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.
Gastric cancer has been identified as one of the most common and fatal cancers in the world [
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 [
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.
A gastric cell line (SGC7901) was purchased from Jennio Biotech Co., Ltd (Guangzhou, China). The cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin, in a humidified 5% CO2 incubator at 37°C.
Male BALB/c nude mice (4–6 weeks old) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). 2 × 106 SGC7901 cells were suspended in 100
Paraffin-embedded tumor tissue sections were deparaffinized and rehydrated. To assess cellular apoptosis, the sections were incubated with proteinase K working solution (15
Sections were immersed in 3% hydrogen peroxide for 15 min. Nonspecific antigen blocking was performed in 2% bovine serum albumin (BSA) for 1 hour. The sections were then incubated overnight at 4°C with rabbit anti-CD31 (1 : 200, Bioworld) or rabbit anti-Ki67 (1 : 100, Abcam) and then incubated with biotinylated anti-rabbit IgG (1 : 100, Santa Cruz) for 1 hour at 37°C and with the avidin-biotin-peroxidase complex (1 : 100, Vector Laboratories) for 1 hour at 37°C. Immunoreactivity was visualized with diaminobenzidine staining and imaged under a microscope.
Tissues were homogenized in RIPA plus buffer containing a cocktail of EDTA-free protease inhibitors. The 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. The 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. Then, 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. The immunopositive bands were visualized using an enhanced chemiluminescent substrate (Thermo Fisher) and Bio-Rad ChemiDoc XRS digital documentation system. The amount of protein expression is presented relative to the levels of
The results of the experiments were expressed as the mean ± standard deviation. Comparisons between groups were assessed by one-way ANOVA, and a
To explore whether ETSJC enhanced the chemosensitivity of 5-FU
Compared with the control group, the tumor volume (a) and tumor weight (b) of each treatment group were lower. The tumor volume and weight of the 5-FU + ETSJC group were lower than those of the 5-FU group.
The 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. The number of proliferating cells diminished after the intervention of ETSJC. This diminishing effect of ETSJC is dose dependent (Figure
The number of Ki67+ cells in the 5-FU + ETSJC group was lower than that in the 5-FU group.
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
The number of TUNEL+ cells in the 5-FU + ETSJC group was higher than that in the 5-FU-treated group.
Compared with the 5-FU group, the expression of Bcl-2 protein decreased in the 5-FU + ETSJC group, while the expression of Bax and caspase-3 protein increased.
The 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. The antiangiogenic effect was enhanced with the increase in dose of ETSJC (Figure
CD31+ microvessels in the 5-FU + ETSJC group were fewer than that in the 5-FU group.
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. This effect was enhanced with the increasement of ETSJC concentration (Figure
Compared with the 5-FU group, the expression of Notch1 and Hes1 protein in the 5-FU + ETSJC group was reduced. This effect was enhanced by increasing the concentration of ETSJC.
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 of gastric cancer with its “multicomponent-multitarget” characteristics [
Angiogenesis refers to the proliferation and migration of normally static vascular endothelial cells [
The study first screened the therapeutic effect of ETSJC on gastric cancer in animal study. The results demonstrated that ETSJC enhanced the chemosensitivity of 5-FU in the subcutaneous xenograft model of human gastric cancer cells. This 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 are available upon request to the corresponding author.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Jing Zhou, Shulan Hao, and Hao Guo contributed equally to this work as first authors.
This work was supported by the Natural Science Foundation of Shanxi Province (No. 201801D121300), the Project of Administration of Traditional Chinese Medicine of Shanxi Province (No. 2020ZYYC022), and the Project of Health Commission of Shanxi Province (No. 2018078).