Liposome-Encapsulated Bacillus Calmette–Guérin Cell Wall Skeleton Enhances Antitumor Efficiency for Bladder Cancer In Vitro and In Vivo via Induction of AMP-Activated Protein Kinase

SIMPLE SUMMARY: We engineered novel nanoparticles consisting of liposome-encapsulated Bacillus Calmette–Guérin cell well skeleton (BCG-CWS) for intravesical instillation in bladder cancer. The liposome-encapsulated BCG-CWS nanoparticles had antitumoral effects in an orthotopic bladder cancer mouse model, and the BCG-CWS nanoparticles can be further developed as a non-toxic substitute for live BCG with improved dispensability, stability, and size compatibility. This is significant because we succeeded in the intravesical delivery of BCG-CWS through the intravesical route using a catheter in an orthotopic bladder cancer mouse model to specifically target tumor cells. This is the first study on the BCG-CWS-induced activation of AMPK in urothelial carcinoma cells, suggesting that AMPK-mediated reactive oxygen species (ROS) production and ER stress is a cellular signaling pathway in tumors sensitive to BCG-CWS. These results have the potential for significant ramifications in targeted therapy using a predictive marker for bladder cancer. ABSTRACT: The Mycobacterium Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS), the main immune active center of BCG, is a potent candidate non-infectious immunotherapeutic drug and an alternative to live BCG for use against urothelial carcinoma. However, its application in anticancer therapy is limited, as BCG-CWS tends to aggregate in both aqueous and non-aqueous solvents. To improve the internalization of BCG-CWS into bladder cancer cells without aggregation, BCG-CWS was nanoparticulated at a 180 nm size in methylene chloride and subsequently encapsulated with conventional liposomes (CWS-Nano-CL) using an emulsified lipid (LEEL) method. In vitro cell proliferation assays showed that CWS-Nano-CL was more effective at suppressing bladder cancer cell growth compared to nonenveloped BCG-CWS. In an orthotopic implantation model of luciferase-tagged MBT2 bladder cancer cells, encapsulated BCG-CWS nanoparticles could enhance the delivery of BCG-CWS into the bladder and suppress tumor growth. Treatment with CWS-Nano-CL induced the inhibition of the mammalian target of rapamycin (mTOR) pathway and the activation of AMP-activated protein kinase (AMPK) phosphorylation, leading to apoptosis, both in vitro and in vivo. Furthermore, the antitumor activity of CWS-Nano-CL was mediated predominantly by reactive oxygen species (ROS) generation and AMPK activation, which induced endoplasmic reticulum (ER) stress, followed by c-Jun N-terminal kinase (JNK) signaling-mediated apoptosis. Therefore, our data suggest that the intravesical instillation of liposome-encapsulated BCG-CWS nanoparticles can facilitate BCG-CW cellular endocytosis and provide a promising drug-delivery system as a therapeutic strategy for BCG-mediated bladder cancer treatment.