, the treated mice were treated with 20 mg/kg of CVC three times a week for three weeks by intraperitoneal injection [15]. Control mice were injected with PBS. After 21 days, an anesthesia procedure was performed by using a ketamine/ xylazine recipe and intraperitoneal injection of ketamine (100 mg/kg) plus xylazine (10 to 5 mg/kg), and later, tumor tissue in each group was removed and processed for the experiments. For this study, the Local Ethics Committee approval was obtained (IR.UMSHA.REC.1401.048).

Tumor Size Measurement.
Tumor size in two groups was measured by a caliper. Tumor imaging and measurement were performed on days 0-7-21 after the frst injection.
2.11. Real-Time PCR for Animal Tissues. After 21 days, tissues were collected, and in the next step, RNA extraction was done using the Universal RNA purifcation kit. Ten, cDNA synthesis was performed. Finally, a real-time PCR was performed to determine the changes in gene expression.

Statistical Analysis.
Graph Pad Prism 8.0 software was used to analyze the data. All the data were presented as a mean ± standard error of the mean. Analysis of variance (One-way ANOVA) was used to compare the data. A p value <0.05 was considered statistically signifcant.

Treatment with CVC Reduced Cell Viability on CT26 Cells.
MTT assay was performed to evaluate the cytotoxic efects of CVC on CT26 cell viability. Diferent concentrations of CVC (20-40-60-80-100 μM) were prepared, and IC 50 was determined (40 μM). Finally, this IC 50 concentration was used for further experiments (Figure 1).  (Figure 2(b)). Tese data showed that CVC could expressively diminish the expression of CCR2, CCL2, VEGF, vimentin, c-Myc, NF-κB, and IL33 genes in all treatment times. Te results obtained from this experiment showed that the amount of these proteins in the treated cells had a major reduction compared to the untreated cells (45% reduction for CCR2, 62% for NF-κB, 34% for c-Myc, 55% for vimentin, and 44% for IL33), at the optimum time (Figure 3(a)). To assess the changes in the soluble form of CCL2 and VEGF proteins in the conditioned medium, ELISA was performed at the optimum time point of 48 h. Te results obtained from this evaluation showed a considerable reduction in the level of CCL2 and VEGF proteins in the conditioned medium of the treated cells compared with the control (45.34% reduction for CCL2 and 60.94% for VEGF) (Figure 3(b)). Te changes in protein levels confrmed the fndings of our targets in mRNA levels.

Treatment with CVC Increased Apoptosis in CT26 Cells.
To evaluate the changes in the apoptosis rate, Annexin V/PI staining was done in the CT26 cells. Te results showed that the apoptosis rate in treated cells was considerably increased (26.64%) versus the control ( * * * p < 0.001) (Figure 4).

Treatment with CVC Reduced Mice Tumor Size.
To evaluate the efect of CVC on tumor growth, tumor size measurement was done in the treated and control groups on the 1st, 7th, and 21st days after the frst treatment. Te results obtained from this measurement showed that the tumor size in the treated groups on the frst day of injection did not change signifcantly compared to the control (12.48% reduction) (Figure 5(a)). Nevertheless, on the 7th and 21st day, there was a considerable decrement in the tumor size in the treated mice compared to the untreated ones (54.57% reduction on the 7th day and 70.57% reduction on the 21st day) (Figures 5(b) and 5(c)) ( * * * p < 0.001).

Discussion
CRC is one of the foremost common neoplasms. Factors such as genetics and environmental factors are efective in the development of this disease [25].
Chemokine signaling can promote cancer progression [2]. Among these chemokines, CCL2, together with its main receptor, CCR2, can increase cancer progression [5]. Te expression of this chemokine is increased in CRC [26]. Te CCR2_CCL2 signaling pathway signifcantly increases the survival of cancerous cells, proliferation, and metastasis. Tis signaling pathway leads to the accumulation of TAMs and MDSCs in the tumor site [5]. TAMs increase malignancy, tumor progression, and angiogenesis and inhibit T cells, which ultimately cause cancer progression and metastasis [9].   MDSCs cause tumor growth through T-cell suppression [4] and also lead to increased angiogenesis and metastasis [27].
Numerous types of research have been done to study the efect of CCR2_CCL2 signaling pathway inhibition in preventing cancer progression [6]. CVC is CCR2 and CCR5 inhibitor [28]. Since CVC inhibits the CCR2_CCL2 signaling pathway, in this research, we used this compound to investigate the efect of CVC on the progression of CRC in mouse models and cell lines.
In this study, the IC 50 value of CVC was 40 μM, and it was determined by MTT assay, and then, the cells were treated with this concentration in the later steps of the study.
After that, we studied the efect of CVC on the expression of CCR2, CCL2, VEGF, c-Myc, IL33, NF-κB, and vimentin in mRNA and protein levels. Treatment with CVC in CT26 cells dramatically decreased the expression of CCR2, CCL2, and VEGF mRNA levels compared to the control groups in all times points, CCR2 (72% reduction for 24 h, 50% for 48 h, and 31% for 72 h), CCL2 (67% reduction for 24 h, 55% for 48 h, and 66% for 72 h), and VEGF (43% for 24 h, 75% for 48 h, and 73% for 72 h). Also, results showed a signifcant reduction in mRNA levels of vimentin (22% reduction for 24 h, 50% for 48 h, and 70% for 72 h), c-Myc (90% reduction for 24 h, 71% for 48 h, and 25% for 72 h), NF-κB (42% reduction for 24 h, 61% for 48 h, and 64% for 72 h), and IL33 (60% reduction for 24 h, 50% for 48 and 72 h) in all treated time points. Also, the amount of these proteins in the treated cells had a major reduction compared to the untreated cells (45% reduction for CCR2, 62% for NF-κB, 34% for c-Myc, 55% for vimentin, 44% for IL33, 45.34% for CCL2, and 60.94% for VEGF), at the optimum time. Te results of the treatment of animal models showed that CCR2, CCL2, VEGF, vimentin, c-Myc, NF-κB, and IL33 genes expression were reduced dramatically in treated mice compared with the control group (43% reduction for CCR2, 45% for CCL2, 38% for VEGF, 38% for NF-κB, 59% for c-Myc, 28% for vimentin, and 18% for IL33). Since the  Evidence-Based Complementary and Alternative Medicine CCR2_CCL2 signaling pathway can increase the survival of cancer cells, proliferation, and metastasis, CCR2 is considered a potential therapeutic target in cancer treatment [5]. In this context, Gage Brummer et al. reported that CCR2 knockdown can reduce the expression of CCR2 and CCL2 in breast cancer tumors [29]. Also, CCR2 inhibitor can suppress CCL2-mediated lung cancer cell invasion by downregulating MMP-9 expression [30].

Evidence-Based Complementary and Alternative Medicine
Te binding of CCL2 to the CCR2 receptor can lead to the activation of several downstream signaling pathways, one of these pathways is the PI3K/AKT/IKK/NF-κB signaling pathway, which can lead to an increase in the expression of CCL2 [31]. Herein, based on our results, we understood that CVC inhibits CCR2, and inhibition of this receptor subsequently can decrease the activity of the PI3K/ AKT/IKK/NF-κB signaling pathway, which in turn downregulates the expression of CCL2.
VEGF is a key mediator in angiogenesis [32]. Some researchers have proven that CCL2 can increase angiogenesis by recruiting TAMs and increasing the expression of VEGF in cancer cells [7]. Lien et al. reported that CCL2 caused modulation of VEGF-A expression and angiogenesis through the CCR2/ILK (integrin-linked kinase)/MEK1/2 signaling pathway in OSCC (oral squamous cell carcinoma) [33]. Terefore, it seems that in this study, CVC may reduce the expression of VEGF by afecting CCR2 and its ligand.
Vimentin is considered an important marker for EMT in cancer. It also has an increased expression in several cancers including colorectal [34]. Zhuang et al. reported that the CCL2_CCR2 signaling pathway stimulates invasion and EMT in HCC through the activation of the Hh pathway and up-regulation of Snail and Vimentin. Also, CCR2 silencing causes the inactivation of the Hh pathway, downregulates Snail and Vimentin, and prevents the reduction of Ecadherin [35]. Consequently, it seems that inhibition of the CCR2_CCL2 signaling pathway can downregulate EMT and reduces cancer progression through the inhibition of transcription factors such as Vimentin. NF-κB signaling is related to the development of CRC [17]. Previous studies have shown that the downregulation of the CCL2_CCR2 signaling pathway by Celecoxib can inhibit the NF-κB pathway [36].
Aberrant expression of c-Myc has been observed in CRC [37]. Teng et al. reported that the inhibition of HCC growth by targeting CCL2 is through the inhibition of STAT3, NF-κB, and c-MYC [38].
IL33 plays a role in many diseases, including cancer. Also, studies have shown that abnormal expression of IL33 has been observed in CRC [39]. Hu et al. reported that IL33 causes the up-regulation of CCL2_CCR2 by activating the NF-κB and extracellular signal-regulated kinase (ERK1/2) signaling in DSCS (decidual stromal cells) [40].
Altogether, we suggested that CVC can reduce NF-κB, c-Myc, and IL33 genes and proteins through inhibition of the CCR2_CCL2 signaling pathway. In addition, as mentioned, one of the signaling pathways activated during the binding of CCL2 to CCR2 is the PI3K/AKT/IKK/NF-κB signaling pathway, which leads to an increase in the expression of CCL2. According to the obtained results, it can be concluded that inhibition of CCR2 by CVC can lead to a decrease in the expression and activity of NF-κB involved in this signaling pathway.
In addition, in this study, the rate of apoptosis in CT26 cells and the efect of CVC on it were investigated by fow cytometry. Te results of the analysis showed that CVC could increase the rate of apoptosis in CT26 cells (26.64%). Te same result was reported by Lu et al. arguing that treatment of the U251 glioma cell line with CCR2 inhibitor increases apoptosis through the downregulation of phosphorylation levels of p38 and ERK1/2 [41]. In another study, Abd-Rabou and Ahmed showed that the use of CCR2 inhibitor (CR) nanotreatments on the A549 lung cancer cell line increases the apoptosis ratio [42]. Another study demonstrated that CCR2 inhibition improves apoptosis by infuencing PI3K/AKT and p38MARK signaling pathway in DLBCL (difuse large B-cell lymphoma) cell lines such as SUDHL-2 and OCI-Ly8 [43]. Te PI3K/AKTand JAK/STAT signaling pathways are downstream pathways that are activated by the binding of CCL2 to CCR2 and lead to the inhibition of apoptosis [6]. Terefore, our fndings suggest that inhibition of CCR2 by CVC can lead to a decrease in the activity of these pathways and lead to an increase in apoptosis.
In the animal experiment, we investigated the efect of CVC on tumor size changes in treated mice compared to the control group. Our result showed that CVC reduced tumor size signifcantly on the 7th and 21st days after treatment (54.57% reduction on the 7th day and 70.57% reduction on the 21st day). In agreement with our result, it has been reported that the knockdown of CCR2 by siRNA inhibits tumor growth in breast cancer [29]. Another study showed that CCR2 antagonist, named 747, can inhibit liver tumor growth by relieving TAMs mediated immunosuppression [44]. Also, the blockade of CCR2 expression reduces tumor growth and inhibits the proliferation of cancer cells by infuencing the PI3K/Akt and p38 MAPK signaling pathway in DLBCL (difuse large B-cell lymphoma) [43]. JAK/STAT and PI3K/AKT signaling pathways are activated during the binding of CCL2 to CCR2, which ultimately leads to an increase in the proliferation of cancer cells [31]. Considering that the proliferation of cancer cells can lead to tumor growth and increase in size, it can be obtained from the results that by inhibiting the CCR2 receptor by CVC, the activity of these signaling pathways is reduced, and subsequently, cell proliferation and tumor size are reduced.
Terefore, taken together, it may be suggested that CVC can be a promising agent to prevent the progression of the tumor through inhibition of the CCR2_CCL2 signaling pathway.

Conclusion
Te main aim of this investigation was to determine the efect of CVC on the main biomarkers involved in the progression of CRC. We demonstrated that this compound can efectively afect these biomarkers and modulate their expression. In addition, it can boost apoptosis and cause tumor growth inhibition. It can be suggested that CVC possesses the potential to efectively modulate the expression of biomarkers downstream of CCR2_CCL2 signaling pathways which are basically linked to the process of invasion and metastasis. Overall, our fndings from this research ofer that CVC can be considered a promising compound to reduce the progression of CRC through inhibition of the CCR2_CCL2 signaling pathway. Terefore, it should be brought up that this fnding provides novel insights into cancer immunotherapy by targeting the tumor microenvironment, and the interactions occurred between tumor cells and cellular and noncellular components. We could present the appropriate infuence of CVC action in the prevention of cancer by afecting basic and pivotal targets Evidence-Based Complementary and Alternative Medicine 9 involved in cancer invasion and metastasis. Further experiments can investigate the CVC efect on biomarkers for cancer cell stemness, EMT, important signaling pathways including PI3K/AKT and JAK/STAT and infltration and activity of TAMs and MDSCs in tumor microenvironment.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.