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.

Young Mi Whang,Da Hyeon Yoon,Hoyub Yoon,Young Wook Choi,In Ho Chang,Serk In Park,Gwang Yong Hwang

doi:10.3390/cancers12123679

Abstract

Simple SummaryWe 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.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.

Highlights

Urothelial carcinoma is more prevalent in the United States and other Western countries than in Asian countries, the incidence of bladder cancer in Korea has increased two-fold from101,032 cases in 1999 to 218,017 cases in 2011 [1]
We recently reported an intracellular delivery system for Bacillus Calmette–Guérin (BCG) cell wall skeleton (BCG-CWS) involving liposome-encapsulated BCG-CWS nanoparticles that circumvented the limitations of the intravesical instillation of BCG-CWS in bladder cancer [12]
Our findings show that the antitumor activity of BCG-CWS induces reactive oxygen species (ROS) production and AMPK activation, which elicits Jun N-terminal kinase (JNK) signaling-mediated apoptosis, owing to the stimulation of the endoplasmic reticulum (ER) stress pathway such as the phosphorylation of both inositol-requiring enzyme 1α (IRE1α) and protease-activated receptor-4 (PAR-4)

Summary

Introduction

101,032 cases in 1999 to 218,017 cases in 2011 [1] With this increase in bladder cancer incidence, there is an increased need for an effective drug-delivery system targeting non-muscle-invasive bladder cancer (NMIBC); the intravesical instillation of Bacillus Calmette–Guérin (BCG) is one such promising system. The intravesical use of live BCG can induce side effects due to infections, even if these events are reported to have a lower than 5% incidence [4,5]. To apply intravesical BCG-CWS instillation into the bladders of animals and humans, the development of an appropriate delivery system has been required, as its large size and insolubility in both aqueous and organic solvents limit its use [10]

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Conclusion