Abstract
Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark, exhibits antitumor effects against solid malignancies and triggers autophagy and/or apoptosis in human cancer cells. Nonetheless, the relationship between autophagy and apoptosis and the potential modulatory actions of BA on autophagy-dependent bladder cancer cell death remain unclear. The present study showed that BA exposure significantly suppressed viability, proliferation, and migration of EJ and T24 human bladder cancer cells. These effects reflected caspase 3-mediated apoptosis and could be attenuated or abolished by inhibiting ROS production with N-acetyl-L-cysteine, inhibiting autophagy with chloroquine, or silencing ATG7 with targeted siRNA. BA-induced autophagy was evidenced by epifluorescence imaging of lentivirus-induced expression of mCherry-GFP-LC3B and increased expression of two autophagy-related proteins, LC3B-II and TEM. Moreover, enhanced AMPK phosphorylation and decreased mTOR and ULK-1 phosphorylation suggested BA activates autophagy via the AMPK/mTOR/ULK1 pathway. Accordingly, exposure to dorsomorphin (Compound C), an AMPK inhibitor, and AICAR, an AMPK activator, respectively inhibited and stimulated BA-induced autophagy in EJ and T24 cells. The effects of Bmi-1 overexpression in vitro and decreased Bmi-1 expression in BA-treated T24 cell xenografts in nude mice suggested that downregulation of Bmi-1 is the underlying mechanism in BA-mediated, autophagy-dependent apoptosis.
Highlights
Bladder cancer is the 2nd commonest malignancy of the urinary system and the 10th most frequent cancer globally [1]
Using in vitro and in vivo approaches, in this study we evaluated the effects of Betulinic acid (BA) on human bladder cancer cell proliferation and migration and examined the role of reactive oxygen species (ROS), the AMPK-mTOR-ULK1 cascade, and Bmi-1 in autophagy-dependent apoptosis triggered by BA exposure
EJ and T24 cells were exposed to different BA doses, stained with Annexin V-FITC/PI, and apoptosis rates determined by flow cytometry (Figure 2A)
Summary
Bladder cancer is the 2nd commonest malignancy of the urinary system and the 10th most frequent cancer globally [1]. The global cancer report by the International Agency for Research on Cancer estimated that 549,393 new bladder cancer cases and 199,922 deaths from bladder cancer occurred in 2018 [1]. Bladder cancer is mainly treated with surgery combined with postoperative bladder perfusion chemotherapy. Up to 50% of patients with non-muscle invasive bladder cancer relapse, whereas 15%-20% of those progress to muscle invasive bladder cancer, characterized by a high propensity for spread to other organs [2, 3]. There is a critical need of new bladder cancer treatments. BA induces cell death, autophagy, and apoptosis in various cancer and noncancer human cell lines [7,8,9]. BA suppresses www.aging-us.com human multiple myeloma growth by triggering reactive oxygen species (ROS) overproduction [10]
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