3,3′-Diindolylmethane Augments 5-Fluorouracil-InducedGrowth Suppression in Gastric Cancer Cells through Suppression of the Akt/GSK-3β and WNT/Beta-Catenin

Gastric cancer (GC) is one of the most lethal cancers in South Korea, and it is a cancer of concern worldwide. 5-fluorouracil (5-Fu) is commonly used as the first-line therapy for advanced GC; however, its side effects often limit the dosage range and impair patients' quality of life. Due to the limitations of current chemotherapy, new anticancer therapies are urgently needed. 3,3′-diindolylmethane (DIM) has been reported to have the ability to protect against various types of cancer. Our study aimed to elucidate the anticancer effect of DIM in GC when treated with the chemotherapeutic agent 5-Fu. In our results, combined treatment with DIM and 5-Fu resulted in higher apoptosis and lower cell proliferation than treatment with 5-Fu in SNU484 and SNU638 cell lines. Furthermore, when DIM and 5-Fu were administered together, cell invasion was diminished by mediated E-cadherin, MMP-9, and uPA; p-Akt and p-GSK-3β levels were reduced more significantly than when 5-Fu was administered alone. Moreover, in the Wnt signaling pathway, combined treatment of DIM and 5-Fu diminished β-catenin levels in the nucleus and inhibited cyclin D1and c-Myc protein levels. The Akt inhibitor, wortmannin, further inhibited the levels of β-catenin and c-Myc that were inhibited by DIM and 5-Fu. Furthermore, an animal xenograft model demonstrated that DIM combined with 5-Fu considerably reduced tumor growth without any toxic effects by regulating the Akt/GSK-3β and β-catenin levels. Our findings suggest that DIM significantly potentiates the anticancer effects of 5-Fu by targeting the Akt/GSK-3β and WNT/β-catenin because the combination therapy is more effective than 5-Fu alone, thereby offering an innovative potential therapy for patients with GC.


Introduction
Gastric cancer (GC) has a high incidence rate and is one of the most lethal cancers worldwide [1]. Moreover, GC is a prominent mortality malignant neoplasm in South Korea [2]. Although the morbidity of GC has recently declined globally, it remains a major killer worldwide [3]. Due to the lack of an early GC diagnostic index, only 28% of patients with GC can be diagnosed in the local stage, and more than 62% of patients were initially discovered to have regional or distant GC [4]. Tus, the 5-year survival rate of patients with GC is merely 37.9% [5]. Surgery remains the preferred method for treating patients with GC because of the limitations of chemotherapy [6]. However, many patients are diagnosed late with advanced-stage disease because of the low rate of early diagnosis [7]. Consequently, most patients with GC miss the surgical time window; the treatment for late-stage is the combination of neoadjuvant chemotherapy, radiotherapy, specifc-targeted therapy, and others [7]. Terefore, GC remains a serious burden on global health because therapies for this disease are limited, and novel therapies are desperately needed. 5-fuorouracil (5-Fu) acts by interrupting the action of DNA replication [8]. Over 50 years, 5-Fu has been extensively used as systemic combination chemotherapy in treating colorectal [9], gastrointestinal [10], anal [11], breast [12], head, and neck cancers [13,14]. Although 5-Fu is recommended as a principal chemotherapeutic reagent for GC [15], various acute problems, such as dose-limiting toxicity, low efcient conversion rate, drug resistance, and serious side efects, will decrease its efcacy [16,17]. Tus, developing natural substances that can alleviate the side efects of the existing anticancer action and increase the efciency of 5-Fu are imperative.
Akt is a serine/threonine kinase and acts as a key player in the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway involved in normal cellular processes [32]. Akt is often highly activated in many cancers [33] and functions as a central point in various signaling pathways [34]. PI3K/Akt pathway has been suggested to be associated with cell invasion, autophagy, and apoptosis regulation in GC [35]. Moreover, Yu et al. have demonstrated that Akt is involved in the chemoresistance that causes drug resistance in GC [36,37]. Hence, targeting Akt seems to be an important approach for preventing and treating GC. Te Wnt pathway elicits a pivotal role in GC [38]. Approximately 30% of gastric adenocarcinomas show a direct correlation with β-catenin nuclear localization through the Wnt signaling pathway [39]. Although DIM can regulate cell proliferation via Akt [29] or β-catenin [40], whether DIM can enhance the efects of chemotherapeutic drugs on GC via the Akt and Wnt signaling pathways remains uncertain. Terefore, our study was designed to investigate the efects of DIM combined with 5-Fu and the probable underlying biological mechanisms in GC. We hypothesize that combination treatment of DIM and 5-Fu has a strong and safe inhibitory efect on GC and efectively improves the susceptibility of GC to DIM by inhibiting Akt/GSK-3β and β-catenin. Terefore, DIM may be a feasible targeted therapy for GC.

Soft Agar Colony Formation Assay.
Moreover, 1% agarose gel was dissolved and added to a 6-well dish until solidifcation. Following this, 0.7% agarose gel was gently mixed with 1 × 10 5 cells in 2 × RPMI-1640 media and 20% FBS plus 2% antibiotics and covered with medium with or without drug, respectively. Te dishes were incubated for 3 weeks and fed fresh medium with or without drug twice or three times a week. Colonies with more than 30 cells were counted as one positive colony, and images were taken using a microscope.

Annexin V-FITC
Analysis. An Annexin V-FITC Assay Kit (Becton Dickinson Biosciences, CA, USA) was used according to the manufacturer's recommended procedure. After 48 h of DIM and 5-Fu treatment, the cells were washed twice with ice-cold DPBS and further resuspended in the annexin-binding bufer containing annexin V-FITC and propidium iodide (PI) solution. Following the incubation of stained cells in a CO 2 incubator at 37°C for 30 min, fow cytometry analysis was performed immediately (Becton Dickinson, New York, NY).

Cell Cycle Analysis.
To analyze the efect of drugs on the cell cycle, SNU484 and SNU638 were cultured in 60 mm dishes and incubated with DIM and 5-Fu for 48 h. After collecting the cells, they were rinsed twice with ice-cold DPBS and soaked for 2 h in 75% ethanol. Subsequently, the cells were washed with DPBS to remove the ethanol. Te cell cycle counts were estimated by incubating at 37°C in a CO 2 incubator for 15 min with RNase, which was followed by nucleus staining with propidium iodide (Sigma Chemical, St. Louis, MO, USA). Ten, cell counts were further analyzed using CytExpert analysis software (Beckman Coulter, Indianapolis, Indiana, USA).

Statistical
Analysis. GraphPad Prism version 7.00 (GraphPad Software, Inc., La Jolla, CA, USA) was used to calculate half-maximal inhibitory concentration (IC50), the drug concentration at which 50% growth inhibition was achieved. One-way analysis of variance with Tukey's posthoc analysis was used as the statistical comparison. Te data are expressed as the means ± standard error (SE). p-values of <0.05 or <0.01 were used to indicate statistical signifcance.

DIM Enhances 5-Fu-Inhibited Proliferation of GC Cells.
We performed WST-1 and colony formation assays using SNU-484 and SNU-638 GC cell lines to measure cell viability.  (Figure 1(b)). Tese results indicated that combined treatment with DIM and 5-Fu inhibited GC cell proliferation more signifcantly than DIM or 5-Fu alone.

DIM Enhances 5-Fu-Inhibited Migration of GC Cells.
Wound-healing assay was performed to test the inhibitory abilities of DIM and 5-Fu on the metastasis in GC cells. Cell migration rates were measured at 24 h and 48 h (Figure 3(a)). While the migration rate in SNU484 and SNU638 cells declined by approximately 50% 24 h and 48 h after treatment with DIM or 5-Fu alone, the migration rate reduced by approximately 70% 24 h and 48 h after DIM and 5-Fu treatment in GC cells. Tis result indicated that DIM and 5-Fu strongly decreases the migration rate of GC cells. Subsequently, we examined how the combined therapy of DIM and 5-Fu afects cell metastasis. Combined treatment of DIM and 5-Fu signifcantly promoted E-cadherin levels but suppressed MMP-9 and uPA levels in SNU484 and SNU 638 cells (Figure 3(b)). Moreover, combined treatment of DIM and 5-Fu considerably upregulated the E-cadherin mRNA and downregulated the MMP-9 mRNA (Figure 3(c)). Terefore, these fndings suggested that combined treatment of DIM and 5-Fu signifcantly diminishes the migration ability of GC cells by regulating Ecadherin, MMP-9, and uPA.

DIM Enhances 5-Fu-Inhibited Akt Signaling in GC Cells.
Akt kinase is a signal molecule for the typical PI3K efector in the PI3K/Akt signaling pathway, and its activation is associated with the pathogenesis of GC [35]. We evaluated the alterations in the Akt signaling and determined the suppressive GC cell growth's efect of DIM and 5-Fu. Combination treatment of DIM and 5-Fu considerably diminished the p-Akt, whereas no signifcant changes were seen in the Akt in SNU484 and SNU638 cells (Figure 4(a)). Furthermore, p-GSK-3β (Ser9), a downstream gene of Akt, greatly decreased after DIM and 5-Fu treatment. We further investigated whether the combined treatment of DIM and 5-Fu in the presence of wortmannin, a PI3K/Akt inhibitor, resulted in a more efective inhibitory efect in SNU484 and SNU638 cells. Te combined treatment of DIM and 5-Fu signifcantly suppressed Akt, p-Akt, and p-GSK-3β (Ser9) levels in SNU484 and SNU638 cells with wortmannin treatment (Figure 4(b)), suggesting that wortmannin further accelerated the efcacy of the inhibitory efect of combination treatment of DIM and 5-Fu on the Akt pathway.

DIM Enhances 5-Fu-Inhibited Wnt Signaling in GC Cells.
As a downstream signaling pathway of PI3K/Akt signaling, the Wnt signaling pathway has been considered to play a critical role in cancer [38]. We further examined whether combination treatment of DIM and 5-Fu can regulate the Wnt singling pathway using western blotting. Te main role of Wnt signaling stimulation is the transfer of β-catenin in the cytosol to the nucleus [42]. Hence, we separated the cytoplasm and nuclear protein to measure β-catenin nuclear translocation after combination treatment of DIM and 5-Fu. Despite no obvious change in the cytoplasm, the β-catenin levels in the nucleus signifcantly decreased after the combined treatment of DIM and 5-Fu compared to that after single treatment ( Figure 5(a)). Furthermore, in the presence of wortmannin, combination treatment of DIM and 5-Fu dramatically inhibited β-catenin nuclear translocation ( Figure 5(b)). Combined treatment with DIM and 5-Fu inhibited the β-catenin, cyclin D1, and c-Myc ( Figure 5(c)). Moreover, combined treatment of DIM and 5-Fu signifcantly diminished the c-Myc and cyclin D1 in the presence of wortmannin ( Figure 5(d)). Tese results suggested that combined treatment of DIM and 5-Fu not only inactivates the Akt signaling pathway through the Akt phosphorylation with p-GSK-3β but also inhibits the Wnt signaling through downregulating β-catenin in GC cells.

DIM Enhances 5-Fu-Inhibited Tumor Growth in Animal
Models. To test the role of DIM and 5-Fu in tumorigenesis in vivo, xenograft mouse models were established. Referring to the previous study that revealed that DIM suppressed gastric tumorigenesis in a xenograft model [30], after subcutaneous injection with SNU484, the mice were classifed into three groups, namely, the control, 5-Fu treatment, and DIM and 5-Fu combination treatment groups. After 2 weeks of treatment, tumor size was measured. In the DIM and 5-Fu combined group, the tumor weight, volume, and size were substantially inhibited compared to control or 5-Fu treatment group     Journal of Oncology  . Te gray value of bands was detected using ImageJ. * p < 0.05, * * p < 0.01 compared to control; # p < 0.05, ## p < 0.01 compared to DIM + 5-Fu. 8 Journal of Oncology ( Figures 6(b)-6(d)). To determine whether there was drug toxicity in mice, we evaluated the toxic efects of combination treatment of DIM and 5-Fu in xenograft mice in vivo. No signifcant diferences in the body weight (Figure 6(a)), heart, liver, and kidney (Figure 7(a)) functions were observed between the groups. Serum biochemical analysis with liver function markers showed no signifcant diferences among the three groups (Figure 7(b)). Regarding nephrotoxicity, BUN and creatinine (Cr) levels were examined and exhibited no diferences among the groups. Overall, these results suggested that combined treatment of DIM and 5-Fu more efectively suppressed tumorigenesis without any toxic efects in the xenografted mouse model in vivo.

DIM Enhances 5-Fu-Inactivated Akt and Wnt Signaling
Pathways In Vivo. Histological tumor sections revealed a remarkable amount of necrosis in the tumors after combination treatment of DIM and 5-Fu (arrow in Figure 8(a)). In the control group, it was seen that the tumor was growing vigorously, and it highly expressed Ki   Journal of Oncology in Akt, GSK-3β, and β-catenin protein levels were similar to the in vitro experiment results. Te p-Akt, p-GSK-3β (Ser9), c-Myc, and cyclinD1 levels were substantially diminished, whereas those of p-β-catenin were greatly increased in the DIM and 5-Fu combined treatment group in vivo (Figures 8(b) and 8(c)). Tese results indicated that DIM augments the anticancer efect of 5-Fuin vivo via the Akt/ GSK-3β and β-catenin signaling pathways.

Discussion
GC is a common malignant neoplasm with a prominent mortality rate in South Korea [2]. Poor diagnosis and the limitations of therapy lead to high mortality of GC worldwide [3]. Terefore, GC remains a serious burden on global health due to the limitations of therapy, and the discovery of new chemotherapy strategies is urgently needed. , and c-myc were detected after DIM (30 μM), 5-Fu (10 μM), or combination treatment for 48 h (d) combination treatment of DIM and 5-Fu with or without wortmannin signifcantly inactivated the Wnt signaling pathway. Lamin B and GAPDH was used as internal nuclear and cytoplasmic controls, respectively. CONT, control; D or DIM, 3,3′-diindolylmethane; 5-Fu, 5-Fluorouracil. Each point represents the mean ± standard error (SE). Te gray value of bands was detected using ImageJ. * p < 0.05, * * p < 0.01 compared with control; # p < 0.05, ## p < 0.01 compared with DIM + 5-Fu. 5-Fu has been difusely used for more than 50 years as an antitumor drug in various tumors, especially in GC [43]. Presently, 5-Fu remains the clinically recommended treatment for GC [4,44]. However, drug resistance, toxicity, and low-level response hampered the application of 5-Fu in clinical settings [13]. DIM was a natural compound extracted from cruciferous vegetables [45]. DIM has several benefcial biological activities against numerous cancers [20,46]. In our previous experiments, DIM arrested the progression of GC in terms of cell apoptosis, migration, and metastasis [29,30]. Additionally, recent studies revealed that DIM and 5-Fu have a synergistic efect on colon and cervical cancer [47,48]. However, the antitumor efects of DIM with classic chemotherapeutics 5-Fu in GC are still indistinct. In this study, we selected two gastric cancer cell lines, SNU484 and SNU638. SNU484 and SNU638 have been established and reported as poorly diferentiated adenocarcinomas [49]. Gastric adenocarcinoma is a malignant epithelial tumor that originates from the glandular epithelium of the gastric mucosa. Approximately 90% of gastric cancers are adenocarcinomas [50]. Poorly diagnosed gastric adenocarcinomas are susceptible to metastasis and can grow in lymph nodes even in the early stages [51]. According to NCCN treatment guidelines, systemic chemotherapy should be administered after partial surgical resection for patients with a high recurrence and metastasis risk [52]. Terefore, we have chosen SNU484 and SNU638 cells as poor adenocarcinoma markers to examine the therapeutic efects of an anticancer herbal component-based drug with chemotherapy for treating gastric cancers. We found that DIM greatly augments the All therapies were intraperitoneally injected once every 2 days. All data were shown as the mean ± standard error (SE). * , the DIM + 5-Fu treatment group compared to the control group; # , the 5-Fu treatment group compared to the control group. * p < 0.05, * * p < 0.01, * * * p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001. anticancer efects of 5-Fu by suppressing proliferation and metastasis of GC cells. Te combined treatment of DIM and 5-Fu more signifcantly inhibited cell viability than singledrug treatment. Similar results were observed in the cell colony formation growth. Moreover, we found that in cell cycle analysis, the sub-G1 phase, a maker of apoptosis, was signifcantly induced after DIM and 5-Fu cotreatment compared with that after single-drug treatment. Furthermore, annexin V-FITC staining data showed that the combination treatment induced apoptosis and necrosis compared to the single-drug treatment in GC cells. Members of the Bcl-2 family include Bax and Bcl-2. Furthermore, it is  , β-catenin, p-β-catenin, c-Myc, and cyclin D1 protein levels from mouse tissue were analyzed. Tree samples were randomly selected in each group. Internal control was used as (b) DIM, 3, 3′diindolylmethane;5-Fu, 5-Fluorouracil. Te gray value of bands was detected using ImageJ. * p < 0.05, * * p < 0.01, * * * p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with DIM + 5-Fu.
widely known that Bax's antiapoptotic function can compete with Bcl-2 [53]. Subsequently, essential proteins for apoptosis can be further activated by caspase-3 after Bcl-2 has been activated to trigger apoptosis [54]. Tese results are connected to the regulated expression of PARP and caspase-3, -7, -9. Combined treatment of DIM and 5-Fu improved the cleaved-forms and activated proteins that increase during apoptosis compared with single-drug treatment. Te inhibitory efects of DIM on cell proliferation in breast [27] and colon cancers [25] were similar to those of our fndings. Trough this study, we found that the efcacy of 5-Fu further increased after DIM was added; combined treatment of DIM and 5-Fu signifcantly inhibited the cancer cell growth and induced apoptosis. Tis synergistic efect of 5-Fu and DIM was also shown in cancer metastasis and migration. In the wound-healing assay, combined treatment with DIM and 5-Fu notably inhibited cell migration after 24 h and 48 h, indicating that DIM substantially augmented inhibitory function of 5-Fu in GC migration. Moreover, combined treatment of DIM and 5-Fu signifcantly increased the levels of E-cadherin, which is referred to as the "suppressor of invasion" [55] and downregulated the protein levels of MMP-9 and uPA compared with DIM or 5-Fu single treatment. Moreover, combined treatment of DIM and 5-Fu suppressed the migration ability of GC cells through Ecadherin, MMP-9, and uPA, suggesting that DIM increases the anticancer activity of 5-Fu in GC cells. Terefore, these results ofer substantial evidence that simultaneous treatment of DIM and 5-Fu synergistically suppresses GC cell growth and metastasis. Akt, a PI3K downstream molecule, causes conformational changes through a series of actions, exposing the phosphorylation sites of Tr308 and Ser473 in the kinase domain and C-terminal domain, respectively [56], which promotes the cancer cell growth and provides resistance to cell apoptosis [34]. Te phosphorylated Akt protein regulates a diversity of cell death pathways and cell-cycle transition [33]. Several studies have shown that chemotherapy reagents, such as 5-Fu and cisplatin, induce drug resistance by activating p-Akt and decreasing chemotherapy sensitivity in GC [57][58][59]. Less than 25% of patients with GC respond after 5-Fu treatment, and 5-Fu combined with other anticancer drugs only increased the response rate to 30%-50% [60,61], suggesting that patients with advanced GC demonstrate resistance to 5-Fu-based chemotherapies. Terefore, the reduction of p-Akt expression appears to stimulate apoptosis and reduce the cell growth and chemotherapeutic resistance in GC [62,63]. Furthermore, it has been reported that DIM inhibits Akt activity in many cancers, including GC [64]. In this study, we found that DIM and 5-Fu decreased the protein expression of p-Akt and p-GSK-3β (Ser9), and the combined treatment of the two further decreased the expression of p-Akt and p-GSK-3β (Ser9). In the presence of wortmannin, a nonspecifc covalent PI3K inhibitor [65], combined treatment of DIM and 5-Fu markedly suppressed p-Akt and p-GSK-3β in GC cells. Terefore, these data suggested that DIM and 5-Fu inactivated Akt by dephosphorylation and may cause activation of GSK-3β via phosphorylation.
Te Wnt signaling pathway, a downstream pathway of Akt, seems to be involved in breast carcinoma [66], lung cancer [67], colorectal cancer [68], and GC [69,70]. β-catenin is working as a main mediator of the canonical Wnt pathway [71]. When the Wnt signaling pathway is activated, β-catenin accumulates and translocates into the nucleus to bind with T cell factor, acting as a transcriptional activator to trigger downstream target genes [42]. Numerous studies have shown that Akt activation caused the inactivation of GSK-3β, which repressed the degradation of β-catenin [72][73][74][75]. In this study, combined treatment with DIM and 5-Fu decreased β-catenin levels in the nuclei of GC cells. Our fndings are consistent with those of previous studies, which reported that the activation of GSK-3β via the inactivation of Akt inhibits the translocation of β-catenin from the cytoplasm to the nucleus, suggesting that combination treatment of DIM and 5-Fu inhibits Akt activity, which activates GSK-3β and thereby induces the degradation of β-catenin in GC cells. Furthermore, the decreased amount of β-catenin caused by DIM and 5-Fu was further accelerated by adding wortmannin. Te PI3K inhibitor, wortmannin, considerably prevented the phosphorylation of Akt and GSK-3β, which subsequently diminished the nuclear localization of β-catenin and therefore accelerated the tumor-inhibiting efect of DIM and 5-Fu. Cyclin D1 and c-Myc, as downstreams of Wnt, are the main participants of cell-cycle progression [76], in which aberrance has been associated with cell proliferation and apoptosis [77,78]. DIM and 5-Fu treatment considerably diminished cyclin D1 and c-Myc compared to single treatment alone, and wortmannin further strengthened this decrease in GC cells. Tese similar fndings were also observed in animal in vivo experiments. Combination treatment of DIM and 5-Fu signifcantly reduced tumor size/weight and volume compared with single treatment alone in xenograft animal models. Additionally, histological examination of tumor sections showed that a signifcant amount of necrosis in the tumors of mice treated with combined DIM and 5-Fu treatment signifcantly decreased the protein expression of p-Akt and p-GSK-3β (Ser9) in xenograft tumor tissue. Furthermore, combination treatment of DIM and 5-Fu induced β-catenin degradation and signifcantly reduced c-Myc and cyclin D1 levels compared with 5-Fu treatment alone in xenograft tumor tissues. Terefore, combination treatment of DIM and 5-Fu inhibited tumorigenesis more efectively without any toxic efects in vivo through the Akt and Wnt signing pathways. Our results suggested that combined treatment with DIM and 5-Fu not only inactivated the Akt signaling pathway through the dephosphorylation of Akt and its downstream protein p-GSK-3β but also inhibited the Wnt signaling through inactivation of β-catenin in GC.
In conclusion, our study revealed that DIM enhances the 5-Fu-inhibited cell growth and induced apoptosis by targeting the Akt/GSK-3β and Wnt/β-catenin signaling pathways; moreover, DIM suppressed cell migration by regulating E-cadherin, MMP-9, and uPA in GC. Tese anticancer efects were verifed in cell lines and animal experiments, thereby indicating that DIM contributes to enhance the efcacy of 5-Fu, which is a widely used as anticancer drug. Terefore, our results provide convincing evidence that simultaneous treatment with DIM and 5-Fu synergistically suppresses the GC cell growth and tumorigenesis and could be a new potential therapy for treating GC.

Data Availability
Te data presented in this study are available within the article.

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