1振興醫療財團法人振興醫院 泌尿外科；2國立台灣海洋大學 生物科技研究所
Ceramide promotes TNF-α-induced cell death via decreasing AKT activity in bladder cancer cell
Hsiao-Hsien Wang1, Yu-Chi Tsou2, Shye-Jye. Tang2
1Section of Urology, Cheng-Hsin Rehabilitation Medical Center, Taipei; 2Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung
Purpose: Bladder cancer, a malignant urinary system tumor, is in the ninth position of the international cancer charts. The cancer shows a highly recurrent rate that is around 50%~60%. Therefore, investigating a new therapy to improve the curing rate of the cancer is important. Nowadays, Bacillus- Calmette-Guerin (BCG) immune therapy is the most common way for the treatment of bladder cancer. However, there are around 20 % patients unable to get any benefit from this, and the main mechanism of the effect of BCG is still unclear. Since BCG treatment have reported, that macrophages might be recruited into the bladder to induce cancer cell death by the pro-inflammatory response, we further study the mechanism of BCG. We used lipopolysaccharide to induce macrophage Raw264.7 for the generation of condition medium (CM-LPS). Our results demonstrated that CM-LPS might cause cell death via a caspase-dependent manner in MBT2 bladder cancer cells. As compared with CM-LPS, TNF-α involved the cell death. Moreover, TNF-α-induced AKT activation was found in MBT2 cell, implying that the activating AKT may have anti-apoptotic activity. Since ceramide is generated after inflammation, we propose that ceramide may increase TNF-α-induced cell death in bladder cancer.
Materials and Methods: MBT-2 cell were treated with different concentrations of TNF-α, ceramide, and then the cell death were analyzed with MTS analysis and Hoechst3334.2 staining for chromatin condensation. The mechanisms of the cell death of TNF-α combining with ceramide were investigated by using western blotting to detect AKT activation.
Results: In our study exhibits that TNF-α had slightly to induce MBT2 cell death. Moreover, using western blot to analyze AKT expression in bladder cell were exhibited that ceramide decrease AKT activation. In our study, ceramide reduced AKT activity, promoted mitochondrial disruption and dephosphorylated Bad, a BH3 containing pro-apoptotic protein. TNF-α combining with ceramide exhibited chromatin condensation and DNA fragmentation by Hochest33342 staining assay. Our findings suggest that CM-LPS has the cytotoxic activity via ceramide and TNF-α to elicit cell death in MBT2 cell.
Conclusions: In this study, we suggest that ceramide were promoted TNF-α-induced cell death via decreasing AKT activity in bladder cancer cell. These results are demonstrated that ceramide-mediated AKT inactivation may play an important role in BCG--induced cell death in MBT2 bladder cancer cells. Our findings suggest that improvement of bladder cancer therapy will be able to decrease the activation of AKT.