| 研究実績の概要 |
The objective was to determine the effects of chemical agents that modify intracellular redox balance and ER stress mediated induction of distinct cell death types in alone or combined with radiation.It was found that CDDO-Me decreases cell viability in HCT-116 colorectal cancer cells. CDDO-Me induced extensive cytoplasmic vacuolation, which was triggered by CDDO-Me induced ROS generation. As pre-treatment with an antioxidant NAC alleviated vacuolation-mediated death. Mechanistic investigation revealed that cytoplasmic vacuolation was brought out by lack of caspase activation or PARP cleavage, and DNA fragmentation. In addition, CDDO-Me persistently induces the expression of autophagy marker LC3-II, along with endoplasmic (ER) stress markers, Bip and CHOP. However, in contrast failure of both apoptosis- and autophagy- inhibitors to reversed cytoplasmic vacuolation, suggest that vacuolation-mediated cell death is different from classical apoptotic and autophagic cell death. Cycloheximide treatment prevent CDDO-Me vacuolation and suggest the induction of paraptosis. Intrestingly, it was also found that these effects are concentration and cell death type dependent, as CDDO-Me induces apoptosis not paraptosis in HCT-15 colorectal cancer cells. In addition, in HeLa cells it was confirmed that combined used of CDDO-Me and glycolysis inhibitors enhanced cell death. Similarly, it was also confirmed that the combined used of romidepsin (FK228), a novel histone deacytylase inhibitor and hyperthermia also enhanced cell death in U937 and MOLT-4 cells.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Both the apoptosis and autophagy inhibitors fail to reverse the cytoplasmic vacuolation and it was found that cycloheximide prevents the cytoplasmic vacuolation, which confirms the involvement of paraptosis. NAC pre-treatment also protects the vacuolation mediated death and the expression of ER stress markers Bip and CHOP showed that intracellular redox modification and ER stress was involved in paraptosis by CDDO-Me. Research is progressing smoothly, in line with the purpose of research.
|
| 今後の研究の推進方策 |
In continuation of last year research, the expression of specific markers associated with paraptosis cell death will be examined by western blot and immunofluorescence imaging methods. Further, its confirmation will be done using positive marker for paraptosis. The involvement of calcium, ER stress and unfolded protein response in paraptosis will be investigated by using specific siRNAS and inhibitors. Mainly the effects will be observed using human colorectal cancer HCT-116 cells but the comparative examination will be done using other colon cancer cell lines. Next, the enhancement of radiation-induced cancer cell death combined with drugs will be investigated. The aim is to elucidate the molecular mechanism for the enhancement of radiation-induced cancer cell death by modifying intracellular redox balance and ER stress. The results will be summarized and published as a research paper.
|
