otein in AsPC-1 and Panc-1 cells had no impact on re- sponsiveness to HSP90 inhibition. We found that sorafenib, a multiple kinase inhibitor, could paradoxically up-regulate the phosphorylated 5 ? 6 JOURNAL OF SURGICAL RESEARCH: 2011 FIG. 4. Sorafenib Gemcitabine and7-AAG have antagonistic effects in AsPC-1 and Panc-1 cells. (A), (B) AsPC-1 and Panc-1 cells had similar responses to sorafenib, with IC 50 values of about1.82 m M. Sorafenib at 5 m M had a negligible impact on the sensitivity of AsPC-1 cells but greatly in- creased the resistance of Panc-1 cells to7-AAG, according to the IC 50 values. Bars, SD. The CI curves of sorafenib and7-AAG for (C) AsPC-1 cells and (D) Panc-1 cells

simulated with CalcuSyn software, showed antagonism, except at high concentrations in AsPC-1 cells. Red squares indicate actual measurements. (E), (F) p-HSP90 (T4/5) as a percentage of total HSP90 changed in a similar way in AsPC-1 and Panc-1 cells after7-AAG and sorafenib treatments, except at the highest concentration of7-AAG in AsPC-1 cells. (F) Signal intensities cal- culated from (E).?,7-AAG treatments; Gemcitabine Cancer sorafenib treatments. components in multiple kinase pathways. To conm our conclusion that the sensitivity of AsPC-1 and Panc-1 cells to HSP90 inhibitors was related with their intrinsic multiple kinase pathways, we determined the combinatorial effects of7-AAG and sorafenib on both AsPC-1 and Panc-1 cells. As expected, sorafenib, at a low concentration (5 m M), greatly increased the resis- tance of Panc-1 cells to7-AAG, according to the IC 50 values, but had a negligible impact on the sensitivity of AsPC-1 cells to7-AAG. The CIs of7-AAG and sor- afenib also indicated that they acted antagonistically in AsPC-1 and Panc-1 cells, except at high concentrations in AsPC-1 cells.

At these concentrations, combined7- AAG and sorafenib treatments markedly reduced total Akt levels in AsPC-1 cells, a likely explanation for the synergism of7-AAG and sorafenib in AsPC-1 cells. Thus, these data further demonstrate that HSP90 reg- ulates the kinase cascades differently in AsPC-1 cells than in Panc-1 cells and that this difference is responsi- ble for the different cytotoxicity of7-AAG in these two cell lines. Although the molecular mechanism underly- ing the different HSP90 functions in AsPC-1 and Panc-1 cells remains elusive at present, our research represents an important and signiant attempt in this area. Our data may be of clinical application for predicting the Gemcitabine 122111-03-9 patients?responses to HSP90 inhibition. ACKNOWLEDGMENTS The authors thank Yue Lu for help in preparing the manuscript. Yafang Li helped with some statistical analyses and the CI calcula- tion. Dr. Bingliang Fang kindly provided the positive control (H460/ TaxR) for the P-glycoprotein. The authors also thank Karen R. Muller, Kristine K.

Ash, and Yolanda Brittain for editing and submit- ting this manuscript. This work was supported by the Kanzius Cancer Research Foundation. SUPPLEMENTARY DATA Supplementary data related to this article can be found online at doi:0.1016/j.jss.2011.09.017 . REFERENCES 1. Blume-Jensen P, Hunter T. Oncogenic kinase signaling. Nature 2001;411:355. 2. Whitesell L, Mimnaugh EG, De Costa B, et al. Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: Essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci U S A994;91:8324. 6 In organocatalytic aza-Michael reactions, the acceptors are activated either by hydrogen bonding of the organocatalysts to the carbonyl group of the acceptors 9 or by imminium formation between R , β -unsaturated aldehydes and the orga- nocatalysts.0 Jørgensen reported the successful use of proline-derived organocatalysts in the addition of nitrogen- containing heterocycles such as triazoles and tetrazoles to R , β -unsaturated aldehydes.1 We envisioned that the exten- sion of Jørgensen’s chemistry1 to the aza-Michael addition of substituted pyrazoles6 or 20 to a