5-Fluorouracil enhances the chemosensitivity of gastric cancer to TRAIL via inhibition of the MAPK pathway

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has the ability to selectively trigger cancer cell apoptosis and can be used as a target for tumor therapy. However, gastric cancer cells are usually insensitive to TRAIL so reducing this drug resistance may improve the treatment of gastric cancer. In this study, we used Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) experiments to determine the effects of 5-uorouracil (5-FU) and TRAIL on the proliferation of gastric cancer cells. An Annexin V/propidium iodide (PI) staining experiment was used to detect apoptosis, and Western blotting was used to analyze the expression levels of apoptosis-related proteins and mitogen-activated protein kinase (MAPK) pathway proteins. The antitumor effects of 5-FU and TRAIL were veried in vivo using a nude mouse tumorigenesis experiment, and a TUNEL assay was performed to evaluate apoptosis in tumor tissue from the nude mice.


Introduction
Gastric cancer is one of the most common malignant tumors in the world and the third leading cause of cancer-related death (1). Because the early symptoms are not obvious, approximately 30% of patients have late-stage disease when they are diagnosed (2), at which point surgical treatment is not an option.
At this time, chemotherapy plays an important role in the overall treatment of patients (3). However, patients with gastric cancer often experience treatment failure due to resistance to chemotherapeutic drugs, resulting in disease worsening. Therefore, the search for new and more effective treatment methods has aroused great interest.
Research on the regulation of tumor-speci c apoptosis has always been a focus of attention. TRAIL is a member of the tumor necrosis factor (TNF) superfamily and can selectively trigger cancer cell death with little toxicity to normal cells (4). TRAIL promotes the formation of a death-inducing signal complex and the activation of caspases to initiate apoptosis by binding to two cell-surface death receptors (DRs), DR4 (also known as TRAIL-R1) and DR5 (also known as TRAIL-R2) (5). The ability of TRAIL to speci cally kill cancer cells has made it a new target of extensive research in the eld of cancer therapy in recent years.
However, during the application process, it was found that some cancer cells, including gastric cancer cells, develop resistance to TRAIL (6). Therefore, how to optimize and improve the e ciency of TRAIL is a hot topic in cancer treatment.
5-FU and its derivatives, such as capecitabine, tegio, carmo uor, etc., are currently the most common drugs used in rst-line chemotherapy for gastric cancer (7), and these drugs prevent tumors by inhibiting the activity of thymidylate synthase and the production of DNA in cells (8). In some cancer types, researchers have explored the use of 5-FU as a TRAIL sensitizer to synergistically exert an antitumor effect (9)(10)(11). However, the mechanism by which 5-FU induces gastric cancer cells sensitivity to TRAILinduced apoptosis has not been reported.
In this study, we investigated whether 5-FU treatment can sensitize gastric cancer cells to TRAIL-induced growth inhibition and apoptosis both in vitro and in vivo. The results showed that the combination of 5-FU and TRAIL could synergistically exert an antitumor effect through the activation of caspases, the upregulation of DR expression and the downregulation of antiapoptotic protein expression and revealed for the rst time that 5-FU sensitizes cells to TRAIL partly due to inactivation of the MAPK pathway.
Therefore, the combination of 5-FU and TRAIL may become a new treatment strategy for patients with gastric cancer.

Determination of cell viability
CCK-8 (Dojindo, Tokyo, Japan) was used to measure cell viability. Brie y, cells were seeded in 96-well plates at a density of 2-4×10 3 cells/well and allowed to attach for 24 hours. After 24 or 48 hours of treatment with the speci ed concentrations of TRAIL and 5-FU, a CCK-8 solution (10 µl) was added to each well, and then a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) was used to measure the optical density (OD) at 450 nm. The following formula was used to calculate the cell growth inhibition rate: 1-OD experiment / OD control (13). The cells were seeded in a six-well plate and were incubated overnight, after which EdU detection reagent was added, and cell proliferation was observed with a uorescence inverted microscope (Olympus, Japan).

Apoptosis analysis by ow cytometry
Flow cytometry was used to analyze cell apoptosis (14). Cells (5x10 5 ) were seeded in 35-mm 2 petri dishes and treated with TRAIL or/and paclitaxel (PTX) at the speci ed concentration for 24 hours. The cells were harvested by trypsin digestion at speci c time points. Collected cells were washed once with 1× Annexin V binding buffer (eBioscience, San Diego, California, USA) and then incubated in a buffer containing FITC-conjugated Annexin V (eBioscience). After incubating in the dark at room temperature for 30 minutes, the cells were washed once with a binding buffer and then resuspended in 500 μl of binding buffer containing a PI solution (0.5 μg/ml).

siRNA transfection
C-IAP1-speci c siRNA and interfering siRNA were purchased from Thermo Fisher Scienti c (Waltham, Massachusetts, USA). Cells (2x10 5 ) were incubated in a 6-well plate for 24 hours according to the manufacturer's instructions, and then siRNA (100 nmol/l) and Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) in serum-free Opti-MEM were added six hours after seeding. The medium was replaced with fresh medium. Forty-eight hours after transfection, the knockdown effect on C-IAP1 expression was veri ed by Western blotting. siRNA transfection was performed as described previously (16).

Western blot assay
GC cells were inoculated into a 6-cm Petri dish, treated for 48 hours, scraped and collected. The cells were completely lysed using RIPA cell lysis reagent (Solarbio, Beijing, China) containing protease and phosphatase inhibitors for 30 minutes. The cell lysate was centrifuged at 12,000 g and 4°C for 20 minutes, and the protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China). The supernatant containing the total protein was then mixed with a corresponding volume of 5x SDS loading buffer, and the mixture was heated at 95°C for 5 minutes. Subsequently, 20 μg of total protein from each sample was separated on a 12% premade gel. A constant current of 300 mA was used for wet electrotransfer of the proteins to a 0.22-μm polyvinylidene uoride (PVDF) membrane. The membrane was blocked with 5% skim milk powder in TBST for 2 hours and incubated with an appropriate primary antibody (1:1,000) overnight. The next day, the membrane was washed 3 times with TBST for 10 minutes each time. At room temperature, the membrane was incubated with an HRPconjugated secondary antibody (1:8,000) for 2 hours and then washed with TBST 3 times for 10 minutes each time. A chemiluminescence kit (Life Technologies, Shanghai, China) was used to observe the bound antibody complexes on a Bio-Rad infrared gel imager system (ChemiDoc XRS+). Western blotting was performed as described previously (15). 6. Tumor xenograft study using athymic nude mice Animal research was conducted strictly in accordance with institutional guidelines. Six-week-old male nude mice were used in the experiment after 7 days of adaptive feeding. At the beginning of the experiment, AGS cells in a healthy logarithmic growth state were inoculated into the right armpit skin of the mice. The tumors were allowed to grow for approximately 1 week (this time was determined to be when the tumor diameter reached 3-5 mm), and then the nude mice were randomly divided into 4 groups, with 3 mice in each group, and drug administration was started. The groups were i) control (vehicle only); ii) TRAIL (100 μg/kg intratumoral injection); iii) 5-FU (10 mg/kg intraperitoneal injection); and iv) TRAIL (100 μg/kg intratumoral injection), followed by 5-FU (10 mg/kg intraperitoneal injection) 24 hours later.
All groups were treated every 3 days for a total of 28 days. Tumor size and weight were monitored three times per week. Tumor volume (V) was calculated as V = 0.5×length×width2. To evaluate tumor growth inhibition (TGI), the formula [1-(T-T0)/(C-C0)]×100, where T and C are the average tumor volumes of the treatment group and the control group, respectively, at the end of the experiment and T0 and C0 are the average tumor volumes of the same groups on the 0th day, was used (17).

TUNEL assay to evaluate apoptosis
The tissue was dehydrated using gradient alcohol. After sectioning, a 2 mg/ml Proteinase K solution was diluted with deionized water at a ratio of 1:100 to a nal concentration of 20 μg/ml and incubated at room temperature for 20 min. Dilute 5×Equilibration Buffer with deionized water at a ratio of 1:5. Add 100μl of 1×Equilibration Buffer to each sample to cover the area of the sample to be tested, and incubate at room temperature for 15 minutes. Wash off most of the 100 μl 1×Equilibration Buffer with absorbent paper around the equilibrated area, place the slides in a wet box, and incubate at 37°C for 60 min. Then wash with PBS 3 times, 5min each time. DAPI was added dropwise and incubated for 5 min in the dark to stain the specimen, and the excess DAPI was washed away with PBST 5 min × 4 times. Mount with a mounting solution containing an anti-uorescence quencher, and then observe and collect the image under a uorescent microscope. Under the uorescence microscope, the apoptotic cells on the tissue section showed red uorescence, and the nucleus showed blue uorescence.

Statistical analysis
All statistical tests were performed with SPSS 19.0 (SPSS, Inc., Illinois, USA). Data are expressed as the mean ± standard deviation and were compared by Student's t test or analysis of variance. P <0.05 was considered signi cant. Results 1. Combination of 5-FU and TRAIL enhances the inhibition of gastric cancer cell proliferation by TRAIL First, we analyzed the inhibition of normal gastric cell and gastric cancer cell proliferation by TRAIL using the MTT method. As shown in Figure 1a, after TRAIL treatment (0.5 µg/ml, 1 µg/ml, or 2 µg/ml) of normal gastric cells and gastric cancer cells for 24 hours, the cell proliferation rate of HGC27 cells was less than 50% of the untreated rate, while those of GES-1 and AGS cells were not signi cantly different; this result indicated that AGS cells were not sensitive to the growth inhibitory effect of TRAIL, so we chose AGS cells for further experiments. AGS cells were treated with 5-FU (0, 50, 100, or 200 ng/ml), which was approximately the half maximal inhibitory concentration (IC50) of 5-FU in this cell line, and TRAIL (1 µg/ml, no effect on AGS cells) either alone or in combination. Compared with the use of 5-FU or TRAIL alone, the combination of 5-FU and TRAIL signi cantly inhibited the cell viability of AGS cells but did not cause growth inhibition of GES-1 cells (Figure 1b and c)..Then we chose TRAIL with a concentration of 1μg/ml and 5FU with 100ng/ml for the next experiment. When using the CCK-8 assay to evaluate the cell viability for 3 days, we found that the cell viability in the combination group was signi cantly lower than in other groups (Fig. 1d). Subsequently, we conducted an EdU experiment, and the results showed that the number of proliferative cells imaged by uorescence microscopy was lowest in the experimental group given combined treatment (Fig. 1e). These data suggest that 5-FU can make gastric cancer cells sensitive to TRAIL-induced proliferation inhibition.

Effect of 5-FU on gastric cancer cell apoptosis induced by TRAIL
We evaluated the effect of 5-FU on TRAIL-induced apoptosis in AGS cells by ow cytometry. As shown in Figure 2a and b, the lower right and upper right quadrants represent early and late apoptotic cells, respectively. The apoptosis rate of AGS cells treated with TRAIL (1 μg/ml) alone was 9.88%, while after treatment with 5-FU (100 ng/ml), an apoptosis rate of 49.83% was observed in AGS cells. Western blot analysis showed that under treatment with 5-FU and TRAIL, the protein expression levels of cleaved caspase-3/-8/-9 and PARP were signi cantly higher than those observed with 5-FU or TRAIL alone ( Figure  2c and d). These data suggest that the combination of 5-FU and TRAIL can induce more gastric cancer cell apoptosis than 5-FU or TRAIL alone.

5-FU upregulates TRAIL receptor expression and downregulates antiapoptotic protein expression
As TRAIL DRs, DR4 and DR5 bind with TRAIL to activate apoptosis-related pathways and lead to apoptosis. To study the potential mechanism of apoptosis induction by 5-FU and TRAIL, we examined the expression levels of DR4 and DR5. As shown in Figure 3a and b, the expression levels of DR4 and DR5 in 5-FU-treated cells were signi cantly increased. To further reveal the role of 5-FU in regulating apoptosis, we studied changes in apoptosis-related proteins. Notably, combination therapy signi cantly inhibited the expression of antiapoptotic proteins, including c-IAP1, c-IAP2, Bcl-2 and Mcl-1 (Fig. 3c and d). In addition, C-IAP1 was selected for functional veri cation. Silencing C-IAP1 enhanced TRAIL-mediated cytotoxicity and 5-FU-mediated sensitization to TRAIL (Fig. 3e). These data suggest that 5-FU makes gastric cancer cells sensitive to TRAIL-induced apoptosis by upregulating TRAIL receptor expression and downregulating antiapoptotic protein expression.

5-FU promotes TRAIL-mediated apoptosis in gastric cancer cells by inducing inactivation of the MAPK pathway
It has been reported that the regulation of TRAIL resistance is related to the MAPK pathway and the MAPK pathway is related to sensitivity to 5-FU therapy (18). We hypothesized that the regulation of MAPK activity by 5-FU may help to improve the sensitivity of gastric cancer cells to TRAIL. As shown in Figure   4a and b, ERK, JNK and p38 were inhibited to varying degrees by treatment with 5-FU, TRAIL or 5-FU combined with TRAIL, with JNK being the most strongly inhibited. Next, we used U0126 (ERK inhibitor), SP600125 (JNK inhibitor) and SB202190 (p38 inhibitor) to study the effect of MAPK pathway disruption on TRAIL sensitivity. Similar to 5-FU, these inhibitors signi cantly enhanced TRAIL-induced cytotoxicity and apoptosis (Figure 4c and d). Among the three inhibitors, SP600125 showed the strongest sensitizing effect. In general, these results suggest that 5-FU enhances the sensitivity of gastric cancer cells to TRAILinduced apoptosis by inducing inactivation of the MAPK pathway.

5.5-FU enhances the antitumor effect of TRAIL in vivo
To evaluate the therapeutic effects of 5-FU and TRAIL in vivo, we established a nude mouse model with AGS tumor xenografts. After 28 days of monotherapy or combination therapy, we found that the tumor volume and weight of the mice treated with the combination therapy were signi cantly lower than those of the mice treated with 5-FU or TRAIL alone (Fig. 5a, b, and c). In addition, we also performed TUNEL analysis of tumor tissues to evaluated whether 5-FU and TRAIL induced AGS cell apoptosis in vivo. Our results showed that the number of apoptotic cells in tissues treated with 5-FU and TRAIL was signi cantly higher than that in tissues treated with 5-FU or TRAIL alone (Fig. 5d). These data support the results of the in vitro studies and further con rm that 5-FU enhances the antitumor effect of TRAIL.

Discussion
Gastric cancer is the fth most common malignant tumor in the world and the third leading cause of cancer-related death (1). Chemotherapy is used to treat a large proportion of patients, but because of the high toxicity, low sensitivity and many adverse reactions of chemotherapeutic drugs (19), there is an urgent need to nd a new treatment. TRAIL has wide application prospects because of its speci c killing effect on tumor cells. TRAIL can induce apoptosis in human tumor cells (20)(21)(22). A recombinant TRAIL protein has been tested in clinical trials and shown promising antitumor activity and mild side effects (23,24). However, the resistance of gastric cancer cells to TRAIL limits its clinical application. It has been reported that many chemotherapeutic agents can make tumor cells sensitive to TRAIL-mediated apoptosis (25)(26)(27)(28). However, there are few data to clarify the synergistic effect of 5-FU and TRAIL on gastric cancer cells, and the molecular pathways involved have not been reported. Clarifying the mechanism of apoptosis induction by TRAIL is very important to develop strategies to maximize the potential effectiveness of TRAIL in clinical applications. In this study, we evaluated the antitumor potential of 5-FU combined with TRAIL in gastric cancer cells in vivo and in vitro and analyzed the molecular mechanism by which 5-FU increases TRAIL sensitivity.
TRAIL, a member of the TNF superfamily, can selectively trigger cancer cell death but has almost no toxicity to normal cells (29). TRAIL mainly induces apoptosis through two pathways: the activation of BID (30), a member of the "only BH3 domain" family, and the direct activation of caspase proteins to form death-induced signaling complexes (31). In this study, we found that compared with that of cells treated with 5-FU or TRAIL alone, the viability of gastric cancer cells treated with 5-FU and TRAIL was signi cantly inhibited, and the apoptosis rate increased signi cantly after treatment with 5-FU and TRAIL. TRAIL alone could only slightly activate caspase-3, caspase-8, caspase-9 and PARP in AGS cells.
However, the cleavage of all caspase proteins increased sharply after the combined application of 5-FU and TRAIL. Notably, the expression level of BID decreased after combined administration, which is a key event in mitochondrial apoptosis signal transduction, indicating that 5-FU enhances the sensitivity of gastric cancer cells to TRAIL by mediating mitochondrial apoptosis.
TRAIL initiates apoptotic signaling by binding to two cell DRs, DR4 and DR5, and TRAIL-targeting ligand compounds increase speci city mainly by increasing the expression of DR4 and DR5 (32). We found that 5-FU could increase the expression of DR4 and DR5 in gastric cancer cells, so the induction of DR4 and DR5 may be an effective way to enhance the potential antitumor effect of TRAIL on gastric cancer.
Another key way to increase the sensitivity of gastric cancer cells to TRAIL is to inhibit antiapoptotic proteins. We found that 5-FU itself downregulated the protein expression of c-IAP1 and c-IAP2 in AGS cells to some extent, while 5-FU combined with TRAIL signi cantly inhibited the expression of c-IAP1, c-IAP2, Bcl-2 and Mcl-1. In addition, the cytotoxicity of TRAIL can be enhanced by knocking down C-IAP1 expression by siRNA, which indicates that the inhibition of antiapoptotic proteins mediated by 5-FU is related to sensitization to TRAIL.
Notably, we found that the MAPK pathway is involved in 5-FU-mediated TRAIL sensitization. The MAPK pathway is mainly composed of ERK, JNK, and p38 MAPK members. It is an important signal transduction system in organisms and participates in various physiological processes, such as cell growth, division, differentiation, death, and functional synchronization (33). In this study, we found that MAPKs play a negative regulatory role in TRAIL-induced apoptosis. 5-FU inhibited the activation of ERK, JNK and p38 to varying degrees, with JNK being the most inhibited. After using MAPK-speci c inhibitors, TRAIL sensitivity was partially restored, and the JNK inhibitor SP600125 showed the strongest TRAIL sensitization activity. This nding is consistent with the results of Xu et al., who found that downregulation of JNK enhances TRAIL-induced apoptosis in human gastric cancer cells (34). All these results indicate that the inhibition of MAPK is related to the TRAIL-sensitizing effect of 5-FU. Finally, we established a nude mouse model of tumor xenografts to verify whether 5-FU enhances the antitumor effect of TRAIL in vivo. The results showed that compared with control treatment, the combination of 5-FU and TRAIL inhibited tumor growth to the greatest extent. The TUNEL assay showed that the number of apoptotic cells in the combination treatment group was the largest.

Conclusion
In conclusion, the results of this study showed that compared with the use of TRAIL or 5-FU alone, the combination of two drugs can inhibit gastric cancer cell proliferation and induce cell apoptosis in vivo and in vitro to the greatest extent. We also found for the rst time that the MAPK pathway may be involved in the sensitization to TRAIL induced by 5-FU. These ndings provide a theoretical basis for the synergistic antitumor effect of 5-FU as a sensitizer of TRAIL and provide guidance for the selection of new and effective therapeutic targets in gastric cancer.