YM155, which inhibits the anti-apoptotic protein survivin, is known to exert anti-tumor effects in various cancers, including prostate and lung cancer. of a previous report . Tang et al. reported that YM155 downregulates Mcl-1 in various malignancy cell types, but not in pancreatic cancer cells. This might reflect the cell line-specific responses of Rabbit Polyclonal to CRY1 pancreatic cancer cells to YM155. While YM155 induced a concentration-dependent decrease in Bid, p-Bad, and Bad levels in most pancreatic cancer cell lines, Bid was unaffected in BxPC-3 cells. These results indicate that YM155 affects apoptotic protein levels, a result that contrasts with previous reports . Previous study showed that a phosphorylation of EGFR was induced by ionizing radiation in a ligand-independent manner and ionizing radiation or cisplatin without EGF induced EGFR transport into the nucleus . They showed that the mechanism for radiation-induced EGFR import into the nucleus was associated with a karyopherin . However, we do not know the exact mechanism of nuclear translocation of EGFR by YM155 without EGF yet. Thus, further studies are needed to find out the mechanism by YM155 in nuclear translocation of EGFR. Liccardi et al. reported that nuclear translocation of EGFR is usually important in modulating the repair of DNA damage following chemotherapy . In this study, 10 nM YM155 induced nuclear translocation of EGFR and increased EGFR transcript levels. EGFR translocates to the nucleus, where EGFR might activate genes 1403764-72-6 associated with repair as a transcription factor . However, higher concentrations of YM155 (100 nM) reduced EGFR transcript levels and enhanced EGFR degradation. Therefore, increased transcription and translocation of EGFR at low concentrations (10 nM) of YM155 might protect cells from apoptosis, whereas high concentrations (100 nM) decrease cell survival by reducing EGFR transcription and increasing EGFR degradation. Levkowitz et al. reported that binding of EGF to EGFR causes EGFR degradation through binding with c-Cbl at the pY1045-EGFR . Ahsan et al. reported EGFR phosphorylation, ubiquitination and degradation in cisplatin-induced cytotoxicity . Pangbum et al reported that sulindac metabolite also induces the ubiquitination of EGFR . Similarly, we found that EGFR phosphorylation, and EGFR ubiquitination and degradation after treatment with YM155 were induced. However, additional research is usually needed to investigate At 1403764-72-6 the3 ubiquitin ligase to YM155. XIAP has been reported to induce the downregulation of survivin through XAF1 (XIAP associated factor 1) . XIAP has also been identified as a cofactor of survivin in the inhibition of apoptosis . Survivin released from mitochondria in response to apoptotic stimuli interacts with XIAP through an XIAP-binding site corresponding to Lys15-Met38, producing in increased XIAP stability against ubiquitination/proteasomal degradation and inhibition of apoptosis , . Phosphorylation of survivin in the cytoplasm inhibits the assembly of the survivinCXIAP complex, abolishing its anti-apoptotic function . Our results showed that the effect of YM155 on XIAP manifestation differed in the context of survivin knockdown. YM155 induced an increase in XIAP transcript levels and promoted XIAP protein degradation. YM155 decreased the conversation of survivin with XIAP, slightly enhanced ubiquitination of XIAP, and induced lysosomal degradation of XIAP. Therefore, YM155 affects the degradation of XIAP as well as survivin, and interferes with the assembly of the survivinCXIAP complex. The YM155-induced decrease in XIAP levels is usually unlikely due to a reduction in survivin levels. In this study, we did not examine phosphorylation of survivin by YM155 or investigate other factors that might affect the survivinCXIAP complex. Accordingly, additional in-depth mechanistic studies on YM155 modulation of XIAP should be performed. In conclusion, we found that YM155, known as a survivin inhibitor, promotes downregulation of PI3K, p-ERK, and p-STAT3 through degradation of EGFR in pancreatic cancer cells. Our data suggest that YM155 has therapeutic potential in pancreatic cancer and provide support for clinical trials of YM155 in this context. Materials and Methods Cell lines, compounds, plasmid, and antibodies The human pancreatic cancer cell lines PANC-1, MIAPaCa-2, and BxPC-3 were obtained from the American Type Culture Collection (Manassas, VA, USAA). YM155 1403764-72-6 was obtained from Hanmi Pharmaceuticals (Seoul, Korea). Other reagents used include LY294002 (Calbiochem, Darmstadt, Philippines), AZD6244 (Chemizon Korea), MG132 (Calbiochem), chloroquine (Sigma-Aldrich, St.