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.
Highlights
Bladder cancer, known as bladder urinary tract carcinoma, is one of the most commonly diagnosed malignancies [1,2]
Betulinic acid (BA) does not affect cell growth in normal cell lines including RAW 264.7 immortalized mouse macrophages and C2C12 immortalized mouse myoblasts (Figure 1D). These results suggest that BA has more potential effect on the suppression of cell proliferation of human bladder carcinoma cells than normal cells
We found that the suppression of cell proliferation following BA treatment was involved in cell death, including apoptosis and necrosis, which is accompanied by sub-G1 phase and partial gap 2/mitosis (G2/M) phase arrest it may differ slightly depending on the cell lines
Summary
Known as bladder urinary tract carcinoma, is one of the most commonly diagnosed malignancies [1,2]. Radical cystectomy is the standard therapy for bladder cancer, and various strategies such as radiation therapy, immunotherapy, chemotherapy, and their combination therapy are widely used in the clinic to treat bladder cancer [6,7]. Drugs, such as doxorubicin, cisplatin, vincristine, and methotrexate are used for bladder cancer chemotherapy, but they have serious side effects and are ineffective or less tolerated [8,9]. There is an urgent need for developing new therapeutic agents with low side effects and high efficiency for treating bladder cancer
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