As mentioned above, ATRA not only induced

the morphologic change (rounded-up cells) in GIST-T1 cells Selleckchem BTK inhibitor after 3-day treatment, but also induced detachment of the cells from the dishes after 6-day treatment (data not shown). To check whether detached cells show the features of apoptosis, cells were collected and fixed onto slides by using a cytospin before performing Wright-Giemsa staining. The result showed that detached cells showed shrunk and fragmented nuclei, the apoptotic features, compared with control cells (Figure 2A right), the fragmented nuclei were confirmed by DNA fragmentation assay (Figure 2B). As expected, DNA fragmentation was observed after 2-day treatment and increased in a time dependent manner. Figure 2 ATRA induces apoptotic cell death in GIST-T1

cells. Panel A shows the shrinkage and fragmentation of nuclei in GIST-T1 cells after 6-day treatment with 180 μM ATRA compared with the control cells. Panel B shows the result of DNA fragmentation after 2-, 4- or 6-day treatment with 180 μM ATRA. Panel C shows the presence of cleaved caspase-3 and cleaved PARP after 2-, 4- or 6-day treatment with 180 μM ATRA. Moreover, to clearly demonstrate that ATRA causes apoptosis in GIST-T1 cells, we assessed the molecular aspects of apoptosis, such as caspase-3, well recognized as a marker of apoptosis, and PARP, considered as a biochemical marker of necrosis

selleck when it is hyperactivated [27], by western blot. After 2-day treatment with 180 μM ATRA, cleaved Cediranib (AZD2171) caspase-3 and PARP were observed (Figure 2C). This result is consistent with the data of DNA fragmentation, demonstrating that ATRA induced apoptosis in GIST-T1 cells. Overall, our results demonstrated that ATRA induced apoptotic cell death in GIST-T1 cells. The similar result was also confirmed in GIST-882 cells (data not shown). ATRA affected on expression of survivin, XIAP and Bax protein It is well known 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 (Figure 3A) and up-regulation of Bax (Figure 3B). These results were consistent with the appearance of cleaved caspase-3 and PARP in GIST-T1 cells (Figure 2C). However, ATRA did not affect on XIAP expression in GIST-T1 cells by western blot analysis (Figure 3C). All together, the apoptosis induced by ATRA treatment may be regulated at least by down-regulation of survivin and up-regulation of Bax proteins. Figure 3 ATRA affects on the expression of survivin and Bax. Panel A shows the down-regulated expression of survivin after 2-, 4- or 6-day treatment with 180 μM ATRA. Panel B shows the up-regulated expression of Bax after 2-, 4- or 6-day treatment with 180 μM ATRA.