Investigation of possible anticancer effects of betulinic acid mediated by mTOR

Endometrial cancer is one of the most common gynecological cancers worldwide, and an average of 42,000 women die each year. Chemotherapy, radiotherapy, and surgery are among the treatments available for endometrial cancer. Currently, drugs used for chemotherapy have had limited success in increasing the cure rate. Betulinic acid, a lupane-type triterpene widely found in the plant kingdom, has attracted attention for cancer treatment in recent years due to its ability to inhibit tumor growth and induce cell apoptosis selectively. The aim of this study is to investigate the mTOR pathway-mediated anti-cancer effects of betulinic acid in human endometrial cancer cells. The effect of betulinic acid on Ishikawa cell viability was determined by the CCK-8 method, the effect on the expression of genes involved in apoptosis and mTOR pathway, real-time PCR, the effect on the expression of proteins in the mTOR pathway, immunohistochemistry, and Western blot, and the effects on apoptosis with Annexin V. The IC50 dose of betulinic acid in Ishikawa cells was determined as 50 µM at 48th hour. Betulinic acid administration caused a significant decrease in Bcl2 (P=0.008) expression and increased caspase 8 (P=0.001) expression in Ishikawa cells. The results of Annexin V supported that betulinic acid administration triggered apoptosis in Ishikawa cells. The mean rate of apoptotic cells in the betulinic acid group was 22±3.23% (P=0.02), while it was 2.31±0.2% in the control group. Betulinic acid caused a significant decrease in the expression of AKT1 (P=0.0001) and a significant increase in the expression of RAPTOR (P=0.00002). Betulinic acid administration also significantly decreased the expression of proteins involved in the mTOR pathway. The percentage of p-PI3K, p-AKT, and p-mTOR positive cells in Ishikawa cells was 89.39 ± 5.19%, 74.84% ± 5.07, and 82.02% ± 6.14 in the control group, respectively. In the betulinic acid group, these values were 49.12 ± 19.12%(P=0.002), 44.46 ± 7.39% (P<0.001), and 53.70 ± 8.94% (P<0.001), respectively. The results of this study showed that betulinic acid decreased cell proliferation and triggered apoptosis by targeting mTOR signaling pathway in Ishikawa cells, and betulinic acid may be a potential anti-cancer agent in the treatment of endometrial cancer. Keywords: Apoptosis, betulinic acid, Ishikawa, mTOR