Erteng-Sanjie Capsule Enhances Chemosensitivity of 5-Fluorouracil in Tumor-Bearing Nude Mice with Gastric Cancer by Inhibiting Notch1/Hes1 Signaling Pathway

Gastric cancer is one of the most common cancers worldwide. This study investigated the chemosensitivity-enhancing effects of Erteng-Sanjie capsule (ETSJC) in combination with 5-fluorouracil (5-FU) on gastric cancer and its possible underlying mechanisms. The study established a subcutaneous xenograft model of human gastric cancer. The animals were divided into five groups: the control group, the 5-FU group, the 5-FU + ETSJC low-dose group, the 5-FU + ETSJC medium-dose group, and the 5-FU + ETSJC high-dose group. The tumor volume and tumor weight were calculated. TUNEL staining was used to evaluate cell apoptosis. Immunohistochemical analysis was used to detect the expression of Ki67+ cells and the CD31+ microvessel density in tumors. Simultaneously, western blot analysis was applied to detect the expression of caspase-3, Bax, Bcl-2, Notch1, and Hes1 proteins. Compared with the control group, tumor volume and weight in the 5-FU and 5-FU + ETSJC groups were inhibited. Moreover, compared with the 5-FU group, tumor volume and weight were significantly inhibited in the 5-FU + ETSJC groups. The numbers of Ki67+ cells, CD31+ microvessel density, and the expression of Bcl-2, Notch1, and Hes1 proteins were markedly decreased in the combination group when compared with the chemotherapy alone group. The numbers of TUNEL+ cells and the expression of Bax and caspase-3 proteins were significantly increased in the 5-FU + ETSJC groups when compared with the 5-FU group. The therapeutic effects were demonstrated to be dose dependent. In conclusion, the findings of the study showed that ETSJC improved the chemosensitivity of 5-FU by blocking Notch1/Hes1 signaling pathway in gastric cancer-bearing mice.


Introduction
Gastric cancer has been identified as one of the most common and fatal cancers in the world [1]. More than 70% of the cases (about 677,000) are found in developing countries; of this number, around half are in East Asia [2]. e five-year survival rate for gastric cancer remains very low [3]. For patients with advanced or metastatic gastric cancer, chemotherapy is still the main form of treatment. 5-Fluorouracil (5-FU) is the commonly used drug in chemotherapy [4]; however, chemoresistance and chemotherapy-induced toxicity and side effects make it intolerable for patients [5]. erefore, therapeutic drugs that can enhance chemotherapeutics but reduce the side effects of chemotherapy are urgently needed.
Traditional Chinese medicine (TCM), which is characterized as "multicomponent-multitarget," shows its potential in the treatment of gastric cancer. Accumulated evidence has demonstrated that TCM can improve the patients' quality of life as well as prolonging it. It can also prevent the recurrence and metastasis of gastric cancer [6]. Erteng-Sanjie capsule (ETSJC), a patented Chinese medicine, is independently developed by the Shanxi Province Academy of Traditional Chinese Medicine. e main ingredients are: chinensis, Prunella vulgaris L, concha ostreae, Rhizoma curcumae, Areca catechu L, and Glycyrrhiza uralensis Fisch. Preliminary clinical studies have shown that ETSJC combined with chemotherapy has significant advantages over chemotherapy alone in alleviating clinical symptoms and reducing the side effects of chemotherapy in patients with advanced gastric cancer [7].
In this study, a subcutaneous xenograft tumor model of gastric cancer was established. ETSJC was administered to investigate its effect in enhancing 5-FU chemosensitivity in gastric cancer.

TdT-Mediated dUTP Nick End Labeling (TUNEL)
Staining. Paraffin-embedded tumor tissue sections were deparaffinized and rehydrated. To assess cellular apoptosis, the sections were incubated with proteinase K working solution (15 μg/mL in 10 mM Tris/HCl, pH 7.5) for 30 min at 37°C. After washing three times with PBS buffer, they were then incubated with 50 μL of TUNEL reaction mixture, covered with a lid, and incubated for 2 hours at 37°C in the dark. en, the slides were incubated with 100 μL stopping buffer for 10 minutes and then rinsed in PBS three times. DAPI was applied to detect the nuclei. Images were observed via a fluorescence microscope, and the percentage of the dUTP-positive cells was detected.

Western
Blot. Tissues were homogenized in RIPA plus buffer containing a cocktail of EDTA-free protease inhibitors. e homogenate was centrifuged at 12,000 rpm for 30 min at 4°C. Protein concentration was assayed using the BCA method, then loaded and subjected to electrophoresis in 10% SDS-PAGE gels, and transferred onto PVDF membranes. e membranes were then blocked in 5% BSA for 2 hour and then incubated with one of the following primary antibodies: rabbit anti-caspase-3 (1 : 500, Proteintech) or mouse anti-Bax (1 : 100, Proteintech) or rabbit anti-Bcl-2 (1 : 500, Proteintech) or mouse anti-Notch1(1 : 500, Santa Cruz) or mouse anti-Hes1 (1 : 1000, Santa Cruz), with gentle shaking at 4°C overnight. en, horseradish peroxidase conjugated goat anti-mouse IgG (1 : 50000, Proteintech) or goat anti-rabbit IgG (1 : 50000, Proteintech) secondary antibodies were incubated with the membranes for 2 hours at room temperature. e immunopositive bands were visualized using an enhanced chemiluminescent substrate ( ermo Fisher) and Bio-Rad ChemiDoc XRS digital documentation system. e amount of protein expression is presented relative to the levels of β-actin.

Statistical Analysis.
e results of the experiments were expressed as the mean ± standard deviation. Comparisons between groups were assessed by one-way ANOVA, and a p value < 0.05 was considered statistically significant.

Impact of ETSJC on Tumor Growth in the Subcutaneous
Xenograft. To explore whether ETSJC enhanced the chemosensitivity of 5-FU in vivo, the study established a subcutaneous xenograft model. As shown in Figure 1, tumor volume (Figure 1(a)) and tumor weight (Figure 1(b)) of the 5-FU and 5-FU + ETSJC groups were significantly reduced when compared with those in the control group. Moreover, the combination of 5-FU and ETSJC produced a significant inhibition of tumor growth compared with animals treated with 5-FU alone. is enhancement effect of ETSJC is dose dependent.

Effect of ETSJC on the Proliferation and Apoptosis in the Experimental Gastric Cancer Model.
e study investigated the effects of ETSJC on tumor cell proliferation and apoptosis. Immunohistochemical analysis showed that a large number of Ki67 + cells proliferated in the tumor tissues of the 5-FU group. e number of proliferating cells diminished after the intervention of ETSJC. is diminishing effect of ETSJC is dose dependent (Figure 2). Evidence-Based Complementary and Alternative Medicine Tumor cell apoptosis determined by TUNEL expression assay was used. TUNEL + cells can be found in the tumor tissues of the 5-FU group. Compared with the 5-FU group, the number of TUNEL + apoptotic cells increased in the 5-FU + ETSJC group ( Figure 3). Further, the expression of apoptosis-related Bcl-2, Bax, and caspase-3 proteins was also detected.
e expression of Bcl-2 protein from the 5-FU + ETSJC group decreased when compared with that of the 5-FU group (Figure 4). However, when compared with the 5-FU group, the levels of Bax and caspase-3 proteins dramatically increased in the combination groups. e apoptotic effect was enhanced when the ETSJC concentration was increased.   Evidence-Based Complementary and Alternative Medicine

Effect of ETSJC on Angiogenesis.
e results showed that the numbers of CD31 + microvessels could be observed in the 5-FU group. After combination with ETSJC, the density of CD31 + microvessels decreased. e antiangiogenic effect was enhanced with the increase in dose of ETSJC ( Figure 5).

Impact of ETSJC on Notch1/Hes1 Signaling Pathway.
To further investigate the underlying antitumor mechanisms of ETSJC, the levels of Notch1 and Hes1 proteins were evaluated. Compared with the 5-FU group, the expression of Notch1 and Hes1 proteins in the 5-FU + ETSJC group was reduced. is effect was enhanced with the increasement of ETSJC concentration (Figure 6).

Discussion
Gastric cancer is one of the most common malignant tumors of the digestive system in the world, and it has attracted more attention in recent years. Chemotherapy remains the main form of treatment for locally advanced and advanced gastric cancer. In chemotherapy, 5-FU is widely used in treating patients with gastric cancer. However, the side effects of chemotherapy often make it difficult for patients to tolerate. For this reason, finding better treatment methods aimed at improving the efficacy of existing anticancer drugs and reducing the toxic side effects is imperative.
Based on the complicated pathological mechanisms of gastric cancer, TCM has shown its efficacy in the treatment * * * * * * Microvessel density (%) of gastric cancer with its "multicomponent-multitarget" characteristics [6,8] Angiogenesis refers to the proliferation and migration of normally static vascular endothelial cells [9,10]. e aggressive nature of gastric cancer appears to be closely related with the degree of angiogenesis [11]. An increasing number of antiangiogenesis therapy has been extensively developed for cancer treatment, effectively suppressing tumor growth [12,13]. Apoptosis is a complex multistep process that involves the regulation of the Bcl-2 family, mitochondrial signal transduction, and caspase activation [14,15]. e caspase protease family is responsible for cell apoptosis. After activation, they further cleave substrates, such as PARP, causing them to lose their normal functions, and increase the activity of the inhibited endonucleases, cleaving the DNA between nucleosomes which eventually lead to apoptosis [16]. e antiapoptotic protein Bcl-2 and the proapoptotic protein Bax are two representative proteins of the Bcl-2 family. Bax and Bcl-2 form a dimer in normal cells. e expression of the two proteins in the cell is relatively stable. When Bax expression increases and Bcl-2 expression decreases, the balance between the two is broken, and apoptosis is induced [17,18]. e current study found that, after the combined application with ETSJC, the 5-FU + ETSJC group has showed lower CD31 + microvessel density than the chemotherapy alone group. e antiangiogenesis effect is obvious. Moreover, the number of TUNEL + cells and the expression of apoptosisrelated proteins Bcl-2, Bax, and caspase-3 indicated the apoptosis promoting effects of ETSJC. e combined effects enhanced chemotherapy sensitivity to 5-FU of human gastric cancer cells bearing nude mice. Notch1 signaling pathway is known to regulate the expression of its target genes and plays a key role in cell proliferation, angiogenesis, apoptosis, and differentiation [19][20][21]. It is well documented that Notch signaling pathway participates in gastric cancer progression [22]. e abnormal activation of Notch signaling is closely associated with tumorigenesis of gastric cancer. Previous study has demonstrated that c-secretase inhibitors (GSIs), which function as Notch signaling inhibitors, combined with 5-FU could be applicable to gastric cancer therapy [23]. In the current study, the expressions of Notch1 and Hes1 were dramatically decreased after combination therapy. is suggested that Notch1/Hes1 signaling pathway was involved in the ETSJC's anticancer feature.

Conclusions
e study first screened the therapeutic effect of ETSJC on gastric cancer in animal study. e results demonstrated that ETSJC enhanced the chemosensitivity of 5-FU in the subcutaneous xenograft model of human gastric cancer cells. is phenomenon may be related to the inhibition of Notch1/Hes1 signaling pathway; it provides a potential therapeutic method to improve the chemosensitivity of 5-FU in gastric cancer.

Data Availability
Data are available upon request to the corresponding author.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.