| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 100 === Lipid metabolism has been recognized for participation in membrane functions and signaling that control cellular activities. Ceramides are one of sphingoil lipid, hydrophobic molecule; composited by sphingosine with long or short chain fatty acid, and it has bioative to promote cell death and growth arrest. Cellular ceramides, long N-acyl chains ranging from 16 to 26 carbons in length depending on the different ceramide synthase isoform. Moreover, the short chain ceramide analogs: N-acetyl-sphingosine (C2-ceramide); N-hexanoylsphingosine (C6-ceramide) and N-octanoylsphingosine (C8-ceramide) are also used due to more membrane permeable.
Our results show that C2-ceramide induces cell death in MBT2 cell line, mouse bladder cancer cells, and HET293T cell line, transformed human cells. To identify whether ceramide-induced cell death is apoptosis or necrosis in MBT2 cells, the membrane potential was measured by flowcytometry with Rhodamine 123 staining. This result shows that ceramide induce mitochondrial dysfunction. To recognize the role of mitochondrial dysfunction, RNA expressions level was measured by RT-PCR. The results show that the Bad, pro-apoptotic Bcl family, mRNA level increase and anti-apoptotic Bcl family such as Bcl-xL and Bik mRNA level decrease after treatment of ceramide. Moreover, ROS was detected after mitochondrial dysfunction measured by flowcytometry with H2DCFDA staining. These results prove that ceramide-induced mitochondrial dysfunction regulated by Bcl family; however, our result shows that the caspase inhibitor can't inhibit ceramide-induced cell death. According the results, we suggest that the mechanism of ceramide-induced cell death is caspase-independent manner in MBT2 cell lines. Although the roles of ceramide-related Bcl family regulation are still not fully understand, recent studies show that ceramide can induce cellular starvation because ceramide decrease cellular nutrient receptor and autophagy causing severe metabolic stress. According these studies, we suggest that ceramide-induced cell death may be related by energy metabolism which correlates AMPK activity.
AMPK is serine-theronine kinase, thought as energy sensor, and keep homeostasis. Our data shows that compound C, AMPK inhibitor, can inhibit ceramide-induced cell death. Although we suggest activate AMPK without ceramide may also induced cell death, the data shows that AICAR, AMPK activator, activate AMPK without ceramide by can't induce cell death. To investigate the mechanism that compound C inhibit ceramide-induced cell death, we detected autophagy in cells by Acridine Orange staining. The result shows that compound C induce autophagy predominately which may explain the effect of compound C which inhibit ceramide-induced cell death. Moreover, Hydroxychloroquine, autophagy inhibitor, can mask the effect of compound C causing ceramide-induced cell death which result supports our suggestion.
In conclusion, our study shows that ceramide can also induce cell death in MBT2 cell line by regulating Bcl family causing mitochondrial dysfunction in caspase-independent apoptosis manner. Our findings show that AMPK activity is essential but not enough for induce ceramide-induced cell death. While autophagy rescues ceramide-induced cell death since AMPK activity is inhibited by compound C.
|
|---|
