The so similar result was also confirmed in GIST 882 cells. ATRA affected on expression of survivin, XIAP and Bax protein It is well known Inhibitors,Modulators,Libraries that apoptotic process is regulated by many factors. We investigated the expression of inhibitors of apoptosis, survivin, XIAP, and pro apoptosis Bax. The results showed down regulation of survivin and up regulation of Bax. These results were consistent with the appearance of cleaved caspase 3 and PARP in GIST T1 cells. However, ATRA did not affect on XIAP expression in GIST T1 cells by western blot analysis. All together, the apoptosis induced by ATRA treatment may be regulated at least by down regulation of survivin and up regulation of Bax proteins. ATRA suppressed the phosphorylation of KIT protein KIT protein is one of the most important molecules in the pathogenesis of GISTs.
Despite Inhibitors,Modulators,Libraries clinicopathological difference, most GISTs have a similar genetic profile, gain of function mutations of KIT or PDGFRA. Upon the importance of KIT protein, we examined whether ATRA can suppress KIT activity in GIST T1 cells. We treated GIST T1 cells with 180 uM ATRA for the indicated duration. Total cell lysates were subjected to western blot analysis. Inhibitors,Modulators,Libraries Interestingly, ATRA treatment resulted in suppression of KIT activity after 4 day treatment in GIST T1 cells and GIST 882 cells. The suppression of KIT activity in GIST T1 and GIST 882 cells by ATRA required longer time compared with other reagents such as imatinib or EGCG. In addition, ATRA treatment also suppressed the AKT activity but not MAPK activ ity in GIST T1 cells.
Interestingly, the suppression of KIT and AKT activity by ATRA treatment was enhanced in serum free media. However, suppression of MAPK activity was not observed even in serum free media. The similar results were observed in GIST 882 cells. ATRA prevented the migration of GIST T1 cells Next, to study the migration of GIST T1 cells in vitro, the scratch assay Inhibitors,Modulators,Libraries was performed. This method is based on the observation that, upon creation of a new artificial gap, so called a scratch on a confluent cell monolayer, the cell on the edge of the newly created gap will move toward the opening to close the scratch until cell to cell contacts are established again. In this study, GIST T1 cells were seeded with Inhibitors,Modulators,Libraries or without ATRA in plates. After 24 hour incubation to get the confluence, a scratch was created.
The images of GIST T1 cells at the beginning selleck products and 24 hour later were compared to assess the migration of GIST T1 cells. The result revealed that 90 uM ATRA inhibited completely migration of GIST T1 cells compared with the non ATRA treated dishes. However, at a lower concentra tion, ATRA inhibited but not completely the migration of these cells. All together, the data suggested that ATRA may be useful to prevent the invasion or metastasis of GIST cells. Cytotoxic effect of combination with ATRA and imatinib The result of isobologram was showed in Figure 5B.