Betulinic acid triggers apoptosis and inhibits migration and invasion of gastric cancer cells by impairing EMT progress

Gastric cancer (GC) is one of the most prevalent types of malignancies. Betulinic acid (BA) is a natural pentacyclic triterpene with a lupine structure. However, to the best of our knowledge, there is no research study on the anti‐tumour and anti‐metastasis effect of BA on GC. In this study, we assessed the anti‐cancer effect of BA on human GC cells in vitro and in vivo. We first investigated the cytotoxicity and anti‐proliferation effect of BA on GC cells of SNU‐16 and NCI‐N87. The results indicated that BA had significant cytotoxic and inhibitory effects on GC cells in a dose‐ and time‐dependent manner. To further study the cytotoxic action of BA on GC cells, we assessed the apoptotic induction effect of BA on SNU‐16 cells and found that BA distinctly induced apoptosis in SNU‐16 cells. In addition, BA inhibited the migratory and invasive abilities of SNU‐16 cells. Western‐blot analysis revealed that BA suppressed the migration and invasion of GC cells by impairing epithelial‐mesenchymal transition progression. Furthermore, in vivo experiments showed that BA could delay tumour growth and inhibit pulmonary metastasis, which is consistent with the results of in vitro studies. Overall, we evaluated the anti‐cancer effect of BA on human GC cells in vivo and in vitro, and the present study provides new evidence on the use of BA as a potential anti‐cancer drug for GC treatment. Significance of the study BA significantly suppressed proliferation and triggered apoptosis in GC cells. Additionally, BA remarkably inhibited migration and invasion of GC cells by impairing the epithelial‐mesenchymal transition signalling pathway. It is worth noting that BA drastically retarded tumour growth in the xenograft mouse model of GC. Our results indicated that BA can be considered a candidate drug for GC therapy.

metastases at the time of diagnosis, and their 5-year survival rates are extremely low. 7 In 2018, 679 000 new cases and 498 000 cases of GC-related mortality were reported by the Chinese National Cancer Center. 8 With the use of multimodal treatments for GC, the overall 5-year survival rate has been found to stabilize at 40% worldwide. [9][10][11] Therefore, it is crucial to understand the molecular mechanism of GC progression and metastasis in early diagnosis and treatment. Currently, modern treatment of GC, including surgery combined with radiotherapy, chemotherapy and targeted therapy still have some drawbacks such as low therapeutic effect, high toxicity, recurrence and even metastasis. 12,13 Therefore, it is important to find safe and effective drug candidates for GC treatment.
Natural products have been used to treat human diseases since ancient times and are they are vital to drug discovery and development. 14,15 Various anti-infection and anti-cancer drugs have been derived from natural products. [16][17][18][19] Additionally, the rapid development of new drugs that are more effective and have fewer adverse effects is a common goal shared by scientists and clinicians. 20,21 Traditional Chinese medicine is a huge treasury of remedies with low toxicity and high sensitivity and the ability to stabilize tumour growth and improve GC prevention and treatment.
Betulinic acid (BA) ( Figure 1A) (3β, hydroxyl-lup-20 [29]-en-28-oic acid) is a natural pentacyclic triterpene with a lupine structure, which is derived from plant sources such as acuminatissima leaves, white birch bark and wild jujube seeds. 22,23 Various studies have reported that BA has a variety of valuable medicinal effects, including anti-bacterial, anti-cancer, anti-malarial, anti-viral and anti-inflammatory. [24][25][26] Furthermore, BA has shown tumorigenesis inhibition in many kinds of cancers, including lung, colon, breast, prostate and pancreatic cancer. [27][28][29][30] In the present study, our results showed that BA distinctly triggered apoptosis and suppressed the proliferation, migration and invasion of GC cells in vitro. The in vivo anti-tumour efficacy was consistent with that reported in vitro studies.

| Cell lines and cell culture
SNU-16 and NCI-N87 cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured in RPMI1640 medium (Gibco, Grand Island, New York) containing 10% fetal bovine serum F I G U R E 1 BA inhibits viability and proliferation of gastric cancer cells. A, The chemical structure of BA. B and C, The growth curves of SNU-16 and NCI-N87 cells treated with different concentrations of BA for 48 and 72 hours. D and E, The colony formation of SNU-16 and NCI-N87 cells treated with different concentrations of BA. Significant differences were indicated as *P ≤ .05; **P ≤ .01; ***P ≤ .001. B, betulinic acid (FBS) (Gibco, Grand Island, New York). All cells were kept in an atmosphere of 5% CO 2 at 37 C.

| Colony-formation assays
Approximately 500 GC cells were seeded into a six-well plate and cultured in media with various concentrations of BA (0, 2.5, 5, 10, 20, 40 and 80 μM). The cells were incubated at 37 C for 8 days.
When larger clones were found in the control group, the incubation was stopped. The culture medium was discarded, and cells were washed with phosphate-buffered saline (PBS), fixed with 800 μL methanol per well for 10 to 20 minutes, and stained with 0.1% crystal violet at 37 C for 10 minutes. Finally, the stained cell clones in dishes were imaged and counted. Each treatment was repeated in three times.

| Western blot analysis
Total protein was separated from the treated cells using 500 μL of radio immune precipitation assay buffer with 1 mM of phenylmethanesulfonylfluoride. Samples with the cells were immediately sonicated for 2 minutes to break the cell membrane, and were then centrifuged. The supernatant containing the proteins was collected and stored at −20 C for further use. The proteins were separated by 10% polyacrylamide gels and then transferred onto a polyvinylidene fluoride membrane. Then, the proteins on the membranes were washed with TBS containing 0.1% Tween-20 (TBST) and blocked by 5% nonfat milk (wt/vol) for 1 hour at room temperature.
After washing with PBST three times, the membranes were incubated with antibodies (E-cadherin, N-cadherin and β-actin) overnight at 4 C.
Next, the membranes were washed again with PBST and incubated with horseradish peroxidase-labelled immunoglobulin G (dilution, 1:5000) at room temperature for 1 hour. Finally, the proteins bands were visualized using enhanced chemiluminescence by western blot analysis.

| Xenograft tumour model in nude mice
The animal experiment in this study was approved by the Institutional

| Immunohistochemistry
The tumour samples were resected from the mice, fixed in 10% formaldehyde, embedded in paraffin, and sectioned (5 μm thickness). The paraffin tumour sections were analysed by Immunohistochemistry (Ki-67 and MMP-2). Then, the sections were dehydrated and fixed, and the slides were sealed with neutral gum. Pictures were obtained using the Leica microscope (Leica, DM4000B).

| Statistical analysis
All experiments were conducted at least three times. Data are showed as mean ± SD (SD). Two-tailed student's t test and one-way analysis of variance (ANOVA) test were used for statistical analysis of the data.
Significant differences of the P-values are as follows: * P < .05; ** P < .01; *** P < .001. Furthermore, colony-formation assays were carried out to study the anti-proliferation effect of BA on SNU-16 and NCI-N87 cells. As shown in Figure 1D,E, the colony-formation ability of SNU-16 and NCI-N87 cells was significantly inhibited by BA treatment. Taken together, the results show that BA exerted sufficient anti-proliferation effect on GC cells.

| DISCUSSION
GC is one of the most prevalent types of malignancies. 1 GC is a malignancy with high morbidity and mortality, and is the third leading cause of cancer-related mortality worldwide. 2,3 Therefore, the need for new and effective anti-GC drug treatments is urgent. Even though chemical drugs such as Dox and PTX can effectively destroy the DNA in tumour cells, severe side effects are inevitable. 31 Natural products from plants and animals exhibit high efficiency, low toxicity and other benefits. 32 It is widely reported that BA is cytotoxic to various types of human cancer cells. [28][29][30] In the present study, we assessed the anti-  33 Lewinska A showed that BA-mediated changes in glycolytic pathway promote cytotoxic autophagy and apoptosis in phenotypically different breast cancer cells. 34 Furthermore, BA proved to possess immunomodulatory activity by producing pro-inflammatory cytokines and activation of macrophages. 35 Therefore, the other anti-tumour mechanisms of BA should be further investigated in future.
In addition, the migration, invasion and EMT process of cancer breast cancer metastasis by targeting GRP78-mediated glycolysis and ER stress apoptotic pathway. 36 Similarly, it is reported that BA inhibits stemness and EMT of pancreatic cancer cells via activation of AMPK signalling. 37 Furthermore, Nuclear Factor-κB is responsible for antiproliferation and anti-migration of BA in Urothelial Tumorigenesis. 38 In conclusion, we assessed the anti-tumour effect of BA on human GC cells by conducting in vitro and in vivo experiments and found that BA induced apoptosis and suppressed the migration and invasion of GC cells. Overall, the present study shows that BA might serve as an anti-tumour drug for GC therapy.

ETHICS STATEMENT
All of the animal experiments in this study were performed according to the National Institutes of Health (Bethesda, MD, USA) guidelines and were approved by the Ethical Committee of First Affiliated Hospital of Gannan Medical College (Ganzhou, China).

CONFLICT OF INTEREST
The authors declare no conflicts of interest.

Yun Chen and Yun Zhou designed the experiments; Xiongjian Wu and
Chi Liu were involved in performing the designed experiments; Chi Liu and Yun Zhou were responsible for analysing the data and writing and approving the manuscript.

DATA AVAILABILITY STATEMENT
The data sets used or analyzed in this study are available from the corresponding author on reasonable request.