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

Abstract

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.

Keywords: apoptosis; betulinic acid; epithelial-mesenchymal transition; gastric cancer; migration.

FIGURE 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

FIGURE 2

BA induces apoptosis in gastric cancer cells. A, Apoptosis of SNU‐16 cells treated with different concentrations of BA was analysed by Hoechst33258 staining. B, Apoptosis of SNU‐16 cells treated with different concentrations of BA was analysed by Annexin V/PI dual staining. Significant differences were indicated as *P ≤ .05; **P ≤ .01; ***P ≤ .001. BA, betulinic acid

FIGURE 3

BA suppresses migration and invasion in gastric cancer cells. A and B, Wound healing assay was used to evaluate migration of SNU‐16 cells after treatment with different concentrations of BA. C and D, Transwell migration and invasion assay were exploited to assess migration and invasion of SNU‐16 cells after treatment with different concentrations of BA. E and F, The variation of epithelial‐mesenchymal transition associated metastatic proteins in SNU‐16 cells were analysed by western‐blot after treatment with different concentrations of BA. Significant differences were indicated as *P ≤ .05; **P ≤ .01; ***P ≤ .001. BA, betulinic acid

FIGURE 4

Betulinic acid inhibits gastric cancer cells growth and pulmonary metastasis in vivo. A, Representative images of tumour in different groups after different treatment at 21 days. B, Curve of tumour growth in the process of treatment. C, Tumour weight in different groups after different treatment at 21 days. D, Number of metastasis nodules in lungs of different treated groups. E, The weight of lungs from different treated groups. F, H&E analysis of lung sections of different treated groups. Significant differences were indicated as *P ≤ .05; **P ≤ .01; ***P ≤ .001

FIGURE 5

Betulinic acid inhibits proliferation and metastasis in gastric cancer cells tumour. A, Immunohistochemical analysis of Ki‐67 and MMP‐2 of tumour sections from different treated groups. B, Statistical analysis of immunohistochemical results of Ki‐67 and MMP‐2 of tumour sections from different treated groups. Significant differences were indicated as *P ≤ .05; **P ≤ .01; ***P ≤ .001