Betulinic Acid Restricts Human Bladder Cancer Cell Proliferation In Vitro by Inducing Caspase-Dependent Cell Death and Cell Cycle Arrest, and Decreasing Metastatic Potential

Abstract

Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid and generally found in the bark of birch trees (Betula sp.). Although several studies have been reported that BA has diverse biological activities, including anti-tumor effects, the underlying anti-cancer mechanism in bladder cancer cells is still lacking. Therefore, this study aims to investigate the anti-proliferative effect of BA in human bladder cancer cell lines T-24, UMUC-3, and 5637, and identify the underlying mechanism. Our results showed that BA induced cell death in bladder cancer cells and that are accompanied by apoptosis, necrosis, and cell cycle arrest. Furthermore, BA decreased the expression of cell cycle regulators, such as cyclin B1, cyclin A, cyclin-dependent kinase (Cdk) 2, cell division cycle (Cdc) 2, and Cdc25c. In addition, BA-induced apoptosis was associated with mitochondrial dysfunction that is caused by loss of mitochondrial membrane potential, which led to the activation of mitochondrial-mediated intrinsic pathway. BA up-regulated the expression of Bcl-2-accociated X protein (Bax) and cleaved poly-ADP ribose polymerase (PARP), and subsequently activated caspase-3, -8, and -9. However, pre-treatment of pan-caspase inhibitor markedly suppressed BA-induced apoptosis. Meanwhile, BA did not affect the levels of intracellular reactive oxygen species (ROS), indicating BA-mediated apoptosis was ROS-independent. Furthermore, we found that BA suppressed the wound healing and invasion ability, and decreased the expression of Snail and Slug in T24 and 5637 cells, and matrix metalloproteinase (MMP)-9 in UMUC-3 cells. Taken together, this is the first study showing that BA suppresses the proliferation of human bladder cancer cells, which is due to induction of apoptosis, necrosis, and cell cycle arrest, and decrease of migration and invasion. Furthermore, BA-induced apoptosis is regulated by caspase-dependent and ROS-independent pathways, and these results provide the underlying anti-proliferative molecular mechanism of BA in human bladder cancer cells.

Potential roles and molecular mechanisms of phytochemicals against cancer

“…Betulinic acid decreases the levels of Snail, Slug and MMP-9 to inhibit the wound healing and invasion ability in bladder cancer cells. 86 Pristimerin, a triterpenoid isolated from celastrus and maytenus spp, reduces the expression of vimentin, MMP-2, F-actin, integrin β1, and Snail to suppress EMT-mediated H1299 cell migration and invasion, and it also suppresses cancer cell proliferation and drives cell apoptosis; 87 pristimerin inhibits the (Ephrin type-B receptor 4) EphB4/cdc42/N-WASP signals to promote ROS generation, cleavage of caspase-3/4/9, and expression of ER stress-associated proteins, thus consequently inducing mitochondrion-mediated intrinsic apoptosis and repressing cell viability, migration, and angiogenesis in conditionally reprogrammed patient-derived lung adenocarcinoma cells; 88 pristimerin could increase caspase-3/7 activity and reduce Bcl-2 expression to induce cell apoptosis. It also upregulates the expression of autophagy-associated proteins (p62 and LC3-II) as well as the unfolded protein response (UPR) to induce incomplete autophagy in breast cancer.…”

The inhibitory effect of betulinic acid on epithelial–mesenchymal transition pathway in renal cell carcinoma

“…On the other hand, it has been reported that betulinic acid impairs the metastatic capability of cancer cells by inhibition of EMT. Studies showed that betulinic acid repressed EMT by decreasing SNAIL, SLUG, Vimentin, and N-cadherin expression and by increasing E-cadherin expression in pancreatic [38], colorectal [39] and bladder cancer [40] cells. With our ndings, the inhibitory effect of betulinic acid on SDC-2 and its relationship with renal cancer were determined for the rst time in the literature.…”

Recent Advances Regarding the Molecular Mechanisms of Triterpenic Acids: A Review (Part II)

“…The cells blocked in the G2/M phase further underwent apoptosis, which was confirmed by characteristic modifications, such as the up-regulation of BAX, the activation of caspase-8, -9, -3 cascade, and an increase in cleaved PARP concentrations ( Figure 4 ). The decrease of Snail, Slug, and matrix metalloproteinase-9 (MMP-9), three factors involved in cell migration, was also observed, suggesting that BA could inhibit cell migration by a yet unexplored mechanism of action [ 43 ]. Contrary to these findings, Zhang et al found that apoptosis induction in EJ and T24 human bladder cell lines was abolished or attenuated when an antioxidant was used, thus suggesting that BA is able to induce apoptosis in a ROS-dependent manner.…”