Investigation of anti-proliferative effects of cucurbitacin B and cisplatin on the cell death pathways in bladder cancer

Bladder cancer is a malignancy that is responsible for a significant proportion of cancer deaths around the world. Cisplatin (Cis)-based chemotherapy is the standard regimen for bladder cancer patients, but its effectiveness is limited by high toxicity and the development of drug resistance. It has been reported in many studies that Cucurbitacin B (CuB) has anti-carcinogenic effects by stimulating apoptosis and autophagy. Here we explored the potential role of CuB on MB49 bladder syngeneic mouse tumor model. Single and combined doses of CuB and Cis were applied to MB49 cell line and the cell viability was determined by WST-1 method. After the tumor model was established, the mice were divided into four groups and administered CuB and cis at the indicated doses and times.The expression levels of apoptosis (Bcl-2, Bax, Caspase-3, cleaved Caspase-3) and autophagy proteins (Beclin-1 and LC3I, LC3II) were detected by Western Blot. Phospho-protein array analysis was performed to determine the relative levels of phosphorylation of proteins which are associated with the PI3K-Akt signaling pathway. After agent applications, tumor tissues and organs were analyzed by staining hematoxylin-eosin. Our findings showed that CuB inhibits Bcl-2 expression and increases the expression of Bax and cleaved caspase 3. LC3II is markedly up-regulated in CuB-treated cells. CuB reduced the phosphorylation of p27, PRAS40, and Raf-1 proteins. CuB and Cis combination synergistically decreased phosphorylation of AKT, ERK1/ERK2, mTOR, BAD levels and increased the level of AMPKα. PI3K/AKT/mTOR has been found to be a signaling pathway that is one of the targets of CuB. The combination of CuB and Cis significantly reduced tumor growth and also had no toxic effects on the lung, liver, kidney, heart and bladder. Our results show that CuB may be a new agent that can support traditional treatment for bladder cancer. Our study is important in enlightening new ways in the treatment of bladder cancer and developing new treatment methods.